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Toxicology SOP - Houston Forensic Science

1. 5 2 GC MS Agilent 7890A Initial Temperature 80 C Hold for 0 5 minutes 30 C min to 290 C Hold for 5 minutes Total Run Time 12 5 minutes 5 3 Wash solvents for autosampler Methanol and ethyl acetate are used as the wash solvents A minimum of 6 pre and 6 post rinses are performed Each rinse cycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 6 EXTRACTION PROCEDURE 6 1 Sonication 15 minutes and centrifugation 15 minutes at 4 000 rpm is permitted for biological samples such as blood tissue homogenates urine or controls as needed 6 2 BLOOD 6 2 1 In around bottom glass centrifuge tube add 1 mL blood and 25 uL Internal Standard Solution 10 ng uL while vortex mixing Final concentration of internal standard in each sample is 250 ng mL NOTE IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALL VOLUME ADD BUFFER AS THE DILUENT AND DOCUMENT IN THE CASE FOLDER 6 2 2 Fortify all controls and calibrators while vortex mixing using the appropriate amount of working standard The typical calibration range for quantitative analysis is 20 1000 ng mL Refer to the fortification guide for guidance on preparation of calibrators CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS An in house control Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 136 of 171 6 2 3 6
2. Evidence that has been outsourced for testing and has been returned must be verified and documented prior to being sealed and returned to the submitting agency This is accomplished by Verifying correct evidence case number Adding a barcode label or manually transferring evidence custody in LIMS Document receiving of evidence through chain of custody in LIMS Photographing evidence Sealing evidence 8 RETURNING OF EVIDENCE 8 1 All submitted items will be returned to submitting agency 8 2 Before evidence is sealed the contents will be checked for proper labeling and one or more pictures will be taken of the tested specimen to record the initials of analysts having opened the container to conduct analysis 8 3 Outer evidence containers will be properly sealed and labeled with initials of the individual placing the seal on the item and date the seal was placed before returning evidence to the submitting agency A part of the initials or date must extend over the edge of the seal onto the container Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 8 of 171 Houston Forensic Science Center ih Y Forensic Analysis Division Toxicology Technical and Administrative Review 1 PURPOSE This document outlines the technical and administrative review process for batch analysis of human toxicology specimens 2 SCOPE This procedure is used to manage th
3. Forensic Analysis Division Toxicology 5 4 1 2 In Process stability if it is expected that a break may need to take place during sample preparation the stability of the analyte over that time period should be evaluated 5 4 1 2 1 Determine the point in the procedure at which a break may occur and how long the break may be In the case of procedures where solid phase or liquid extractions are performed the studied step should be the final extraction or elution solvent 5 4 1 2 2 Prepare the cutoff calibrator negative control and positive control according to the SOP 5 4 1 2 3 Prepare a second set of positive controls in duplicate but delay completion of preparation at the predetermined step and for the correct time 5 4 1 3 Auto sampler stability Both the analyte s and the internal standard in processed QC samples will be tested for stability This will demonstrate that extracts are stable while in the auto sampler awaiting analysis In addition determination of stability beyond 12 hours will allow for the re injection of samples in the event of instrument malfunction 5 4 1 4 Determine the maximum residence time of the extracts in the auto sampler to be tested Include the time a run may sit unanalyzed over a weekend in the auto sampler due to an instrument malfunction A different approach may be necessary for unstable molecules 5 4 1 5 Prepare a cutoff calibrator curve along with at least 4 positive controls and 1 nega
4. Wash column with 1 mL hexane Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 113 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 6 12 Wash column with 1 mL ethyl acetate 6 13 Wash column with 1 mL methanol 6 14 Dry column on full vacuum pressure for 5 minutes 6 15 Replace waste tubes reservoir with conical collection tubes If the Teflon inserts vacuum manifold only appear dirty replace them 6 16 Elute basic drugs with 1 mL elution solvent 2 ammonium hydroxide in methylene chloride isopropyl alcohol 80 20 ELUTION SOLVENT MUST BE MADE FRESH 6 17 Turn on vacuum or apply positive pressure for a few seconds to ensure all elution solvent has drained from the column 6 18 Evaporate to dryness under nitrogen for approximately 10 minutes at 50 60 C 6 19 Reconstitute in 30uL ethyl acetate or appropriate volume vortex and transfer to autosampler vials 6 20 Inject 2 uL onto the GC MS using METHADONE EDDP M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 114 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 7 INSTRUMENTAL ANALYSIS 7 1 Ensure that the daily QC and tune verification have been completed 7 2 SIM acquisition Methadone EDDP M Retention Time RT v
5. 4 9 2 Data Analysis 4 9 2 1 Use the established calibration curve to calculate the concentration of the analyte of interest 4 9 3 Acceptance Criteria 4 9 3 1 All storage stability samples must quantify within control acceptance criteria typically 20 of the method compared to the average concentration on Day O 4 9 3 2 All in process and auto sampler stability samples must quantify within control acceptance criteria of the average value obtained in the Bias and Precision studies Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 163 of 171 Houston Forensic Science Center j IP 4 Forensic Analysis Division Toxicology Validation of Reportable Qualitative Analytical Methods 1 PURPOSE This procedure is intended to define the minimum parameters and sets of experiments to validate a reportable qualitative method 2 SCOPE This procedure applies to all qualitative bio analytical methods which produce reportable results Method validation is required to verify the performance parameters of a method are fit for purpose Validation is required after the following events occur 2 1 Development of a new method 2 2 Modification of a validated method to improve its performance or extend its use beyond that for which it was originally validated 2 3 Transfer of a validated method to a new instrument Modifications to existing methods or transfer of a va
6. Check the foreline pump oil level 3 1 2 1 1 4 Monthly Check the o ring and change as needed 3 1 2 1 1 5 Every six months to one year 312 1151 Replace the foreline pump oil 3 1 2 1 1 5 2 Check calibration vial and refill PFTBA as necessary S 1221 1 5 5 Check diffusion pump oil and replace if necessary 3 1 2 1 1 6 As needed depending upon instrument and sample throughput 3 1 2 1 1 6 1 Change the septum 3 1 2 1 1 6 2 Check and replace the inlet liner 3 1 2 1 1 6 3 Check and replace the gold seal 3 1 2 1 1 6 4 Clip the column 3 1 2 1 1 6 5 Replace Switch filament s 3 1 2 1 1 6 6 Replace gas cylinders 3 1 2 1 1 6 7 Clean the ion source 3 1 3 Methods Electronic backups of the methods and data files are recommended for each instrument An electronic copy of the method is located in the Instrument Method Folder or equivalent Methods are updated regularly following routine instrument maintenance It is recommended that the examiner report initial and date any updated methods 3 1 4 Sample Preparation and Sequence Set up 3 1 4 1 Samples should be prepared for analysis according to the section s standards or specific SOP 3 1 4 2 The data file path must clearly identify the location and storage of the data The convention for the data file storage should include the date and name of the examiner 3 1 4 3 Retain GC MS analysis data in the case record 4 PIPETTES 4 1 Method of Use Refer to appropriate manuals for proper handling and use Vers
7. Quantitative results are determined using drug standards of known concentration Quantitative analyses are not performed on urine samples For blood samples drug free blood is fortified with the drugs of interest Limits of detection for selected basic acidic and neutral drugs can be found in the respective procedure s validation 9 QUANTITATIVE ANALYSIS 9 1 Specific targeted quantitation procedures utilizing selected ion monitoring SIM and deuterated internal standards are available for many commonly encountered drugs 9 2 Mepivacaine pentobarbital d5 and prazepam are used as internal standards for full scan acquisition Other internal standards can be used for the quantitation of other basic acidic or neutral drugs using either full scan or SIM analysis Deuterated internal standards are preferred if available Refer to the drug inventory for a current list of deuterated internal standards 9 3 If selected ion monitoring SIM is used refer to the appropriate Method File for the GC MS For SIM all three characteristic ions must be present lon intensity and ratios should be taken into consideration lon ratios should be within 20 of the appropriate calibrator or QC If ion ratios are out of range quantitative data may be reported if the ratios are within 20 of the nearest calibrator or QC However ions of low intensity may not be within this range for all case samples These are evaluated on a case by case basis and are subject
8. Toxicology Analytical Manual Forensic Analysis Division Houston Forensic Science Center Forensic Analysis Division Toxicology ENAA EEN EE E A EEEE ee ee eee E EA EE AEE E rer ere rere 4 E E A A E A 4 PEENE E PEI OES ETA E EENT I E EEE OE E E E T 5 Technical and Administrative Review cccccccsssssseccccccceansseeecceceeseuesseeececeessaaeusseeeeeeessaaaaseseceesessaaaaesss 9 Preparation of Drug Free Matrix ccccccccssccccssseccceesececeeecceeeesececeeececeeneceeeeuecceseusecessuecesseneceesaneeeetas 11 Verification of Relative Concentrations of Stock Standards cccccccccccccceseseseeeeceeeessaeeseeeeeeeeeeaaas 13 Preparation and Validation of Drug Standards Spiking Solutions Controls and Reagents 14 In Process Calibration and Quality Control for Drug Screening Confirmation Testing 00 19 General Guidelines for Instruments and Equipment cccccccsssccceessececeesececeeeeceeeeusecessuaeceesenecessenes 21 Cleaning and Deactivating GC MS Injection Liners Silanizing Glassware 27 Operation and Maintenance of Zymark Turbo Vap LV cccccccssecccceeseceeeenecceceeeceeeeeseceseegeceeeeeeeeeeas 29 Operation and Maintenance of CEREX Pressure Processors ccccccesecccesececeesceeecseeececeeeceeeneeseeneeees 31 Operation and Maintenance of the TECAN Freedom Evo 75 cccccccsecccsecceeseceeeeceeeeeceseeecesees
9. 2015 Uncontrolled When Printed Page 146 of 171 9 Houston Forensic Science Center Forensic Analysis Division Toxicology FLOWCHART FOR ZOLPIDEM ANALYSIS To 1 mL specimen add 100 uL I S 1 ng L while vortex mixing VL Fortify calibrators controls according to fortification guide while vortex mixing NV Add 2 mL 100 mM sodium phosphate buffer Vortex 7 Centrifuge samples for at least 10 minutes at 4000 rpm 7 Add sample to Polychrom Clin II SPE column Use sufficient vacuum pressure to draw sample through column VY Wash 1 mL deionized water 7 Wash 1 mL 1 M acetic acid VL Dry column on full vacuum pressure for 5 minutes VL Wash 1 mL hexane VY Wash 1 mL ethyl acetate Vy Wash 1 mL methanol Vv Dry column on full vacuum pressure for 5 minutes N7 Elute Basic Drugs with 1 mL methylene chloride isopropyl alcohol 80 20 with 2 ammonium hydroxide PREPARE FRESH NY Evaporate to dryness under nitrogen at 50 60 C for 10 minutes NY Reconstitute in 30 uL ethyl acetate Transfer to GC vial and Inject 2 uL onto GC MS using ZOLPIDEM M Version 2 0 Issue Date August 3 2015 Page 147 of 171 Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed Houston Forensic Science Center Forensic Analysis Division Toxicology Volume of Blood Target Concentration Working Standard Volume Added uL mL ng mL Concentration ng uL 11 LITERATURE AND SUPPORTING DOCU
10. 80 20 with 2 ammonium hydroxide PREPARE FRESH NY Evaporate to dryness under nitrogen at 50 60 C for 10 minutes NY Reconstitute in 30 uL ethyl acetate Transfer to GC vial and inject 2 uL onto GC MS using PCP M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 133 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 18 LITERATURE AND SUPPORTING DOCUMENTATION 18 1 Kunsman G W et al Phencyclidine Blood Concentrations in DRE Cases J Anal Toxicol 1997 October Vol 21 pp 498 502 18 2 Grieshaber A et al Stability of Phencyclidine in Stored Blood Samples J Anal Toxicol 1998 October Vol 22 pp 515 519 18 3 Pestaner J P et al Sudden Death During Arrest and Phencyclidine Intoxication American J Forensic Medicine and Pathology 2003 June Vol 24 No 2 pp 119 122 18 4 R C Baselt Phencyclidine Disposition of Toxic Drugs and Chemicals in Man 8th edition Biomedical Publications Foster City CA pp 1223 1226 18 5 Method file PCP M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 134 of 171 Houston Forensic Science Center j oP 4 Forensic Analysis Division Toxicology Stimulants Confirmation by Gas Chromatography Mass Spectrometry GC MS 1 PURPOSE A targeted analysis is per
11. All evidence must be protected from loss cross transfer contamination or deleterious change 5 1 Upon receipt of evidence into the Toxicology Section 5 1 1 Toxicology personnel will examine evidence container s to ensure that proper seals which are further detailed in the Quality Manual are in place 5 1 2 All evidence transfers must be documented electronically as part of the chain of custody and include comments pertaining to any evidence processing excluding storage If needed a paper chain of custody will suffice using the HFSC Chain of Custody Form 513 The outer most container e g a bag envelope or box containing specimens for a case must be marked with a unique case identifier This may be accomplished by adding a LIMS barcode label associated with the evidence to the container 5 1 4 A submission form or an electronic equivalent must be filled out for all submitted evidence and contain the following information agency case number subject name s and description s of evidence If date s of birth is used as an identifier on the evidence the date of birth must be included in the submission information If any of the above information is missing the evidence will be returned and a rejection report will be issued Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 50f171 Houston Forensic Science Center Forensic Analysis Division
12. Tiscione NB Shan X Alford Yeatman DT Quantitation of Benzodiazepines and in Whole Blood by Electron Impact Gas Chromatography Mass Spectrometry J Anal Toxicol 2008 Nov Dec Vol 32 8 pp 644 52 12 5 Method file BENZOSIM M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 77 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Cannabinoid Confirmation by Gas Chromatography Mass Spectrometry GC MS 1 PURPOSE A targeted analysis is performed for A9 tetrahydrocannabinol THC and 11 nor 9 carboxy A9 tetrahydrocannabinol THCA Drugs are isolated from the matrix using solid phase extraction SPE Trimethylsilyl derivatives are prepared and analyzed using gas chromatography mass spectrometry GC MS deuterated internal standards and selective ion monitoring SIM in electron ionization El mode 2 SCOPE Confirmatory analysis of target analytes from toxicology specimens including but not limited to blood and urine Urine confirmations are reported only qualitatively 3 STANDARDS AND SOLUTIONS 3 1 THC THCA Working Standards Three working standards are used Each contains THC and THCA at either 10 1 or 0 1 ng uL respectively 3 1 1 Preparation of 10 ng uL THC THCA Working Standard Add 1000 uL of THC and THCA 0 1 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerat
13. Toxicology 6 ACCESSIONING All specimens related to a case will be compared with the submission form and evidence packaging documentation for proper accessioning inventory The accessioner will mark all inner evidence packaging with unique case identifier and initials as well as sub itemize and mark specimens with the sub item number unique case identifier date and initials This may also be accomplished by printing LIMS mini barcode labels which include the sub item number unique case identifier date and unique employee number 6 1 Discrepancies Fatal discrepancies will result in a report to the customer indicating the evidence has been rejected for analysis due to standard phrase highlighted in bold for each discrepancy or an equivalent statement and the evidence being returned All fatal discrepancies will be documented in the case record minor discrepancies will be captured in the photographs 6 1 1 Fatal discrepancies include 6 1 1 1 Inconsistent subject name this includes instances where the name is missing on any pieces of evidence when the name is not exactly the same throughout all pieces of evidence or subject name between the evidence and submission information do not match 6 1 1 2 Date of birth of subject given on evidence contradicts date of birth given on submission information 6 1 1 3 Inconsistent agency case numbers this includes instances where the case number varies between any pieces of evidence or the case num
14. Vortex mixer Centrifuge Tissue homogenizer Sonicator Heating block Vacuum pump or house vacuum Agilent 7890 GC 5975 MSD 5 INSTRUMENTAL PARAMETERS 5 1 Capillary Column 30 m HP 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 1 2 mL min with an injection volume of 2 uL in split mode 10 1 5 2 GC MS Agilent 7890A Initial Temperature 150 C Hold for 0 5 minutes Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 63 of 171 5 3 Houston Forensic Science Center Forensic Analysis Division Toxicology 20 C min to 180 C Hold for 2 minutes 5 C min to 230 C Hold for 1 minute 10 C min to 250 C Hold for 0 5 min 30 C min to 290 C Hold for 6 min 24 833 minutes Wash solvents for autosampler Methanol and ethyl acetate are used as the wash solvents A minimum of 6 pre and 6 post rinses are performed Each rinse cycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 6 EXTRACTION PROCEDURE BLOOD URINE AND ALL OTHER MATRICES 6 1 In around bottom glass culture or centrifuge tube add 2 mL of blood and 100 uL Internal Standard Solution while vortex mixing NOTE 1 IT IS RECOMMENDED TO CENTRIFUGE SPECIMENS IN PREPARATION FOR EXTRACTION NOTE 2 IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALLER VOLUME ADD BUFFER A
15. if applicable Note At this time HFSC does not employ Liquid Chromatography Tandem Mass spectrometry for any analysis lonization suppression enhancement is not applicable to GCMS testing 4 VALIDATION EXPERIMENTS 4 1 Calibration Model Linearity The most appropriate calibration model should be determined during method development For methods using a detector that is theoretically linear over the covered concentration range it is recommended that initially a straight line concentration response model be adopted using weighted linear regression with inverse concentration weighting to estimate slope and Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 157 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology intercept Other procedures may be probed preferably in the following sequence Straight line fit using weighted linear regression with inverse concentration squared weights Straight line fit forced through the origin for single point calibrations only Quadratic fit with inverse concentration using a weighted 2nd order polynomial There are three components to validation of the calibration model linearity verification of weighting scheme validation of linearity across the analytical measurement range and validation of calibration 4 1 1 Procedure Once the best calibration model has been determined the follow
16. replace them Elute basic drugs with 1mL of elution solvent 2 ammonium hydroxide in methylene chloride isopropyl alcohol 80 20 ELUTION SOLVENT MUST BE MADE FRESH Turn on vacuum or apply positive pressure for a few seconds to ensure all elution solvent has drained from the column Evaporate to dryness under nitrogen for 15 minutes at 50 60 C Reconstitute in 30 uL ethyl acetate or an appropriate volume vortex and transfer to autosampler vials Inject 2 uL onto the GC MS using ZOLPIDEM Version 2 0 Issue Date August 3 2015 Page 145 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 7 INSTRUMENTAL ANALYSIS 7 1 Ensure that the daily QC and tune verification have been completed 7 2 SIM acquisition ZOLPIDEM M Use this method to perform targeted analysis for zolpidem Zolpidem D7 242 314 223 Zolpidem 235 307 219 Retention Time RT varies with column length Deuterated D7 internal standards are used throughout Corresponding ions for internal standards are M 7 8 INTERPRETATION OF RESULTS 8 1 Assay performance including but not limited to precision accuracy limit of detection limit of quantitation and linearity are summarized in the validation documentation 8 2 Limit of detection and limit of quantitation of zolpidem in whole blood are 20 ng mL and 25 ng mL respectively Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3
17. 0 1 mg mL morphine and 125 uL of 0 1 mg mL phencyclidine PCP Bring to volume with methanol Store ina silanized glass vial in the refrigerator 6 months expiration EIA Blood Drug Standard Mix Using methanolic drug standards in a 25 mL volumetric flask add the following 100 uL of 0 1 mg mL of I THCA 200 uL of 0 1 mg mL of d methamphetamine 500 uL of 0 1 mg mL of benzoylecgonine BE 500 uL of 0 1 mg mL of oxazepam 200 uL of 0 1 mg mL of morphine and 100 uL of 0 1 mg mL of phencyclidine PCP Bring to volume with methanol Store in a silanized glass vial in the refrigerator 6 months expiration 5 EQUIPMENT 5 1 5 2 5 3 5 4 5 5 5 6 5 7 5 8 TECAN Freedom Evo 75 TECAN HydroFlex Plate Washer TECAN Sunrise Plate Reader Vortex mixer Air Displacement pipettes 100 1000 uL 20 200 uL 2 20 uL Centrifuge Sonicator Homogenizer 6 PROCEDURE FOR BLOOD AND URINE 6 1 6 1 1 6 1 2 6 1 3 6 1 4 Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed In each run the following controls are included Positive control in duplicate fortified with target drugs at the specified threshold Negative control in duplicate drug free matrix Low QC in duplicate fortified with target drugs at half the concentration found in the positive control High QC in duplicate fortified with target drugs at five times the concentration found in the positive control Vers
18. 2 3 4 3 3 4 3 3 1 4 3 3 2 4 3 3 3 4 4 4 4 1 4 4 1 1 4 4 1 2 4 4 2 4 4 2 1 4 4 2 2 4 4 3 4 4 3 1 4 4 3 2 time peak shape mass spectral ion ratios Limit of Quantitation LOQ Procedure Houston Forensic Science Center Forensic Analysis Division Toxicology Achieves acceptable predefined detection and identification criteria e g retention Prepare a pooled fortified matrix sample at the same concentration as the lowest calibrator Analyze a minimum of three replicates a run in five different runs Data analysis Use the established calibration curve to quantify the analyte of interest Calculate the bias Bias Nominal Concentration Calculate the within run and between run Precision Within RunImprecision Between RunImprecision Acceptance Criteria Bias Bias lt 25 Within Run Precision CV lt 25 Between Run Precision CV lt 25 Bias and Precision Procedure Average of calculated concentrations Nominal concentration Standard deviation of single run Standard deviation of all runs x x 100 Average of single run 100 Average of allruns Prepare pooled fortified matrix samples using a minimum of three replicates per run at three control concentrations low medium and high Analyze a minimum of three replicates per run in five different runs Data Analysis Use the established calibration curve to calculate the concentration of the
19. 2 4 6 2 5 6 2 6 6 2 7 6 2 8 6 2 9 6 2 10 6 2 11 6 2 12 6 2 13 6 2 14 6 2 15 6 2 16 6 2 17 6 2 18 6 2 19 6 2 20 6 2 21 6 2 22 6 2 23 6 3 6 3 1 6 3 2 6 3 3 6 3 4 6 3 5 6 3 6 Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed Houston Forensic Science Center Forensic Analysis Division Toxicology containing 100 ng mL of each analyte is recommended Use appropriate external controls e g UTAK Whole Blood Controls when available Add 2 mL of phosphate buffer pH 6 0 Vortex Centrifuge samples at 4 000 rpm for at least 10 minutes Place collection tubes or reservoir for biological waste into vacuum manifold rack Pour sample onto SPE columns If sample quality is poor decant the supernatant layer into a clean tube and avoid putting solid material onto the column Use sufficient vacuum to draw blood samples through the column approximately 5 psi Wash column with 1 mL deionized water Remove collection tubes or reservoir for biological waste and replace with collection tubes or reservoir for organic waste Dispose of biological waste Wash column with 1 mL 1 M acetic acid Dry column on full vacuum pressure for 5 minutes Wash column with 1 mL hexane Wash column with 1 mL ethyl acetate Wash column with 1 mL methanol Place conical collection tubes into the rack If using Teflon inserts and they appear dirty replace them Elute drugs
20. 5 2 Using forceps gently place the liners in the chromic acid solution with the top of liner pointing up 5 3 Using a Pasteur pipette continuously fill the liners with the chromic acid solution Allow the solution to move through the liners cleaning and removing interior soiling Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 27 of 171 5 5 5 6 5 5 8 5 9 Houston Forensic Science Center Forensic Analysis Division Toxicology When all soiling appears to be removed from the interior carefully transfer the liners to a clean beaker to finish draining Heavily soiled liners may need more rinses in the chromic acid solution to become clean Carefully rinse the beaker with the drained liners with deionized water in order to remove all traces of the chromic acid solution Individual liners may be rinsed with deionized water to facilitate this process Sonicate the liners in deionized water for 10 minutes Repeat with fresh deionized water Sonicate the liners in methanol for 10 minutes Briefly allow the liners to dry Cleaned and deactivated liners should be handled only with gloves and should be stored in a location to protect them from possible sources of contamination Liners will be silanized prior to use 6 PROCEDURE FOR SILANIZATION 6 2 6 3 6 4 6 5 6 6 6 7 6 8 6 9 6 10 6 11 6 12 6 13 Place glassware items to be sil
21. 6 3 1 Drug free blood or urine is evaluated in triplicate for each relevant assay as negative control samples with internal standard if applicable The samples should not trigger positive results or affect performance of the assays When relevant signal abundance after corrected by internal standard should be less than 10 of that of LOQ Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 12 of 171 Houston Forensic Science Center j P 4 Forensic Analysis Division Toxicology Verification of Relative Concentrations of Stock Standards 3 1 3 1 PURPOSE This procedure may be performed to compare a new stock standard solution with an old stock standard solution The following experiment is done when required in order to verify the relative concentrations of the calibration and the control stock standards SCOPE This SOP is applicable to all laboratory staff and this procedure is not routinely required to be performed unless specified in a protocol method or SOP PROCEDURE This procedure may be performed when two separate stock standard solutions for a compound are used one for the calibrators and another for the controls and or validation standards and the nature of the study requires a defined relative accuracy between the two stock solutions or the nature of the study requires a defined accuracy between the calibrators and controls validat
22. 85 and 1 15 and either 5 3 3 2 2 It is between 0 95 and 1 05 or 53 3 23 The uncertainty range 95 confidence interval contains 1 D333 The y intercept of the best fit line is acceptable if 5 3 3 3 1 The uncertainty range 95 confidence interval contains 0 5 3 3 4 The QC values when calculated vs the new calibrator must be within the acceptance used when validating the particular QC 5 3 3 5 If criteria are met the pass review fields will read Pass 5 3 3 6 If the criteria are not met the pass review fields will read Review 5 3 3 7 Further supplemental information is available to assist in evaluating how the calibrators compare Version 2 0 Issue Date 08 03 2015 Page 16 of 171 Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed Houston Forensic Science Center Forensic Analysis Division Toxicology The difference between the two calibrator results will be calculated and will be shaded if the new calibrator is more than 15 different than the current calibrator 5 3 3 7 2 The bias from the new calibrator to current calibrator is calculated Note The calculations used in the excel spreadsheet are described in the calculations Tab 5 3 4 Review approval process 5 3 4 1 After all appropriate data has been entered to the file it is to be saved and the appropriate Supervisor Team Leader or designee is to be notified 5 3 4 2 If all criteria are
23. Analysis Division Toxicology 1 PURPOSE A targeted analysis is performed for confirmatory analysis of fentanyl by solid phase extraction SPE and gas chromatography mass spectrometry GC MS Fentanyl is isolated from the matrix using a basic extraction Deuterated internal standard and selective ion monitoring SIM are used in electron impact El mode 2 SCOPE Confirmatory analysis of fentanyl from toxicology specimens including but not limited to blood This procedure is recommended if quantitative analysis is desired Qualitative results may also be reported For routine full scan qualitative analysis however refer to Basic Acidic and Neutral Drugs by GC MS 3 REAGENTS AND STANDARDS 3 1 Fentanyl Working Standards 3 1 1 Preparation of 1 ng uL Fentanyl Working Standard Add 100 uL of Fentanyl 0 1 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 1 2 Preparation of 0 1 ng uL Fentanyl Working Standard Add 1 mL of Fentanyl 1 ng uL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 1 3 Preparation of 0 5 ng uL Internal Working Solution Add 50 uL of fentanyl Ds deuterated drug standard 0 1 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 2 1M Acetic Acid 3 3 100 mM pH 6 0 Phosphate Buffer 3 4 Elution Solvent 2 ammonium hydroxide concentrate
24. Clear Bath Add more water until liquid level is at standard operating height Refer to user manual for other maintenance issues including but not limited to fuse replacement leak checks and troubleshooting 7 LITERATURE AND SUPPORTING DOCUMENTATION Operator s Manual TurboVap LV Evaporator Workstation Zymark Corporation P N 71467 Rev 3 Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 30 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Operation and Maintenance of CEREX Pressure Processors 1 PURPOSE During solid phase extraction SPE samples require processing and drying which may be carried out using positive pressure The CEREX Pressure Processor is equipped with 48 individual restricted gas ports to allow for positive pressure processing of solid phase extraction columns It is also equipped with on off switches in order to preserve gas and save time by not having to plug ports not in use 2 SCOPE Each of two positive pressure manifolds CEREX System 48 and CEREX System 48 II will be used routinely during solid phase extraction in the toxicology section of the laboratory Analysts should be knowledgeable regarding use and maintenance of these processors 3 EQUIPMENT 3 1 CEREX System 48 System 48 or CEREX System 48 II CEREX 48 3 2 Collection tube rack 16x100mm 3 3 SPE Rack 6CC
25. Discard 6 months 8 1 2 Working solution A 1 ng L Add 100 uL of 0 1 mg mL stock solution to a 10 mL volumetric flask and bring to volume with methanol Storage Store refrigerated Discard 3 months 8 1 3 Working Solution B 0 1 ng L Add 1000 ul of 1 ng uL working solution A to a 10 mL volumetric flask and bring to volume with methanol Storage Store refrigerated Discard 3 months Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 127 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 9 INTERNAL STANDARD This solution consists of Phencyclidine D5 9 1 I S Working Solution 1 ng pL Add 100 uL of the deuterated drug standard 0 1 mg mL to a 10 mL volumetric flask and bring to volume with methanol Storage Store refrigerated Discard 6 months 10 Quality Control Solutions NOTE Calibrators and controls must be prepared from separate lots or sources of certified reference material Controls may be purchased or prepared in house In house prepared controls shall be verified according to SOP Preparation and Validation of Drug Standards Spiking Solutions Controls and Reagents 10 1 QC Stock Solution 10 ng pL Transfer 250 uL of PCP reference material 1000 ng uL into a 25 mL volumetric flask containing approximately 15 mL of methanol Bring to volume with methanol Storage Store frozen in amber vial
26. Division Toxicology Introduction This Toxicology Standard Operating Procedures manual manual or SOP manual is intended to consolidate the policies and procedures specific to the toxicology section of the Houston Forensic Science Center HFSC Any conflict between this document and any governing policies established by HFSC such as the overall quality assurance system shall be resolved in favor of the HFSC policy This manual is part of an overall quality assurance system in the Houston Forensic Science Center Any policies established in the governing quality system or in the policies established by the Houston Forensic Science Center will supersede any requirements stated in this manual However this document may add additional guidance that supplements what has already been established This document is an amalgamation of multiple existing policies and procedures specific to the toxicology section This document supersedes any existing policies and procedures that are not incorporated into this document Safety These procedures must be conducted in accordance with the existing Health and Safety Manual and the current Quality Assurance Manual All biological samples shall be treated with universal precautions Appropriate personal protective equipment should be worn at all times Flammable liquids and vapors may cause eye skin and respiratory tract irritation Derivatization reagents are toxic and must be handled in a
27. L Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 62 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Ina 10 mL volumetric flask add 1 0 mL of BAN Working Mix 20 ng uL Bring to volume with methanol Store refrigerated 3 month expiration Refer to fortification guide below for guidance on preparation of calibrators and controls 3 2 3 3 3 4 3 5 3 6 3 7 BAN Internal Standard Solution This solution consists of mepivacaine 0 01 mg mL prazepam 0 01 mg mL and pentobarbital D 0 01 mg mL Preparation of BAN Internal Standard Solution In a 10 mL volumetric flask add 1 0 mL mepivacaine 0 1 mg mL 1 0 mL prazepam 0 1 mg mL and 1 0 mL of pentobarbital D 0 1 mg mL and bring to volume with methanol Store refrigerated 6 month expiration 1M Acetic Acid 100 mM pH 6 0 Phosphate buffer Methylene Chloride Isopropanol 80 20 Elution Solvent 2 ammonium hydroxide concentrated in 80 20 methylene chloride isopropanol Acidic Methanol 1 HCI in methanol 4 EQUIPMENT AND MATERIALS 4 1 4 2 4 3 4 4 4 5 4 6 4 4 8 4 9 4 10 4 11 4 12 4 13 Air displacement pipettes 100 1000 uL 20 200 uL 2 20 uL Cerex PolyChrom Clin II SPE columns Black ones Vacuum manifold with disposable Teflon inserts or positive pressure SPE manifold Pan balance pH meter Evaporator
28. Screen EIA if positive gt Confirmation 6 4 2 DWI Felony Blood Specimens ALC if lt 0 10 gt Drug Screen EIA BAN if positive gt Confirmation 6 4 3 DUID Blood Specimens ALC if lt 0 10 gt Drug Screen EIA if positive gt Confirmation Urine Specimen Drug Screen EIA BAN if positive gt Confirmation 6 4 4 Vehicular Homicide or Death Due to Accident Blood Specimens ALC gt Drug Screen EIA BAN if positive gt Confirmation 6 4 5 Sexual Assault Toxicology Kit usually contains both blood and urine specimens Blood Specimens only ALC gt Drug Screen EIA BAN if positive gt Confirmation Blood Specimens ALC Urine Specimen Drug Screen EIA BAN if positive gt Confirmation 6 4 6 Other Assignment will be made based on client request or consult 7 OUTSOURCED CASES 7 1 Evidence to be outsourced to an external laboratory must be processed in the following manner 7 1 1 Verify correct evidence case number 7 1 2 Add a barcode label or manually transfer evidence custody in LIMS 7 1 3 Document outsourcing of evidence through chain of custody in LIMS Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 7 of 171 7 2 1 7 2 2 723 7 2 4 7 2 5 Houston Forensic Science Center Forensic Analysis Division Toxicology Verify the photograph of the evidence has been taken Seal the evidence for shipment
29. Understanding Mass Spectra A Basic Approach 2nd Edition John Wiley and Sons Inc Hoboken NJ 2004 Watson D Mass Spectrometry in Clarke s Analysis of Drugs and Poisons 3rd Edition Moffat AC Ossleton MD Widdop B Ed The Pharmaceutical Press London 2004 pp 379 391 Watson JT Introduction to Mass Spectroscopy Biomedical Environmental and Forensic Applications Raven Press Books New York NY 1976 Gilson Microman Pipette Manual Middleton WI 2006 International Organization for Standardization ISO 8655 Piston Operated Volumetric Apparatus 2002 VWR VWR Signature Ergonomic High Performance Pipettor Manual West Chester PA Beckman Instruments Inc Fullerton CA 92835 Users manual A59443AA May 2008 Dickson SC3 Temperature Recorder Quick Start Guide VWR VWR Chromatography Refrigerator Manual 2007 Mettler Toledo Operating Instructions AB S FACT 2007 Mettler Toledo Operating Instructions PB S FACT 2007 Version 2 0 Issue Date 08 03 2015 Page 26 of 171 Houston Forensic Science Center j oP 4 Forensic Analysis Division Toxicology Cleaning and Deactivating GC MS Injection Liners Silanizing Glassware 1 PURPOSE The use of silanized glassware may be necessary for some toxicological procedures A silanizing reagent is used to deactivate the surface of the glass and reduce adsorptive losses that may occur with polar drugs and or metabolites particularly those that may be present at very lo
30. Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 70 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 6 1 11 Centrifuge for 5 minutes at 4000 rpm 6 1 12 Transfer the organic upper layer into a clean conical bottom glass tube 6 1 13 Evaporate to dryness under nitrogen for 10 minutes at 50 C 6 1 14 IN THE FUME HOOD add 10 uL ethyl acetate and 25 uL BSTFA vortex and cap tightly Heat at 70 C for 15 minutes 6 1 15 Remove from heat block and cool to room temperature 6 1 16 Transfer to autosampler vials Reconstitution volumes may be adjusted with ethyl acetate prior to injection when necessary 6 1 17 Inject 2 uL onto the GC MS using BENZOSIM M 6 2 BLOOD DECONJUGATION OPTIONAL This procedure is recommended when the EIA screen results for Benzodiazepines is high but confirmation by GCMS is negative If deconjugation of glucuronidated benzodiazepines in blood is required proceed as follows 6 2 1 In a round bottom glass centrifuge tube add 1 mL of sample NOTE IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALLER VOLUME AND DOCUMENT IN THE CASE FOLDER For qualitative analysis include a minimum of one negative and one positive control in each run For preparation of a 100 ng mL positive control add 100 uL of BENZO Working Standard Solution 1 ng uL to 1 mL blood while vortex mixing For hydrolyzation of glucuro
31. acetate NV Drv column on full vacuum pressure for 5 minutes v Elute THCA with 2 mL 3 glacial acetic acid concentrated in 90 10 hexane ethyl acetate PREPARE FRESH NY Evaporate to dryness under nitrogen at 50 60 C NY Add 30 uL of BSTFA cap tightly and heat at 70 C for 15 minutes v Allow to cool to room temperature Transfer to GC vial and inject 2 uL onto GC MS using THC M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 83 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 11 FLOWCHART FOR CANNABINOID ANALYSIS IN URINE To 1 mL urine fortify calibrators controls according to fortification guide while vortex mixing NY Add 100 uL of 10 M KOH and vortex NY Cap and heat samples for 15 minutes at 60 C Cool to room temperature Vv Add 100 ul I S 1 ng L while vortex mixing VL Add sample to Cerex THC SPE column and allow to flow under gravity VL Wash 1 ammonium hydroxide concentrated in 85 15 H2O acetonitrile PREPARE FRESH 7 Dry column on full vacuum pressure for 5 minutes VL Elute THC with 2 mL ethyl acetate Vy Dry column on full vacuum pressure for 5 minutes VL Elute THCA with 2 mL 3 glacial acetic acid concentrated in 90 10 hexane ethyl acetate PREPARE FRESH NY Evaporate to dryness under nitrogen at 50 C NY Add 30 uL
32. analyte of interest Calculate bias within run and between run precision Acceptance Criteria Bias Bias lt 20 Within Run Precision CV lt 20 Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed Version 2 0 Issue Date August 3 2015 Page 160 of 171 100 Houston Forensic Science Center Forensic Analysis Division Toxicology Between Run Precision CV lt 20 4 5 Carryover 4 5 1 Procedure 4 5 1 1 Analysis of blank matrix samples with IS following the high calibrator in each validation run 4 5 2 Data Analysis 4 5 2 1 Use the established calibration curve to calculate the concentration of the analyte of interest 4 5 2 Acceptance Criteria 4 5 2 1 Quantitative result must be lt 25 of LOQ 4 6 Interference Studies 4 6 1 Procedure 4 6 1 1 Matrix interference Analyze a minimum of 10 different sources of blank matrix without IS 4 6 1 1 Interference from stable isotope internal standards Analyze a blank matrix fortified with IS but NO analyte of interest in each validation run 4 6 1 2 Interference from commonly encountered exogenous analytes Analyze blank matrix fortified with analytes of interest at the low control concentration and potential interferences at high therapeutic or lethal concentrations 4 6 2 Data analysis 4 6 2 1 Matrix interference Evaluate response of any peak at the retention time of the analyte of interest 4 6 2
33. approved Vendor Spiking Solution Solution prepared by diluting drug standard to a pre determined concentration and used to prepare calibration or QC Samples Reagent a chemical chemical mixture or dilution of a chemical substance used in toxicological analysis DRUG STANDARDS PURCHASING STORAGE AND EXPIRATION Vendors should supply a Certificate of Analysis that contains specific free and formula molecular weights purity storage conditions solubility and a lot number Information may include an expiration or re test date Drug standards purchased as liquids in sealed ampoules expire on the date indicated by the manufacturer Solid drug standards expire on the date indicated by the manufacturer Once a drug standard solution is prepared transferred or diluted it expires within one year unless specified otherwise in the SOP Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 14 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 4 4 A drug standard may not be used for quantitative analysis if the drug control or standard in question is beyond the expiration date Expired drug standards may be used only for qualitative analysis beyond their expiration date if they are verified prior to use GC MS is the preferred technique for qualitative verification purposes These verified standards are clearly identified as Quali
34. control should be included in each run Use appropriate external controls e g UTAK Whole Blood Controls when available An in house quantitative QC of 50 ng mL is recommended 6 1 4 Add 2 mL of 100 mM pH 6 0 sodium phosphate buffer to each tube Vortex 6 1 5 Centrifuge all samples for at least 10 minutes at 4000 rpm Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 144 of 171 6 1 8 6 1 9 6 1 10 6 1 11 6 1 12 6 1 13 6 1 14 6 1 15 6 1 16 6 1 17 6 1 18 6 1 19 6 1 20 Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed Houston Forensic Science Center Forensic Analysis Division Toxicology Place collection tubes or reservoir for waste into vacuum manifold rack or positive pressure apparatus Pour sample onto Polychrom Clin Il columns Use sufficient vacuum pressure to draw samples through the columns Wash column with 1 mL deionized water Remove waste tubes reservoir and replace with fresh waste tubes reservoir Wash column with 1 mL of 1 M acetic acid Dry column on full vacuum pressure for 5 minutes Wash column with 1 mL hexane Wash column with 1 mL ethyl acetate Wash column with 1 mL methanol Dry column on full vacuum pressure for 5 minutes Replace waste tubes reservoir with conical collection tubes If the Teflon inserts vacuum manifold only appear dirty
35. dilution results of an alcoholic beverage or other liquid case specimens 10 SAMPLE PREPARATION 10 1 Allow calibrators controls and case specimens to come to room temperature prior to sampling 10 2 Mix all calibrators controls and case specimens well prior to sampling by gentle inversion or rocking Avoid shaking 10 3 The DI Water Control consisting of 50 uL deionized DI water is left open for the duration of the sampling process When all sampling is completed add internal standard cap and crimp tightly 10 4 Dilutions will be performed with deionized DI water prior to sampling 10 4 1 When alcoholic beverages or other liquids are analyzed which is infrequent fluid from the container is diluted appropriately based on suspected alcohol content prior to analysis From the dilution a 50 uL aliquot is analyzed 10 4 2 Example For a 4 alcoholic beverage a 1 20 dilution is appropriate 10 4 3 A DQC using the same dilution as the case specimen if possible must be included in the batch when diluted alcoholic beverages or other liquids are analyzed 10 4 4 Commonly used dilutions are as follows Beverage uL Deionized Water pL 10 5 Using the pipettor dilutor aliquot 50 uL of the calibrators controls and case specimens The pipettor dilutor will deliver 500 uL of the I S working solution along with the 50 uL sample aliquot into the appropriately labeled headspace vial Cap and crimp tightly Note Samples must be pre
36. for 1 6 minutes Wash solvents for autosampler Methanol and ethyl acetate are used as the wash solvents A minimum of 6 pre and 6 post rinses are performed Each rinse cycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 6 EXTRACTION PROCEDURE Blood Urine and all Other Matrices 6 1 In a round bottom glass culture tube add 1 mL of specimen and 100 uL WORKING Internal Standard Solution 1 ng uL while vortex mixing total concentration 100 ng mL NOTE IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALLER VOLUME AND DOCUMENT IN THE CASE FOLDER 6 2 Fortify all controls and calibrators while vortex mixing using the appropriate amount of working standard Refer to the fortification guide for guidance on preparation of calibrators CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS Use appropriate external controls e g UTAK Whole Blood Controls when available 6 3 Add 2 mL of 100 mM pH 6 0 sodium phosphate buffer to each tube Vortex 6 4 Place collection tubes or reservoir for waste into vacuum manifold rack or positive pressure apparatus 6 5 Pour sample onto Polychrom Clin Il columns Use sufficient vacuum pressure to draw samples through the columns 6 6 Wash column with 1 mL deionized water 6 7 Remove waste tubes reservoir and replace with fresh waste tubes reservoir 6 8 Wash column with 1 mL 1 M acetic acid 6 9 Dry column on full vacuum pressure for 5 minut
37. for the High QC Mean Binding for the High QC Mean absorbance for the Low QC Mean Binding for the Low QC The spreadsheet calculates the running mean Binding for all assays CVs and displays results on a control chart for evaluation of trends Binding is calculated numerically as follows B A positive control A negative control x 100 Example A positive control produces a mean absorbance reading of 0 66 Apositive control 0 66 the mean absorbance for the negative control is 2 0 A negative contro 2 0 The Binding is 0 66 2 0 x 100 33 ogy Standard Operating Procedures Version 2 0 By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 48 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 10 1 3 The control charts are reviewed with the EIA batch file Only a copy of the EIA results is placed in the case folder EIA results are reviewed and stored in batch files in the laboratory 10 2 Acceptance Criteria 10 2 1 During technical review of the EIA batch file the following acceptance criteria apply to all assays 10 2 1 1 Binding for the POSITIVE CONTROL must be lt 60 and 10 2 1 2 CV for the NEGATIVE CONTROL n 2 must be lt 20 10 2 2 Assays not meeting both of these criteria should be repeated Repeat analysis may be warranted for other reasons Variations in assay performance are expected between lot numbers Changes in procedure may be
38. mL 1 M acetic acid 6 1 10 Dry column on full vacuum pressure for 5 minutes 6 1 11 Wash column with 1 mL hexane 6 1 12 Wash column with 1 mL ethyl acetate 6 1 13 Wash column with 1 mL methanol 6 1 14 Dry column on full vacuum pressure for 5 minutes 6 1 15 Replace waste tubes reservoir with conical collection tubes If the Teflon inserts vacuum manifold only appear dirty replace them 6 1 16 Elute basic drugs with 1mL elution solvent 2 ammonium hydroxide in methylene chloride isopropyl alcohol 80 20 ELUTION SOLVENT MUST BE MADE FRESH 6 1 17 Turn on vacuum or apply positive pressure for a few seconds to ensure all elution solvent has drained from the column 6 1 18 Evaporate to dryness under nitrogen for approximately 10 minutes at 50 60 C Reconstitute in 30uL ethyl acetate or an appropriate volume vortex and transfer to autosampler vials 6 1 19 Inject 2 uL onto the GC MS using KETAMINE M FOR URINE SAMPLES ONLY USE KETAMINE_URINE M 6 2 Dilutions If the sample is known to contain a high concentration of drug it should be diluted prior to analysis Aliquot the desired volume of blood and make up to volume using phosphate buffer DOCUMENT THESE CHANGES IN THE CASE FOLDER Then proceed from step 4 of the blood procedure 7 INSTRUMENTAL ANALYSIS 7 1 Ensure that the daily QC and tune verification have been completed 7 2 SIM acquisition KETAMINE_URINE M Use this method to perform targeted analyses for ketamine in
39. met the supervisor team leader or designee can enter yes in the approval field and enter their name and date in the review area thereby approving the new calibration lot 5 3 4 3 If any criteria are not met review of the new lot of calibrator can only be performed by the Supervisor 5 3 4 4 If upon further review the supervisor decides that the new calibration lot is acceptable appropriate comments are to be placed in the Comments field specifying why it was accepted The supervisor must then enter yes in the approval field along with their name and date in the review area thereby approving the calibration lot 5 3 4 5 If upon further review the supervisor decides that the new calibration lot is not acceptable appropriate comments are to be placed in the Comments field specifying the appropriate steps to be taken The supervisor must then enter no in the approval field along with their name and date in the review area thereby verifying that the review is complete 6 VALIDATING AND RE VALIDATING INTERNAL STANDARDS 6 1 Validating a New Lot of Internal Standard 6 1 1 Add the amount of internal standard noted in the bench procedure to blank matrix Run in duplicate 6 1 2 Compare the area s or height s of the new internal standard to the area or height of the current internal standard in a blank matrix sample 6 1 3 The two results should match within 30 For results outside this range consult a Supervisor o
40. ng L 3 2 1 Preparation of 10 ng uL Internal Standard Solution Add 1000 uL of each deuterated drug standard 0 1 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 3 1M Acetic Acid 3 4 Acidic Methanol 1 HCI in methanol 3 5 Phosphate Buffer pH 6 0 3 6 Elution Solvent 2 ammonium hydroxide concentrated in ethyl acetate plus 1 mL of methanol 3 7 PFPA pentafluoropropionic anhydride for derivatization Store at room temperature Discard if there is a change in color 4 EQUIPMENT AND MATERIALS 4 1 Air displacement pipettes 100 1000 uL 20 200 uL 2 20 uL 4 2 Cerex PolyChrom Clin II SPE columns 4 3 Vacuum manifold with disposable Teflon inserts or positive pressure SPE manifold 4 4 Pan balance Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 135 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 4 5 pH meter 4 6 Evaporator 4 7 Vortex mixer 4 8 Centrifuge 4 9 Tissue homogenizer 4 10 Sonicator 4 11 Heating block 4 12 Vacuum pump or house vacuum 4 13 Agilent 7890 GC 5975 MSD 4 14 Stir plate and stir bars 5 INSTRUMENTAL PARAMETERS 5 1 Capillary Column 30 m HP 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 1 1 mL min with an injection volume of 2 uL in split mode 10 1
41. ng uL while vortex mixing Fortify calibrators controls according to fortification guide while vortex mixing Add 1 mL 100mM pH 6 8 Phosphate Buffer Deconjugation Buffer Add 2 mL KCI NaOH and vortex Add 6 mL n butylchloride cap and rotate for 5 min Centrifuge for 5 min at 4000 rpm Transfer upper layer to clean round bottom centrifuge tube Add 1 mL of 1 10 KCI NaOH pH 12 Do not centrifuge or vortex Cap and rotate for 5 min Centrifuge for 5 min at 4000 rpm Transfer upper layer to clean conical bottom centrifuge tube Evaporate to dryness under nitrogen at 50 C Add 25 uL of ethyl acetate and 25 uL BSTFA cap tightly and heat at 70 C for 15 min Transfer to GC vial and inject 2 UL onto GC MS using BENZOSIM M th Mi Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 75 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 10 FLOWCHART FOR DECONJUGATED BENZODIAZEPINE ANALYSIS To 1 mLurine or blood OPTIONAL fortify calibrators controls according to fortification guide while vortex mixing NY Add 1 mL 100mM of pH 6 8 Phosphate Buffer Deconjugation Buffer vortex Add 100 uL of deconjugation enzyme vortex Cap and heat samples for 1 hour at 37 C Cool to room temperature Add 100 uL I S 2 ng L while vortex mixing Add 2 mL KCI NaOH and vortex Add 6 mL n butylchloride cap and rotate for 5 min C
42. specifically directed otherwise by the submitting agency or client Results are qualitative in nature Quantitative results are not possible due to the non linear binding characteristics of the assay and antibody specificity EIA reports display a numerical value for all cases calculated as follows A positive control A sample x C positive control Example A case sample produces an absorbance reading of 0 5 for the cannabinoid assay A sample 0 5 the mean absorbance for the positive control is 1 0 A positive control 1 0 The concentration of THCA in the positive control is 10 ng mL C positive control 10 so the numerical value assigned to the sample is 1 0 0 5 x 10 20 ng mL Note that these numerical values are not quantitative but can be used to differentiate low and high concentrations in case samples These values should be considered when performing confirmatory analyses 10 QUALITY ASSURANCE 10 1 10 1 1 10 1 1 1 10 1 1 2 10 1 1 3 10 1 1 4 10 1 1 5 10 1 1 6 10 1 1 7 10 1 1 8 10 1 2 Toxicol Issued EIA QC Logs After each run the following data is entered into the Blood EIA QC Log or Urine EIA QC Log Mean absorbance for the positive control Mean Binding for the positive control CV for duplicate analyses for the positive control Mean absorbance for the negative control CV for duplicate analyses for the negative control The mean Binding for the negative control is 100 by default Mean absorbance
43. the result is reported as None Detected 16 LIMITATIONS Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 131 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Validation was performed using human blood without preservatives for the preparation of calibrators and controls Evaluation of samples collected in gray top tubes indicates that one of the additives in these tubes causes an elevated m z 200 ion As a result the qualifier ion ratio in samples containing lt 10 ng mL does not fall within 20 of the average calibrator ion ratio The quantitative results are not significantly affected Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 132 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 17 Flow Chart for PCP Preparation To 1 mL specimen add 50 uL I S 1 ng L while vortex mixing NY Fortify calibrators controls according to fortification guide while vortex mixing NY Add 2 mL 100 mM sodium phosphate buffer Vortex VL Add sample to Polychrom Clin II SPE column Use sufficient vacuum pressure to draw sample through column NY NY NY NY NY NY NY Dry column on full vacuum pressure for 5 minutes Elute Basic Drugs with 1 mL methylene chloride isopropyl alcohol
44. to each use The value should be within 5 C of the optimal temperature for relevant analytical assays This is documented on Form LAB 69 Temperature Log or equivalent method 10 LITERATURE REFERENCES 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 10 15 10 16 10 17 10 18 10 19 10 20 Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed Agilent Technologies Agilent G1701EA GC MSD ChemStation Getting Started Part Number G1701 90062 2007 Agilent Technologies Agilent 5975 Mass Selective Detector Hydrogen Safety Part Number G3170 90010 2005 Agilent Technologies Agilent 5975 Series MSD Operation Manual Part Number G3170 90030 2010 Agilent Technologies Agilent 7890A Gas Chromatography Quick Reference Part Number G3430 90009 2007 Agilent Technologies Agilent MSD ChemStation Software User Information CD Part Number G1701 60125 Agilent Technologies Evaluate Tune System Verification Report Dawling S Gas Chromatography in Clarke s Analysis of Drugs and Poisons 3rd Edition Moffat AC Ossleton MD Widdop B Ed The Pharmaceutical Press London 2004 pp 425 499 McLafferty FW Interpretation of Mass Spectra 3rd Edition University Science Books Mill Valley CA 1980 Skoog DA Ed Principles of Instrumental Analysis 3rd Edition Saunders College Publishing New York 1985 pp 523 535 554 Smith RM
45. urine 7 3 SIM acquisition KETAMINE M Use this method to perform targeted analyses for ketamine in blood Retention Time RT varies with column length Deuterated D4 internal standards are used throughout Corresponding ions for internal standards are M 4 Refer to attached method for acquisition parameters 8 INTERPRETATION OF RESULTS Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 100 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 8 1 Assay performance including but not limited to precision accuracy limit of detection limit of quantitation and linearity are summarized in the validation documentation 8 2 Limits of detection and quantitation of ketamine in blood are 10 ng mL Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 101 of 171 9 Houston Forensic Science Center Forensic Analysis Division Toxicology FLOWCHART FOR KETAMINE ANALYSIS Vv Vv NV Vy to draw sample through column Vy Vy V Vv Vv VL NY NY Elute Basic Drugs with 1 mL methylene chloride isopropyl alcohol 80 20 with 2 ammonium hydroxide PREPARE FRESH NY Evaporate to dryness under nitrogen at 50 C for 10 mins NY Reconstitute in 30 uL ethyl acetate Transfer to GC vial and inject 2 uL onto GC MS usi
46. with 1 mL elution solvent 2 ammonium hydroxide in ethyl acetate plus 1 mL methanol Turn on vacuum pressure for a few seconds to ensure all elution solvent has drained from the column Remove tubes from rack and add 30 uL of acidic methanol Evaporate to dryness under nitrogen at a temperature not to exceed 50 C avoid over drying samples IN THE FUME HOOD add 50 uL ethyl acetate and 50 uL PFPA Cap tightly Vortex Heat at 70 C for 30 minutes Allow samples to come to room temperature Evaporate to dryness under nitrogen at a temperature not to exceed 50 C Reconstitute in 20 uL ethyl acetate or appropriate volume vortex and transfer to autosampler vials Inject 2 uL onto the GC MS using STIMSIM M URINE In a round bottom centrifuge tube add 1 mL urine and 25 uL Internal Standard Solution 10 ng uL while vortex mixing The total concentration of internal standard is 250 ng mL For qualitative analysis include a minimum of one negative and one positive control in each run For preparation of a 200 ng mL positive control add 20 uL of Stimulant Working Standard Solution 10 ng uL to 1 mL urine while vortex mixing Add 2 mL phosphate buffer pH 6 0 Vortex If sample is cloudy or contains particulates centrifuge for 15 minutes 4000 rpm Proceed from step 4 through step 20 of blood procedure Evaporate to dryness under nitrogen at a temperature not to exceed 50 C Reconstitute in 40 uL ethyl acetate or appropriate vo
47. 00 g 100 mL r Ethanol calibrators must be run with the first batch of case specimens prepared by an individual analyst each day Subsequent batches prepared by the same analyst during the same work shift must contain controls prepared along with the case specimens 7 1 2 Purchased e g Cerilliant Absolute or equivalent NIST certified reference ethanol standards or mixed volatile standards that include ethanol are used as calibrators for ethanol calibration See manufacturer s Certificate of Analysis for storage and expiration information The following calibrator concentrations are used for ethanol calibration unless otherwise specified in the case record Level 1 0 010 g 100 mL Level 2 0 050 g 100 mL Level 3 0 100 g 100 mL Level 4 0 200 g 100 mL Level 5 0 400 g 100 mL Level 6 0 500 g 100 mL 7 1 3 Purchased calibrator solutions typically come in vials containing approximately 1 mL of solution Once opened the contents of the vial may be transferred to a labeled container sealed and stored in the refrigerator Opened and transferred solutions can be used for ethanol calibration for 1 month 7 2 Methanol Isopropanol Acetone M I A Calibration 0 010 0 400 g 100 mL 12k If historical calibration is used for M I A it must be re established or re verified at least once every six months OR when one of the following occurs 7 2 1 1 New n propanol internal standard 1 S stock solution is prepared 7 2 1 2 Significant instrumen
48. 00 mL obtained values from FID1 A BAC1 must be within 5 of target value For volatile concentrations lt 0 050 g 100 mL obtained values from FID1 A BAC1 must be within 10 of target value The calibration curve must yield an R value of 0 99 or greater If a control or calibrator is not within the given range The associated control s or calibrator s will be re evaluated a second time If after the second analysis the result remains outside the allowable range the section Manager or Supervisor will be consulted Any case specimens bracketed by a control not within the acceptable range must be re analyzed Internal Standard Recovery For each batch the consistency of internal standard must be evaluated The internal standard recovery as indicated by the area counts of the integration must be within to 2 times the average of the calibrators and controls within the batch If an unknown sample is not within this range the sample may be re injected once If it is still not within this range the sample must be re aliquotted Chromatography All chromatography must be well resolved from any interfering peaks and symmetric Peak symmetry will be evaluated for every peak associated with an analyte of interest The instrument software will generate and report the peak symmetry A number between 0 5 and 2 indicates acceptable symmetry Peak resolution will be evaluated for every peak associated with an analyte of interest
49. 00 uL of Urine Drug Standard Mix 6 2 4 2 Positive Control To 1960 uL of drug free urine add 40 uL of Urine Drug Standard Mix 6 2 4 3 Low QC To 1980 uL of drug free urine add 20 uL of Urine Drug Standard Mix Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 43 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Positive Control oxazepam 00 50o LTHCA a ee a Pheneyeldine 28 ass d methamphetamine 200 10 100 0o morphine 20 109 10 0o 6 3 Subsampling 6 3 1 Blood 1 10 dilution with forensic diluent phosphate buffered saline 6 3 1 1 Pipette 100uL of controls negative blood and the unknowns into subsample tubes Note If blood samples are clotted remove clots and decant sample into new subsample tube Sonication centrifugation or other sample preparation steps may be necessary for degraded specimens 6 3 1 2 Add 900 uL of Forensic diluent PBS buffer to each subsample tube and vortex 6 3 2 Urine 1 10 dilution with forensic diluent phosphate buffered saline 6 3 2 1 Pipette 100uL of controls negative urine and the unknowns into subsample tubes Note If samples contain particulate material centrifugation may be performed before transfer into the subsample tube 6 3 2 2 Add 900 uL of Forensic diluent PBS buffer to each subsample tube and vortex 6 4 Placement
50. 10 uL acetic anhydride Transfer to GC vial and inject 2 uL onto GC MS using KETO M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 109 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Volume of Blood Target Concentration Working Standard Volume Added uL mL ng mL Concentration ng uL 11 LITERATURE AND SUPPORTING DOCUMENTATION 11 1 Kerrigan S Goldberger BA Opioids in Principles of Forensic Toxicology 3 Edition Levine B Ed AACC Press Washington DC 2009 pp 225 244 11 2 Method file KETO M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 110 of 171 Houston Forensic Science Center j oP Forensic Analysis Division Toxicology Methadone EDDP Confirmation by Gas Chromatography Mass Spectrometry GC MS 1 PURPOSE A targeted analysis is performed for confirmatory analysis of methadone and its metabolite 2 ethylidene 1 5 dimethyl 3 3 diphenylpyrrolidine EDDP by solid phase extraction SPE and gas chromatography mass spectrometry GC MS Drugs are isolated from the matrix using a basic extraction Deuterated internal standards and selective ion monitoring SIM are used in electron ionization El mode 2 SCOPE Confirmatory analysis of Methadone EDDP from toxicology specimens including but not
51. 2 Interference from stable isotope internal standards Evaluate response of any peak at the retention time of the analyte of interest 4 6 2 3 Interference from commonly encountered exogenous analytes Use the established calibration curve to calculate the concentration of the analyte of interest 4 6 3 Acceptance Criteria 4 6 3 1 Matrix interference Response of blank matrix must be lt 10 of the average response of LOQ 4 6 3 2 Interference from stable isotope internal standards Response of blank matrix must be lt 10 of the average response of LOQ 4 6 3 3 Interference from commonly encountered exogenous analytes Concentration of analytes of interest must meet acceptance criteria of the method 4 7 Matrix Matching If matrices other than that used to create the calibration will be analyzed the ability of the method to accurately quantify analytes in that matrix must be evaluated Example a method uses blood calibrators but will be used to analyze serum samples Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 161 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 4 7 1 Procedure 4 7 1 1 Perform bias and precision studies in each matrix that will be analyzed using the method 4 7 1 2 Ideally multiple sources of each matrix should be used for these studies 4 7 2 Data Analysis 4 7 2 1 Use the
52. 6 pp 375 380 12 4 Awad T DeRuiter J Clark CR GC MS Analysis of Acylated Derivatives of the side chain and ring regioisomers of methylenedioxymethamphetamine J of Chromatographic Science 2005 July Vol 43 6 pp 296 303 12 5 Baselt RC Disposition of Toxic Drugs and Chemicals in Man 8 Edition Biomedical Publications Foster City CA 2008 12 6 Method file STIMSIM Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 142 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 1 PURPOSE A targeted analysis is performed for confirmatory analysis of zolpidem by solid phase extraction SPE and gas chromatography mass spectrometry GC MS Drugs are isolated from the matrix using a basic extraction Zolpidem D7 is used as the internal standard and selective ion monitoring SIM is used in electron ionization El mode 2 SCOPE Confirmatory analysis of zolpidem from toxicology specimens including but not limited to blood Urine confirmations are reported only qualitatively and are typically reported using Basic Acidic and Neutral Drugs Confirmation by GC MS 3 REAGENTS AND STANDARDS 3 1 Zolpidem Working Standard 3 1 1 Preparation of 10 ng uL Zolpidem Working Standard Add 100 uL of zolpidem 1 0 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 1 2 Pr
53. 6 1 3 Precipitate proteins by addition of 2 mL cold acetonitrile while vortex mixing 6 1 4 Centrifuge for 10 minutes at 4000 rpm 6 1 5 Pour off the supernatant layer into a clean round bottom glass tube Add 1 mL 0 1 M HCI 6 1 6 Place collection tubes or reservoir for waste into vacuum manifold rack or on positive pressure apparatus 6 1 7 Pour acidified sample onto SPE columns Samples should flow under gravity Do not use the vacuum or pressure unless it is necessary 6 1 8 Wash column with 1 mL deionized water 6 1 9 Wash column with 1 mL 0 1 M HCI 6 1 10 Wash column with 1 mL methanol 6 1 11 Wash column with 1 mL ethyl acetate 6 1 12 Dry column on full vacuum pressure for 5 minutes 6 1 13 Place conical glass collection tubes into the rack 6 1 14 Elute drugs with 1 mL elution solvent 2 ammonium hydroxide in methylene chloride isopropyl alcohol 80 20 Samples should flow under gravity Do not use vacuum or pressure unless it is necessary ELUTION SOLVENT MUST BE MADE FRESH DAILY 6 1 15 Turn on vacuum for a few seconds to ensure all elution solvent has drained from the column 6 1 16 Remove tubes from rack and evaporate to dryness approximately 10 minutes under nitrogen at 50 C 6 1 17 IN THE FUME HOOD add 75 uL PFPA and 75 uL HFIP Cap tightly Heat at 70 C for 15 minutes 6 1 18 Evaporate to dryness under nitrogen for 10 minutes at 50 C Reconstitute in 30 uL ethyl acetate vortex and transfer to autosa
54. AACC Press Washington DC 2009 pp 225 244 13 2 Isenschmid DS Cocaine in Principles of Forensic Toxicology 3 Edition Levine B Ed AACC Press Washington DC 2009 pp 245 268 13 3 Grinstead GF A closer look at acetyl and pentafluoropropionyl derivatives for quantitative analysis of morphine and codeine by gas chromatography mass spectrometry J Anal Toxicology 1991 Nov Dec Vol 15 6 pp 293 8 13 4 Adejan RE Schmitt G Wu M Meyer C Determination of cocaine and benzoylecgonine by derivatization with iodomethane D3 or PFPS HFIP in human blood and urine using GC MS El or PCI mode J Anal Toxicology 1993 Jan Feb Vol 17 1 pp 51 5 13 5 Method File OPICOC M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 125 of 171 Houston Forensic Science Center ay Y Forensic Analysis Division Toxicology Phencyclidine Confirmation by Gas Chromatography Mass Spectrometry GC MS 1 4 4 1 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 5 1 PURPOSE This document outlines the confirmatory procedure for quantitative blood and qualitative urine analysis of Phencyclidine PCP by Solid Phase Extraction SPE and Gas Chromatography Mass Spectrometry GC MS Drugs are isolated from the matrix using basic extraction Deuterated Internal Standard and Selective lon Monitoring SIM are used in Electron lonization mode SCOPE This procedure is u
55. Analysis Division Toxicology 317 272 196 345 317 182 Retention Time RT varies with column length Deuterated D3 internal standards are used throughout Corresponding ions for internal standards are M 3 Refer to attached method for acquisition parameters OPICOCSIM M 9 INTERPRETATION OF RESULTS 9 1 Assay performance including but not limited to precision accuracy limit of detection limit of quantitation and linearity are summarized in the validation documentation 9 2 Limits of detection in blood and urine are as follows LOD and LOQ l Drug Bood nemi LOD Urine ng mL MODNIE Ee a E Tl 6 AM 6 acetyl Morphine a E O E E ae Cocaethylene 5 S O0 Ecgonine Methyl Ester l o 5 20 9 3 Qualitative identification is determined using characteristic retention time RT and mass spectral characteristics For Selected lon Monitoring SIM all three characteristic ions must be present lon intensity and ratios should be taken into consideration lon ratios should be within 20 of the calibrator or QC However ions of low intensity may not be within this range for all case samples to approval of the technical leader or section manager If drugs other than the target analytes are suspected samples may be re injected using alternative methods of acquisition If keto opioids hydrocodone oxycodone hydromorphone or oxymorphone are present they should be re extracted and analyzed using the appropriate pro
56. D 6 1 1 In around bottom glass centrifuge tube add 1 mL of blood NOTE IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALLER VOLUME AND DOCUMENT IN THE CASE FOLDER For qualitative analysis include a minimum of one negative and one positive control in each run For preparation of a 100 ng mL positive control add 100 uL of BENZO Working Standard Solution 1 ng uL to 1 mL blood while vortex mixing 6 1 2 Add 50 uL of Internal Standard 2 ng uL while vortex mixing Final concentration of internal standard in each sample is 100 ng mL 6 1 3 Fortify all controls and calibrators while vortex mixing using appropriate amount of working standard Refer to the fortification guide for preparation of calibrators CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS A minimum of one in house control should be included in each run Use appropriate external controls eg Utak Whole Blood Controls when available 6 1 4 Add 1 mL of 100 mM pH 6 8 phosphate buffer deconjugation buffer and vortex 6 1 5 Add 2 mL KCI NaOH Working Solution to each tube Vortex 6 1 6 Add 6 mL n butyl chloride to each tube Cap tightly and mix on tube rotator for 5 minutes 6 1 7 Centrifuge for 5 minutes at 4000 rpm 6 1 8 Transfer the organic upper layer into a clean round bottom centrifuge tube 6 1 9 Add 1 mL 1 10 KCI NaOH pH 12 NOTE DO NOT VORTEX 6 1 10 Rotate tubes for 5 minutes Toxicology Standard Operating Procedures
57. Discard 12 months 10 2 PCP HIGH Blood QC Samples 80 ng mL Transfer 750 uL of PCP Stock QC solution 10 ng uL into 100 mL volumetric flask containing approximately 50 mL of room temperature blank blood Bring to volume with blank blood Transfer 1 2 mL aliquots to appropriately labeled 2 0 mL snap cap conical tubes Assign lot number and expiration date Storage Store frozen Discard 6 months 10 3 PCP LOW BLOOD QC Samples 15 ng mL Transfer 150 uL of PCP Stock QC solution 10 ng uL into 100 mL volumetric flask containing approximately 50 mL of room temperature blank blood Bring it to volume with blank blood Transfer 1 2 aliquots to appropriately labeled 2 0 mL snap cap conical tubes Assign lot number and expiration date Storage Store frozen Discard 6 months 10 4 PCP Negative Urine QC Samples 10 ng mL Transfer 50 uL of PCP Stock QC solution 10 ng uL into 100 mL volumetric flask containing approximately 50 mL of room temperature blank urine Bring it to volume with blank urine Transfer 1 2 aliquots to appropriately labeled 2 0 mL snap cap conical tubes Assign lot number and expiration date Storage Store frozen Discard 6 months 10 5 PCP Positive Urine QC Samples 20 ng mL Transfer 200 uL of PCP Stock QC solution 10 ng L into 100 mL volumetric flask containing approximately 50 mL of room temperature blank urine Bring it to volume with blank urine Transfer 1 2 aliquots to appropriately labeled 2 0 mL snap cap conical t
58. Disposition of Toxic Drugs and Chemicals in Man 8th edition Biomedical Publications Foster City CA pp 616 619 11 2 Bronwyn F et al Determination of subnanogram concentrations of Fentanyl in plasma by gas chromatography mass spectrometry comparison with standard radioimmunoassay J Chrom B 1997 688 pp 79 85 11 3 Carson H J et al A fatality involving an unusual route of Fentanyl delivery Chewing and aspirating the transdermal patch Legal Med 2010 Vol 12 pp 157 159 11 4 Kuhlman J J Jr et al Fentanyl use misuse and abuse a summary of 23 postmortem cases J Anal Toxicol 2003 Vol 27 pp 499 504 11 5 Watts V et al Determination of Fentanyl in Whole Blood at Subnanogram Concentrations by Dual Capillary Column Gas Chromatography with Nitrogen Sensitive Detectors and Gas Chromatography Mass Spectrometry J Anal Toxicol 1988 October Vol 12 pp 246 254 11 6 Method file FENTANYL M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 97 of 171 Houston Forensic Science Center j oP 4 Forensic Analysis Division Toxicology Ketamine Confirmation by Gas Chromatography Mass Spectrometry GC MS 1 PURPOSE A targeted analysis is performed for confirmatory analysis of ketamine by solid phase extraction SPE and gas chromatography mass spectrometry GC MS Drugs are isolated from the matrix using a basic extraction A deute
59. E IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALLER VOLUME ADD BUFFER AS THE DILUENT AND DOCUMENT IN THE CASE RECORD 6 2 Fortify all controls and calibrators while vortex mixing using the appropriate amount of working standard Refer to the fortification guide for guidance on preparation of calibrators CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS A minimum of one negative and one positive control must be included in each run for qualitative analysis For quantitative analysis a minimum of one quantitative QC must be included in each run An in house whole blood QC of 100 ng mL is recommended Enter results into the appropriate QC Log after each run Use appropriate external controls e g UTAK Whole Blood Controls when available Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 57 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 6 3 Add 2 mL of 100 mM pH 6 0 sodium phosphate buffer to each tube Vortex Sonication and or centrifugation may be used if necessary Document any additional sample pretreatment in the case folder 6 4 Place collection tubes or reservoir for waste into vacuum manifold rack or positive pressure apparatus 6 5 Centrifuge samples for at least 10 minutes at 4000 rpm 6 6 Pour sample onto Polychrom Clin Il columns Use sufficient
60. EME d3 CE d3 1ng uL and 6 AM 0 5 ng uL in acetonitrile 3 2 1 Preparation of 10 ng uL Internal Standard Stock Solution Add 1000 uL of each deuterated drug standard 0 1 mg ml to a 10 mL volumetric flask and bring to volume with acetonitrile Store refrigerated 3 month expiration 3 2 2 Preparation of 1 ng uL Internal Standard Solution Add 1000 uL of 10 ng uL internal standard stock solution to a 10 mL volumetric flask and bring to a volume with acetonitrile Store refrigerated 3 month expiration 3 3 0 1 M HCI 3 4 1M HCI 3 5 Cold Acetonitrile HPLC grade acetonitrile used in this procedure should be stored in the refrigerator at 0 C to 10 C prior to use 3 6 Elution Solvent 2 ammonium hydroxide concentrated in 80 20 methylene chloride isopropanol 3 7 Methylene Chloride lsopropanol 80 20 3 8 PFPA pentafluoropropionic anhydride and HFIP 1 1 1 3 3 3 hexafluoro 2 propanol for derivatization Store both reagents at room temperature Discard if there is a change in color Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 118 of 171 3 10 3 11 3 12 Houston Forensic Science Center Forensic Analysis Division Toxicology Deconjugation Enzyme E coli Type IX A B Glucuronidase 125 000 Units To one vial of 125 000 units add 25 mL of deconjugation buffer and mix gently Store frozen expiration of reconstituted enzyme rea
61. In case of instrument malfunction inform Laboratory Management immediately and document the problem on the lab form LAB 62 EIA Troubleshooting Log or using an equivalent method Take any necessary correction action Note For further instructions on the Sunrise Absorbance Reader refer to the operating manual TECAN instructions for the Use of Sunrise Absorbance Reader Option Touchscreen Color 5 REFERENCES 5 1 TECAN Sunrise Plate Reader Operating Manual Document Part No 30041769 2008 11 Document Revision No 1 5 Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 39 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 5 2 TX 03 01 Drugs of Abuse Screening by ELISA Standard Operating Procedure 5 3 TX 07 02 Operation and Maintenance of the Plate Washer Standard Operating Procedure 5 4 Lab form LAB 62 EIA Troubleshooting Log Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 40 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Enzyme Linked Immunosorbent Assay ELISA 1 PURPOSE Preliminary screening of blood or urine samples for certain drugs or classes of drugs may be performed using Enzyme Linked Immunosorbent Assay ELISA ELISA relies on drug specific antibodies which are att
62. MENTATION 11 1 Baselt Randall C Disposition of Toxic Drugs and Chemicals in Man 7 Ed Biomedical Publications Foster City CA p 1212 1214 2004 11 2 Timothy P Rohrig and Christine M Moore Zolpidem Review Forensic Science Medicine and Pathology Human Press 2005 11 3 Method file ZOLPIDEM M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 148 of 171 Houston Forensic Science Center j P 4 Forensic Analysis Division Toxicology Analysis of Alcohol and Other Volatiles by Headspace GC FID 1 PURPOSE This document outlines the procedures for the qualitative or quantitative analysis of ethanol methanol acetone and isopropanol in toxicology specimens using headspace sampling and dual column gas chromatography with flame ionization detection GC FID 2 SCOPE This procedure is used for the qualitative or quantitative analysis of ethanol methanol acetone and isopropanol in toxicology specimens 3 SAFETY This procedure must be conducted in accordance with the HFSC Health and Safety Manual and Quality Manual All case specimens should be treated with Universal Blood borne Pathogen Precautions Appropriate personal protective equipment must be worn during sample and reagent preparation and when handling volatile or caustic chemicals Material Safety Data Sheets MSDS are available in the laboratory 4 REAGENTS 4 1 Ethyl Alcoh
63. ON OF RESULTS 8 1 Assay performance including but not limited to precision accuracy limits of detection quantitation and linearity are summarized in the validation documentation 8 2 The limit of detection in blood and urine are as follows LOD and LOQ Blood 8 3 Quality Controls For quantitative analysis use external QCs e g UTAK QCs when available UTAK Benzodiazepines Plus 100 ng mL QC contains 100 ng mL of flurazepam nordiazepam oxazepam diazepam lorazepam temazepam clonazepam and alpha hydroxyalprazolam Record the calculated concentrations in the appropriate QC Log NOTE Although present in the UTAK QC clonazepam cannot be quantitated due to an interference caused by prazepam also in the UTAK QC with the target ion Qualitative identification is determined using characteristic retention times and mass spectral characteristics lon ratios should be within 20 of the calibrator or QC However ions of low intensity lt 10 may not be within this range and are acceptable if they fall within 4 absolute of the established ion ratio These are evaluated on a case by case basis Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 74 of 171 9 Houston Forensic Science Center Forensic Analysis Division Toxicology FLOWCHART FOR BLOOD BENZODIAZEPINE ANALYSIS NO DECONJUGATION To 1 mL blood add 50 uL I S 2
64. ONTROL OF QUALITATIVE ASSAYS 7 1 Each analytical batch of a qualitative assay must include a matrix blank and at least one positive control i e a sample of the control matrix fortified to a concentration within 3 times that of the reporting limit for the assay with all drug classes or individual compounds for which the assay is designed to detect For immunoassay methods the positive control may be spiked at a concentration 200 above the positivity threshold 7 2 Each analytical batch must include a low QC sample fortified at no less than 50 of the cutoff calibrator concentration 7 3 A run may be accepted if system suitability if applicable is acceptable if all QC samples perform as expected i e negative controls and reagent blank give a negative response and positive control s gives a positive response Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 20 of 171 Houston Forensic Science Center j oP 4 Forensic Analysis Division Toxicology General Guidelines for Instruments and Equipment 1 PURPOSE Instrumentation and equipment must be regularly maintained to ensure precision and accuracy in the various assays used by the toxicology section 2 SCOPE These guidelines are intended to describe proper operation maintenance and performance verification procedures for key instrumentation 3 MASS SPECTROMETERS 3 1 Tune Verification and Autotu
65. Quantitative drug determinations in urine cannot be interpreted pharmacologically due to differences in total urine volume and elimination rate 8 1 2 Unless specifically requested by the client it is the policy of the laboratory not to report certain substances that are routinely encountered in toxicological specimens including caffeine nicotine and their metabolites and byproducts 8 1 3 Blood is the preferred specimen for quantitative drug determination Blood drug results may be reported either qualitatively or quantitatively The laboratory reserves the right to determine whether qualitative or quantitative results are reported as determined by 8 1 3 1 Class of drug 8 1 3 2 Case type offense type 8 1 3 3 Other toxicological findings 8 1 3 4 Data and or specimen quality 8 1 3 5 Quantity of sample 8 1 3 6 Technical abilities of the laboratory 8 2 In general it is the policy of the laboratory not to perform quantitative analyses that have no interpretive value For example drugs or drug classes that are non impairing e g fluoxetine are reported only qualitatively Qualitative reports may be issued if insufficient sample exists for quantitation Qualitative results may also be reported if the quantity of drug is very low e g between the limit of detection and the limit of quantitation or determined to have little importance compared to other results The laboratory may only report qualitative and or quantitative results for procedur
66. S THE DILUENT AND DOCUMENT IN THE CASE RECORD 6 2 Fortify all controls and calibrators while vortex mixing using the appropriate amount of working standard Refer to the fortification guide for guidance on preparation of calibrators CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS A minimum of one in house control should be included in each run Use appropriate external controls e g UTAK Whole Blood Controls when available 6 3 Add 4 mL of 100 mM pH 6 0 sodium phosphate buffer to each tube Vortex Centrifuge for at least 10 minutes at 4000 rpm 6 4 Place collection tubes or reservoir for waste into vacuum manifold rack positive pressure apparatus 6 5 Pour sample onto SPE columns Use sufficient vacuum pressure to draw samples through the columns 6 6 Wash column with 1 mL deionized water 6 7 Remove waste tubes reservoir and replace with new 6 8 Wash column with 1 mL of 1 M acetic acid 6 9 Dry columns for 5 minutes at full vacuum 6 10 Wash column with 1 mL hexane 6 11 Place conical collection tubes into the vacuum manifold positive pressure apparatus If the Teflon inserts appear dirty vacuum manifold replace them Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 64 of 171 6 18 6 19 6 20 6 21 6 22 Houston Forensic Science Center Forensic Analysis Division Toxicology Elute acidic and neutr
67. S Laboratory Information Management System LLC lower than the lowest calibrator LLE liquid liquid extraction LMQC low aqueous mixed volatile control LOD limit of detection LOQ limit of quantitation METH methamphetamine MeOH methanol methyl alcohol mcg ug or ug microgram mg milligrams min minute mins minutes MS mass spectrometer MU measurement uncertainty N A NA or na not applicable Neg negative NF not found ng nanogram OB outer box OFC or Ofc officer OPICOC Opiates cocaine OZ ounce PCP phencyclidine Pkg package Pos positive QC quality control QNS quantity not sufficient Qs quantum satis bring to volume RL reporting limit RRT relative retention time RT retention time SPE solid phase extraction SS mixed volatile system suitability control STIM stimulants THC tetrahydrocannabinol THCA tetrahydrocannabinol carboxylic acid TN true negative TP true positive UNK or unk unknown w with w out without Version 2 0 Issue Date August 3 2015 Page 171 of 171 Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed
68. SIS To 1 mL blood urine add 50 ul I S 1 ng ul while vortex mixing NY Fortify calibrators controls according to fortification guide while vortex mixing NY Add 2 mL 100 mM sodium phosphate buffer Vortex NU Add 2 mL cold acetonitrile while vortex mixing blood only and centrifuge for 10 minutes 4000 rpm NY Transfer supernatant to new tube and add 1 mL of 0 1 M HCI for blood Add 1 0 mL of 1 M HCI for urine Add sample to Cerex Clin II SPE column and allow to flow under gravity Wash 1 mL deionized water Ny NV NV Wash 1 mL 0 1 M HCI NV Wash 1 mL ethyl acetate NY Dry column on full vacuum pressure for 5 minutes v Elute drugs with 1 mL methylene chloride isopropyl alcohol 80 20 with 2 ammonium hvdroxide PREPARE FRESH NY Evaporate to dryness under nitrogen at 60 C NY Reconstitute in ethyl acetate 30 uL Blood 50 uL for urine Transfer to GC vial and inject to 2 uL onto GC MS using OPICOCSIM M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 124 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Volume of Blood Target Concentration OPICOC Working Volume Added uL mL ng mL Standard Conc ng uL pg mL 13 LITERATURE AND SUPPORTING DOCUMENTATION 13 1 Kerrigan S Goldberger DA Opioids in Principles of Forensic Toxicology 3 Edition Levine B Ed
69. Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 61 of 171 Houston Forensic Science Center j oP 4 Forensic Analysis Division Toxicology Basic Acidic and Neutral Drugs by Gas Chromatography Mass Spectrometry GC MS 1 PURPOSE Basic acidic and neutral drugs are extracted from the matrix using Cerex Polychrom Clin II solid phase extraction SPE columns and gas chromatography mass spectrometry GC MS analysis in electron ionization El mode This procedure is used to identify a wide range of basic acidic and neutral drugs Mepivacaine pentobarbital d5 and prazepam are used as internal standards 2 SCOPE 2 1 This procedure functions as an initial screen for multiple drugs of interest from toxicology specimens including but not limited to blood and urine Urine specimens are always reported qualitatively 2 2 This procedure may be suitable for the analysis of benzodiazepines barbiturates anticonvulsants antihistamines tricyclic antidepressants non tricyclic antidepressants such as selective serotonin reuptake inhibitors SSRIs and related compounds muscle relaxants narcotic analgesics dissociative anesthetics and other abused drugs Other targeted procedures are available for several basic acidic and neutral drugs For samples containing benzodiazepines Alprazolam Confirmation and Benzodiazepines Confirmation are available 2 3 Targeted methods of analysis with greater sensitivity are availa
70. Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 45 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 7 2 To edit or create a method 7 2 1 Go to the main menu 7 2 2 Click on create edit a method 7 2 3 Click OK 7 2 4 Click open 7 2 5 Click on the ELISA kit test number 7 2 6 Click on Endpoint measurement 7 2 7 Click on define evaluation The plate map is displayed 7 2 8 Click on the boxes that need to be changed 7 2 9 Select the identifiers Acronym 7 2 10 Click Fill Selection 7 2 11 Click Finish 7 2 12 Click Save Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 46 of 171 7 4 Houston Forensic Science Center Forensic Analysis Division Toxicology Volume Stop Solution Volume Volume Volume TMB Blood Conjugate Substrate oxazepam Diluted matrix 1 10 in PBS or Forensic Diluent The instrument dispenses the following volumes for urine assays Volume Volume Volume TMB Stop Conjugate uL Substrate uL Solution Volume Urine oxazepam 7 5 7 6 Diluted matrix 1 10 in TF or Forensic Diluent Samples incubate with the drug enzyme conjugate for 1 hour and with the enzyme substrate for 30 minutes The EIA operator is respons
71. State University Standard Operating Procedure 11 6 TECAN Freedom Evo 75 Operators Manual 11 7 TECAN HydroFlex Plate Washer Operators Manual 11 8 TECAN Sunrise Plate Reader Operation Manual Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 49 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Reagents for Drug Screening Confirmation Analyses 1 3 1 3 2 3 3 3 4 3 5 3 6 a4 3 8 3 9 4 2 4 3 5 2 5 3 6 PURPOSE This document outlines the preparation of reagents used in drug screening confirmation analysis SAFETY This procedure must be conducted in accordance with the HFSC Health and Safety Manual and Quality Manual All case specimens should be treated with universal blood borne pathogen precautions Appropriate personal protective equipment must be worn during sample and reagent preparation and when handling caustic chemicals Flammable liquids and vapors may cause eye skin and respiratory tract irritation Derivatization reagents are toxic and must be handled in a chemical safety hood or well ventilated area Material Safety Data Sheets MSDS are available in the laboratory REAGENTS Organic solvents are HPLC grade or higher and inorganic reagents e g salts acids are ACS grade Deionized water is prepared using a Millipore Direct QUV3 water system or equivalent Preparations of
72. The instrument software will generate and report the peak to peak valley ratio for peaks that have an apparent shoulder or interfering peak A number greater than 10 indicates acceptable resolution Any samples with unresolved or asymmetric peaks may be re injected once If the same condition is found the sample must be re aliquotted Case specimens Ethanol Results from FID1 A BAC1 and FID2 B BAC2 per aliquot must be within 5 of each other If the average truncated to 3 decimals of aliquot 1 and aliquot 2 from FID1 A BAC1 lt 0 050 g 100 mL both aliquot values must be 10 of the average Aliquot 1 Aliquot 2 Aliquot 1 Aliquot 2 ii A E x Acceptable Range 7 x 0 9 to 7 Version 2 0 Issue Date August 3 2015 Page 154 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 12 4 1 3 If the average truncated to 3 decimals of aliquot 1 and aliquot 2 from FID1 A BAC1 gt 0 050 g 100 mL both aliquot values must be 5 of the average Aliquot 1 Aliquot 2 Aliquot 1 Aliquot 2 Acceptable Range a 0 95 to a 1 05 12 4 1 4 If a case specimen is not within the given range that case specimen must be re analyzed If after two analyses the duplicate difference for each run exceeds the acceptable range the lowest obtained blood alcohol value from all four results FID1 A will be used Any further attempts should be investigated and discussed with the section Manager Supervisor
73. UTION MUST BE MADE FRESH 6 1 9 Dry column on full vacuum pressure for 5 minutes 6 1 10 Place conical glass collection tubes into the rack 6 1 11 Elute THC with 2 mL ethyl acetate 6 1 12 Dry column on full vacuum pressure for 5 minutes 6 1 13 Elute THCA with 2 mL SPE Elution solvent 3 glacial acetic acid in hexane ethyl acetate 90 10 ELUTION SOLVENT MUST BE MADE FRESH 6 1 14 Turn on vacuum or apply pressure for a few seconds to ensure all elution solvent has drained from the column 6 1 15 Remove tubes from rack and evaporate to dryness approximately 20 minutes under nitrogen at 50 60 C 6 1 16 IN THE FUME HOOD add 30 uL BSTFA Cap tightly Heat at 70 C for 15 minutes 6 1 17 Allow samples to cool to room temperature Transfer to auto sampler vials 6 1 18 Inject 2 uL onto the GC MS using THC M Note Ethyl acetate HPLC grade may be added to samples that require dilution i e overloaded peaks if necessary 6 2 URINE USE SALINIZED GLASSWARE 6 2 1 In around bottom centrifuge tube add 1 mL urine For qualitative analysis include appropriate negative and positive controls For preparation of a 50 ng mL positive control add 50 uL of THC working Standard Solution 1 ng uL to 1 mL urine while vortex mixing A glucuronidated control should also be included For preparation of a 50 ng mL THC glucuronide control add 50 uL of THCA glucuronide 1ng uL to 1 mL urine while vortex mixing 6 2 2 Add 100 uL of 10 M KOH an
74. WORKING Internal Standard Solution 1 ng uL while vortex mixing total concentration 100 ng mL NOTE IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALLER VOLUME ADD BUFFER AS THE DILUENT AND DOCUMENT IN THE CASE FOLDER 6 1 2 Fortify all controls and calibrators while vortex mixing using the appropriate amount of working standard Refer to the fortification guide for guidance on preparation of calibrators CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS A minimum of one in house control should be included in each run Use appropriate external controls e g UTAK Whole Blood Controls when available An in house quantitative QC of 100 ng mL is recommended 6 1 3 Add 2 mL of 100 mM pH 6 0 sodium phosphate buffer to each tube Vortex 6 1 4 Centrifuge samples for at least 10 minutes at 4000 rpm 6 1 5 Place collection tubes or reservoir for waste into vacuum manifold rack or positive pressure apparatus Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 99 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Pour sample onto Polychrom Clin Il columns Use sufficient vacuum pressure to draw samples through the columns 6 1 7 Wash column with 1 mL deionized water 6 1 8 Remove waste tubes reservoir and replace with fresh waste tubes reservoir 6 1 9 Wash column with 1
75. ached to polystyrene wells on a 96 well microliter plate The unknown sample is fortified to the plate with a drug enzyme conjugate horseradish peroxidase Any free drug in the sample competes with the conjugate for antibody binding sites on the surface of the well After the well is washed a chromogenic substrate is added and a color is produced by catalysis by horseradish peroxidase The enzymatic reaction is stopped by using dilute hydrochloric acid and then the absorbance is measured at 450 and 620 nm The intensity of the color is inversely proportional to the concentration of drug in the sample 2 SCOPE This protocol describes usage of ELISA as a preliminary qualitative screen for certain drugs or classes of drugs 3 REAGENTS AND KITS SUPPLIED BY THE VENDOR 3 1 Antibody coated polystyrene microliter plates These are purchased as commercial kits Immunalysis Corporation 3 1 1 Cannabinoids THCA Direct ELISA Kit 3 1 2 Cocaine Metabolite BE Specific Direct ELISA Kit 3 1 3 Opiates Direct ELISA Kit 3 1 4 PCP Direct ELISA Kit 3 1 5 Methamphetamine Direct ELISA Kit 3 1 6 Benzodiazepines Direct ELISA Kit 3 2 Enzyme conjugate Horseradish peroxidase labeled drug and diluted in a protein matrix with protein stabilizers 3 3 Substrate Reagent Each bottle contains 3 3 5 5 tetramethylbenzidine and urea peroxidase in buffer 3 4 Stop Reagent Each bottle contains 1N hydrochloric acid HCI 3 5 Forensic Diluent pH 7 0 Phosphate buffe
76. age Refrigerated 6 3 Discard 6 months 7 80 20 Methylene Chloride isopropanol 7 1 In a500 mL volumetric flask add 100 mL of isopropanol to 400 mL of methylene chloride 7 2 Storage Room temperature 7 3 Discard 6 months 8 2 Ammonium hydroxide concentrated in 80 20 methylene chloride isopropanol Elution Solvent 8 1 Ina25 mL volumetric flask add 0 5 mL concentrated ammonium hydroxide Bring to volume with 80 20 methylene chloride isopropanol 8 2 Storage Room temperature 8 3 Discard after each daily use 9 2 Ammonium hydroxide concentrated in ethyl acetate with 1 mL of methanol 9 1 Ina50mLvolumetric flask partially filled with ethyl acetate add 0 5 mL concentrated ammonium hydroxide Bring volume to 25 mL with ethyl acetate Add 1 mL of Methanol Mix vigorously 9 2 Storage Room temperature 9 3 Discard after each daily use 10 1 Hydrochloric acid in methanol Acidic Methanol 10 1 Add 1 mL of concentrated hydrochloric acid into a 100 mL volumetric flask using a volumetric pipette Bring to volume with methanol 10 2 Storage Room temperature 10 3 Discard 3 months 11 0 1 M Hydrochloric acid 11 1 Add 8 4 mL concentrated hydrochloric acid to a 1 liter volumetric flask containing deionized water Bring to volume with deionized water 11 2 Storage Room temperature 11 3 Discard 12 months 12 1M Hydrochloric acid Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date A
77. al drugs with 1 mL ethyl acetate Turn on vacuum for a few seconds to ensure all elution solvent has drained from the column Remove conical collection tubes and replace with waste tubes reservoir Wash column with 1 mL methanol Dry column for 5 minutes at full vacuum or pressure as appropriate Replace the conical collection tubes containing the acidic neutral fraction into the vacuum manifold positive pressure apparatus If the Teflon inserts appear dirty replace them Elute basic drugs with 1 mL elution solvent 2 ammonium hydroxide in methylene chloride isopropyl alcohol 80 20 ELUTION SOLVENT MUST BE MADE FRESH Turn on vacuum or apply pressure for a few seconds to ensure all elution solvent has drained from the column Remove tubes from rack and add 30 uL of acidic methanol Evaporate to dryness under nitrogen for 15 minutes at 50 60 C Reconstitute in 30 uL ethyl acetate or an appropriate volume vortex and transfer to autosampler vials Inject 2 uL onto the GC MS using BAN M 7 INSTRUMENTAL ANALYSIS Tak 7 2 Ensure that the daily QC and tune verification have been completed FULL SCAN acquisition BAN M 8 INTERPRETATION OF RESULTS 8 1 8 2 Qualitative identification is determined using relative retention time RRT and mass spectral characteristics Relative retention times must be within 2 of the standard or QC and acceptable mass spectral library fit See Current Working RRT List for RRTs of common drugs
78. alibration document 3 before it may be used for casework 4 2 9 Follow manufacturer s instructions for troubleshooting maintenance if needed 4 2 10 If a pipette fails a performance verification check or if an analyst has reason to believe that a pipette is not working properly they must 4 2 10 1 Perform a pipette verification and if the pipette is not in proper working order 4 2 10 1 1 Clearly mark the pipette OUT OF SERVICE 4 2 10 1 2 Inform the laboratory manager No laboratory case work will be performed using the pipette until the problem is corrected 4 2 10 1 3 Repair or send out the pipette for repairs 4 2 10 1 4 Verify that the pipette is working correctly and falls with in the proper tolerances following routine repair maintenance or calibration 4 2 10 1 5 Maintain the appropriate documentation and update the log book 4 2 10 2 Occasionally a pipette may be out of service even if no problem has been identified newly purchased pipettes pending verification or calibration In the event a pipette is out of service inactive in repair etc the physical pipette shall be marked OUT OF SERVICE and the appropriate dates for the period documented LAB 41 Pipette Performance Verification Check or an equivalent form 4 2 10 3 A volumetric or positive displacement pipette is intended for the quantitative transfer of a liquid On occasion however pipettes are used only for qualitative purposes e g transfer st
79. anized into the vacuum oven Ensure that glassware does not obstruct the flow of gases or vapors Heat the oven to 160 C Ensure that both vent valves are closed Turn on the vacuum and slowly open the vacuum valve to allow the vacuum to rise The needle on the gauge will move When the vacuum reaches 15 psi close the vacuum valve Turn off the vacuum Prepare the injection by drawing up 100 uL of 1 1 1 3 3 3 Hexamethyldisilazane into a Hamilton gas tight syringe Carefully open the injector valve just until the vacuum begins to steadily fall Inject the 1 1 1 3 3 3 Hexamethyldisilazane The oven must keep a vacuum of at least 10 psi during silanization Close the injector valve Silanize glassware for at least 1 hour Open the vacuum valve so that the vacuum starts to fall gradually to zero Vent the oven by opening the injection valve Turn off the oven and allow the contents to cool to room temperature Remove the glassware Store silanized glassware in a clearly marked receptacle 7 LITERATURE AND SUPPORTING DOCUMENTATION 7 1 Operator Manual NAPCO Vacuum Oven Thermo Scientific Manual P N 3177871 Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 28 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Operation and Maintenance of Zymark Turbo Vap LV 1 PURPOSE During toxicological extraction samples
80. aries with column length Deuterated D3 and D9 internal standards are used throughout 8 INTERPRETATION OF RESULTS 8 1 Assay performance including but not limited to precision accuracy limit of detection limit of quantitation and linearity are summarized in the validation documentation 8 2 The limits of detection and quantitation for methadone and EDDP are 20 ng mL in blood Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 115 of 171 9 Houston Forensic Science Center Forensic Analysis Division Toxicology FLOWCHART FOR METHADONE EDDP ANALYSIS To 1 mL specimen add 50 uL I S 5 ng uL while vortex mixing NY Fortify calibrators controls according to fortification guide while vortex mixing NY Add 2 mL 100 mM sodium phosphate buffer pH 6 0 Vortex NY Centrifuge samples for at least 10 minutes at 4000 rom NY Add sample to Polychrom Clin II SPE column Use sufficient vacuum pressure to draw sample through column NY Wash 1 mL deionized water NY NY NY NY NY NY NY Elute Basic Drugs with 1 mL methylene chloride isopropyl alcohol 80 20 with 2 ammonium hydroxide PREPARE FRESH NY Evaporate to dryness under nitrogen at 50 60 C for 10 mins NY Reconstitute in 30 uL ethyl acetate Transfer to GC vial and inject 2 uL onto GC MS using METHADONE EDDP M Toxicology Standard Operating Procedures Versio
81. ate 08 03 2015 Uncontrolled When Printed Page 31 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 5 5 2 CEREX 48 Simultaneously push the blue buttons on each side of the front of the unit until the manifold moves down onto the rack assembly Note Releasing the blue buttons too soon will cause the depression mechanism to automatically open and return to the manifold to its original position 5 6 Using the 4 toggle switches on the top of the manifold turn on the rows that will be needed If rows are not needed they may be turned off if desired to save nitrogen 5 7 To adjust the flow use the SPE flow rate knob located on the front of the unit Three different settings are available to use during extraction 5 7 1 Off No gas flows to the manifold This setting should be used when compression and decompression of the manifold are taking place 5 7 2 Adjust Flow The gas is delivered through the ADJ FLOW regulator and then through the rotameter which is controlled by the needle valve located at its base This flow is optimal when precise slow flow is required for the columns This is the most gentle limiting flow setting 5 7 3 Max Flow The gas delivery system provides rapid gas flow to the manifold which can be controlled by adjusting the regulator located below the Max Flow pressure gauge on the front of the unit This pressure range can be used to maximize flow t
82. ated 4 EQUIPMENT AND MATERIALS 4 1 Air displacement pipettes 100 1000 uL 20 200 uL 2 20 uL 4 2 Cerex PolyChrom Clin II SPE columns 4 3 Vacuum manifold with disposable Teflon inserts or positive pressure SPE manifold 4 4 Pan balance 4 5 pH meter 4 6 Evaporator 4 7 Vortex mixer 4 8 Centrifuge 4 9 Tissue homogenizer 4 10 Sonicator 4 11 Heating block 4 12 Vacuum pump or house vacuum 4 13 Agilent 7890 GC 5975 MSD Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 79 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 5 INSTRUMENTAL PARAMETERS 5 1 Capillary Column 30 m HP 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 0 8 mL min with an injection volume of 2 uL in splitless mode 5 2 GC MS Agilent 7890A Initial Temperature 160 C Hold for 0 5 minutes 40 C min to 230 C Hold for 6 minutes 30 C min to 290 C Hold for 8 minutes 5 3 Wash solvents for autosampler Methanol and ethyl acetate are used as the wash solvents A minimum of 6 pre and 6 post rinses are performed Each rinse cycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 6 EXTRACTION PROCEDURE BLOOD USE SALINIZED GLASSWARE 6 1 1 In around bottom glass centrifuge tube add 1 mL blood and 50 uL Internal Standard Solution 1 ng L while vortex m
83. ation It is recommended that internal performance verification be performed at least annually This is documented using form LAB 12 Balance and Weights Performance Check or an equivalent method General Maintain a log book or electronic equivalent with the results of performance verifications of balances and weight sets maintenance and certification If the result from a performance check is outside of the acceptable range the balance will be immediately taken out of service until maintenance and or certification are performed by an approved vendor Laboratory management should be notified and the problem is to be documented in the instrument log book or an electronic equivalent Since the tolerances of electronic balances vary instrument specifications must be checked to determine the appropriate criteria for satisfactory performance The following general specifications may be used Balance Cass Weights Acceptable Range f e o oo f e O Top Loading 0 95 1 05 g 9 HEATING BLOCK Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 25 of 171 9 1 oa 9 2 Houston Forensic Science Center Forensic Analysis Division Toxicology Method of Use Refer to the appropriate operating instructions for proper handling and use Performance Verification and Maintenance Temperature of heating blocks should be measured with a thermometer prior
84. ation and Maintenance of the TECAN Sunrise Plate Reader 1 PURPOSE The TECAN Sunrise Plate Reader is a spectrophotometer used to measure absorbance of 96 well microtiter plates in the Enzyme Linked Immunosorbent Assay ELISA 2 SCOPE This document describes the operation and maintenance of the TECAN Sunrise plate reader Further operational instruction is found in the ELISA SOP 3 EQUIPMENT TECAN Sunrise Plate Reader 4 PROCEDURE 4 1 4 2 4 3 4 4 4 5 4 6 4 4 8 4 9 4 10 4 11 4 12 4 13 4 14 4 15 4 16 4 17 4 18 4 19 Double click on Magellan 7 2 icon on the desk top Click the Start Measurement Wizard Click on the green button Click the Use Predefined Method Click on blood or urine folder depending on the samples Click on the respective plate that needs to be read e g thc_b mth for THC blood samples Similarly THC_U mth for urine samples Verify that the date is correct in the workspace Verify the correct method is selected Under the Sample ID List click on Insert Click on import Select Tecan Files from the files drop down box Select the microplate that needs to be read Click on open The plate map with the list of samples that has been loaded in the racks is displayed Click OK Click on Start The UV spectrophotometer measures the absorbance at two wavelengths 450nm and 620nm Click on Finish Click Save to save the data
85. ber between the evidence and submission information do not match 6 1 1 4 Specimens missing affixed labels or specimens with affixed labels missing pertinent information this includes at least two of the following subject name agency case number or date of birth 6 1 1 5 Outer most evidence container not being properly sealed 6 1 1 6 All specimens are compromised e g leaking cracked or tampered container s 6 1 1 7 Inconsistent evidence descriptions between evidence received submission form and evidence documentation 6 1 2 Minor discrepancies Minor discrepancies include any missing or contradicting information not pertaining to fatal discrepancies 6 2 Photographs Evidence must have representative images uploaded into LIMS The naming scheme of Item Picture e g Item1 1 may be used Pictures may include 6 2 1 Outer most container including all sides 6 2 2 Inner plastic box 6 2 3 Blood vials in plastic tubing 6 2 4 Blood vials at all angles 6 2 5 Urine specimen containers at all angles 6 2 6 Any other exhibits Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 6 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 6 3 Sub itemizing A description of specimens within a case along with the corresponding sub item number must be documented on the Evidence Description and Review Form LAB 32 or an electronic
86. ble for many commonly encountered drugs Thirty drugs were selected to represent the wide range of drugs and drug classes that are commonly detected using this procedure 3 STANDARDS AND SOLUTIONS 3 1 BAN Working Mix The BAN Working Standard is a methanolic solution of common basic acidic and neutral BAN drugs Refer to the Reagent QC Log for composition of the current BAN mix Typical drugs in the BAN working standard include benzodiazepines barbiturates muscle relaxants opioids tricyclic antidepressants selective serotonin reuptake inhibitors or selective norepinephrine reuptake inhibitors dissociative anesthetics and other abused drugs For example butalbital secobarbital phenobarbital benzylpiperazine meperidine meprobamate ketamine diphenhydramine carisoprodol PCP tramadol methadone propoxyphene amitriptyline nortriptyline imipramine cyclobenzaprine hydrocodone oxycodone zolpidem diazepam nordiazepam flurazepam alprazolam venlafaxine and sertraline 3 1 1 Preparation of BAN Working Mix 20 ng uL Separate calibrator and quality control working mixes are prepared at 20 ng ml Drug standards are purchased in 1 0 mg mL ampoules In a 10 mL volumetric flask add 200 uL of undiluted drug standard 1 0 mg mL Bring to volume with methanol Store refrigerated 3 month expiration Refer to fortification guide below for guidance on preparation of calibrators and controls 3 1 2 Preparation of BAN Working Mix 2 ng
87. but delay completion of preparation at the predetermined step and for the correct time 4 9 1 3 Auto sampler stability Both the analyte s and the internal standard in processed QC samples will be tested for stability This will demonstrate that extracts are stable while in the auto sampler awaiting analysis In addition determination of stability beyond 12 hours will allow for the re injection of samples in the event of instrument malfunction 4 9 1 3 1 Determine the maximum residence time of the extracts in the auto sampler to be tested Include the time a run may sit unanalyzed over a weekend in the auto sampler due to an instrument malfunction A different approach may be necessary for unstable molecules 4 9 1 3 2 Set aside a validation run that includes calibrators and at least 2 low and 2 high controls to be re injected These samples will be added to another validation run with a freshly prepared calibrator 4 9 1 3 3 Prepare a calibration curve along with at least 4 low and 4 high controls 4 9 1 3 4 Inject the calibration curve only and one set at least 2 low and 2 high controls 4 9 1 3 5 Store the remaining vials in the auto sampler for at least 12 hours The interval chosen should represent the longest period that the processed extracts will remain at auto sampler conditions during the course of an analytical run 4 9 1 3 6 At the end of the predetermined time period inject all controls half will be unanalyzed half will be re injects
88. calculated by instrument software 13 2 2 All calibrators must back calculate to within 20 of target 13 2 3 A maximum of 1 calibrator may be dropped from the curve Ifa calibrator is not used this must be documented on the Calibration Curve along with an explanation as to why it was not included 13 3 Urine 13 3 1 The Limit of Detection LOD and Limit of Quantitation LOQ in blood are10 ng ml 13 3 2 Urine is calibrated using a 1 point calibration curve 14 QUALITY CONTROL ACCEPTANCE CRITERIA 14 1 All Blood QC samples must quantify within 20 of target 14 2 All urine controls must give the expected result negative must be negative positive must be positive 14 3 All blood QCs must pass to report blood results 14 4 All urine QCs must pass to report urine results 15 REPORTING RESULTS 15 1 Blood 15 1 1 All ion ratios must be within 20 of the average ion ratio of the calibrators 15 1 2 Quantitative results are reported by rounding to the ones place ex 5 6 ng mL reported as 6 ng mL 9 2 ng mL reported as 9 ng mL 10 7 ng mL reported as 11 ng mL 15 2 Urine 15 2 1 All ion ratios must be within 20 of the ion ratio of the cutoff calibrators 15 2 2 If the response ratio of the target ion for PCP is greater than the response ratio of the 10 ng mL cutoff calibrator the result is reported as Positive 15 2 3 If the response ratio of the target ion for PCP is less than the response ratio of the 10 ng mL cutoff calibrator
89. cedure 9 4 For quantitative analysis calibrators are typically prepared at concentrations between 20 and 1000 ng mL Although the limit of quantitation for some drugs is lower than 20 ng ml concentrations in the range between the limit of quantitation and the lowest calibrator may be reported as less than 20 ng mL See Quantitative Analysis below An independent control is included in each run 10 QUALITY CONTROLS 10 1 For quantitative analysis use external QCs e g UTAK QCs when available Record the calculated concentration in the appropriate QC Log 10 1 1 UTAK Drugs of Abuse Level QC contains 100 ng mL of morphine codeine cocaine BE and CE 10 1 2 UTAK Drugs of Abuse Level 2 QC contains 500 ng mL of morphine codeine cocaine BE and CE Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 122 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 10 2 If total morphine is to be determined both the working standard and deuterated internal Standard are glucuronidated morphine 3 glucuronide and morphine 3 glucuronide d3 respectively Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 123 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 11 FLOWCHART FOR OPIATE COCAINE ANALY
90. chemical safety hood or well ventilated area Material Safety Data Sheets MSDS are available in the laboratory Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 4 of 171 Houston Forensic Science Center j oP 4 Forensic Analysis Division Toxicology Evidence Handling 1 PURPOSE This document outlines the handling procedures of submitted evidence into the Toxicology Section 2 SCOPE This procedure is used for the handling and storage of evidence within the Toxicology Section 3 SUBMISSION OF EVIDENCE Evidence to be analyzed by the Toxicology Section is received from law enforcement agencies or the judicial system 4 STORAGE OF EVIDENCE Toxicology evidence is routinely stored in refrigerators within the toxicology section Freezer storage is an acceptable alternative All refrigerators and freezers in the toxicology section are monitored using TempAlert which is further detailed in the Quality Manual or equivalent system Acceptable refrigerator temperature range gt 0 10 C Acceptable freezer temperature range lt 0 C If a refrigerator freezer stops functioning and exceeds the acceptable temperature range evidence will be moved to another functioning refrigerator freezer and transfer documented 5 RECEIVING EVIDENCE It is the responsibility of Toxicology personnel to maintain the integrity of evidence at all times while in their custody
91. d in 80 20 methylene chloride isopropanol 3 5 Methylene Chloride lsopropanol 80 20 4 EQUIPMENT AND MATERIALS 4 1 Air displacement pipettes 100 1000 uL 20 200 uL 2 20 uL 4 2 Cerex PolyChrom Clin II SPE columns 4 3 Vacuum manifold with disposable Teflon inserts or positive pressure SPE manifold 4 4 Pan balance 4 5 pH meter 4 6 Evaporator 4 7 Vortex mixer 4 8 Centrifuge 4 9 Tissue homogenizer 4 10 Sonicator 4 11 Heating block Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 92 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 4 12 Vacuum pump or house vacuum 4 13 Agilent 7890 GC 5975 MSD 5 INSTRUMENTAL PARAMETERS 5 1 Capillary Column 30 m HP 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 1 3 mL min with an injection volume of 2 uL in split mode 10 1 5 2 GC MS Agilent 7890A Initial Temperature 140 C Hold for 0 5 minutes 30 C min to 290 C Hold for 2 5 minutes 50 C min to 310 C Hold for 2 6 minutes 5 3 Wash solvents for autosampler Methanol and ethyl acetate are used as the wash solvents A minimum of 6 pre and 6 post rinses are performed Each rinse cycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 6 EXTRACTION PROCEDURE Blood Urine and all Other matrices 6 1 In around bottom glass cult
92. d in 80 20 methylene chloride isopropanol 4 EQUIPMENT AND MATERIALS 4 1 Air displacement pipettes 100 1000 uL 20 200 uL 2 20 uL 4 2 Cerex PolyChrom Clin II SPE columns 4 3 Vacuum manifold with disposable Teflon inserts or positive pressure SPE manifold 4 4 Pan balance 4 5 pH meter 4 6 Evaporator 4 7 Vortex mixer Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 98 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 4 8 Centrifuge 4 9 Tissue homogenizer 4 10 Sonicator 4 11 Heating block 4 12 Vacuum pump or house vacuum 4 13 Agilent 7890 GC 5975 MSD 5 INSTRUMENTAL PARAMETERS 5 1 Capillary Column 30 m HP 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 1 2 mL min with an injection volume of 2 uL in split mode 10 1 5 2 GC MS Agilent 7890A Initial Temperature 140 C Hold for 0 5 minutes 30 C min to 290 C Hold for 2 5 minutes 50 C min to 310 C Hold for 2 6 minutes 5 3 Wash solvents for auto sampler Methanol and ethyl acetate are used as the wash solvents A minimum of 6 pre and 6 post rinses are performed Each rinse cycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 6 EXTRACTION PROCEDURE BLOOD URINE AND OTHER MATRICES 6 1 1 In a round bottom glass culture tube add 1 mL of specimen and 100 uL
93. d By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 126 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Round bottom tubes conical tubes GCMS vials and caps 6 INSTRUMENT PARAMETERS 6 1 Capillary Column 30 m HP 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 1 3 mL min with an injection volume of 2 uL in split mode 10 1 6 2 GC MS Agilent 7890 Initial Temperature 140 C Hold for 0 5 min 30 C min to 290 C Hold for 2 5 min 50 C min to 310 C Hold for 2 6 min Total Run Time 11 min Injector Temperature 250 C Interface Temperature 280 C MS Quads 150 C MS Source 230 C 6 3 Wash solvents for autosampler METHANOL and ETHYL ACETATE are used as the wash solvents A minimum of 6 pre and 6 post injection rinses are performed Each rinse cycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 7 MAINTENANCE For detailed instructions on the operation of the GC MS refer to TX 07 07 Maintenance and Use of the HP 5975 MSD 8 CALIBRATION Calibrators and controls are prepared using drug free urine or drug free blood Preparation of stock solutions and working standards are documented in a retrievable format 8 1 Calibrator Solutions 8 1 1 Stock Solution 0 1 mg mL Add 1000 uL of Phencyclidine 1 0 mg mL to a 10 mL volumetric flask and bring to volume with methanol Storage Store refrigerated
94. d the end of the sequence 5 3 7 Enter sample IDs reflecting the order of the samples in the sample tray no two boxes can have the identical name i e High High2 5 3 8 Click Save Sample List print sequence by clicking Print Samples List and save run by clicking Save Job 5 3 9 Take a printout of the sample list and place in batch folder 5 3 10 Review the sequence and sample IDs under the Review tab 5 3 11 Click the Run Evo button to start run The dialogue box displays if the machine detects any error The instrument pipettes the indicated amount of controls and samples into the wells in each plate and 100 uL of the conjugate solution A timer automatically pops up Allow the timer to proceed through the incubation countdown 5 3 12 Incubate for 1 hour away from light 5 3 13 After incubation a command box appears instructing to wash the plates in the plate washer and replace them on the deck See procedures for operation of plate reader 5 3 14 After washing the plates place them back into their respective places and pour the TMB substrate into the TMB substrate container Place the TMB substrate container into its respective position on the instrument Click OK 5 3 15 The instrument pipettes 100 uL of TMB substrate into the washed wells Incubate for 30 minutes away from light 5 3 16 Place the plates back into their respective places on the instrument prior to the completion of the 30 m
95. d vortex 6 2 3 Heat samples at 60 C for 15 minutes Let cool to room temperature 6 2 4 Add 100 uL Internal Standard Solution 1 ng uL while vortex mixing The total concentration of internal standard is 100 ng mL 6 2 5 If sample is cloudy or contains particulates centrifuge for 10 minutes at 4000 rpm 6 2 6 Proceed from step 6 through 18 of blood procedure It is acceptable to increase the reconstitution volume for urine extracts prior to injection to prevent sample overload Ethyl acetate may be used for sample dilution or reconstitution of derivatized extracts The presumptive screening test results can be used as a guide Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 81 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 7 INSTRUMENTAL ANALYSIS 7 1 Ensure that the daily QC and tune verification have been completed THC 386 6 67 THCA d3 A76 374 491 a Retention Time RT varies with column length Deuterated D3 internal standards are used throughout Corresponding ions for internal standards are M 3 Refer to attached method for acquisition parameters THC M 7 2 SIM acquisition THC M Use this method to perform targeted analysis for THC and THCA 8 INTERPRETATION OF RESULTS 8 1 Assay performance including but not limited to precision accuracy limit of detection limit of quantitation a
96. dor If blood does not contain the preservative and anticoagulant it can be prepared in house at the specified concentrations Store drug free blood at 0 10 C 3 month expiration from date of receipt for toxicology testing purposes 6 2 Drug Free Urine for Immunoassay and GC MS qualitative analyses 6 2 1 Human urine from drug free individuals is collected into a disposable plastic specimen cup or other collection containers and refrigerated 6 2 2 Once approximately 500 mL of urine has been collected it should be pooled and tested by immunoassay and or GCMS Screen to ensure it is drug free 6 2 3 Verified drug free urine shall be transferred into individual plastic containers each containing approximately 10 mL and frozen Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 11 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 6 2 4 On day of use thaw sufficient urine to complete assay and combine before use 6 2 5 Alternatively commercial drug free human urine products can be used once they are demonstrated not to interfere with the analytical assays in service 6 2 6 Store drug free urine at lt O C 12 month expiration 6 3 Quality Assurance Quality Control All drug free matrices are appropriately tested using screening or confirmatory drug testing prior to use Documentation is maintained in a retrievable format
97. e A minimum of 2 low and 2 high controls must be included in every run that contains blood a minimum of 2 positive and 2 negative controls must be included in every run that contains urine Blood Urine Calibrator 1 Cutoff Calibrator Calibrator 2 Negative Calibrator 3 Positive QC Calibrator 4 Negative QC Calibrator 5 10 Case Samples Negative Negative QC High QC 10 Case Samples e g 1 2 3 4 5 Low QC Positive QC 10 Case Samples 10 Case Samples Low QC Positive QC 10 Case Samples e g 1 2 3 4 5 Negative QC High QC 10 Case Samples High QC Low QC Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed Version 2 0 Issue Date August 3 2015 Page 130 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 13 METHOD OF CALCULATION See SOP In Process Calibration and Quality Control for Toxicology Testing for general information on calibration and control of assays Retention Time RT varies with column length Deuterated D5 internal standards are used throughout Refer to the method for acquisition parameters of PCP M 13 1 Blood 13 2 The Limit of Detection LOD and Limit of Quantitation LOQ in blood are 5 ng ml 13 2 1 Analyte concentrations in blood are determined by linear regression y mx b with 1 x weighting based on the ratio of the peak area of the calibrator divided by the peak area of the internal standard The calibration curve is
98. e Buffer 3 5 Methylene Chloride lsopropanol 80 20 3 6 Elution Solvent 2 ammonium hydroxide concentrated in 80 20 methylene chloride isopropanol 3 7 Acetic anhydride GC Grade for derivatization Store at room temperature 4 EQUIPMENT AND MATERIALS 4 1 Air displacement pipettes 100 1000 uL 20 200 uL 2 20 uL Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 104 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Cerex PolyChrom Clin II SPE columns Vacuum manifold with disposable Teflon inserts or positive pressure SPE manifold Pan balance pH meter Evaporator Vortex mixer Centrifuge Tissue homogenizer Sonicator Heating block Vacuum pump or house vacuum Agilent 7890 GC 5975 MSD Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed Version 2 0 Issue Date August 3 2015 Page 105 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 5 INSTRUMENTAL PARAMETERS sml 5 2 5 3 Capillary Column 30 m HP 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 1 2 mL min with an injection volume of 2 uL in split mode 10 1 GC MS Agilent 7890A Initial Temperature 140 C Hold for 0 5 minutes 30 C min to 290 C Hold for 2 5 minutes 50 C min to 310 C Hold
99. e Confirmation by Gas Chromatography Mass Spectrometry GC MS 118 Phencyclidine Confirmation by Gas Chromatography Mass Spectrometry GC MS 0 126 Stimulants Confirmation by Gas Chromatography Mass Spectrometry GC MS 0 ee 135 Zolpidem Confirmation by Gas Chromatography Mass Spectrometry GC MS 0 0cccceceeee 143 Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 2 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Analysis of Alcohol and Other Volatiles by Headspace GC FID ccccccsssssssssssssssssssssssssesseeesseeeens 149 Quantitative Method Validation cccccccsccccccccccseessseeccccessaeesseeeccceessseaseeeceeessseuusseeeceeeesaaaaaseeeseees 156 Validation of Reportable Qualitative Analytical Methods ccccccccccccsseceesececeeeceeeeeseeeeseeeeeees 164 Validation of Manufactured Kits used for Screening Analysis cccccsseccccssscceeeesececeeseceeeeneceeseees 168 Appendix 1 Abbreviations eseeeeseseenseeeeessrrrsrrererrrresrrerssrrressreresrrressrerosreresereresetreserereserreserereseerese 170 Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 3 of 171 Houston Forensic Science Center Forensic Analysis
100. e technical and administrative review of toxicology documentation 3 REVIEW PROCESS Following batch casework analysis a batch file will be created by the casework analyst conducting the analysis The batch file will then be technically reviewed initially by the casework analyst and afterwards by another qualified analyst using the appropriate Batch Review Checklist Upon successful completion of the batch technical review the casework analyst will then enter case related information into the Laboratory Information Management System LIMS resulting in case reports Each case will then be reviewed by three qualified analysts using the appropriate Case File Review Checklist in the following order casework analyst technical reviewer and administrative reviewer Casework analyst review of each case file consists of both a technical and administrative review Below is a schematic of the overall review process LIMS data entry Batch analysis ie Case files Case files Batch file Batch file report writing a Generate final and generate f technical administrative analyst review technical review and analyst 3 reports batch file review review A B review A c D D A Casework analyst B Any qualified analyst not A C Any qualified analyst not A D Any qualified analyst not Aor C Any errors caught during technical or administrative review must be addressed before proceeding to the next task in the review process If an
101. ed 3 month expiration 3 1 2 Preparation of 1 ng uL Working Standard Add 1000 uL of the 10 ng uL THC THCA Working Standard to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 1 3 Preparation of 0 1 ng uL Working Standard Add 1000 uL of the 1 ng uL THC THCA Working Standard to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 1 4 Preparation of 10 ng uL THCA Glucuronide Stock Standard Add 1000 uL of THCA Glucuronide 0 1 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 1 5 Preparation of 1 ng uL THCA Glucuronide Working Standard Add 1000 uL of THCA Glucuronide 10 ng uL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 2 Internal Standard Solution This solution consists of THC d3 and THCA d3 in methanol at 1 ng L 3 2 1 Preparation of 10 ng uL Internal Standard Stock Solution Add 1000 uL of each deuterated drug standard 0 1 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 12 month expiration 3 2 2 Preparation of 1 ng uL Internal Standard Solution Add 1000 uL of 10 ng L internal standard stock solution to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 months expiration 3 3 Cold Acetonitrile HPLC grade acetonitrile used in this procedu
102. ed at room temperature refrigerated temperature and frozen If other types are expected to be received they should also be investigated NOTE If analytes are determined to be unstable at all storage conditions an investigation of the effects of light protection should be considered 4 9 1 1 2 Prepare enough control material at low and high control concentrations to complete Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed entire stability experiment Version 2 0 Issue Date August 3 2015 Page 162 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 4 9 1 1 3 Store specimens at determined temperatures 4 9 1 1 4 Analyze samples in triplicate after storage for 0 7 14 and 30 days and 6 months NOTE It is permissible to use Day 1 validation QC results as Day 0 for stability purposes 4 9 1 2 In Process stability if it is expected that a break may need to take place during sample preparation the stability of the analyte over that time period should be evaluated 4 9 1 2 1 Determine the point in the procedure at which a break may occur and how long the break may be In the case of procedures where solid phase or liquid extractions are performed the studied step should be the final extraction or elution solvent 4 9 1 2 2 Prepare a calibration curve and controls according to the normal procedure 4 9 1 2 3 Prepare a second set of controls in duplicate
103. eir mass to charge ratio m z The results obtained from GC MS analysis must be evaluated to determine the acceptability of the results This applies to both quantitative and qualitative results 2 SCOPE This policy applies to all GC MS analysis 3 QUALITY CONTROLS 3 1 Positive and negative QCs must be included in each run Each run must end with a negative and positive control For qualitative analysis each batch must contain a negative drug free extract and an extracted drug standard 3 2 For quantitative analysis an in house or external QC must be included Record the calculated concentrations in the appropriate QC Log 4 BLOOD AND URINE Blood is the preferred specimen for impairment cases as it reveals what compounds may be affecting the individual and the concentration of the compounds may be associated with impairment Urine is an acceptable specimen but is typically not quantitated due to variations in volume 5 TISSUES AND OTHER MATRICES 5 1 Appropriate sample preparation steps must be used for tissues or alternative matrices that may be encountered Tissues are routinely homogenized using deionized water 1 1 prior to analysis Additional water may be added to achieve full homogenization Document the conditions for homogenization in the case folder Following homogenization tissues and other solids are analyzed in accordance with blood samples 5 2 Sample preparation for alternative matrices may include homogenization filtrati
104. ent 7890 GC 5975 MSD 5 INSTRUMENTAL PARAMETERS 5 1 Capillary Column 30 m HP 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 1 2 mL min with an injection volume of 2 uL in split mode 10 1 5 2 GC MS Agilent 7890A Initial Temperature 140 C Hold for 0 5 minutes 30 C min to 290 C Hold for 2 5 minutes 50 C min to 310 C Hold for 2 6 minutes 5 3 Wash solvents for autosampler Methanol and ethyl acetate are used as the wash solvents A minimum of 6 pre and 6 post rinses are performed Each rinse cycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 6 EXTRACTION PROCEDURE 6 1 Blood Urine and all Other matrices 6 1 1 In a round bottom glass culture tube add 1 mL of specimen and 100 uL WORKING Internal Standard Solution 1ng uL while vortex mixing total concentration is 100 ng mL 6 1 2 Dilutions If the sample is known to contain a high concentration of drug it should be diluted prior to analysis Aliquot the desired volume of blood and make up to volume using phosphate buffer DOCUMENT THESE CHANGES IN THE CASE RECORD Then proceed from step 4 of the blood procedure 6 1 3 Fortify all controls and calibrators while vortex mixing using the appropriate amount of working standard Refer to the fortification guide for guidance on preparation of calibrators CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS A minimum of one in house
105. entrifuge for 5 min at 4000 rpm Transfer upper layer to clean round bottom centrifuge tube Add 1 mL of 1 10 KCI NaOH pH 12 Cap and rotate for 5 min Centrifuge for 5 min at 4000 rom Transfer upper layer to clean conical bottom centrifuge tube Evaporate to dryness under nitrogen at 50 C Add 25 uL of ethvl acetate and 25 uL BSTFA cap tightlv and heat at 70 C for 15 min Transfer to GC vial and inject 2 uL onto GC MS using BENZOSIM M ihi Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 76 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Working Standard Volume Added Concentration uL ng uL g mL 12 LITERATURE AND SUPPORTING DOCUMENTATION 12 1 Gunnar T Ariniemi K Lillsunde P Determination of 14 benzodiazepines and hydroxyl metabolites zaleplon and zolpidem as tert butyldimethylsilyl derivatives compared with other common silylating reagents in whole blood by gas chromatography mass spectrometry J Chrom B 2005 Nov Dec Vol 818 2 pp 175 189 12 2 Jufer Phipps RA Levine B Benzodiazepines in Principles of Forensic Toxicology 3 Edition Levine B Ed AACC Press Washington DC 2009 pp 191 205 12 3 Siek TJ Specimen Preparation Liquid Liquid Extraction in Principles of Forensic Toxicology 3 Edition Levine B Ed AACC Press Washington DC 2009 pp71 77 12 4
106. eparation of 1 ng uL Zolpidem Working Standard Add 1 mL of 10 ng uL working standard to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 2 Internal Standard Solution 3 2 1 Preparation of 10 ng uL Internal Standard Stock Solution Add 1 mL of Zolpidem D7 100 ug mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 2 2 Preparation of 1 ng uL Working Internal Standard Add 1 mL of 10 ng uL Zolpidem D7 toa 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 3 1M Acetic Acid 3 4 100 mM pH 6 0 Phosphate Buffer 3 5 Elution Solvent 2 ammonium hydroxide concentrated in 80 20 methylene chloride isopropanol 3 6 Methylene Chloride lsopropanol 80 20 4 EQUIPMENT AND MATERIALS 4 1 Air displacement pipettes 100 1000 uL 20 200 uL 2 20 uL 4 2 Cerex PolyChrom Clin II SPE columns 4 3 Vacuum manifold with disposable Teflon inserts or positive pressure SPE manifold 4 4 Pan balance 4 5 pH meter 4 6 Evaporator 4 7 Vortex mixer 4 8 Centrifuge Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 143 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 4 9 Tissue homogenizer 4 10 Sonicator 4 11 Heating block 4 12 Vacuum pump or house vacuum 4 13 Agil
107. eps during derivatizations All pipettes are subject to external calibration and internal performance verification unless they are identified as OUT OF SERVICE or QUALITATIVE ONLY 5 PH METER Three Point Calibration Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 23 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 5 1 Refer to the User s Manual for detailed instructions on proper handling and use 5 2 A three point performance check is a more accurate and preferred method for measuring pH of a solution when pH accuracy better than 0 1 is required It is recommended that this check is performed monthly or as needed depending on use 5 3 At time of use if a three point check has not been done the pH meter should be verified using a pH buffer as close to the sample pH as possible 6 REFRIGERATORS 6 1 Maintenance and Performance Verification 6 1 1 1 Refrigerators and freezers should remain clean and organized at all times If a spill occurs appropriate cleaning procedures should be performed 6 1 1 2 Evidence must be kept separately from drug standards reagents and other analytical substances by storage in a separate refrigerator or freezer 6 1 1 3 Toxicology evidence is routinely stored in refrigerators within the toxicology section Freezer storage is an acceptable alternative All refrigerato
108. equivalent Evidence must be sub itemized in LIMS and correspond with the documentation For cases with multiple evidence items separate evidence forms will be used for each item 6 3 1 Common parent item descriptions in LIMS include ONE TOXICOLOGY KIT TWO BLOOD VIALS ONE BLOOD VIAL or ONE URINE SPECIMEN 6 3 2 Common sub item descriptions include 6 3 2 1 one color top tube 6 3 2 2 one color top tube with replacement top 6 3 2 3 one replacement top tube 6 3 2 4 one plastic container 6 3 3 For cases that have multiple color top tubes the best suited one will be analyzed as follows grey gt lavender gt pink gt tan gt royal blue if it contains anticoagulant The following color top tubes require discussion with manager supervisor and client to decide the appropriateness of testing in the event they are the only type provided gold or red grey orange light green or green grey white red royal blue if it contains a clot activator green light blue Yellow top BD brand tubes will not be analyzed 6 4 Assignments tasks Based on type of offense and type of evidence submitted the following assignments tasks will be added to LIMS unless otherwise requested For cases with multiple subjects associated with separate evidence items an assignment task will be added for each subject Three types of reports may be issued alcohol negative screening and confirmation 6 4 1 DWI Misdemeanor Blood Specimens ALC if lt 0 10 gt Drug
109. erformance verification and maintenance should be performed following the manufacturer s guidelines and the schedule described below Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 24 of 171 8 2 1 1 3 8 2 1 1 4 8 2 1 2 8 2 1 2 1 8 2 1 2 2 8 2 1 2 3 8 2 1 2 4 8 2 1 3 8 2 1 3 1 8 2 1 3 2 8 2 1 3 3 Houston Forensic Science Center Forensic Analysis Division Toxicology Balances Balances must be calibrated and certified with a traceable certificate by an external vendor once a year An internal performance check must be conducted prior to each use using NIST traceable reference weights If an internal performance check on a balance has been completed on a particular day the balance does not need to be checked again for the day This is documented using form LAB 12 Balance and Weights Performance Check or an equivalent method Additional performance verifications may be performed as necessary Balances should be checked for accuracy each time the balance is moved and after maintenance is performed Weights Weights used to performance check the balance shall be sent to a vendor for re certification every 3 years Laboratory weights should be inspected after the annual recertification of the balance Laboratory weights should be stored transported and handled using precautions to protect them from contamination and deterior
110. error is caught during administrative review of a case file the error must be corrected and the case file technically and administratively reviewed once more If the batch file is printed the casework analyst will scan and store the documentation in the proper digital location following successful batch file technical review If the batch file exists digitally it will be moved if not already to the proper digital location Printed or digital case specific documentation will then be moved to each respective case record 4 BATCH FILE A batch file will be created printed or digitally by the casework analyst 4 1 Batch files must include 4 1 1 Batch Review Checklist 4 1 2 Work list 4 1 3 Sequence 4 1 4 Calibration Controls 4 1 5 Data 4 1 6 Worksheet summarizing the data and detailing other information concerning testing Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 9 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 4 2 Batch files may include 4 2 1 Method 4 2 2 Blood and urine batch report 4 2 3 Any corrective action documentation 5 CASE FILE A case file is generated printed or digitally upon submission of evidence to the Houston Forensic Science Center Each case file must include the following 5 1 Examination Documentation 5 1 1 Data relevant to the case 5 1 2 Worksheet summarizing the res
111. es 6 10 Wash column with 1 mL hexane 6 11 Wash column with 1 mL ethyl acetate 6 12 Wash column with 1 mL methanol 6 13 Drycolumn on full vacuum pressure for 5 minutes Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 106 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 6 14 Replace waste tubes reservoir with conical collection tubes If the Teflon inserts vacuum manifold only appear dirty replace them 6 15 Elute basic drugs with 1 mL elution solvent 2 ammonium hydroxide in methylene chloride isopropyl alcohol 80 20 ELUTION SOLVENT MUST BE MADE FRESH DAILY 6 16 Turn on vacuum or apply positive pressure for a few seconds to ensure all elution solvent has drained from the column 6 17 Evaporate to dryness under nitrogen for 15 minutes at 60 C 6 18 Reconstitute in 10uL ethyl acetate and 10 uL acetic anhydride or other appropriate volume of 1 1 ethyl acetate acetic anhydride vortex and transfer to autosampler vials 6 19 Inject 2 uL onto the GC MS using KETO M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 107 of 171 7 re Z 8 Houston Forensic Science Center Forensic Analysis Division Toxicology INSTRUMENTAL ANALYSIS 1 Ensure that the daily QC and tune verification have been c
112. es demonstrated to be scientifically valid 8 3 Screening and Confirmatory Tests 8 3 1 It is the policy of the laboratory not to report positive immunoassay screening results in the absence of confirmation All presumptive positive drug test results by immunoassay are subject to confirmatory analysis using gas chromatography mass spectrometry GC MS or equivalent provided there is sufficient specimen 8 3 2 Preliminary screening tests such as immunoassay have limited scope and utility They are predominantly used to direct the scope of other analytical testing Immunoassay results are not reported unless they are negative and no additional tests are performed In those instances the report clearly states the scope of testing was limited to immunoassay and lists the drug classes and appropriate cutoffs Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 55 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Alprazolam Confirmation by Gas Chromatography Mass Spectrometry GC MS 1 PURPOSE A targeted analysis is performed for confirmatory analysis of alprazolam by solid phase extraction SPE and gas chromatography mass spectrometry GC MS Quantitative and or qualitative analysis are performed using deuterated internal standard and selective ion monitoring SIM in electron ionization El mode 2 SCOPE This procedure
113. established calibration curve to calculate the concentration of the analyte of interest 4 7 2 2 Calculate bias within run and between run precision 4 7 3 Acceptance Criteria 4 7 3 1 Bias Bias lt 20 4 7 3 2 Within Run Precision CV lt 20 4 7 3 3 Between Run Precision CV lt 20 4 8 Dilution integrity if applicable 4 8 1 Procedure 4 8 1 1 Dilute the high control with blank matrix evaluate dilution ratios that may be used on case specimens Common dilutions are 2x 1 part sample 1 part diluent 5x 1 part sample 4 parts diluent and 10x 1 part sample 9 parts diluent 4 8 1 2 Analyze a minimum of two replicates of each dilution per run for at least three runs 4 8 2 Data Analysis 4 8 2 1 Calculate bias and precision as described above 4 8 2 2 Note The average control concentrations from the bias and precision experiments can be used as the target for evaluating dilution integrity 4 8 3 Acceptance Criteria 4 8 3 1 All diluted samples must quantify within control acceptance criteria typically 20 of the average value obtained in the Bias and Precision studies 4 9 Stability 4 9 1 Procedure 4 9 1 1 Storage stability A literature search should be performed to determine if storage Stability for the analyte of interest has been reported If no information can be found then follow the procedure below 4 9 1 1 1 Determine what conditions will be evaluated At minimum stability should be investigated in gray top tubes stor
114. f detection limit of quantitation and linearity are summarized in the validation documentation Limits of detection in blood and urine are as follows Drug LOD and LOQ Blood mg L In the event of a co extractive interference that influences the ion ratios of the internal standard meprobamate D7 it is permissible to use the second internal standard carisoprodol D7 to Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 88 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology report meprobamate subject to review and approval by the Laboratory Manager In doing so all qualitative and quantitative reporting criteria must be met Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 89 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 9 FLOWCHART FOR CARISOPRODOL MEPROBAMATE DETERMINATION To 0 25 mL Specimen add 2 mL of 100mM Sodium Phosphate Buffer pH 6 0 and vortex NY Add 100 uL I S 10 ng uL while vortex mixing NY NY Fortify calibrators controls according to fortification guide while vortex mixing Centrifuge samples for at least 10 minutes at 4000 rpm NU Add sample to Cerex Clin II SPE column and draw sample with minimum vacuum or pressure if necessary NV Wash 1
115. for 48 Place Processor 3 4 Waste bin s and waste bin rack 3 5 Nitrogen and Air compressed gas cylinder supply 4 PRECAUTIONS 4 1 The SPE Processor does not include a filter on the source gas input A clean oil free gas source is essential to prevent sample contamination 4 2 Compressed nitrogen is the recommended pressure source If necessary the unit can operate using high purity filtered air Note The positive pressure processor must be used in a fume hood in order to prevent inhalation of aspirated biological fluids and organic solvents 5 PROCEDURE 5 1 Once samples are ready for extraction place SPE columns into SPE rack 5 2 Place a waste bin in the waste bin rack and set the SPE rack on top The black marker on the rack leg should be in the back right corner for correct alignment of racks column openings and gas flow 5 3 Turn on gas flow to the unit 5 4 To compress the manifold place the assembled collection and column racks on the slide platform Slide rack to the back of the unit using the small handle on the front of the slide platform The platform will stop when it reaches the stop located at the back of the unit under the manifold 5 5 To begin applying pressure to the rack assembly 5 5 1 System 48 Simultaneously lift the black switches on each side of the unit This will move the rack assembly up and against the manifold Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue D
116. formed for methamphetamine amphetamine ephedrine pseudoephedrine 3 4 methylenedioxymethamphetamine MDMA methylenedioxy amphetamine MDA and 3 4 methylenedioxy N ethylamphetamine MDEA Drugs are isolated from the matrix using solid phase extraction SPE Perfluoroacyl derivatives are prepared and analyzed using gas chromatography mass spectrometry GC MS deuterated internal standards and selective ion monitoring SIM in electron ionization El mode 2 SCOPE Confirmatory analysis of target analytes from toxicology specimens including but not limited to blood and urine Urine confirmations are reported only qualitatively 3 REAGENTS AND STANDARDS 3 1 Stimulant Working Standards Two working standards are used Each contains methamphetamine amphetamine ephedrine pseudoephedrine MDEA MDA and MDMA at either 10 ng uL or 1 ng uL respectively 3 1 1 Preparation of 10 ng uL Stimulant Working Standard Add 1000 uL of each drug standard 0 1 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 1 2 Preparation of 1 ng uL Stimulant Working Standard Add 1000 uL of the 10 ng uL Stimulant Working Standard to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 2 Internal Standard Solution This solution consists of methamphetamine d14 amphetamine d11 ephedrine d3 pseudoephedrine d3 MDEA d5 MDA d5 and MDMA d5 in methanol at 10
117. gent is 30 days Deconjugation Buffer 100 mM sodium phosphate buffer pH 6 8 Conjugated Morphine Working Standard 10 ng uL For total morphine determination only a morphine 3 glucuronide working standard is used Add 1000 uL of 0 1 mg ml morphine 3 glucuronide drug standard to a 10 mL volumetric flask Bring to volume with acetonitrile Store refrigerated 6 month expiration Conjugated Morphine Internal Standard 10 ng uL For total morphine determination only a deuterated morphine 3 glucuronide internal standard is used Add 1000 uL of a 0 1 mg ml morphine 3 glucuronide d3 drug standard to a 10 mL volumetric flask Bring to volume with acetonitrile Store refrigerated 6 month expiration 4 EQUIPMENT AND MATERIALS 4 1 4 2 4 3 4 4 4 5 4 6 4 4 8 4 9 4 10 4 11 4 12 4 13 Air displacement pipettes 100 1000 uL 20 200 uL 2 20 uL Cerex PolyChrom Clin II SPE columns Vacuum manifold with disposable Teflon inserts or positive pressure SPE manifold Pan balance pH meter Evaporator Vortex mixer Centrifuge Tissue homogenizer Sonicator Heating block Vacuum pump or house vacuum Agilent 7890 GC 5975 MSD 5 INSTRUMENTAL PARAMETERS 5 1 Capillary Column 30 m HP 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 1 1 mL min with an injection volume of 2 uL in split mode 10 1 5 2 GC MS Agilent 7890A Initial Temperature 100 C Hold for O minutes 30 C min
118. he GC MS using ALPRAZOLAM M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 58 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 7 INSTRUMENTAL ANALYSIS 7 1 Ensure that the daily QC and tune verification have been completed Alprazolam D5 313 284 278 o Alprazolam 308 279 273 9 10 Retention Time RT varies with column length Deuterated D5 internal standards are used throughout Corresponding ions for internal standards are M 5 Refer to attached method for acquisition parameters Alprazolam M 8 INTERPRETATION OF RESULTS 8 1 Assay performance including but not limited to precision accuracy limits of detection quantitation and linearity are summarized in the validation documentation 8 2 The limit of detection in whole blood is 10 ng mL and the limit of quantitation is 20 ng mL Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 59 of 171 9 Houston Forensic Science Center Forensic Analysis Division Toxicology FLOWCHART FOR ALPRAZOLAM ANALYSIS VL VY NV Vv Add sample to Polychrom Clin II SPE column Use sufficient vacuum pressure to draw sample through column Ny Wash 1 mL deionized water NV Wash 1 mL 1 M acetic acid v v Vv Vv v v Elute drug with 1mL Methylene Chl
119. hen necessary 11 7 Wash column with 1 mL of deionized water 11 8 Wash column with 1 mL of 1M acetic acid 11 9 Dry column on full vacuum pressure for 5 minutes 11 10 Wash column with 1 mL of hexane 11 11 Wash column with 1 mL ethyl acetate 11 12 Wash column with 1 mL methanol 11 13 Dry column on full vacuum pressure for 5 minutes 11 14 Replace waste tubes reservoir with labelled conical collection tubes Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 129 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 11 15 Elute Basic Drugs with 1 mL methylene chloride isopropyl alcohol 80 20 with 2 ammonium hydroxide PREPARE FRESH Preparation 2 ammonium hydroxide concentrated in 80 20 methylene chloride isopropanol For instance in a 25 mL volumetric flask add 0 5 mL concentrated ammonium hydroxide Bring to volume with 80 20 methylene chloride isopropanol 11 16 Turn onvacuum pressure for a few seconds to ensure all elution solvent has drained from the column 11 17 Remove tubes from rack and evaporate to dryness approximately 10 minutes under nitrogen at 50 C 60 C 11 18 Reconstitute in 30 uL of ethyl acetate or an appropriate volume vortex and transfer to autosampler vials 11 19 Inject 2 uL onto the GC MS using PCP M method and SIM acquisition 12 SEQUENCE TABLE Example only Not
120. hrough the columns and during column drying Note The max flow regulator is a locking knob It must be adjusted by pulling out the knob Once the desired pressure is set it can be locked in by pushing in on the knob 5 8 Decompression of the manifold is done by the following procedure 5 8 1 System 48 Simultaneously push down the black switches on each side of the unit This will move the rack assembly down and away from the column assembly 5 8 2 CEREX 48 Simultaneously push the blue buttons on each side of the front of the unit until the manifold moves up away from the rack assembly 6 MAINTENANCE Both System 48 and CEREX 48 are constructed of anodized aluminum stainless steel and solvent resistant plastics However the following items should be observed during the operation 6 1 Solvent spillage or overflow should be cleaned immediately in order to prevent instrument damage 6 2 The column seal is silicone rubber and should be cleaned with methanol after each use 7 LITERATURE Operating Instruction System 48 II CEREX Pressure Processor CEREX 48 II manual rev B Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 32 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Operation and Maintenance of the TECAN Freedom Evo 75 1 PURPOSE The TECAN Freedom Evo 75 is an instrument programmed to aliqu
121. ible for completing EIA QC Logs for blood and urine EIA batch reports and submitting data for review Once reviewed EIA results are placed in folders and depending on the results requests for confirmatory analysis are placed 8 OTHER MATRICES 8 1 Appropriate sample preparation steps must be used for tissues or other alternative matrices that may be encountered Tissues are routinely homogenized using deionized water 1 1 prior to analysis Additional water may be added to achieve full homogenization Document the conditions for homogenization in the case folder Following homogenization tissues and solids are analyzed in accordance with blood samples 8 2 Sample preparation for alternative matrices may include homogenization filtration sonication centrifugation or dilution Liquid based alternative matrices are analyzed in accordance with urine samples 8 3 Document all sample preparation steps in the case folder Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 47 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 9 INTERPRETATION OF RESULTS 9 1 9 2 9 3 9 4 9 4 1 9 4 2 9 4 3 9 4 4 9 4 5 All samples with absorbance readings BELOW the positive control mean of duplicate analysis are presumptive positive Confirmatory analyses are requested on all presumptive positive samples unless
122. ing experiment is to be performed to evaluate the effectiveness of the model 4 1 1 1 Prepare a series of standards covering the concentration range of interest with typically no more than a 5 fold increase between sequential calibrators At least six nonzero concentration levels should be used 4 1 1 2 A minimum of 5 replicates per concentration is required The replicates shall be in separate runs The origin shall not be included as a calibration point 4 1 2 Data Analysis 4 1 2 1 Use data from all five runs to produce combined calibration curves with no weighting 1 x weighting and 1 x weighting and process data calibration data using all three calibration curves This can be done by exporting the data and using a validated excel worksheet designed for this purpose 4 1 2 1 1 These combined curves will be used to verify the weighting scheme 4 1 2 2 Using the data from the appropriate weighting scheme performs an unweighted linear regression Of Crominal V Ccalculated Where Crominal is nominal labeled or established concentration of calibrator and Ceatculatead is Concentration obtained from calibration curve 4 1 2 2 1 These data will be used to evaluate the linearity of the analytical measurement range 4 1 2 3 Process each individual run using the selected weighting scheme 4 1 2 3 1 These data will be used to validate the calibration range 4 1 2 4 If fewer calibrators will be used to analyze case samples than were used in validatio
123. inute incubation period 5 3 17 After 30 minutes of incubation the instrument automatically pipettes 100 uL of STOP solution into the wells The plates are ready to be read on the plate reader 5 4 For analyzing individual assays 5 4 1 To enter the aliquots manually for blood and urine runs select Unlock 5 4 2 Right click the panel to add or delete a microplate 5 4 3 Microplate names can be changed by clicking in the associated white box Verify that the amount to be aliquoted is correct 5 4 4 Click on the white drop down box and select the ELISA kit test numbers based on the drug that needs to be tested on the microplates 5 4 5 Check the TMB on the deck box Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 34 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Click the Lock button when complete and saving the panel is optional by clicking Save Panel 6 MAINTENANCE OF THE TECAN EVO 75 6 1 Follow the preventative maintenance procedure below as needed or before running case work 6 1 1 Thoroughly flush the system with deionized water from the system fluid container 6 1 2 Remove racks from the instrument surface Carefully clean the work surface using a chloride disinfectant wipe and a KimWipe 6 1 3 Empty all waste containers 6 1 4 Clean the Teflon sample tip by gently wiping it with a lin
124. ion 2 0 Issue Date 08 03 2015 Page 22 of 171 Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed Houston Forensic Science Center Forensic Analysis Division Toxicology 4 2 Performance Verification and Maintenance Each pipette should be externally calibrated and certified by an approved calibration vendor once per calendar year 4 2 2 Pipettes that dispense volumes greater than 25 uL are evaluated twice per calendar year using a combination of external calibration by an approved vendor and internal performance verification procedures 4 2 3 Due to the inability to gravimetrically verify the small volumes 2 0 uL associated with low volume pipettes these are not subject to in house verification These pipettes are calibrated and certified annually by an outside vendor 4 2 4 Pipette verifications are also performed following any routine maintenance or cleaning 4 2 5 To check for accuracy room temperature deionized water should be pipetted into a weighing vessel on an analytical balance 4 2 6 The pipette should be checked using 3 points over the full range with 5 replicates at each volume 4 2 7 The weight of the water delivered should be recorded on the form LAB 41 Pipette Performance Verification Check or by an equivalent method 4 2 8 The average value of each pipette Air and Positive Displacement should fall within the verification tolerance ranges set by the external vendor s c
125. ion 2 0 Issue Date August 3 2015 Page 42 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 6 2 Positive Control Concentrations 6 2 1 Samples with absorbance readings BELOW the positive control are considered PRESUMPTIVE POSITIVE Confirmatory analysis is routinely indicated for all presumptive positive cases 6 2 2 Note Confirmatory analysis may be requested on any sample regardless of preliminary screening results Positive Control Blood Urine ng mL ng mL 50 300 20 200 20 200 Preparation of QCs 6 2 3 Blood QCs PREPARED DAILY FROM THE BLOOD DRUG STANDARD using drug free blood preserved with 1 NaF Sodium Fluoride and 0 2 K2C204 Potassium Oxalate proceed as follows 6 2 3 1 High QC To 1 0 mL of drug free blood add 125 uL of Blood Drug Standard Mix 6 2 3 2 Positive Control To 1950 uL of drug free blood add 50 uL of Blood Drug Standard Mix 6 2 3 3 Low QC To 1975 uL of drug free blood add 25 uL of Blood Drug Standard Mix Positive Control ng mL Low QC ng mL High QC ng mL Negative Control ng mL Oxazepam SO TO y o O THCA ee a e Benzoylecgonine 5o 23 230 Phencyclidine 0 5 o o d methamphetamine 20 10 10 Morphine 2 1 too 6 2 4 Urine QCs PREPARED DAILY FROM THE URINE DRUG STANDARD Using drug free urine preserved with 1 NaF Sodium Fluoride proceed as follows 6 2 4 1 High QC To 900 uL drug free urine add 1
126. ion This solution consists of carisoprodol D and meprobamate D in methanol The final concentration is 10 ng L Working Internal Standard Solution 10 ng uL Add 1000 uL of carisoprodol D 0 1 mg mL and 1000 uL meprobamate D 0 1 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 3 100 mM sodium Phosphate Buffer pH 6 0 4 EQUIPMENT AND MATERIALS 4 1 Air displacement pipettes 100 1000 uL 20 200 uL 2 20 uL 4 2 Cerex PolyChrom Clin II SPE columns 4 3 Vacuum manifold with disposable Teflon inserts or positive pressure SPE manifold 4 4 Pan balance 4 5 pH meter 4 6 Evaporator 4 7 Vortex mixer 4 8 Centrifuge 4 9 Tissue homogenizer 4 10 Sonicator 4 11 Heating block 4 12 Vacuum pump or house vacuum 4 13 Agilent 7890 GC 5975 MSD Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 86 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 5 INSTRUMENTAL PARAMETERS 5 1 5 2 5 3 Capillary Column 30 m HP 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 1 3 mL min with an injection volume of 2 uL in split mode 10 1 GC MS Agilent 7890A Initial Temperature 140 C Hold for 0 5 minutes 30 C min to 290 C Hold for 2 5 minutes 50 C min to 310 C Hold for 2 6 minutes Wash s
127. ion standards prepared from these stock solutions It will be done before the preparation of batches of controls or validation standards that are to be used in a method validation or characterization Once a method is established correct preparation of calibrators and controls can be verified using the SOP Preparation and Validation of Drug Standards Spiking Solutions Controls and Reagents Each of the two stock standards are to be precisely diluted in triplicate in an appropriate solvent so that they may be analyzed directly by the instrumental technique that will be used for analysis of subject specimens Each dilution is to be analyzed in duplicate giving a total of 12 analyses 1 For example if the compound of interest is to be analyzed by GC MS dilutions will be prepared from each stock solution by diluting each stock solution with reconstitution solvent and then analyzing by GC MS Each dilution will be prepared in triplicate and injected in duplicate giving a total of 12 injections These dilutions must be prepared so that they are within the linear response range of the GC MS instrument A typical procedure would read 3 1 2 Prepare triplicate dilutions of each of the 100 ng uL stock standard solutions by adding 20 uL SZ of the stock standards into conical tubes For instance using a 25 uL syringe add 2 0 mL of freshly prepared reconstitution solvent to each 13mm x 100mm culture tube and vortex briefly to mix Transfer to aut
128. is Division Toxicology PURPOSE This procedure is intended to define the minimum parameters and sets of experiments to validate a quantitative method SCOPE This procedure applies to all quantitative bio analytical methods Method validation is required to verify the performance parameters of a method are fit for purpose Validation is required after the following events occur Development of a new method Modification of a validated method to improve its performance or extend its use beyond that for which it was originally validated Transfer of a validated method to a new instrument Modifications to existing methods or transfer of a validated method to new instruments may not require re validation of all parameters Determination of validation parameters which must be re evaluated will be dependent upon the changes made to the method VALIDATION PARAMETERS The following validation parameters are recommended by the Scientific Working Group for Forensic Toxicology SWGTOX Standard Practices for Method Validation in Forensic Toxicology Please refer to the document published May 20 2013 for detailed information on each parameter Calibration Model Linearity Limit of Detection LOD Limit of Quantification LOQ Bias and Precision Carryover Interference Studies Matrix Matching if applicable not included in SWGTOX guidelines Dilution integrity if applicable Stability if applicable 3 10 lonization suppression enhancement
129. itive or negative 5 3 2 2 Interference from stable isotope internal standards determine if sample is positive or negative 5 3 2 3 Interference from commonly encountered exogenous analytes determine if sample is positive or negative 5 3 3 Acceptance Criteria All samples must test negative 5 4 Stability 5 4 1 Procedure 5 4 1 1 Storage stability A literature search should be performed to determine if storage stability for the analyte of interest has been reported If no information can be found then follow the procedure below Determine what conditions will be evaluated At minimum stability should be investigated in plastic urine cups stored at room temperature refrigerated temperature and frozen If other types are expected to be received they should also be investigated NOTE If analytes are determined to be unstable at all storage conditions an investigation of the effects of light protection should be considered 5 4 1 1 1 Prepare enough positive control material to complete entire stability experiment 5 4 1 1 2 Store specimens at determined temperatures 5 4 1 1 3 Analyze samples in triplicate after storage for 0 7 14 and 30 days and 6 months NOTE It is permissible to use Day 1 validation QC results as Day 0 for stability purposes Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 166 of 171 Houston Forensic Science Center
130. ium hydroxide 1 ml methanol v Vy over drying samples 7 IN THE FUME HOOD add 50 uL of ethyl acetate and 50 uL PFPA Cap tightly Vortex Heat at 70 C for 30 minutes Allow to come to room temperature Evaporate to dryness under nitrogen at a temperature not to exceed 50 C Reconstitute in 20 uL for blood or 40 uL for urine of ethyl acetate vortex and transfer to autosampler vials Inject 2 uL onto the GC MS using STIMSIM M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 141 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Volume of Blood Target Concentration Stimulant Working Volume Added uL mL ng mL Standard Concentration ng uL 12 LITERATURE AND SUPPORTING DOCUMENTATION 12 1 Villamor JL Bermejo AM Fernandez P Tabernero MJ Anew GC MS method for the determination of five amphetamines in human hair J Anal Toxicol 2005 March Vol 29 2 pp 135 139 12 2 Churley M Robandt PV Kuhnle JA Lyons TP Bruins MR Extraction of amphetamine and methamphetamine from urine specimens with Cerex Polycrom Clin II Solid Phase Extraction columns and the Speedisk 48 Pressure Processor J Anal Toxicol 2002 September Vol 26 6 pp 347 354 12 3 Valtier S Cody JT Evaluation of internal standards for the analysis of amphetamine and methamphetamine J Anal Toxicol 1995 October Vol 19
131. ivision Toxicology Opiates and Cocaine Confirmation by Gas Chromatography Mass Spectrometry GC MS 1 PURPOSE A targeted analysis is performed for morphine codeine 6 acetylmorphine 6 AM cocaine benzoylecgonine BE ecgonine methyl ester EME and cocaethylene CE Drugs are isolated from the matrix using solid phase extraction SPE Perfluoroacyl derivatives are prepared and analyzed using gas chromatography mass spectrometry GC MS deuterated internal standards and selective ion monitoring SIM in electron impact El mode 2 SCOPE Confirmatory analysis of target analytes from toxicology specimens including but not limited to blood and urine Urine confirmations are reported only qualitatively 3 STANDARDS AND SOLUTIONS 3 1 Opiate and Cocaine OPICOC Working Standards Two working standards are used Each contains morphine codeine 6 AM cocaine BE EME and CE at either 10 ng uL or 1 ng ul respectively 3 1 1 Preparation of 10 ng ul OPICOC Working Standard Add 1000 uL of each drug standard 0 1 mg mL to a 10 mL volumetric flask and bring to volume with acetonitrile Store refrigerated 3 month expiration 3 1 2 Preparation of a 1 ng uL OPICOC Working Standard Add 1000 uL of the 10 ng uL OPICOC working standard to a 10 mL volumetric flask and bring to volume with acetonitrile Store refrigerated 3 month expiration 3 2 Internal Standard Solution This solution consists of morphine d3 codeine d3 cocaine d3 BE d3
132. ixing Final concentration of internal standard in each sample of 50 ng mL NOTE IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALLER VOLUME AND DOCUMENT IN THE CASE RECORD 6 1 2 Fortify all controls and calibrators while vortex mixing using the appropriate amount of working standard Refer to the fortification guide for guidance on preparation of calibrators CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS A minimum of one in house control e g 20 ng mL THC THCA should be included in each run Use appropriate external controls e g UTAK Whole Blood Controls when available 6 1 3 Precipitate proteins by addition of 2 mL cold acetonitrile while vortex mixing 6 1 4 Centrifuge for 10 minutes at 4000 rpm 6 1 5 Pour off the supernatant layer into a clean round bottom glass tube Add 2 mL of deionized water 6 1 6 Place collection tubes or reservoir for waste into vacuum manifold rack or on positive pressure apparatus 6 1 7 Pour sample onto SPE columns Samples should flow under gravity Do not use the vacuum or pressure unless it is necessary Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 80 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Wash column with 1 mL SPE wash solution 1 ammonium hydroxide concentrated in 85 15 H2O Acetonitrile WASH SOL
133. king Standard Volume Added mL ng mL Concentration ng uL 12 LITERATURE AND SUPPORTING DOCUMENTATION 12 1 Chen Xiao Hua et al Isolation of Acidic Neutral and Basic Drugs from Whole Blood Using A Single Mixed Mode Solid Phase Extraction Column J Anal Toxicol 1992 Nov Dec Vol 16 6 pp 351 355 12 2 Method File BAN M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 67 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Benzodiazepine Confirmation by Gas Chromatography Mass Spectrometry GC MS 1 PRINCIPLE A targeted analysis is performed for desalkylflurazepam flurazepam nordiazepam oxazepam diazepam lorazepam temazepam clonazepam and alpha hydroxyalprazolam Drugs are isolated from the matrix using liquid liquid extraction LLE Trimethylsilyl derivatives of each drug and deuterated internal standards are prepared and analyzed using GC MS selective ion monitoring SIM and electron El ionization 2 SCOPE Confirmatory analysis of target analytes from toxicology specimens including but not limited to blood and urine Urine confirmations are reported only qualitatively 3 REAGENTS AND STANDARDS 3 1 1 3 1 2 3 2 3 3 3 4 3 5 3 6 BENZO Working Standards Two working standards are routinely used Each contains desalkylflurazepam flurazepam nordiazepam oxazepam diaze
134. l range 4 1 3 3 1 1 RE of each calibrator as determined by the weighted linear regression of the individual run must be lt 20 4 1 3 4 Validation of casework calibration 4 1 3 4 1 The linear regression of Cvaical VS Ccasecal MUSt Meet the following requirements 4 1 3 4 1 1 95 Cl of slope must include 1 4 1 3 4 1 2 95 Cl of intercept must include 0 NOTE All remaining validation experiments should be evaluated using results from the data analysis which includes only the calibrators which are to be used in the analysis of case samples 4 2 Limit of Detection LOD 4 2 1 Procedure Analyze a minimum of three different sources of blank matrix fortified at decreasing concentrations in duplicate 4 2 2 Data Analysis 4 2 2 1 Visually inspect the chromatograms to evaluate the retention time peak shape mass spectral ion ratios and any other criteria used to identify the analyte of interest 4 2 2 2 Use the instrument software to evaluate the signal to noise ratio in the blanks and the LOD samples 4 2 3 Acceptance Criteria The LOD is the lowest concentration that 4 2 3 1 Yields a reproducible instrument response greater than or equal to three times the noise level of the background signal from the negative samples Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 159 of 171 4 3 4 3 1 4 3 1 1 4 3 1 2 4 3 2 4 3 2 1 4 3 2 2 4 3
135. les in alcoholic beverages or other liquid specimens results are reported in grams of ethanol per 100 milliliters of liquid For serum plasma specimens the report should state Item x x was found to contain x xxx grams of ethanol per 100 milliliters of serum plasma The equivalent whole blood concentration was calculated to be x xxx x xxx grams of ethanol per 100 milliliters of blood using a conversion factor range of 1 15 to 1 2 or equivalent statement For specimens that required homogenization the report should state This sample was homogenized prior to analysis or an equivalent statement For alcoholic beverages or liquid specimens the results are reported in grams per 100 milliliters of liquid If no volatiles are detected or if results are below the lowest calibrator 0 010 g 100 mL the report should state no alcohol detected or an equivalent statement If there is insufficient sample the report should state Insufficient sample for analysis or equivalent statement If the sample is unsuitable for analysis the report should state Sample unsuitable for analysis or equivalent statement Quantitative Method Validation Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed Version 2 0 Issue Date August 3 2015 Page 156 of 171 2 1 2 2 2 3 3 1 3 2 3 3 3 4 3 5 3 6 3 7 3 8 3 9 Houston Forensic Science Center Forensic Analys
136. lick on Flush 5 2 7 Make sure path is clear for moving the tips 5 2 8 Click Start Flushing Tips The instrument flushes both tips with deionized water before starting the run 5 2 9 Click Ok 5 2 10 Repeat flushing for at least 4 cycles repeat steps 7 amp 8 to prime the instrument Check to see if any bubbles are trapped in the instrument s tubing 5 2 11 When flushing is complete go to File click Exit unload drivers Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 33 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 5 2 12 The display screen stating Should Evoware move all arms to their home positions Click Yes 5 3 To Start Running a Sample Set Using NaviTrak Operating System OS 5 3 1 Double click NaviTrak2 icon and click start 5 3 2 Under the Test Panel tab click Recall Panel 5 3 3 For analyzing blood samples and all assays click blood pnl This automatically brings up the panel that has the aliquots of blood that need to be added into the wells 5 3 4 Similarly for urine samples and all assays click urine pnl 5 3 5 Verify the TMB on the deck is checked 5 3 6 Under the Sample List tab create the sequence by filling out the ID column of the second box controls samples and a high QC after every 10 samples an
137. lidated method to new instruments may not require re validation of all parameters Determination of validation parameters which must be re evaluated will be dependent upon the changes made to the method 3 Definitions Cutoff calibrator matrix sample fortified with the analyte of interest at the reporting limit of the assay Qualitative Negative Control matrix sample fortified with the analyte of interest at a concentration no less than 50 of the cutoff calibrator Qualitative Positive Control matrix sample fortified with the analyte of interest at a concentration no more than 200 of the cutoff calibrator Blind samples matrix matched samples containing the analyte of interest above and below the cutoff concentration True Positive TP sample containing the analyte of interest above the cutoff concentration that gives a positive result True Negative TN sample containing the analyte of interest below the cutoff concentration that gives a negative result False Positive FP sample containing the analyte of interest below the cutoff concentration that gives a positive result Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 164 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology False Negative FN sample containing the analyte of interest above the cutoff concentration that gives a negative res
138. limited to blood This procedure is recommended if quantitative analysis is desired Qualitative results may also be reported For routine full scan qualitative analysis however refer to Basic Acidic and Neutral Drugs Confirmation by GC MS 3 REAGENTS AND STANDARDS 3 1 Methadone EDDP Stock Standard 3 1 1 Preparation of 0 1 mg mL Methadone EDDP Stock Standard Add 1000 uL of methadone and EDDP 1 0 mg mL to a 10mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 2 Methadone EDDP Working Standards 3 2 1 Preparation of 10 ng uL Methadone EDDP Working Standard Add 1000 uL of methadone 0 1 mg mL and 1000 uL of EDDP 0 1 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 2 2 Preparation of a 1 ng uL Methadone EDDP Working Solution Add 1000 uL of Methadone EDDP 10 ng uL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 3 Internal Standard Solution Methadone D9 EDDP D3 5 ng uL 3 3 1 Preparation of 5 ng uL Internal Standard Solution Add 500 uL of Methadone D y and EDDP D3 0 1 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 4 1M Acetic Acid 3 5 100 mM pH 6 0 Phosphate Buffer 3 6 Methylene Chloride lsopropanol 80 20 3 7 Elution Solvent 2 ammonium hydroxide concentrated in 80 20 methylene chloride iso
139. lume vortex and transfer to autosampler vials Version 2 0 Issue Date August 3 2015 Page 137 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Samples suspected of containing high concentrations of drug may be reconstituted ina larger volume of ethyl acetate if necessary Presumptive immunoassay test results can be used as a guide for reconstitution volume for urine samples 6 3 7 Inject 2 uL onto the GC MS using STIMSIM M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 138 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 7 INSTRUMENTAL ANALYSIS 7 1 Ensure that the daily QC and tune verification have been completed 7 2 SIM acquisition STIMSIM M Use this method to perform targeted analysis for amphetamine methamphetamine ephedrine pseudoephedrine MDMA MDA and MDEA MDA 325 Retention Time RT varies with column length Deuterated internal standards are used throughout Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 139 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 8 INTERPRETATION OF RESULTS 8 1 Assay performance including but not limited to precision accuracy limit of detection limit of quantitation and lineari
140. mL deionized water NV Wash 1 mL 1 M acetic acid NV Dry column on full vacuum pressure for 5 minutes VL Wash 1 mL hexane Vy Elute 1 mL ethyl acetate NY Evaporate to dryness under nitrogen at 50 60 C for 10 v Reconstitute in 40 uL ethyl acetate Transfer to GC vial and inject 2 uL onto GC MS using CARISO MEPRO M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 90 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 10 FORTIFICATION GUIDE Volume of Carisoprodol Meprobamate Carios Mepro Volume Added Blood mL Target Conc Target Conc Work Std Conc uL mg L mg L mg mL 0235 A 0o 0 02 0 04 0 02 0 04 0235 20 40 0 02 0 04 11 LITERATURE AND SUPPORTING DOCUMENTATION 11 1 Downey Delisa and Kerrigan Sarah Quantitative Analysis of Carisoprodol and Meprobamate in Whole Blood Using Benzylcarbamate and Deuterated Meprobamate as Internal Standards J Anal Toxicol 2009 June Vol 33 pp 278 282 11 2 Baselt Randall C Disposition of Toxic Drugs and Chemicals in Man 7t Ed Biomedical Publications Foster City CA p 182 183 2004 11 3 Method file CARISO MEPRO M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 91 of 171 Houston Forensic Science Center Forensic
141. mpler vials It is acceptable to increase the Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 120 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology reconstitution volume prior to injection as needed to prevent sample overload The presumptive screening test results can be used as a guide 6 1 19 Inject 2 uL onto the GC MS using OPICOCSIM M 6 2 URINE 6 2 1 In a round bottom glass centrifuge tube add 1 mL urine and 50 uL Internal Standard Solution 1 ng L while vortex mixing Final concentration of internal standard in each sample of 50 ng mL 6 2 2 For qualitative analysis include a minimum of one negative and one positive control in each run For preparation of 200 ng mL positive control add 20 uL of OPICOC Working Standard Solution 10 ng uL to 1 mL urine while vortex mixing 6 2 3 If sample is cloudy or contains particulates centrifuge for 10 minutes 4000 rpm 6 2 4 Add 1 mL 1 M HCI and mix 6 2 5 Proceed from step 6 through 17 of the blood procedure 6 2 6 Evaporate to dryness under nitrogen for 10 minutes at 50 C Reconstitute in 50 uL ethyl acetate vortex and transfer to autosampler vials Samples containing extremely elevated drug concentration based on presumptive immunoassay results may be reconstituted using a larger volume of ethyl acetate if necessary 6 2 7 Inject 2 uL onto the GC MS usi
142. mples BQC2 Up to 10 samples Continue to alternate 10 samples with low and high controls BQC1 LMQC EQC DI Water Control 1 Only necessary if quantifying M I A Only necessary if diluted case specimen s are included in batch NOTE Each sample batch shall end with BQC1 LMQC EQC and DI Water Control 11 2 The order of samples on the autosampler must be verified by a second reviewer before or after analysis This must be documented by initialing and dating the sequence 11 3 Batch size will accommodate all accessioned case specimens or a maximum of 30 case specimens 12 CALCULATION AND ACCEPTANCE CRITERIA Analyte concentrations are determined by linear regression y mx b with 1 x weighting based on the ratio of the peak area of the calibrator divided by the peak area of the internal standard The calibration curve is calculated by instrument software Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 153 of 171 12 1 12 1 1 12 1 2 12 1 3 12 1 4 12 15 12 2 12 2 1 12 2 2 12 2 3 12 3 12 3 1 12 3 2 12 3 3 12 3 4 12 4 12 4 1 12 4 1 1 12 4 1 2 Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed Houston Forensic Science Center Forensic Analysis Division Toxicology Calibrators and Controls Ethanol and M I A For volatile concentrations gt 0 050 g 1
143. n reprocess all data using only the calibrators selected to be used during case analysis Perform an unweighted linear regression Of Cyaical V Ccaseca Where Cvacai is the calculated concentrations of calibrators and controls using all calibrators included in the validation experiments and Ceasecai is the calculated concentrations of calibrators and controls using the calibrators that will be used during analysis of case samples 4 1 3 Acceptance Criteria Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 158 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 4 1 3 1 Verification of weighting scheme 4 1 3 1 1 Correct weighting must be verified by evaluating the X RE for at least unweighted 1 x and 1 x The least complex weighting scheme that minimizes x RE should be used Ceatculatead Guominal Calculate nomina x 100 RE Chominal 4 1 3 2 Validation of linearity 4 1 3 2 1 Visual examination of combined calibration curve with appropriate weighting must verify linearity across analytical range 4 1 3 2 1 1 The linear regression Of Crominal VS Ccakulatea MUSt meet the following requirements 4 1 3 2 1 1 1 95 Cl of slope must include 1 4 1 3 2 1 1 2 95 Cl of intercept must include 0 4 1 3 3 Validation of calibration 4 1 3 3 1 Visual examination of calibration curve must verify linearity across analytica
144. n 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 116 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Working Standard Volume Added uL Concentration ng uL 11 LITERATURE AND SUPPORTING DOCUMENTATION 11 1 Lucas A C S et al Use of solid phase microextraction SPME for the determination of methadone and EDDP in human hair by GC MS Forensic Science International 2000 107 pp 225 232 11 2 Nikolaou P D et al Development and Validation of an El GC MS Method for the Determination of Methadone and its Major Metabolites EDDP and EMDP in Human Breast Milk J Anal Toxicol 2008 Sept Vol 32 pp 478 484 11 3 Nikolaou P D et al Validated method for the simultaneous determination of methadone and its main metabolites EDDP and EMDP in plasma of umbilical cord blood by gas chromatography mass spectrometry J Chrom B 2008 867 pp 219 225 11 4 R C Baselt Disposition of Toxic Drugs and Chemicals in Man 8th edition Biomedical Publications Foster City CA pp 941 944 11 5 Kuhlman J J Jr et al Fentanyl use misuse and abuse a summary of 23 postmortem cases J Anal Toxicol 2003 Vol 27 pp 499 504 11 6 Method file Methadone EDDP M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 117 of 171 Houston Forensic Science Center Forensic Analysis D
145. n of purchased control material must be verified prior to being used with casework Upon verification the verifying analyst shall note the date of verification and initial that date Verification run must be documented and data kept in a retrievable format in the laboratory 5 3 Validation of new lots of spiking solutions calibration samples 5 3 1 Validation batch must include 5 3 1 1 Current calibrator set 5 3 1 2 New calibrator set 5 3 1 3 QC Samples 5 3 2 Evaluation of new spiking solutions calibration samples 5 3 2 1 Calculate batch as normal using current calibrator set as calibrators 5 3 2 2 Treat the new calibrators and quality controls as unknowns and determine their calculated values 5 3 2 3 If possible repeat steps using new calibrator set as calibrators 5 3 2 4 Open the Calibrator Validation Excel Soreadsheet Template 5 3 2 5 Open template and save with name of procedure and new calibrator lot number 5 3 2 6 Right click on the worksheet tab select move or copy check create a copy and select move to end Repeat for every analyte included in the calibration samples 5 3 2 7 Rename each sheet to match analyte being evaluated 5 3 3 Acceptance Criteria 5 3 3 1 When the current calibrator lot results and new calibrator lot results are entered to the spreadsheet a chart will be generated comparing the two sets of data 5 3 3 2 The slope of best fit line is acceptable if 5 3 3 2 1 It is between 0
146. n with 1 mL hexane 6 12 Wash column with 1 mL ethyl acetate 6 13 Wash column with 1 mL methanol 6 14 Dry column on full vacuum pressure for 5 minutes 6 15 Replace waste tubes reservoir with conical collection tubes If the Teflon inserts vacuum manifold only appear dirty replace them 6 16 Elute basic drugs with 1 mL elution solvent 2 ammonium hydroxide in methylene chloride isopropyl alcohol 80 20 ELUTION SOLVENT MUST BE MADE FRESH 6 17 Turn on vacuum or apply positive pressure for a few seconds to ensure all elution solvent has drained from the column 6 18 Evaporate to dryness under nitrogen for approximately 10 minutes at 50 60 C 6 19 Reconstitute in 30 uL ethyl acetate or appropriate volume vortex and transfer to autosampler vials 6 20 Inject 2 uL onto the GC MS using FENTANYL M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 94 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 7 INSTRUMENTAL ANALYSIS 7 1 Ensure that the daily QC and tune verification have been completed 7 2 SIM acquisition FENTANYL M Fentanyl d5 250 151 194 Retention Time RT varies with column length Deuterated D5 internal standards are used throughout Corresponding ions for internal standards are M 5 Refer to attached method for acquisition parameters 8 INTERPRETATION OF RESULTS 8 1 A
147. nager Issue Date August 3 2015 Uncontrolled When Printed Page 85 of 171 Houston Forensic Science Center j P 4 Forensic Analysis Division Toxicology Carisoprodol Meprobamate Confirmation by Gas Chromatography Mass Spectrometry GC MS 1 PURPOSE A targeted analysis is performed for confirmatory analysis of carisoprodol and or meprobamate by solid phase extraction SPE and gas chromatography mass spectrometry GC MS Drugs are isolated from the matrix using an acidic neutral extraction Deuterated internal standards and selective ion monitoring SIM are used in electron ionization El mode 2 SCOPE This procedure should be used for confirmatory analysis of carisoprodol and or meprobamate from toxicology specimens typically blood This procedure is recommended if quantitative analysis is desired Qualitative results may also be reported For routine full scan qualitative analysis of carisoprodol and or meprobamate however refer to Basic Acidic and Neutral Drugs Confirmation by GC MS 3 REAGENTS AND STANDARDS 3 1 Carisoprodol Meprobamate Working Standards Note The concentration of carisoprodol in the working standard is half that of the meprobamate Carisoprodol 0 02 mg mL Meprobamate 0 04 mg mL Working Standard Add 200 uL of carisoprodol 1 0 mg mL and 400 uL of meprobamate 1 0 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 2 Internal Standard Solut
148. nd linearity are summarized in the validation documentation 8 2 Limits of detection in blood and urine are as follows LOD and LOQ Blood ng mL LOD Urine ng mL me 9 QUALITY CONTROLS 9 1 Appropriate positive and negative QCs must be included in each run For qualitative urine analysis each batch must contain appropriate negative and positive controls 9 2 For quantitative analysis use appropriate in house or external QCs e g UTAK Whole Blood QCs when available UTAK Drugs of Abuse Level QC contains 50 ng mL of THCA Record the calculated concentrations in the appropriate QC log Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 82 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 10 FLOWCHART FOR CANNABINOID ANALYSIS IN BLOOD To 1mL blood add 50 uL of I S 1 ng uL while vortex mixing NY Fortify calibrators controls according to fortification guide while vortex mixing 7 Add 2 mL cold acetonitrile while vortex mixing blood only and centrifuge for 10 minutes at 4000 rpm NY Transfer supernatant to new tube and add 2 mL of DI H20 v Add sample to Cerex THC SPE column and allow to flow under gravity v Wash 1 ammonium hydroxide concentrated in 85 15 H2O acetonitrile PREPARE FRESH v Dry column on full vacuum pressure for 5 minutes NV Elute THC with 2 mL ethyl
149. necessary to maintain performance Any changes are prominently documented in the EIA batch file 10 2 3 The examiner and technical reviewer will also evaluate 10 2 3 1 Overall reproducibility poor reproducibility is typically due to liquid handling 10 2 3 2 Lot specific changes to assays 10 2 3 3 Trends that are not assay specific which can indicate liquid handling issues or the need for additional preventive maintenance washing etc 10 2 3 4 Separation absorbance difference between the negative and positive controls 10 2 3 5 Absolute absorbance of the negative control low absorbance may result in a high CV 10 2 3 6 Drift or reproducibility across the plate evaluated using the High QC 10 2 3 7 Responses of the Low QC and High QC 11 LITERATURE AND SUPPORTING DOCUMENTATION 11 1 Smith M Immunoassay in Principles of Forensic Toxicology 3 Edition Levine B Ed AACC Press Washington DC 2009 pp 117 138 11 2 Kerrigan S and Phillips Jr W H Comparison of ELISAs for Opiates Methamphetamine Cocaine Metabolite Benzodiazepines Phencyclidine and Cannabinoids in Whole Blood and Urine Clin Chem 47 540 547 2001 11 3 TX 07 01 Operation and Maintenance of Tecan Freedom Evo 75 Sam Houston State University Standard Operating Procedure 11 4 TX 07 02 Operation and Maintenance of Plate Washer Sam Houston State University Standard Operating Procedure 11 5 TX 07 03 Operation and Maintenance of Plate Reader Sam Houston
150. nes 3 1 1 A tune verification must be performed each day the instrument is used to evaluate the instrument s performance and to check for leaks An autotune must be performed after any maintenance and may be done at other intervals as deemed necessary by the analyst During an autotune the MSD is calibrated by tuning the instrument to ensure that the mass to charge ratios m z are assigned correctly and that the scan ratio is set properly This procedure also serves as a check for air leaks 3 1 1 1 Each day that a tune verification or an autotune is performed it should be documented on the GC MS Maintenance log form LAB 24 or an equivalent form A copy of the most recent tune file should be recorded in a retrievable format 3 1 1 2 Following an El autotune or tune verification on the mass spectrometer the tune report should be examined If the tune does not meet the criteria for the application then action should be taken to determine why it does not meet said criteria For example the system may need a refreshed autotune a source cleaning or there may be an air leak 3 1 1 3 If an instrument does not pass the tune verification no casework will be performed using that instrument until the problem is resolved and the tune verification falls within acceptable specifications 3 1 1 3 1 Tune Specifications 3 1 1 3 1 1 The three tuning masses must be within 0 2 amu of 69 00 219 00 and 502 00 3 1 1 3 1 2 The peak widths of the three tuning masse
151. ng KETAMINE M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 102 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Target Concentration Working Standard Volume Added uL ng mL Concentration ng uL 11 LITERATURE AND SUPPORTING DOCUMENTATION 11 1 Baselt Randall C Disposition of Toxic Drugs and Chemicals in Man 7t Ed Biomedical Publications Foster City CA p 586 588 2004 11 2 Jenkins Amanda J Hallucinogens in Principles of Forensic Toxicology 2 Ed Levine Barry Ed AACC Press Washington DC pp 265 283 2003 11 3 Method file KETAMINE M 11 4 Method file KETAMINE_URINE M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 103 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Keto Opioids Confirmation by Gas Chromatography Mass Spectrometry GC MS 1 PURPOSE A targeted analysis is performed for confirmatory analysis of hydrocodone hydromorphone oxycodone and oxymorphone using solid phase extraction SPE and gas chromatography mass spectrometry GC MS Drugs are isolated from the matrix using a basic extraction Deuterated internal standards and selective ion monitoring SIM are used in electron impact El mode 2 SCOPE Confirmatory analysis of keto
152. ng OPICOCSIM M 7 TOTAL MORPHINE DETERMINATION 7 1 In a round bottom glass centrifuge tube add 1 mL blood and 25 uL of 10 ng uL morphine 3 glucuronide d3 Internal Standard Solution to all standards QCs and samples Note This is a different deuterated internal standard from the free opiate analysis Deuteration and glucuronidation of the internal standard ensures that the deconjugation of opiates in the sample is monitored 7 2 Fortify all controls and calibrators while vortex mixing using the appropriate amount of conjugated morphine working standard Refer to the fortification guide for guidance on preparation of calibrators A minimum of one in house control should be included in each run CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS 7 3 Add 0 2 mL deconjugation enzyme 5000 units Incubate at 37 C for 3 hours 7 4 Proceed from step 3 of the blood procedure 8 INSTRUMENTAL ANALYSIS 8 1 Ensure that the daily QC and tune verification have been completed 8 2 SIM acquisition OPICOCSIM M Use this method to perform targeted analysis for morphine codeine 6 AM cocaine BE EME and CE Qualfierions RT Morphine 577 558 414 Codeine 445 282 266 6 AM 414 473 361 10 60 Cocaine 303 182 82 BE ao 318272 6a Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 121 of 171 Houston Forensic Science Center Forensic
153. ng confirmation LAB 74 Case File Review Drug Toxicology Equivalent electronic checklists may also be used Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 10 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Preparation of Drug Free Matrix 1 PURPOSE Preparation and preservation of drug free matrices using sodium fluoride and or sodium azide 2 SCOPE Drug free matrices are used for the preparation of calibrators and controls for screening and confirmatory toxicology tests Drug free blood containing sodium fluoride preservative and potassium oxalate as anticoagulant is purchased from a commercial vendor Drug free urine is prepared in house and preserved using sodium fluoride and or sodium azide Other biological matrices may be prepared as needed 3 SAFETY QUALITY ASSURANCE This procedure must be conducted in accordance with the Safety Manual the Chemical Hygiene Plan and the Quality Manual 4 REAGENTS 4 1 Sodium fluoride ACS grade or better 4 2 Potassium oxalate ACS grade or better 5 EQUIPMENT 5 1 Stirrer 5 2 Glass media bottles 5 3 Stir bars 5 4 Top loading balance 5 5 Homogenizer for preparation of tissues only 6 PROCEDURE 6 1 Drug Free Blood Drug free blood containing sodium fluoride preservative 1 and potassium oxalate 0 2 as anticoagulant is purchased from a commercial ven
154. ng slot in the instrument 5 5 From Program select Start and then select ELISA Press OK on Run 1 ELISA 5 6 Enter the number of strips on the plate The plate washer washes 2 strips at atime S denotes the presence of a strip and N denotes no strip Example If 4 strips need to be washed the screen display should be SSSSNNNNNNNN 5 Click OK 5 8 The plate washer washes the wells 6 times 5 9 After washing is complete place the plates back into its respective slot on the TECAN EVO 75 instrument 6 MAINTENANCE AS NEEDED 6 1 Rinsing Rinsing is performed to flush the liquid system and to prevent needle blockages During the rinse procedures the needles are soaked in the prime tray 6 2 Rinse Menu The rinse menu has the following options 6 2 1 Rinse Day This is done if the instrument is to be left to stand for a short time of up to two hours Rinse day can be performed with wash buffer or deionized water 6 2 1 1 Procedure Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 36 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 6 2 1 1 1 Switch on the Plate Washer 6 2 1 1 2 Press gt key to find Procedures 6 2 1 1 3 Press OK on Procedures 6 2 1 1 4 Select Rinse 6 2 1 1 5 Press OK to enter the Rinse menu 6 2 1 1 6 Select Rinse Da
155. nidated drugs a glucuronidated control must be included For preparation of a 200 ng mL oxazepam glucuronide control add 20 uL of oxazepam glucuronide 10 ng uL to 1 mL blood while vortex mixing Other glucuronidated drugs may be used when available 6 2 2 Add 1 mL of deconjugation buffer and vortex 6 2 3 Add 100 uL deconjugation enzyme and vortex 6 2 4 Cap and heat samples at 37 C for 1 hour Let cool to room temperature then proceed as outlined blow 6 2 5 Add 50 uL Internal Standard Solution 2 ng uL while vortex mixing The final concentration of internal standard is 100 ng mL 6 2 6 Proceed from step 4 of blood procedure above 6 3 URINE ALL SPECIMENS ARE DECONJUGATED 6 3 1 In a round bottom glass centrifuge tube add 1 mL of sample NOTE IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALLER VOLUME AND DOCUMENT IN THE CASE FOLDER For qualitative analysis include a minimum of one negative and one positive control in each run For preparation of a 100 ng mL positive control add 100 uL of BENZO Working Standard Solution 1 ng uL to 1 mL urine while vortex mixing For hydrolyzation of glucuronidated drugs a glucuronidated control must be included For preparation of a 200 ng mL oxazepam glucuronide control add 20 uL of oxazepam glucuronide 10 ng uL to 1 mL urine while vortex mixing Other glucuronidated drugs may be used when available 6 3 2 Add 1 mL of deconjugation buffer to all tubes and vorte
156. nsure 12 Adjust pH as needed Store at room temperature 12 month expiration 1 10 KCI NaOH Working Solution pH 12 Add 100 mL KCI NaOH Working Solution toa 1L volumetric flask Bring to volume with deionized water Store at room temperature 12 month expiration Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 68 of 171 3 10 3 11 Houston Forensic Science Center Forensic Analysis Division Toxicology 1 Chlorobutane BSTFA N O bis trimethylsilyl trifluoroacetamide for derivatization Must be refrigerated Deconjugation Enzyme E Coli Type IX A B Glucuronidase 125 000 Units To one vial of 125 000 Units add 25 mL of deconjugation buffer and mix gently Store frozen aliquots to prevent freeze thaw cycles Expiration of reconstituted enzyme reagent is 30 days Do not re freeze once reconstituted Deconjugation Buffer 100 mM sodium phosphate buffer pH 6 8 Add 7 0 g of dibasic sodium phosphate to a 500 mL volumetric flask and bring to volume with deionized water while stirring In a second 500 mL volumetric flask add 6 0 g of monobasic sodium phosphate and bring to volume with deionized water while stirring Mix the two solutions together until a pH of 6 8 is obtained Refrigerated storage is recommended expiration 6 months Conjugated Oxazepam Working Standard 10 ng uL An oxazepam glucuronide working standard is used Add 100 uL
157. ntrols Follow the manufacturer s instructions for material preparation and then perform 4 separate analytical runs with 3 replicates per run or a minimum of 12 replicates over more than one run to establish the mean Perform these determinations in parallel with the existing controls to verify performance Acceptable criterion is that the mean value is within 15 of nominal The controls should have verified QC result data from the manufacturer whenever possible to designate the nominal value This validation is conducted if the LOT number for commercial controls changes 7 2 In House controls The target is defined to be the average calculated concentration of a total of at least 12 analyses performed in a minimum of 4 separate analytical runs with a minimum of 3 replicates per run The verified target must be within 15 of nominal concentration 7 3 Daily spiked QCs The target is defined as the nominal value 8 ESTABLISHING PERFORMANCE FOR QC SAMPLES FOR QUALITATIVE ASSAYS 8 1 Commercial controls Follow the manufacturer s instructions and perform a minimum of 4 separate analytical runs with a minimum of 3 replicates per run Perform these determinations in parallel with the existing controls to verify performance All samples must provide accepted results 8 2 In House controls 8 3 The target is defined as the average calculated concentration of a total of at least 12 analyses performed in a minimum of 4 separate analytical runs with a minim
158. of 0 1 mg mL oxazepam glucuronide drug standard to a 10 mL volumetric flask Bring to volume with methanol Store refrigerated 6 month expiration Other conjugated benzodiazepines may be used e g lorazepam glucuronide if oxazepam glucuronide is not available 4 EQUIPMENT AND MATERIALS 4 1 Air displacement pipettes 100 1000 uL 20 200 uL 2 20 uL 4 2 Tube rotator 4 3 Pan balance 4 4 pH meter 4 5 Evaporator 4 6 Vortex mixer 4 7 Centrifuge 4 8 Tissue homogenizer 4 9 Sonicator 4 10 Heating block 4 11 Vacuum pump or house vacuum 4 12 Agilent 7890 GC 5975 MSD Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 69 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 5 INSTRUMENTAL PARAMETERS 5 1 Capillary Column 30 m HP 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 1 5 mL min with an injection volume of 2 uL in splitless mode 5 2 GC MS Agilent 7890A Initial Temperature 180 C Hold for 0 25 minutes 15 C min to 240 C Hold for 3 minutes 30 C min to 290 C Hold for 5 minutes 5 3 Wash solvents for autosampler Methanol and ethyl acetate are used as the wash solvents A minimum of 6 pre and 6 post rinses are performed Each rinse cycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 6 EXTRACTION PROCEDURE BLOO
159. of BSTFA cap tightly and heat at 70 C for 15 minutes NY Allow to cool to room temperature Transfer to GC vial and inject 2 uL onto GC MS using THC M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 84 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Volume of Blood Target Concentration THC THCA Working Volume Added uL ng mL Standard Concentration ng uL 13 LITERATURE AND SUPPORTING DOCUMENTATION 13 1 Huestis MA Cannabis in Principles of Forensic Toxicology 3 Edition Levine B Ed AACC Press Washington DC 2009 pp 269 303 13 2 Baselt RC Tetrahydrocannabinol in Disposition of Toxic Drugs and Chemicals in Man 8 Edition Foster City CA Biomedical Publications 2009 pp 1513 1518 13 3 Watanabe Kanako 11 2 2 Cannabinoids and their metabolites in Drugs and poisons in Humans A Handbook of Practical Analysis Springer Verlag Berlin Heidelberg 2005 pp 187 194 13 4 Abraham TT Lowe TH Pirnay SO Darwin WD Huestis MA Simultaneous GC EI MS determination of D9 Tetrahydrocannabinol 11 Hydroxy D9 tetrahydrocannabinool and 11 nor 9 tetrahydrocannabinol in human urine following tandem enzyme alkaline hydrolysis Journal of Analytical Toxicology 31 October 2007 pp 477 485 13 5 Method File THC M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Ma
160. of an analytical procedure 3 3 Carryover An analyte that is retained from one sample into another sample usually the sample immediately following one that has an elevated concentration of the analyte of interest 3 4 Certified Reference Material CRM drug standard purchased from an approved vendor which includes a certificate of analysis verifying the concentration 3 5 Fortified Quality Control Sample A sample of similar matrix to the unknown case sample which has been spiked with a predetermined amount of the analyte s of interest 3 6 Internal Standard An analyte generally of similar chemical structure to an analyte being measured that is added in a known concentration to all samples calibrators QC s unknowns in an analytical method and that functions as a reference marker for that sample against which the analyte of interest can be measured 37 Linear Range Typically the Limit of Quantification LOQ to the Upper Limit of Quantification ULOQ are administratively defined as the concentration of the lowest and highest calibrator used in preparation of the calibration curve 3 8 Matrix The material into which is spiked known amounts of an analyte s of interest in order to calibrate the method or to track method performance 3 9 Neat A systematic representative of an analyte of interest that is free from admixture or dilution 3 10 Negative control matrix fortified with Internal Standard The negative control may also c
161. of samples Place unknowns and QCs in appropriate positions in subsample racks see chart below NOTE A High QC must be run in between every ten case samples and at the end of every run Carpe ps pe ps Te a NEG SAMPLE SAMPLE SAMPLE SAMPLE HIGH QC B NEG SAMPLE SAMPLE SAMPLE SAMPLE SAMPLE p Posac SAMPLE SAMPLE SAMPLE SAMPLE SAMPLE E Lowac SAMPLE SAMPLE SAMPLE SAMPLE SAMPLE F lowac sampte sampLe HiGHOC SAMPLE SAMPLE 6 mGnac samte saMpue saMPue SAMPLE SAMPLE DH HigH ac samte SAMPLE SAMPLE SAMPLE HIGH QC Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 44 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 7 FLOW CHART 1 10 Dilution of sample with Forensic Diluent PBS pH 7 0 is added into the wells See procedure for individual sample volume for different assays Add 100 uL of the enzyme conjugate to each well Incubate plates for one hour at room temperature 4 t RN 4 Wash plates in the plate washer 6 washes deionized water Add 100 uL of the substrate reagent to each well NY Incubate plates for 30 minutes at room temperature v Add 100 uL of 1N HCI Stop solution to each well to change color from blue to yellow v Measure the absorbance within one hour at a dual wavelength of 450 nm and 620 nm Toxicology
162. ol Ethanol CH3CH70H 64 17 5 200 proof 4 2 n Propanol CH3CH7CH7OH 71 23 8 4 3 lsopropanol CH3CH OH CH3 67 63 0 4 4 Acetone CH3COCH3 67 64 1 4 5 Methanol CH30H 67 56 1 4 6 Deionized DI Water 7732 18 5 4 7 Blank Blood Potassium Oxalate and Sodium Fluoride preserved 5 EQUIPMENT 5 1 Instrumentation using a method that is approved and validated for use in the section 5 2 Compressed gas cylinders or equivalent Helium Hydrogen Air and Nitrogen 5 3 Vials caps stoppers and crimpers 5 4 Hamilton pipettor dilutor or equivalent 5 5 Volumetric flasks 50 mL 100 mL 500 mL 1000 mL etc 5 6 Analytical balance 5 7 Homogenizer 5 8 Centrifuge 5 9 Vortex mixer 6 MAINTENANCE A verification run including calibrators and at least 3 replicates of BQC1 BQC2 EQC and LMQC should be performed following any preventative maintenance and or major repairs Verification run must meet acceptance criteria outlined in this procedure If acceptance criteria are not met appropriate measures must be taken to rectify the problem Verification run must be documented in the maintenance log and data kept in a retrievable format in the laboratory Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 149 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 7 CALIBRATION 7 1 Ethanol Calibration 0 010 0 5
163. olvents for autosampler Methanol and ethyl acetate are used as the wash solvents A minimum of 6 pre and 6 post rinses are performed Each rinse cycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 6 EXTRACTION PROCEDURE Blood and All Other Matrices 6 1 In a round bottom glass culture tube add 0 25 mL specimen and 2 mL of 100 mM sodium phosphate buffer pH 6 0 Vortex mix 6 2 Add 100 uL of Working Internal Standard Solution 10 ng uL while vortex mixing The final concentration of internal standard in each sample is 4 ug mL or 4 mg L 6 3 Fortify all controls and calibrators while vortex mixing using the appropriate amount of working standard The typical calibration range for quantitative analysis is 0 20 mg L carisoprodol and 0 40 mg L meprobamate Refer to the fortification guide for guidance on preparation of calibrators CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS NOTE IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALLER VOLUME ADD BUFFER AS THE DILUENT AND DOCUMENT IN THE CASE FOLDER 6 4 A quantitative QC containing 4 mg L carisoprodol and 8 mg L meprobamate must be used Use appropriate external controls e g UTAK Whole Blood Controls if available 6 5 Centrifuge samples for at least 10 minutes at 4000 rpm 6 6 Place collection tubes or reservoir for waste into vacuum manifold rack 6 7 Add the sample to Polychrom Clin II Column Draw the sample through
164. ompleted 2 SIM acquisition KETO M Oxymorphone d3 Oxymorphone 302 299 288 285 360 357 346 343 Retention Time RT varies with column length Deuterated D3 internal standards are used throughout Corresponding ions for internal standards are M 3 INTERPRETATION OF RESULTS Assay performance including but not limited to precision accuracy limit of detection limit of quantitation and linearity are summarized in the validation documentation Dug LOD and LOQ in Blood ng mL LOD in Urine ng mL Hydrocodone 208 Hydromorphone 235 20 Oxycodone 20 Oxymorphone Toxicology Standard Operating Procedures Issued By Section Manager Uncontrolled When Printed Version 2 0 Issue Date August 3 2015 Page 108 of 171 9 Houston Forensic Science Center Forensic Analysis Division Toxicology FLOWCHART FOR KETO OPIOIDS ANALYSIS To 1 mL specimen add 100 uL I S 1 ng L while vortex mixing NY Fortify calibrators controls according to fortification guide while vortex mixing Vy Add 2 mL 100 mM sodium phosphate buffer Vortex Vv Add sample to Polychrom Clin II SPE column Use sufficient vacuum pressure to draw sample through column NY NY NY NY NY NY NY NY Elute Basic Drugs with 1 mL methylene chloride isopropyl alcohol 80 20 with 2 ammonium hydroxide PREPARE FRESH V Evaporate to dryness under nitrogen at 50 60 C for 10 minutes NY Reconstitute in 10 uL ethyl acetate and
165. on sonication centrifugation and or dilution Liquid based alternative matrices are analyzed in accordance with urine samples 5 3 DOCUMENT ALL SAMPLE PREPARATION STEPS IN THE CASE RECORD AND INCLUDE APPROPRIATE MATRIX MATCHED CONTROLS Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 53 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 6 QUALITATIVE ANALYSIS 6 1 6 2 6 2 1 6 2 2 6 2 3 6 3 6 3 1 6 3 2 6 3 3 6 4 All mass spectral libraries and or reference spectra must be approved by the Technical Leader or Toxicology Manager The following acceptance criteria may be used as guidelines but not as absolutes to identify a substance through its mass spectrum Usually the base peak will be the same and isotope clusters will contain the same major ions and relative abundances When compared to a reference spectrum the spectra must contain strong similarities Any differences between the reference spectrum and the unknown spectrum must be carefully evaluated for acceptability The following criteria must be met for qualitative identification of a compound Retention times RT and or relative retention times RRT must be within 2 of the target Case mass spectrum must be of comparable quality to a reference spectrum in mass assignment and intensity Any extraneous ions of significant intensit
166. on Forensic Science Center Forensic Analysis Division Toxicology Negative no more than 50 below decision point and positive controls no more than 50 above decision point are prepared and tested for each qualitative method validation In addition a material of known concentration near the cutoff limit is tested 4 2 1 2 Perform a daily assay testing each of the three materials negative positive and near cutoff over a period of 5 days with five replicates per day Thus 25 data points are generated for each level The same lot of reagents and cutoff material is used during the test period 4 2 2 Data Analysis 4 2 2 1 Determine if each sample is positive or negative using the average OD of the 5 replicate cutoff calibrator 4 2 3 Acceptance Criteria 4 2 3 1 Expected results for the negative and positive controls 4 2 4 Monitored data 4 2 4 1 These data are calculated as part of method validation to identify any potential issues which may be encountered during routine use These are not criteria for acceptance or rejection of the immunoassay validation 4 2 4 1 1 Overall CV of the optical density lt 20 at each of the three levels 4 2 4 1 2 Mean 2 standard deviations of the optical density should not overlap the decision point Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 169 of 171 Houston Forensic Science Center Forensic Analysis Di
167. ontain the analyte of interest at a concentration below the Limit of Quantification or Cutoff of the assay 4 CALIBRATION OF QUANTITATIVE ASSAYS 4 1 Calibration protocol must be performed as validated and described in the analytical method 4 2 A matrix blank must be included in each analytical run but this blank will NOT be used in the generation of the calibration curve unless specifically indicated in the analytical method 4 3 Unless otherwise specified in the analytical procedure no fewer than three calibration levels spanning the linear range of the assay not including the blank may be used for a linear Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 19 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology calibration curve The three concentrations must span the linear range of the assay 4 4 No fewer than 4 non zero points spanning the analytical range of the assay may be used for a quadratic curve at any time 4 5 For the calibration curve to be accepted the back calculated results for each calibrator must calculate to within 20 of its target value A variance of 25 is allowed at the LOQ 4 6 In some situations one point may be eliminated from the calibration curve to improve the quality of the curve fit This does not include the blank Elimination of two points should only be made in exceptional ci
168. ontrol EQC 0 080 g 100 mL 9 3 1 The EQC is a purchased quality control Once open the contents of the vial may be transferred to a labeled container and tightly capped Storage Store refrigerated Discard Expiration date 1 month opened 9 4 High Aqueous Ethanol Control HEQC 0 400 g 100 mL 9 4 1 Certified reference material is purchased to make HEQC Spike 5 1 mL of ethanol into a 100 mL volumetric flask filled with approximately 90 mL of DI water Bring to volume QS with DI water Cap and mix thoroughly Carefully transfer into containers that are pre labeled Tightly cap each container Storage Store in freezer Discard 1 year frozen 1 month thawed 9 5 Mixed Volatile System Suitability Control SS 0 010 g 100 mL ethanol methanol isopropanol and acetone 9 5 1 The system suitability is a qualitative control used to confirm resolution prior to each Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 151 of 171 9 6 9 6 1 Houston Forensic Science Center Forensic Analysis Division Toxicology calibration The concentration should be approximately 0 010 g 100 mL of each analyte This can be a purchased aqueous control or remaining calibration material from the historical calibration Dilution control DQC A dilution of the HEQC or other CRM with known concentration is analyzed in the batch if necessary to verify
169. opioids from toxicology specimens including but not limited to blood Urine confirmations are reported only qualitatively and may also be reported using Basic Acidic and Neutral Drugs Confirmation by GC MS 3 REAGENTS AND STANDARDS 3 1 Keto Opioid Working Standard 3 1 1 Two solutions will be needed for this analysis The solutions contain concentrations of hydrocodone hydromorphone oxycodone and oxymorphone at 10 ng uL and 1 ng L respectively 3 1 2 Preparation of 10 ng uL Keto Opioid Working Standard Add 100 uL of each keto opioid 1 0 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 1 3 Preparation of 1 ng uL Working Standard Add 1 mL of 10 ng uL working standard to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 2 Keto Opioid Internal Standard Solution This solution contains concentrations of hydrocodone D3 hydromorphone D3 oxycodone D3 and oxymorphone D3 3 2 1 Preparation of 10 ng uL Keto Opioid Stock Internal Standard Add 100 uL of each keto opioid 100 g mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 2 2 Preparation of 1 ng uL Working Internal Standard Add 1 mL of 10 ng uL internal standard to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 3 1M Acetic Acid 3 4 100 mM pH 6 0 Phosphat
170. or Quality Manager 12 4 2 M I A 12 4 2 1 These compounds can be reported qualitatively if the calculated concentration is 0 010 g 100 mL and the retention time is within 2 of that obtained for each analyte of interest in the LMQC 12 4 2 2 If a quantitative value is needed for M I A positive specimens must be re analyzed with the inclusion of MQC in the batch and MQC must meet acceptance criteria to report a quantitative result 12 4 2 3 The values from aliquot 1 and aliquot 2 on FID1 A BAC1 must be within 10 of the average truncated to 3 decimals of aliquot 1 and aliquot 2 Aliquot 1 Aliquot 2 Aliquot 1 Aliquot 2 Acceptable Range i x 0 9 to C x 1 1 13 MEASUREMENT UNCERTAINTY MU This is a statistical calculation of all known variables that contribute to the inherent variance of the overall result at a desired confidence level For this procedure the confidence level used is 99 73 k 3 The measurement uncertainty will be evaluated yearly 13 1 Uncertainty components that contribute to the calculated measurement uncertainty MU include 13 1 1 Measurement Reproducibility accounts for the control with the largest Percent Relative Standard Deviation or RSD based on historical data 13 1 2 CRM or Certified Reference Material Uncertainty accounts for the CRM used as a calibrator or control with the largest reported uncertainty 13 1 3 Pipettor Dilutor accounts for the variability of the two syringes used b
171. oride lsopropanol 80 20 with 2 Ammonium Hydroxide MUST BE MADE FRESH 4 Evaporate to dryness under nitrogen at 50 60 C for 10 minutes NY Reconstitute in 30 uL ethyl acetate Transfer to GC vial and inject 2 uL onto GC MS using ALPRAZOLAM M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 60 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Volume of Blood Target Concentration Working Standard Volume Added uL mL ng mL Concentration ng uL 11 LITERATURE AND SUPPORTING DOCUMENTATION 11 1 Cairns Eric R et al Quantitative Analysis of Alprazolam and Triazolam in Hemolysed Whole Blood and Liver Digest by GC MS NICI with Deuterated Internal Standards J Anal Toxicol 1994 Jan Feb Vol 18 1 pp 1 6 11 2 Tiscione Nicholas B et al Quantitation of Benzodiazepines in Whole Blood by Electron Impact Gas Chromatography Mass Spectrometry J Anal Toxicol 2008 Oct Vol 32 8 pp 644 652 11 3 Levine Barry Central Nervous System Depressants in Principles of Forensic Toxicology 2 Ed Levine Barry Ed AACC Press Washington DC pp 173 186 2003 11 4 Baselt Randall C Disposition of Toxic Drugs and Chemicals in Man 7 Ed Biomedical Publications Foster City CA p 35 36 2004 11 5 Method file ALPRAZOLAM M Toxicology Standard Operating Procedures Version 2 0 Issued By
172. osampler vials and analyze using the normal analytical conditions Make duplicate injections of each dilution Data Analysis Using the peak area or peak height response determine the response factor of the stock solutions by dividing the average response of the duplicate injections for each stock solution by the solution concentration Calculate the response factor ratio by dividing the response factors of one of the stock solutions by the response factor for the other stock solution A ratio of 1 0 0 03 gives confidence in the preparation of the stock solutions Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 13 of 171 Houston Forensic Science Center j oP 4 Forensic Analysis Division Toxicology Preparation and Validation of Drug Standards Spiking Solutions Controls and Reagents 3 3 1 3 2 3 3 3 4 3 5 3 6 3 7 4 4 1 4 2 4 3 PURPOSE Organic solvents should be HPLC grade or higher and inorganic reagents e g salts should be ACS grade Deionized water should be prepared using a Millipore Direct Q UV3 water system or from an equivalent source Any internally prepared calibrators or controls may be purchased from an appropriate vendor in lieu of preparation in house All drug standards spiking solutions and quality control preparations must be documented on LAB 68 Reagent Preparation Worksheet or equivalent and incl
173. ositive or negative for PCP or fortified samples 20 of the samples should have concentrations of 0 75 of the cutoff calibrator concentration and 20 should have the analyte of interest at concentrations of 2125 of the cutoff concentration The expected result of these samples should be blinded to the analyst performing the validation 5 2 2 Data Analysis 5 2 2 1 Determine if each sample is positive or negative 5 2 2 2 Calculate Sensitivity and Specificity TP Sensitivity x 100 Y TP EN Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 165 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Specificity TNAFP x 100 5 2 3 Acceptance Criteria 5 2 3 1 Sensitivity 2 90 5 2 3 2 Specificity gt 90 5 3 Interference Studies 5 3 1 Procedure 5 3 1 1 Matrix interference Analyze a minimum of 10 different sources of blank matrix without IS 5 312 Interference from stable isotope internal standards Analyze a blank matrix fortified with IS but NO analyte of interest in each validation run 534 3 Interference from commonly encountered exogenous analytes Analyze blank matrix fortified with analytes of interest at the negative control concentration and potential interferences at high therapeutic or lethal concentrations 5 3 2 Data analysis 5 3 2 1 Matrix interference determine if sample is pos
174. ot fixed quantities of sample and reagent into 96 well microliter plates for use in the Enzyme Linked Immunosorbent Assay ELISA 2 SCOPE This document describes the operation and maintenance of the Tecan Freedom Evo 75 3 EQUIPMENT TECAN Freedom Evo Model FREEDOM Evo 75 4 REAGENTS AND MATERIALS 4 1 Commercial immunoassay kits strips for drugs of abuse testing 4 2 Deionized water 4 3 1M HCI hydrochloric acid Add 84 6 mL concentrated hydrochloric acid HCI to a 1 liter volumetric flask containing deionized water Bring to volume with deionized water Store at room temperature 12 month expiration 4 4 1 M NaOH sodium hydroxide Dissolve 40 0 g sodium hydroxide pellets in 1L deionized water Store at room temperature 12 month expiration 5 INSTRUMENT OPERATION 5 1 1 Place the subsample tubes into racks in accordance with the plate map Place a high QC after every 10 samples and after the last sample in the run Make sure to place the conjugate tubes with the conjugate solution corresponding to its microplate All reagents samples and strips must be at room temperature before the start of the run 5 2 Flushing Tips 5 2 1 Double click on Evoware Standard icon 5 2 2 Enter username and password and click enter 5 2 3 Check Edit and existing script 5 2 4 Click the Start your Selection arrow a2 Click on favorites double click on PP_2TipNO2_96 5 2 6 Click on the Commands tab Double c
175. ouse In house prepared controls shall be verified to establish a target mean before use by replicates of 3 over 4 runs or a minimum of 12 replicates over several runs 9 1 Whole Blood Controls 9 1 1 Purchased whole blood ethanol controls at low BQC1 and high BQC2 concentrations will be used for quantitative ethanol analysis A target mean shall be established for each new lot of BQC1 and BQC2 by replicates of 3 over 4 runs or a minimum of 12 replicates over several runs Storage Store refrigerated Discard Expiration date 1 month opened 9 1 2 If quantitative M I A analysis is required a purchased mixed volatile whole blood control MQC must be used in addition to the ethanol only blood controls BQC1 and BQC2 A target mean for each analyte shall be established for each new lot of MQC by replicates of 3 over 4 runs or a minimum of 12 replicates over several runs Storage Store refrigerated Discard Expiration date 1 month opened 9 2 Low Aqueous Mixed Volatile Control LMQC 0 020 g 100 mL 9 2 1 Certified reference materials are purchased to make the LMQC Spike 25 4 uL each of ethanol methanol isopropanol and acetone into a 100 mL volumetric flask filled with approximately 90 mL of DI water Bring to volume QS with DI water Cap and mix thoroughly Ca refully transfer into containers that are pre labeled Tightly cap each container Storage Store in freezer Discard 1 year frozen 1 month thawed 9 3 Aqueous Ethanol C
176. pam lorazepam temazepam clonazepam and alpha hydroxyalprazolam at either 10 ng uL or 1 ng uL Additional working standards may be prepared as needed Preparation of 10 ng uL BENZO Working Standard Add 1000 uL of each drug standard 0 1 mg mL to a 10mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration Preparation of 1 ng uL Working Standard Add 1000 uL of the 10 ng uL BENZO Working Standard to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 1 month expiration Internal Standard Solution This solution consists of desalkylflurazepam d4 nordiazepam d5 oxazepam d5 diazepam d5 lorazepam d4 temazepam d5 clonazepam d4 and alph hydroxyalprazolam d5 2 ng uL in methanol Preparation of 2 ng uL Internal Standard Solution Add 200 uL of each deuterated drug standard 0 1 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration KCI Stock Solution 200 mg mL Add 20 g KCI to a 100 mL volumetric flask Bring to volume with deionized water Store at room temperature 12 month expiration NaOH Stock Solution 100 mg mL Add 10 g NaOH to a 100 mL volumetric flask Bring to volume with deionized water Store at room temperature 12 month expiration KCI NaOH Working Solution pH 12 Add 4 8 mL NaOH Stock Solution and 18 6 mL KCI Stock Solution to a 1 L volumetric flask Bring to volume with deionized water Check pH to e
177. pared in the same order they will be analyzed see sequence below Between each sample rinse the pipettor dilutor tubing with Dl water at least 2 flushes of the internal standard solution and wipe tubing 10 6 All case specimens are analyzed in duplicate A single aliquot will be taken from each case specimen and recapped Once all case specimens have been singly prepared for analysis they will be re ordered prior to sampling a second time This can be accomplished by preparing case specimens in reverse order Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 152 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 10 7 For cases that have multiple items submitted one specimen from each item will be analyzed Preference will be given in the following order grey gt lavender gt pink gt tan gt royal blue if it contains anticoagulant 10 8 The condition of a case specimen during analysis must be recorded e g normal thick clotted decomposed If a case specimen is clotted the specimen must be homogenized using a tissue grinder or equivalent prior to analysis 10 9 Transfer vials from the sampling rack to the Autosampler using batch sequence 11 SEQUENCE TABLES 11 1 Example sequence SS Calibrators Low to High Negative Control Carryover check HEQC BQC2 EQC Mac DQC Up to 10 samples BQC1 Up to 10 sa
178. propanol 4 EQUIPMENT AND MATERIALS 4 1 Air displacement pipettes 100 1000 uL 20 200 uL 2 20 uL 4 2 Cerex PolyChrom Clin II SPE columns 4 3 Vacuum manifold with disposable Teflon inserts or positive pressure SPE manifold 4 4 Pan balance Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 111 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 4 5 pH meter 4 6 Evaporator 4 7 Vortex mixer 4 8 Centrifuge 4 9 Tissue homogenizer 4 10 Sonicator 4 11 Heating block 4 12 Vacuum pump or house vacuum 4 13 Agilent 7890 GC 5975 MSD Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 112 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 5 INSTRUMENTAL PARAMETERS sml 5 2 5 3 Capillary Column 30 m HP 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 1 3 mL min with an injection volume of 2 uL in split mode 10 1 GC MS Agilent 7890A Initial Temperature 140 C Hold for 0 5 minutes 30 C min to 290 C Hold for 2 5 minutes 50 C min to 310 C Hold for 2 6 minutes Wash solvents for autosampler Methanol and ethyl acetate are used as the wash solvents A minimum of 6 pre and 6 post rinses are performed Each rinse c
179. r manager 6 2 Re validating an Existing Lot of Internal Standard 6 2 1 Prior to the expiration date the current lot of internal standard should be evaluated to determine if the expiration date can be extended Compare the internal standard ina current run to the chromatograms from the original validation 6 2 2 Fortify a blank matrix sample with the normal amount of IS 6 2 3 Evaluate the blank matrix to ensure the internal standard has not degraded or become contaminated 6 2 4 Any peak for the analyte of interest should be lt 10 of the area or height observed in the lowest calibration sample 6 2 5 Using at least two blank matrixes from the current run compare the area or height of the internal standard to the original validation data The internal standard should be within 30 of the validation data 6 2 6 If the internal standard meets acceptance criteria place a new label on the internal standard bottle and attach re validation paperwork to original paperwork 6 2 7 If the internal standard is deemed unacceptable after investigation prepare a new lot of IS Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 17 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology and validate as described above 7 ESTABLISHING TARGET CONCENTRATION AND ACCEPTANCE RANGE FOR QC SAMPLES IN QUANTITATIVE ASSAYS 7 1 Commercial co
180. rated internal standard and selective ion monitoring SIM are used in electron ionization El mode 2 SCOPE This procedure should be used for confirmatory analysis of ketamine from toxicology specimens typically blood This procedure is recommended if quantitative analysis is desired Qualitative results may also be reported For routine full scan qualitative analysis however refer to Basic Acidic and Neutral Drugs Confirmation by GC MS 3 REAGENTS AND STANDARDS 3 1 Ketamine Working Standard 3 1 1 10 ng uL Ketamine Working Standard Add 100 uL of ketamine 1 0 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 1 2 1 ng uL Ketamine Working Standard Add 1 mL of 10 ng uL working standard to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 5 2 Ketamine D4 Internal Standard Solution 3 2 1 10 ng uL Ketamine D4 Stock Internal Standard Add 1 mL of Ketamine D 100 ug mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 12 month expiration 3 2 2 Preparation of 1 ng uL Ketamine Working Internal Standard Add 1 mL of 10 ng uL internal standard to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 3 1M Acetic Acid 3 4 100 mM pH 6 0 Phosphate Buffer 3 5 Methylene Chloride lsopropanol 80 20 3 6 Elution Solvent 2 ammonium hydroxide concentrate
181. ray is filled with liquid and the manifold is lowered into it 6 2 2 1 12 Once the rinse procedure is complete press stop to abort the rinse procedure 6 2 2 1 13 Do not switch off the instrument 6 2 2 1 14 END appears on the display when the procedure is completed The manifold remains immersed in the prime tray until END is pressed Press END to finish the rinse procedure and the firmware returns to the Program menu 6 2 2 1 15 In case of instrument malfunction inform Laboratory Management immediately and 7 REFERENCES document the problem on lab form LAB 62 EIA Troubleshooting Log or use an equivalent method Take necessary corrective action Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 37 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 7 1 TECAN HydroFlex Plate Washer Operating Manual Document Part No 30026397 2008 02 7 2 TX 03 01 Drugs of Abuse Screening by ELISA Standard Operating Procedure 7 3 TX 07 03 Operation and Maintenance of the Plate Reader Standard Operating Procedure 7 4 Lab Form 61 EIA Maintenance Log 7 5 Lab Form 62 EIA Troubleshooting Log Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 38 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Oper
182. rcumstances when other evidence supports the use of analytical data from that particular analysis batch The approval of a supervisor or designee is required when two points are discarded 4 7 When a calibrator is discarded the fact that it was discarded and the reason must be clearly documented with the data for that batch 4 8 The lowest acceptable calibrator for a given batch is the reporting limit unless otherwise specified in the analytical method 4 9 A negative control should be included after the highest calibrator in each analytical run to monitor for carryover 5 CALIBRATION OF QUALITATIVE ASSAYS 5 1 A cutoff calibrator must be included in every analytical run 5 2 Results of unknown samples are determined to be positive or negative when evaluated against the response of the cut off calibrator 6 CONTROL OF QUANTITATIVE ASSAYS 6 1 Each analytical run must contain a matrix blank two low QC sample and two high QC sample 6 1 1 Low QC concentration should be no more than 3x the LOQ of the assay 6 1 2 High QC concentration should be no less than 75 of the highest calibrator 6 2 The number of QC samples must be at least 5 of the total number of unknown samples in the run 6 3 A low QC sample should be injected after the last case specimen for each run 6 4 QC samples must be included for every analyte being quantified by the method 6 5 Acceptance criteria for QC samples should be defined in SOP of each analytical assay 7 C
183. re can be stored in the refrigerator or freezer prior to use Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 78 of 171 3 9 Ld 3 9 2 3 5 3 3 5 4 Houston Forensic Science Center Forensic Analysis Division Toxicology 10 M KOH Add 56 10 g KOH pellets to a 100 milliliter volumetric flask Bring to volume with deionized water Stir Let cool and check volume as exothermic reaction may have an effect on volume Store at room temperature 12 month expiration H20 Acetonitrile 85 15 In a 1 liter volumetric flask add 150 mL acetonitrile Fill to volume with deionized water Store at room temperature expiration 6 months SPE Wash Solution 1 ammonium hydroxide concentrated in 85 15 H20 Acetonitrile For example In a 25 mL volumetric flask add 0 25 mL concentrated ammonium hydroxide Bring to volume with 85 15 H20 Acetonitrile MUST BE PREPARED FRESH Hexane Ethyl Acetate 90 10 In a 1 liter volumetric flask add 100 mL of ethyl acetate Bring to volume with hexane Store at room temperature expiration 6 months Elution Solvent 3 glacial acetic acid concentrated in 90 10 hexane ethyl acetate For example In a 50 mL volumetric flask add 1 5 mL glacial acetic acid Bring to volume with 90 10 hexane ethyl acetate MUST BE PREPARED FRESH BSTFA N O bis trimethylsilyl trifluoroacetamide for derivatization Must be refriger
184. reagents are documented in a retrievable format Glacial acetic acid Hydrochloric acid Ammonium hydroxide Dibasic sodium phosphate Methylene chloride lsopropanol Methanol Ethyl acetate Deionized DI water 7732 18 5 1 M Acetic acid Add 28 6 mL of concentrated glacial acetic acid to a 500 mL volumetric flask half filled with deionized water Bring to volume with deionized water and mix Storage Room temperature Discard 1 year 100 mM pH 6 0 Phosphate buffer Add 7 0 g of dibasic sodium phosphate to a 500 mL volumetric flask and bring to volume with deionized water while stirring In a second 500 mL volumetric flask add 6 0 g of monobasic sodium phosphate and bring to volume with deionized water while stirring Mix the two solutions together until a pH of 6 0 is obtained Storage Refrigerated Discard 6 months 100 mM sodium phosphate buffer pH 6 8 Deconjugation Buffer Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 50 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 6 1 Add 7 0 g of dibasic sodium phosphate to a 500 mL volumetric flask and bring to volume with deionized water while stirring In a second 500 mL volumetric flask add 6 0 g of monobasic sodium phosphate and bring to volume with deionized water while stirring Mix the two solutions together until a pH of 6 8 is obtained 6 2 Stor
185. red saline PBS consisting of 150 mM saline in 100 mM phosphate buffer 3 5 1 PBS may be prepared in house 35 11 Add 7 0 g of dibasic sodium phosphate to a 500 mL volumetric flask and bring to volume with deionized water while stirring 3 5 1 2 In a second 500 mL volumetric flask add 6 0 g of monobasic sodium phosphate and bring to volume with deionized water while stirring 3 5 1 3 Mix the two solutions together until a pH of 7 0 is obtained 3 5 1 4 Add sufficient sodium chloride to bring the concentration to 150mM 35 15 For 1 L phosphate buffer add 8 8 g sodium chloride 3 5 1 6 Store refrigerated expiration 6 months Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 41 of 171 3 6 3 7 Houston Forensic Science Center Forensic Analysis Division Toxicology EIA Stock standards for blood and urine 1 mg mL in methanol NOTE Allow all reagents to come to room temperature before use 4 STANDARDS 4 1 4 1 1 4 1 1 1 4 1 1 2 4 1 1 3 4 1 1 4 4 1 1 5 4 1 1 6 4 1 2 4 2 4 2 1 4 2 1 1 4 2 1 2 4 2 1 3 4 2 1 4 4 2 1 5 4 2 1 6 4 2 2 EIA Urine Drug Standard Mix Using methanolic drug standards in a 10 mL volumetric flask add the following 100 uL of 0 1 mg mL 11 nor 9 carboxy 9 tetrahydrocannabinol THCA 1000 uL of 0 1 mg mL d methamphetamine 1500 uL of 0 1 mg mL benzoylecgonine BE 500 uL of 0 1 mg mL oxazepam 1000 uL of
186. require evaporation in order to concentrate the isolated material The Zymark Turbo Vap LV is a microprocessor controlled evaporator that provides simultaneous and automated concentration of multiple samples unattended operation convenience and speed The Turbo Vap will allow high capacity evaporation at a maximum of 50 Samples processed simultaneously 2 SCOPE The Turbo Vap will be used routinely during extractions in order to concentrate extracts or as needed Analysts should be knowledgeable regarding the use precautions and maintenance of this evaporator 3 EQUIPMENT 3 1 Zymark Tubo Vap LV Workstation and accessories 3 2 Compatible test tube rack 3 3 Inert gas supply nitrogen 3 4 Distilled or deionized water 3 5 Timer 3 6 Siphon tube 3 7 Spectrum Clear Bath 4 PRECAUTIONS 4 1 The workstation must be placed in an appropriate location with available gas and electrical sources as well as adequate ventilation The workstation may either be placed inside a fume hood or the exhaust duct supplied with the unit must be utilized This MUST go to a suitable ventilation system vented outside the laboratory Note Exhaust gases may be hazardous Consult the Material Safety Data Sheets for all solvents used 4 2 The workstation must be used on a flat level stable surface 4 3 The workstation must never be used with hydrogen or other flammable gases which may explode or catch on fire 4 4 DO NOT move the unit when the bath is full of water It is a b
187. rs and freezers in the toxicology section are monitored using TempAlert which is further detailed in the Quality Manual or equivalent system Acceptable refrigerator temperature range gt 0 10 C 7 Acceptable freezer temperature range lt 0 C 7 1 1 1 If a refrigerator or freezer is open for an extended period e g cleaning inventory this should be documented on form LAB 47 Universal Maintenance Repair and or External Calibration log or by an equivalent method 7 1 1 2 If a refrigerator freezer stops functioning and exceeds the acceptable temperature range evidence will be moved to another functioning refrigerator freezer and transfer documented 7 1 2 NIST Traceable Thermometers 7 1 2 1 Temperature measurements for refrigerators freezers and chart recorders should be performed using NIST traceable thermometers 7 1 2 2 If any thermometer fails performance verification or is suspected of not working properly laboratory management should be notified and a record made in the instrument lab book or equivalent The manual may be used for troubleshooting but if the problem is not resolved in a timely manner it should immediately be removed from service Once repaired and verified to be within acceptable specifications the equipment may be returned to service 8 BALANCES 8 1 Method of Use 8 1 1 Refer to the appropriate operating instructions for proper handling and use 8 2 Performance Verification and Maintenance 8 2 1 P
188. s must be within 0 10 amu of 0 60 amu 3 1 1 3 1 3 The ratio of mass 70 to 69 must be from 0 5 1 6 3 1 1 3 1 4 The ratio of mass 220 to 219 must be from 3 2 5 4 3 1 1 3 1 5 The ratio of mass 503 to 502 must be from 7 9 12 3 3 1 1 3 1 6 The ratio of mass 219 to 69 must be gt 40 3 1 1 3 1 7 The ratio of mass 502 to 69 must be gt 2 4 3 1 1 3 1 8 The precursor for mass 69 must be lt 3 3 1 1 3 1 9 The precursor for mass 219 must be lt 6 3 1 1 3 1 10 The abundance of any peaks less than 69 amu must not be greater than 10 of the base peak abundance Peaks at 18 28 and 32 amu are indicative of water nitrogen and oxygen respectively and may indicate an air leak Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 21 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 3 1 2 Maintenance 3 1 2 1 Maintenance should be performed following the manufacturer s guidelines or more frequently as needed Refer to Agilent 5975 Series MSD Operation Manual or equivalent All maintenance and repairs should be documented on the GC MS Maintenance Log Form LAB 24 or an equivalent form 3 1 2 1 1 Before each use 3 1 2 1 1 1 Perform a tune verification 3 1 2 1 1 2 Verify tank pressure 3 1 2 1 1 2 1 Check the wash solvents note The solvent vials may be rinsed and filled or refilled as needed 3 1 2 1 1 3 Weekly
189. sed for confirmatory analysis of PCP in blood and urine SAFETY This procedure must be conducted in accordance with the HFSC Health and Safety Manual and Quality Manual All case specimens should be treated with Universal Blood borne Pathogen Precautions Appropriate personal protective equipment must be worn during sample and reagent preparation and when handling caustic chemicals Flammable liquids and vapors may cause eye skin and respiratory tract irritation Derivatization reagents are toxic and must be handled in a chemical safety hood or well ventilated area Material Safety Data Sheets MSDS are available in the laboratory REAGENTS Refer to SOP Reagents for Drug Toxicology for preparation information 1M Glacial Acetic Acid 100 mM pH 6 0 Phosphate buffer 80 20 Methylene Chloride Isopropanol Concentrated Ammonium Hydroxide Deionized DI Water 7732 18 5 Ethyl Acetate Hexane Methanol Blank Blood Potassium Oxalate and Sodium Fluoride preserved Blank Urine No preservatives added EQUIPMENT Instrumentation using a method that is approved and validated for use in the section 5 1 1 Air Displacement Pipettes 100 1000 uL 20 200 uL 2 20 uL 5 1 2 Cerex PolyChrom Clin II SPE columns 5 1 3 Positive Pressure manifold or equivalent 5 1 4 Analytical Balance 5 1 5 pH Meter 5 1 6 Evaporator FAL Vortex mixer 5 1 8 Centrifuge 5 1 9 Agilent 7890 GC 5975 MSD Toxicology Standard Operating Procedures Version 2 0 Issue
190. seeneeees 33 Operation and Maintenance of the TECAN HydroFlex Plate Washer cc ccscccccsssececeeseeeeeeeeeeeees 36 Operation and Maintenance of the TECAN Sunrise Plate Reader ccccccscecccseeseeeeceeeeceeeneceeeneeees 39 Enzyme Linked Immunosorbent Assay ELISA cccccccccsssseccccceessecceeeeeesecceeeeeeeecceeeeueeeeeeeegaeeeeeeeas 41 Reagents for Drug Screening Confirmation Analyses ccccccccccccccccccccccecceeeceeeeeeeeeeeeeeeeeeseeeeeeeeeeeeeeeess 50 Evaluation of Results from Gas Chromatography Mass Spectroscopy cccccccccccccccccccceeeeeeeeeeeeeeseeees 53 Alprazolam Confirmation by Gas Chromatography Mass Spectrometry GC MS 000000eeeeee 56 Basic Acidic and Neutral Drugs by Gas Chromatography Mass Spectrometry GC MS 62 Benzodiazepine Confirmation by Gas Chromatography Mass Spectrometry GC MSS 68 Cannabinoid Confirmation by Gas Chromatography Mass Spectrometry GC MS 0 ce 78 Carisoprodol Meprobamate Confirmation by Gas Chromatography Mass Spectrometry GC MS 86 Fentanyl Confirmation by Gas Chromatography Mass Spectrometry GC MS 92 Ketamine Confirmation by Gas Chromatography Mass Spectrometry GC MS 98 Keto Opioids Confirmation by Gas Chromatography Mass Spectrometry GC MS 104 Methadone EDDP Confirmation by Gas Chromatography Mass Spectrometry GC MS 111 Opiates and Cocain
191. should be used for confirmatory analysis of alprazolam from toxicology specimens typically blood This procedure is recommended if quantitative analysis is desired Qualitative results may also be reported For routine full scan qualitative analysis refer to Basic Acidic and Neutral Drugs by GC MS 3 REAGENTS AND STANDARDS 3 1 Alprazolam Working Standard 3 1 1 10 ng uL Alprazolam Working Standard Add 100 uL of alprazolam 1 0 mg mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 1 2 1 ng uL Alprazolam Working Standard Add 1 mL of 10 ng uL working standard to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 3 month expiration 3 2 Alprazolam Ds Internal Standard Solution 3 2 1 10 ng uL Alprazolam Ds STOCK Internal Standard Add 1 mL of Alprazolam Ds 100 ug mL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 12 month expiration 3 2 2 1 ng uL Alprazolam Ds WORKING Internal Standard Add 1 mL of Alprazolam Ds 10 ng uL to a 10 mL volumetric flask and bring to volume with methanol Store refrigerated 6 month expiration 3 3 1M Acetic Acid 3 4 100 mM pH 6 0 Phosphate Buffer 3 5 Methylene Chloride Isopropanol 80 20 3 6 Elution Solvent 2 ammonium hydroxide concentrated in 80 20 methylene chloride isopropanol 4 EQUIPMENT AND MATERIALS 4 1 Air displacement pipettes 100 1000 uL 20 200 uL 2 20
192. ssay performance including but not limited to precision accuracy limit of detection limit of quantitation and linearity are summarized in the validation documentation 8 2 The limit of detection and quantitation in whole blood and urine is 2 5 ng mL Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 95 of 171 9 Houston Forensic Science Center Forensic Analysis Division Toxicology FLOWCHART FOR FENTANYL ANALYSIS Vv Vv NY Centrifuge samples for 10 minutes at 4000 rpm NY Add sample to Polychrom Clin II SPE column Use sufficient vacuum pressure to draw sample through column NY NY NY NY NY NY NY Dry column on full vacuum pressure for 5 minutes Elute Basic Drugs with 1 mL methylene chloride isopropyl alcohol 80 20 with 2 ammonium hydroxide PREPARE FRESH NY Evaporate to dryness under nitrogen at 50 C for 10 minutes NY Reconstitute in 30 uL ethyl acetate Transfer to GC vial and inject 2 uL onto GC MS using FENTANYL M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 96 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Target Concentration Working Standard Volume Added uL ng mL Concentration ng uL 11 LITERATURE AND SUPPORTING DOCUMENTATION 11 1 Baselt R C Fentanyl
193. t be within 20 of the expected value When determining the acceptability of a calibrator the concentration range is taken into account If a calibrator needs to be excluded from the curve the action should be described and justified in the Batch file Quantitative values above or below the highest and lowest calibrators should be reported as greater than or less than the appropriate calibrator concentration If the calculated concentration is within 20 of either the lowest or highest calibrator the result may be reported upon approval from the Technical Leader or Toxicology Manager Dilution of a specimen may be necessary for samples that contain an elevated concentration of drug Any Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 54 of 171 7 3 Houston Forensic Science Center Forensic Analysis Division Toxicology changes in sample volume that deviate from the technical procedure must be documented in the case folder For quantitative reporting the calculated value of the in house or external QC should be within 20 of the target concentration Alternatively quantitative results may be reported if the QC is 3 SD of the established mean 8 TOXICOLOGY REPORTING GUIDELINES 8 1 Qualitative vs Quantitative Results 8 1 1 Drugs in urine are reported qualitatively unless specifically noted otherwise in the standard operating procedure
194. t free tissue and deionized water 6 1 5 Check the level of the system fluid container It is good practice to replace system liquid at least once a week and to keep container filled 6 1 6 Check the syringes for leaks bubbles or internal contamination If required clean the syringes taking care in removing syringes If the syringes are leaking replace the caps on the syringe plungers 6 1 7 Check around the valve for signs of moisture 6 1 8 Check the green Teflon coating of the stainless steel pipette tip for any damage 6 1 9 Check for air bubbles or contamination in the pipetting tubing Tighten the tubing connections or replace the tubing as required 6 1 10 Clean the toothed rack with a lint free tissue 6 1 11 Clean the system liquid container with a mild soap Make sure to thoroughly rise out container before filling with deionized water 6 2 Acid Base Wash 6 2 1 Performing an Acid Base wash using 1N HCI and 1N NaOH is recommended for the maintenance of the Tecan Evo 75 This procedure can be done as needed 6 2 2 Double click on the NaviTrak2 Shortcut icon 6 2 3 Click Start 6 2 4 Click on the Utilities tab 6 2 5 Click the Acid Base Wash button 6 2 6 Click the Run button and follow the pop up window instructions 6 2 7 Document in the maintenance log 7 REFERENCES 7 1 TECAN Operating Manual Freedom EVO 75 BG N 30023958 02 7 2 TECAN Freedom Evo 75 Operators Manual Chapter 7 Preven
195. t from the same manufacturer can be used If a drug standard is not available from a different manufacturer or a different lot from the same manufacturer different ampoules should be used to make separate stock solutions for controls spiking solutions and calibration samples 5 2 4 QC samples must be prepared at a different time and by a different analyst than the solutions used for calibration samples 5 2 5 Information regarding preparation must be documented using the appropriate form or an equivalent form or method for example 5 2 5 1 LAB 27 Multi Component Working Stock Standard Preparation Log 5 2 5 2 LAB 28 Toxicology Single Component Working Stock Standard Preparation Log 5 2 5 3 LAB 68 Reagent Preparation Worksheet 5 2 6 All solutions samples must be labeled accordingly The label must contain at a minimum the Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 15 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology solution name and concentration solution preparation date initials of the preparer and expiration date 5 2 7 Spiking solutions QC Samples and IS solutions must be verified to ensure that the solutions and QC samples been correctly prepared before being used in casework Upon verification the verifying analyst shall note the date of verification and initial that date 5 2 8 The concentratio
196. t maintenance has been performed 7 2 1 3 Troubleshooting quality issues 7 2 2 In house prepared purchased e g Cerilliant Absolute or equivalent NIST certified reference mixed volatile standards or a combination of the two are used as calibrators to establish the M I A calibration The following calibrator concentrations are used unless otherwise specified in the case record Level 1 0 010 g 100 mL Level 2 0 050 g 100 mL Level 3 0 100 g 100 mL Level 4 0 400 g 100 mL 8 INTERNAL STANDARD 8 1 n Propanol Internal Standard Stock Solution 1 0 g 100 mL 8 1 1 Weigh out 1 0 g of n propanol in a 100 mL volumetric flask Bring to volume with DI water This will give a 1 0 I S stock solution 1 0 g 100 mL Storage Store refrigerated Discard 6 months Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 150 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 8 2 n Propanol Internal Standard Working Solution 0 010 g 100 mL 8 2 1 Add 10 mL of the 1 I S stock solution to a 1000 mL volumetric flask Bring to volume with DI water This will give a 0 010 I S working solution 0 010 g 100 mL Storage Store at room temperature Discard 9 months 9 CONTROLS NOTE Calibrators and controls must be prepared from separate lots or sources of certified reference material Controls may be purchased or prepared in h
197. tative only 4 5 An Internal Standard prepared from a solid reference material or sealed ampoules may be used beyond the expiration date provided by the manufacturer provided that 4 5 1 It is re verified prior to use and 4 5 2 Documented on LAB 68 Reagent Preparation Worksheet or equivalent and 4 5 3 Provides results within the acceptance criteria specified in the standard operating procedure or by the manufacturer 4 6 Biological controls may be used beyond the manufacturer s expiration date provided that the above criteria are met 4 7 Expired reagents should be discarded or clearly labeled not for casework 5 CALIBRATION SAMPLES Assay Calibration must be performed as validated and described in the analytical method 5 1 QC Samples Types of controls in order of preference 5 1 1 Commercial controls 5 1 2 In house controls prepared in bulk 5 1 3 Controls prepared at the time of analysis using a spiking solution 5 2 Preparation of spiking solutions calibration samples internal standard solutions controls and reagents 5 2 1 Standards must be made and stored in accordance with the SOP An equivalent procedure may be used if it is documented on the appropriate preparation log 5 2 2 In house prepared controls and or spiking solutions should be prepared from a different manufacturer than the CRM used to prepare calibration samples 5 2 3 If a drug standard is not available from a different manufacturer then a different lo
198. ted 2 3 Transfer of a validated method to a new instrument Modifications to existing methods or transfer of a validated method to new instruments may not require re validation of all parameters Determination of validation parameters which must be re evaluated will be dependent upon the changes made to the method 3 VALIDATION PARAMETERS The following validation parameters are recommended by the Scientific Working Group for Forensic Toxicology SWGTOX Standard Practices for Method Validation in Forensic Toxicology Please refer to the document published May 20 2013 for detailed information on each parameter 3 1 Limit of Detection LOD 3 2 Precision at the decision point 3 3 Cross reactivity if applicable Note cross reactivity data is generally provided with commercially available immunoassays thus evaluation of cross reactivity is generally not needed for immunoassay 3 4 Dilution integrity if applicable 3 5 Stability if applicable Note stability is generally evaluated during the validation of confirmation assays and thus is not needed for immunoassay validation 4 VALIDATION EXPERIMENTS 4 1 Limit of Detection LOD Typically the LOD of immunoassay based methods is defined as the decision point concentration 4 2 Precision at the decision point Procedure 4 2 1 Procedure Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 168 of 171 Houst
199. tive Maintenance and Repairs 7 3 Enzyme Linked Immunosorbent Assay ELISA Standard Operating Procedure 7 4 Operation and Maintenance of the Plate Washer Standard Operating Procedure 7 5 Operation and Maintenance of the Plate Reader Standard Operating Procedure 7 6 LAB Form 61 EIA Maintenance Log 7 7 Lab From 62 EIA Troubleshooting Log Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 35 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Washer 1 PURPOSE The TECAN Hydroflex Plate Washer is used to rinse microtiter plates that are used for drugs of abuse screening by Enzyme Linked Immunosorbent Assay ELISA 2 SCOPE This document describes the operation and maintenance of the TECAN HydroFlex Further operational instruction is found in the ELISA SOP 3 EQUIPMENT TECAN HydroFlex Plate Washer 4 REAGENTS AND MATERIALS Deionized Water 5 PROCEDURE 5 1 Switch on the plate washer 5 2 Use the gt key to select between options OK to confirm and o to go back 5 3 Priming Select Procedures then Prime Select Time10 sec and press OK to start priming The system must be primed with the liquid that will be used to wash the plates Repeat the priming steps several times 5 4 After the system is primed place the microplate to be tested into the correspondi
200. tive control 5 4 1 6 Inject the calibration curve negative control and one set at least 2 positive controls 5 4 1 7 Store the remaining vials in the auto sampler for at least 12 hours The interval chosen should represent the longest period that the processed extracts will remain at auto sampler conditions during the course of an analytical run 5 4 1 8 At the end of the predetermined time period inject all controls half will be unanalyzed half will be re injects 5 4 2 Data Analysis Determine if samples are positive or negative 5 4 3 Acceptance Criteria All positive controls must remain positive Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 167 of 171 Houston Forensic Science Center j oP 4 Forensic Analysis Division Toxicology Validation of Manufactured Kits used for Screening Analysis 1 PURPOSE This SOP is intended to define a series of experiments that are relevant elements of a qualitative immunoassay method validation 2 SCOPE This procedure applies to immunoassay based methods using commercially available kits Method validation is required to verify the performance parameters of a method are fit for purpose Validation is required after the following events occur 2 1 Development of a new method 2 2 Modification of a validated method to improve its performance or extend its use beyond that for which it was originally valida
201. to 230 C Hold for 5 minutes 30 C min to 290 C Hold for 5 minutes Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 119 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 5 3 Wash solvents for autosampler Methanol and ethyl acetate are used as the wash solvents A minimum of 6 pre and 6 post rinses are performed Each rinse cycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 6 EXTRACTION PROCEDURE 6 1 Blood Use silanized glassware for blood extracts 6 1 1 In around bottom glass centrifuge tube add 1 mL blood and 50 uL Internal Standard Solution 1 ng uL while vortex mixing Final concentration of internal standard in each sample of 50 ng mL for all target drugs except 6 MAM 25 ng ml NOTE IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALL VOLUME AND DOCUMENT IN THE CASE RECORD 6 1 2 Fortify all controls and calibrators while mixing using the appropriate amount of working standard The typical calibration range for quantitative analysis is 0 1000 ng mL Refer to the fortification guide for guidance on preparation of calibrators CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS A minimum of one in house control 100 ng mL should be included in each run Use appropriate external controls e g UTAK Whole Blood Controls when available
202. to technical and administrative review Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 65 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 10 FLOWCHART FOR BASIC ACIDIC AND NEUTRAL DRUG ANALYSIS L v VL Samples are centrifuged for at least 10 minutes at 4000 rpm g Add sample Cerex Clin II SPE column Use sufficient vacuum pressure to draw sample through column NV Wash 1 mL deionized water Ny Wash 1 mL 1 M acetic acid VL Dry column on full vacuum pressure for 5 minutes VL Wash 1 mL hexane NY Elute acidic and neutral drugs with 1 mL ethyl acetate NY Wash 1 mL methanol NY Dry column under full vacuum pressure for 5 minutes NY Replace conical tubes containing acidic neutral fraction and elute basic drugs with 1 mL methylene chloride isopropyl alcohol 80 20 with 2 ammonium hydroxide PREPARE FRESH DAILY NY Add 30 uL acidic methanol NY Evaporate to dryness under nitrogen at 50 60 C for 15 NY Reconstitute in 30 uL ethyl acetate Transfer to GC vial and inject 2 uL onto GC MS using BAN M Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 66 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology Volume of Blood Target Concentration BAN Wor
203. ty are summarized in the validation documentation 8 2 Limits of detection in blood and urine are as follows Drug LOD LOQ LOD Urine Blood Blood ng mL ng mL ng mL Amphetamine Methamphetamine 8 3 MDEA is reported qualitative only For MDA and MDMA curve is loaded till 250 ng ml as higher concentration causes interferences in the Internal Standard IF the sample contains greater than 500ng ml of MDA and MDMA then dilution is needed 9 QUALITY CONTROLS 9 1 Positive and negative QCs must be included in each run For qualitative analysis each batch must contain a blank drug free extract and an extracted drug standard 9 2 For quantitative analysis use additional external QCs e g UTAK QCs when available UTAK Drugs of Abuse Level QC contains 100 ng mL of amphetamine methamphetamine MDA MDEA and MDMA Record the calculated concentrations in the appropriate QC Log Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 140 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 10 FLOWCHART FOR STIMULANT ANALYSIS NY NY NY Centrifuge samples for at least 10 minutes NY Use sufficient vacuum pressure to draw blood samples through the column approximately 5 PSI NY NY NY NY NY Wash 1 mL ethyl acetate NY Wash 1 mL methanol NY Elute drugs with 1 mL elution solvent ethyl acetate with 2 ammon
204. uL 4 2 Cerex PolyChrom Clin II SPE columns 4 3 Vacuum manifold with disposable Teflon inserts or positive pressure SPE manifold 4 4 Pan balance 4 5 pH meter 4 6 Evaporator Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 56 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 4 7 Vortex mixer 4 8 Centrifuge 4 9 Tissue homogenizer 4 10 Sonicator 4 11 Heating block 4 12 Vacuum pump or house vacuum 4 13 Agilent 7890 GC 5975 MSD 4 14 Stir plate and stir bars 5 INSTRUMENTAL PARAMETERS 5 1 Capillary Column 30 m DB 5MS Agilent J amp W GC Column or equivalent 0 250 mm id X 0 25 um film thickness The flow rate is 1 2 mL min with an injection volume of 2 uL in split mode 10 1 5 2 GC MS Agilent 7890A Initial Temperature 140 C Hold for 0 5 minutes 30 C min to 290 C Hold for 2 5 minutes 50 C min to 310 C Hold for 2 6 minutes 5 3 Wash solvents for autosampler Methanol and ethyl acetate are used as the wash solvents A minimum of 6 pre and 6 post rinses are performed Each rinse cycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 6 EXTRACTION PROCEDURE BLOOD URINE AND OTHER MATRICES 6 1 In a round bottom glass culture tube add 1 mL of specimen and 100 uL Working Internal Standard Solution 1 ng uL while vortex mixing total concentration 100 ng mL NOT
205. ubes Assign lot number and expiration date Storage Store frozen Discard 6 months 10 6 Dilution Control Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 128 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology A 2x dilution of High Blood QC is analyzed in the batch if necessary to verify dilution results of case specimens 11 SAMPLE PREPARATION 11 1 Aliquot 1 mL blank blood controls and case samples into labeled round bottom glass culture tube NOTE Dilution of a specimen may be necessary for samples that contain elevated concentration of drug Any changes in sample volume that deviate from the technical procedure must be documented in the case folder 11 2 Fortify calibrators according to the table below Phencyclidine Target Concentration Volume of Blood Phencyclidine Working Standard Volume Concentration Added ng L uL 11 3 Add 50 uL of Internal Standard Solution 1 ng uL while vortex mixing Final concentration of internal standard in each sample is 50 ng mL 11 4 Add 2 mL of 100 mM pH 6 0 sodium phosphate buffer to each tube Vortex Centrifuge at 4000 RPM for 10 minutes 11 5 Place collection tubes or reservoir for waste into vacuum or positive pressure manifold rack 11 6 Add the sample to Polychrom Clin II Column Draw the sample through using sufficient vacuum pressure w
206. ubes into the evaporator by opening the cover and placing the sample racks into the water bath Close the cover and press START To stop an evaporation run simply press STOP To pause an evaporation run press the START PAUSE pushbutton To shut off the evaporation in any row of test tubes press the corresponding TUBE STATIONS pushbutton When the cycle is complete the gas automatically shuts off and the evaporator buzzer will sound every 30 seconds Lift lid and leave open as soon as possible Remove tubes and blot with absorbent material to remove moisture if desired Note highly volatile samples can be lost if they are allowed to remain in the unit When use of the TurboVap is complete for the day turn the unit power and gas supply off Lift the cover and keep it open 6 MAINTENANCE 6 1 6 2 6 3 6 4 6 5 6 6 6 7 6 8 Routine maintenance of the Turbo Vap LV eliminates the need for frequent cleaning due to cloudy or bacteria infested water Note Cleaning the water bath may cause exposure to bacterial or viral hazards Use good laboratory operating procedures when dealing with liquids Turn the evaporator s AC power OFF and unplug the power cord Open the cover and remove the rack Siphon the water out of the bath Use an appropriate cleaner if desired to wipe any residue from the bath walls Rinse the bath and re siphon the liquid Also clean the rack itself Pour 1 liter distilled or deionized water into bath Add 15 drops of
207. ude key information regarding drug standard and chemical names manufacturers lot numbers for both manufacturer and new solutions verification dates and by whom the solution was prepared Reference drug standards controls and reagents used in the laboratory must be of sufficient quality for their intended use SCOPE This procedure can be used for the qualitative or quantitative analysis of all toxicology specimens DEFINITIONS Certified Reference Material CRM Drug standard purchased from an approved vendor which includes a certificate of analysis verifying the concentration Drug standard any chemical other than the sample used in the preparation of standard solutions for calibrators controls or internal reference CRM should be used as drug standards whenever possible Calibration Sample Analytical standard used to fix set or check the graduations or scale of an analytical procedure Internal Standard IS An analyte generally of similar chemical structure to an analyte being measured that is added in a known concentration to all samples calibrators QC s and unknowns in an analytical method and that functions as a reference marker for that sample against which the analyte of interest can be measured Fortified Quality Control QC sample A sample of similar matrix to the unknown case sample which has been spiked with a predetermined amount of the analyte s of interest QC samples can be prepared in house or purchased from an
208. ugust 3 2015 Uncontrolled When Printed Page 51 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 12 1 Add 84 6 mL concentrated hydrochloric acid to a 1 liter volumetric flask containing deionized water Bring to volume with deionized water 12 2 Storage Room temperature 12 3 Discard 12 months Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 52 of 171 Houston Forensic Science Center j IP 4 Forensic Analysis Division Toxicology Evaluation of Results from Gas Chromatography Mass Spectroscopy 1 PURPOSE The GC MS is composed of two major building blocks the gas chromatograph GC and the mass spectrometer MS The GC utilizes a capillary column which depends on the column s dimensions length diameter film thickness as well as the stationary phase properties The difference in the chemical properties of the different compounds in a sample and their relative affinity for the stationary phase of the column will promote separation of these compounds as the sample travels through the length of the column The compounds are retained by the column and then elude from the column at different times retention time The mass spectrometer is the detector portion of the instrument As the compounds elude off the column and enter into the MS they are ionized fragmented separated and detected based upon th
209. ult 4 VALIDATION PARAMETERS The following validation parameters are recommended by the Scientific Working Group for Forensic Toxicology SWGTOX Standard Practices for Method Validation in Forensic Toxicology Please refer to the document published May 20 2013 for detailed information on each parameter 4 1 Precision around reporting limit RL 4 2 Sensitivity Specificity 4 3 Interference Studies 4 4 Stability if applicable 4 5 lonization suppression enhancement if applicable Note At this time HFSC does not employ Liquid Chromatography Tandem Mass spectrometry for any analysis lonization suppression enhancement is not applicable to GCMS testing 5 VALIDATION EXPERIMENTS 5 1 Precision around reporting limit RL 5 1 1 Procedure 5 1 1 1 Prepare a pooled fortified matrix sample at the negative control cutoff calibrator and positive control concentrations 51 12 Analyze 5 samples a run at each concentration over a minimum of 5 runs 25 samples minimum at each concentration 5 1 2 Data analysis 5 1 2 1 Determine if each sample is positive or negative 5 1 3 Acceptance Criteria 5 1 3 1 At least 22 90 of the negative controls must be negative 5 1 3 2 At least 22 90 of the positive controls must be positive 5 1 3 3 8 16 35 65 of the cutoff calibrators must be positive 5 2 Sensitivity Specificity 5 2 1 Procedure Prepare a total of 40 blind specimens These specimens can be discarded patient specimens know to be p
210. ults and detailing other information concerning testing NOTE For each report generated e g alcohol negative screening and or positive screening confirmation the appropriate Case File Review Checklist will include total number of examination pages associated with the report 5 2 Administrative Documentation 5 2 1 Case File Review Checklist 5 2 2 Evidence Description and Review Form 5 2 3 Submission Form 5 2 4 Photographs of submitted evidence 5 2 5 Administrative documentation may include 5 2 5 1 Report 5 2 5 2 Discovery Order 5 2 5 3 Correspondence phone email and or other types of communication 5 2 5 4 Any recovery or corrective action documentation 5 2 0 5 Other documentation e g outside agency forms 6 BATCH REVIEW CHECKLIST Batch review checklists which are part of the case record detail the aspects of the batch file requiring inspection for technical accuracy These have been developed for alcohol LAB 70 ALC Batch Review Checklist immunoassay screening LAB 75 Immunoassay Batch Checklist and GC MS screening and or confirmation LAB 73 Batch Review Checklist Drug Toxicology Equivalent electronic checklists may also be used 7 CASE FILE REVIEW CHECKLIST Case file review checklists which are part of the case record detail the aspects of the case file requiring inspection for technical and administrative accuracy These have been developed for both alcohol LAB 71 ALC Case File Review Checklist and screeni
211. um of 3 replicates per run All samples must provide expected results 9 VALIDATION OF NEWLY PREPARED REAGENTS 9 1 Run a set of controls using the newly prepared reagent in parallel with controls made using the existing reagent 9 2 The reagent will be considered validated if controls meet the acceptance criteria defined by the method Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 18 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology In Process Calibration and Quality Control for Drug Screening Confirmation Testing 1 PURPOSE This procedure describes the preparation and implementation of a calibration curve and in process quality control QC samples at Houston Forensic Science Center This procedure is designed to provide a means of detecting potential problems with assay performance and to ensure accurate and reliable test results 2 SCOPE These are default procedures for QC of all validated qualitative and quantitative bio analytical assays applicable to all analytical SOPs not having specific QC protocols 3 DEFINITIONS 3 1 Calibration Protocol A written procedure which describes the preparation of calibration samples the processing of these samples and the method specific calculation model that is to be used 3 2 Calibration sample An analytical standard used to fix set or check the graduations or scale
212. ure tube add 2 mL of sample NOTE IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALLER VOLUME ADD BUFFER AS THE DILUENT AND DOCUMENT IN THE CASE FOLDER 6 2 Add 40 uL of Internal Standard 0 5 ng uL while vortex mixing Final concentration of internal standard in each sample is 10 ng mL 6 3 Fortify all controls and calibrators while vortex mixing using the appropriate amount of working standard Refer to the fortification guide for guidance on preparation of calibrators CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS Use appropriate external controls e g UTAK Whole Blood Controls when available It is recommended the controls be at 10 ng mL 6 4 Add 4 mL of 100 mM pH 6 0 sodium phosphate buffer to each tube Vortex 6 5 Centrifuge the samples for at least 10 minutes at 4000 rpm 6 6 Place collection tubes or reservoir for waste into vacuum manifold rack or positive pressure apparatus 6 7 Pour sample onto Polychrom Clin II columns Use sufficient vacuum pressure to draw samples through the columns 6 8 Wash column with 1 mL deionized water 6 9 Wash column with 1 mL 1 M acetic acid Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 93 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 6 10 Dry column on full vacuum pressure for 5 minutes 6 11 Wash colum
213. urn hazard 4 5 DO NOT operate the Turbo Vap LV without water in the bath to avoid the risk of fire or burn injuries 4 6 To avoid injury DO NOT EXCEED 100 psi maximum inlet pressure 5 PROCEDURE 5 1 Turn on the unit and gas supply Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date 08 03 2015 Uncontrolled When Printed Page 29 of 171 5 3 5 4 J 5 6 5 7 5 8 5 9 5 10 Houston Forensic Science Center Forensic Analysis Division Toxicology Check the water bath and fill to the appropriate level with distilled or deionized water The water bath level should be AS HIGH AS the initial level of solvent in your sample tubes unless you are using micro centrifuge racks and tubes Check the gas supply and pressure Set the water bath temperature as specified in the respective procedure The bath will be at the correct temperature when the TEMP light stops blinking in approximately 20 minutes Set the gas pressure and time setting in accordance with the SOP Turn on the rows containing sample tubes by pressing the corresponding tube stations pushbuttons There are 5 rows of 10 stations available for use Partial rows may have unused gas nozzles These should be plugged with supplied stoppers for optimum gas flow and sample protection against moisture Note Leaving empty rows unplugged while in use can cause condensation on the unit lid which may in turn contaminate the sample Load sample t
214. using sufficient vacuum as necessary 6 8 Wash column with 1 mL of deionized water 6 9 Wash column with 1 mL of 1M acetic acid 6 10 Dry column on full vacuum pressure for 5 minutes Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 87 of 171 6 15 6 16 Houston Forensic Science Center Forensic Analysis Division Toxicology Wash column with 1 mL of hexane Elute drugs with 1 mL of ethyl acetate Turn on vacuum for a few seconds to ensure all elution solvent has drained from the column Remove tubes from rack and evaporate to dryness approximately 10 minutes under nitrogen at 50 60 C Reconstitute in 40 uL of ethyl acetate vortex and transfer to autosampler vials An appropriate volume of solvent should be used to prevent overloading Inject 2 uL onto the GC MS using CARISO MEPRO M 7 INSTRUMENTAL ANALYSIS 7 1 7 2 Ensure that the daily QC and tune verification have been completed SIM acquisition CARIO MEPRO M Use this method to perform targeted analysis for carisoprodol and meprobamate Retention Time RT varies with column length Deuterated 7 internal standards are used throughout Corresponding ions for internal standards are M 7 Refer to attached method for acquisition parameters 8 INTERPRETATION OF RESULTS 8 1 8 2 8 3 Assay performance including but not limited to prevision accuracy limit o
215. vacuum pressure to draw samples through the columns 6 7 Wash column with 1 mL deionized water 6 8 Remove waste tubes reservoir and replace with fresh waste tubes reservoir 6 9 Wash column with 1 mL 1 M acetic acid 6 10 Dry column on full vacuum pressure for 5 minutes 6 11 Wash column with 1 mL hexane 6 12 Place conical collection tubes into the SPE manifold If the Teflon inserts vacuum manifold only appear dirty replace them 6 13 Elute drug with 1 mL ethyl acetate 6 14 Turn on vacuum or apply positive pressure for a few seconds to ensure all elution solvent has drained from the column 6 15 Remove conical collection tubes and replace with waste tubes reservoir 6 16 Wash column with 1 mL methanol 6 17 Dry column on full vacuum pressure for 5 minutes 6 18 Replace waste tubes reservoir with conical collection tubes containing the acidic neutral fraction If the Teflon inserts vacuum manifold only appear dirty replace them 6 19 Elute drug with 1 mL elution solvent 2 ammonium hydroxide in methylene chloride isopropy alcohol 80 20 ELUTION SOLVENT MUST BE MADE FRESH 6 20 Turnonvacuum or apply positive pressure for a few seconds to ensure all elution solvent has drained from the column 6 21 Evaporate the combined basic and acidic neutral fractions to dryness under nitrogen for approximately 10 minutes at 50 60 C Reconstitute in 30 uL ethyl acetate vortex and transfer to autosampler vials 6 22 Inject 2 uL onto t
216. vision Toxicology Appendix 1 Abbreviations 6 AM or 6 MAM equal plus or minus less than greater than less than or equal to greater than or equal to 6 acetyl morphine or 6 monoacetyl morphine ADA assistant district attorney ALC test for the presence of alcohol ALP alprazolam BAN basic acid neutral drug screen BCR blood collection report located on inner plastic box BE or BZE benzoylecgonine BH biohazard BZ or BENZO benzodiazepines cor c containing C M carisoprodol meprobamate CNS Central Nervous System COC cocaine CRM certified reference material DI deionized DOB date of Birth DQC dilution control DUID driving under the influence of drugs DWI driving while intoxicated EIA enzyme Immunoassay EME ecgonine methyl ester ENV envelope EQC aqueous ethanol control EtAc ethyl acetate EtOH ethanol ethyl alcohol FN false negative FP false positive g grams GC gas chromatograph GC FID gas chromatograph flame ionization detector GC MS gas chromatograph mass spectrometer H20 water HEQC high aqueous ethanol control HHC higher than the highest calibrator hr hour hrs hours IB inner box Incl including Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Uncontrolled When Printed Issue Date August 3 2015 Page 170 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology I S or IS internal standard KETO keto opioids kg kilogram L liter Ib pound LIM
217. w concentrations Refer to the respective procedures to determine if silanized glassware is necessary 2 SCOPE The continued use of a GC MS liner will eventually result in the accumulation of contaminants in the liner causing a loss in performance and making it unsuitable for use Following routine use GC MS liners can be reused if appropriate cleaning and chemical deactivation steps are followed This SOP describes the cleaning deactivation and silanization process 3 REAGENTS 3 1 Chromic Acid 3 1 1 May be prepared using concentrated sulfuric acid and potassium dichromate 3 1 2 A recommended preparation includes 20 mL potassium dichromate 60 mL concentrated sulfuric acid and 200 mL deionized water Quantity may be increased as needed using appropriate ratios 1 3 10 3 1 3 The solution should be a dark orange red color should it turn green it should be disposed of using proper disposal procedures 3 2 Silanizing reagent 1 1 1 3 3 3 Hexamethyldisilazane CAUTION Flammable liquid extremely harmful when inhaled and ingested extremely corrosive to skin mucous membranes and respiratory tract May cause eye burns 4 EQUIPMENT AND MATERIALS 4 1 Hot Plate 4 2 Beakers 4 3 Forceps 4 4 Pasteur pipettes 4 5 Sonicator 4 6 Vacuum oven 4 7 Hamilton 500 uL syringe or equivalent 5 PROCEDURE FOR CLEANING AND DEACTIVATION 5 1 Carefully place the chromic acid solution on a hot plate in a hood and heat to approximately 80 C
218. x Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 71 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 6 3 3 Add 100 uL deconjugation enzyme to all tubes and vortex 6 3 4 Cap and heat samples at 37 C for 1 hour Let cool to room temperature then proceed as outlined blow 6 3 5 Add 100 uL Internal Standard Solution 2 ng uL while vortex mixing The final concentration of internal standard is 200 ng mL 6 3 6 Proceed from step 4 of blood procedure above Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 72 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 7 INSTRUMENTAL ANALYSIS 7 1 Ensure that the daily QC and tune verification have been completed 7 2 SIM acquisition BENZOSIM M OOo een o e o BSCS Retention Time RT varies with column length Deuterated D4 and D5 internal standards are used throughout Refer to attached method for acquisition parameters Desalkylflurazepam d4 is used for both desalkylflurazepam and flurazepam Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 73 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 8 INTERPRETATI
219. y procedure 6 2 1 1 7 Confirm that the rinsing solution and the tube connecting the solution to the plate washer are connected 6 2 1 1 8 Confirm waste bottle is empty 6 2 1 1 9 Select channel 1 The tube connecting the plate washer to the rinse bottle has the number on it 6 2 1 1 10 Select the duration time or the volume 6 2 1 1 11 Once the rinse procedure is complete press stop to abort the rinse procedure 6 2 1 1 12 END appears on the display when the procedure is completed The manifold remains immersed in the prime tray until END is pressed Press END to finish the rinse procedure and the firmware returns to the Program menu 6 2 2 Rinse Night the instrument is left to stand 12 hours or longer with the manifold soaking in deionized water Rinse night must be performed with deionized water only 6 2 2 1 Procedure 6 2 2 1 1 Switch on the Plate Washer 6 2 2 1 2 Press gt key to find Procedures 6 2 2 1 3 Press OK on Procedures 6 2 2 1 4 Select Rinse 6 2 2 1 5 Press OK to enter the Rinse menu 6 2 2 1 6 Select Rinse Night procedure 6 2 2 1 7 Confirm that the rinsing solution and the tube connecting the solution to the plate washer are connected 6 2 2 1 8 Confirm waste bottle is empty 6 2 2 1 9 Select channel 1 The tube connecting the plate washer to the rinse bottle has the number on it 6 2 2 1 10 Select OK and the wash system is rinsed 6 2 2 1 11 The prime t
220. y that are not found in the reference spectrum must be demonstrated as being from background or known interference by using an extracted ion chromatogram Qualitative identification is determined using characteristic retention time and mass spectral characteristics Deuterated internal standards in each sample are used to identify the correct retention time of the drug For SIM all three characteristic ions must be present lon intensity and ratios should be taken into consideration lon ratios should be within 20 of the calibrator or the QC These are evaluated on a case by case basis and are subjected to technical and administrative review 7 QUANTITATIVE ANALYSIS 7 1 7 2 Concentration of a drug is determined using linear regression analysis At least 3 calibrators and a blank blood sample must be used for each quantitation R values of at least 0 99 should be attained following linear regression analysis If more than three calibrators are included in the run one may be excluded if the response factor is not within 20 of the remaining calibrators The response factor is defined as follows Peak Area of the Internal Standard Peak Area of the Calibrator x Calibrator Concentration If the response factor is within range it may be permissible to exclude the point from the calibration provided that the independent control is still within acceptable limits and the reviewer is in agreement The calculated concentration of a calibrator mus
221. y the Hamilton pipettor dilutor for creating samples for analysis Values are obtained from yearly calibration certificates 13 1 4 Duplicates accounts for the maximum allowed difference between analytically obtained values of duplicate case samples 13 2 The Alcohol Measurement Uncertainty Spreadsheet is available in the laboratory in a retrievable format Toxicology Standard Operating Procedures Version 2 0 Issued By Section Manager Issue Date August 3 2015 Uncontrolled When Printed Page 155 of 171 Houston Forensic Science Center Forensic Analysis Division Toxicology 14 REPORTING 14 1 14 1 1 14 1 2 14 2 14 2 1 14 2 2 14 2 3 14 2 4 14 2 5 14 2 6 14 2 7 14 2 8 14 2 9 Ethanol and M I A Reported concentration will be the average of the results obtained from aliquot 1 and aliquot 2 on FID1 A BAC1 column truncated to three decimal places FID2 B BAC2 column is used as a semi quantitative confirmation only Reported measurement uncertainty MU will be calculated using the following formula and truncated to three decimal places Reported MU Reported concentration x MU Reporting Statements Blood specimen results are reported in grams per 100 milliliters of blood The reported MU must be included on each report using the statement The measurement uncertainty associated with this value is x xxx grams per 100 milliliters or an equivalent statement For volati
222. ycle consists of 3 methanol rinses followed by 3 ethyl acetate rinses 6 EXTRACTION PROCEDURE Blood Urine and all Other matrices 6 1 In a round bottom glass culture tube add 1 mL of sample NOTE IF THE SAMPLE IS KNOWN TO CONTAIN A HIGH CONCENTRATION OF DRUG ALIQUOT A SMALLER VOLUME AND DOCUMENT IN THE CASE FOLDER 6 2 Add 50 uL of Internal Standard 5 ng uL while vortex mixing Final concentration of internal standard in each sample is 250 ng mL 6 3 Fortify all controls and calibrators while vortex mixing using the appropriate amount of working standard The typical calibration range for quantitative analysis is 0 1000 ng mL for Methadone EDDP Refer to the fortification guide for guidance on preparation of calibrators CONTROLS AND CALIBRATORS MUST COME FROM INDEPENDENT STOCK SOLUTIONS An in house control containing 100 ng mL methadone EDDP is recommended Use appropriate external controls e g UTAK Whole Blood Controls when available 6 4 Add 2 mL of 100 mM pH 6 0 sodium phosphate buffer to each tube Vortex 6 5 Centrifuge samples for 10 minutes at 4000 rpm 6 6 Place collection tubes or reservoir for waste into vacuum manifold rack or positive pressure apparatus 6 7 Pour sample onto Polychrom Clin II columns Use sufficient vacuum pressure to draw samples through the columns 6 8 Wash column with 1 mL deionized water 6 9 Wash column with 1 mL 1 M acetic acid 6 10 Dry column on full vacuum pressure for 5 minutes 6 11

Toxicology SOP - Houston Forensic Science

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