USER`S MANUAL
Contents
1. e 3 3 Sample Preparation by SPE and Derivatization Prepare Eluting Medium first refer to section 3 2 for preparation protocol The freshly prepared Eluting Medium vial may be placed in one of the empty slots in the reagent tray 1 Foreach sample line up one glass sample preparation vial in the vial rack Figure 2 Be aware of some variability in vial opening and sorbent tip dimensions which may prevent the tip from reaching to the bottom of the sample preparation vial Note Droplets of liquid in SPE tip or spilled sorbent particles will not affect the precision of the assay in any way EZ faast User s Manual Gass Viar Line Up Ficure 2 For each sample line up one glass sample preparation vial in the vial rack Figure 2 2 Pipette 100uL sample serum plasma urine or other and 1000 Reagent 1 Internal Standard Solution into each sample preparation vial Caution The pH of biological samples is usually around 7 After the addition of Reagent 1 Internal Standard the mixture has the correct pH for successful loading onto the SPE tip as described in the next step With other samples make sure that the sample Reagent 1 mixture has a pH between pH 1 5 and pH 6 0 Note Samples with amino acid concentrations higher than 10mmol L 10umol mL e g dark colored urine should be analyzed by pipetting only 50uL or 25uL sample in the sample preparation vial instead of 100uL Co2. Tip Ficure 4 Wet the sorbent with Eluting Medium and stop before it gets to the filter then eject the liquid and sorbent particles out of the tip 4 0 Gas CuRoMArocRAPHIC ANALYSIS 4 1 Column For EZ faast Physiological Amino Acid Analysis by GC MS The Zebron ZB AAA GC column comes without a cage Connect the ends of the column in the usual manner rest the column coil on the oven bracket Keep the pieces of thermal thread spaced evenly around the column coil The maximum column temperature is 320 340 C Caution Always use safety glasses while installing the GC column 4 2 Instrument Settings GC Injection Split 1 15 250 C 1 5 2uL with hot needle see section 4 5 Carrier Gas Helium 1 1mL min constant flow Oven program 30 C min from 110 to 320 C MS MS Source 240 C MS Quad 180 C Auxiliary 310 C Scan Range 45 450 m z Sampling Rate 22 3 5 scans s When using a Shimadzu GC instrument please increase the injector temperature to 300 C e For your convenience we have included the GC MS methods for Shimadzu QP 2010 Varian Saturn 2000 and Agilent 5973 GC MS systems on the reference CD included with the kit To use these included methods copy the method folder into the appropriate method folder in your software and load EZ aast User s Manual 4 3 Liners Use the best deactivated liners supplied by the instrument manufacturer Good results were obtained with
3. L breaking with tradition in Ca Rc 1x EZ aast Free Physiological Amino Acid Analysis by GC MS For Part Number KG0 7166 o D 2 a gt E lt S E E lt o gt 4 D O qu U N 9 e 5 phenomenex breaking with tradition Phenomenex Ltd Deutschland Zeppelinstr 5 411 Madrid Ave 63741 Aschaffenburg ud Torrance CA 90501 1430 Deutschland USA EZ aast User s Manual The following is a description of the symbols used in the EZ faast manuals on EZ faast packaging and EZ faast kit components Symbol for In Vitro Diagnostic Medical Device Symbol for Manufacturer Symbol for Authorised Representative In The European Community ud T Symbol for Use By and or Expiration Date 9 Symbol for Batch Code and or Lot Number Symbol for Catalogue Number Symbol for Serial Number Symbol for Flammable Substances Symbol for Irritating or Harmful Substances oh Me Symbol for Corrosive Substances TABLE CONTENTS 1 NIE 2 Sample Preparation Procedure 4 Gas Chromatographic Analysis 8 lo ll kee 11 Sample Storage and Stability 12 Cleaning
4. FocusLiners included Phenomenex P N AGO 4680 fits Agilent and Varian 1177 injectors In general the liner should carry a plug of silanized quartz or pesticide grade glass wool 4 4 Injection Split injection at a ratio of 1 10 to 1 20 is recommended e Injection volumes of 1 5 2yL are optimal Quasi splitless injection mode will produce a 5 to 10 fold increase in sensitivity with some instruments In this mode the split valve should be closed for an initial 5 to 7 seconds Before selecting this injection mode it should be checked experimentally that no significant discrimination of late eluting amino acid derivatives takes place in comparison with common split injection Alternatively instruments equipped with EPC AFC can be operated with double initial head pressure for 6 10 seconds 4 5 Sampling Both autosampler and manual sampling can be performed If manual sampling is pre ferred hot needle injection is recommended to prevent discrimination of components with high boiling temperatures With this technique the sample plug is completely drawn into the syringe barrel leaving the needle empty The needle is inserted and kept in the hot injector for about two seconds before injection 4 6 Calibration Standards For quantitation purposes mixtures of amino acid standards should be prepared following the Sample Preparation by SPE and Derivatization procedure described in this manual in Sec tion 3 3 Standard mixtures should be stored
5. and Care of Supplies 12 65 13 Product Limitations 13 Ordering Information 14 15 FACCHI User s Manual 1 0 Kit Components 1 1 Reagents Reagent Ingredients Volume Reagent 1 Norvaline 0 2 mM Internal Standard Solution N propanol 10 50mL Reagent 2 Washing Solution N propanol 90mL Reagent 3A Eluting Medium Component Sodium Hydroxide 60mL Reagent 3B Eluting Medium Component II N propanol 40mL Regent 4 Organic Solution Chloroform 4 vials 6mL each Reagent 5 Organic Solution II Iso octane 50mL Reagent 6 Iso octane 8096 Re dissolution Solvent Chloroform 2096 50mL 0 1 2 amp 3 Please refer to section 4 6 Amino Acid Standard Mixtures in the manual 2 vials of each SD 2mL each 1 2 Supplies Sorbent tips in racks 2 2 Sample preparation EEEE SEEE EEEE Microdispenser 20 700u Syringe OOM ecce ces c e ee eto uei Syringe RTE ZB AAA 10m x 0 25mm Amino Acid Analysis GC Column e Autosampler vials with inserts sss Focusbiners CREER RERUM GEGANGEN RU UO e EZ faast Demo Video and Reference CD i s dE 1 3 Materials Required but Not Supplied In Kit e 100uL 1mL pipette SoftGrip pipette Phenomenex P N AH0 5968 or equivalent 30 300uL pipette SoftGrip pipette Ph
6. in the freezer as some amino acids are not stable in solution Three vials of different standard mixtures are included in the kit SD1 23 amino acids 200 nmoles mL each as follows AAA ASP GLY LEU PHE THR ABA DAD HIS LYS PRO TYR alLE C C HYP MET SAR VAL ALA GLU ILE ORN SER SD2 Complementary amino acids not stable in acidic solution 200 nmoles mL each as follows ASN GLN TRP SD3 Complementary urine amino acids 200 nmoles mL each as follows APA CTH GPR HLY PHP TPR Figure 5 EZ faast User s Manual A typical chromatogram of a mixture of all three amino acid standard solutions included in this kit Column and instrumental settings as specified in section 4 1 4 2 gt Abundance CPS x 105 8 1 50 2 00 2 50 3 00 3 50 4 00 4 50 5 00 5 50 6 00 5 54 3 45 4 95 5 13 5 24 6 26 m C A Om OD O E Figure 5 Elution order tR 1 42min ALA 1 48 SAR 1 53 GLY 1 64 ABA 1 74 VAL 1 83 BAIB 1 88 IS NVAL 1 97 LEU 2 00 alLE 2 03 ILE 2 25 THR 2 29 SER 2 37 PRO 2 47 ASN 2 84 TPR 3 04 ASP 3 08 3 22 3 42 GLU 3 45 PHE 3 73 AAA 4 00 APA 4 08 GLN 4 48 ORN 4 53 GPR 4 75 LYS 4 95 HIS 5 13 HLY 5 24 TYR 5 47 PHP 5 54 TRP 6 03 CTH and 6 26 C C 4 7 Calibration Procedure Use the following standard amino acid mixtures and make duplicate injections of each to generate the des
7. video included with this kit demonstrates the simplicity of the procedure Please be aware that some sample preparation steps described in the video may be different than what is described in this users manual Please use the video as a general guide but follow the exact steps and sequence described in this manual 2 2 Amino Acids and Related Compounds in Physiological Samples The EZ faast method has been developed for the analysis of more than 60 aliphatic and aromatic amino acids including primary and secondary amines Further amino acids and related compounds may be analyzed with this kit A brief adjustment of gas chromatographic conditions may be necessary Please contact Phenomenex for method modifications and other LC and GC amino acid kits Included with the EZ faast kit is a CD that contains spectral librar ies for most amino acid derivatives as run by the EZ faast methodology Spectral libraries are included for Varian Saturn and Agilent Chemstation software systems Table 1 Comprehensive list of amino acids and related compounds prepared by EZ faast for GC MS analysis internal standard listed in bold hemical Nam Abl n Abb Agilent 59 var Ethanolamine 116 117 Alanine ALA A 130 88 130 70 1 0 1 Allin 216 173 129 Sarcosine SAR 130 217 130 88 1 0 1 Glycine GLY G 116 207 116 102 2 0 1 a Aminobutyric acid ABA 144 102 144 102 1 0 2 Valine VAL V 158 116 116 98 158 0 6
8. 0 2 Fluoro alanine B Alanine B ALA 129 158 98 B Aminoisobutyric acid B AiB 158 116 130 84 144 4 0 2 B Amino n butyric acid BABA 88 70 Norvaline NORV 158 72 158 116 Leucine LEU L 172 86 172 130 0 7 0 1 allo Isoleucine alLE 172 130 130 101 0 7 0 1 Isoleucine ILE 172 130 130 101 0 7 0 2 Homoserine HSER 102 128 143 Norleucine NLE 172 86 Threonine THR T 160 101 101 2 0 2 EZ faast User s Manual Table 1 continued EH S N 3 1 Agilent 5973 Varian 2000 ep ms y Amino n butyric acid GABA 130 144 172 Serine SER S 146 203 101 86 2 0 2 Proline PRO P 156 243 156 114 1 0 1 Asparagine ASN N 155 69 113 2 2 5 3 Methyl cysteine 172 259 130 Pipecolic Acid HPRO Thioproline TPR 174 147 174 147 04 0 1 Aspartic acid ASP D 216 130 216 130 0 9 0 1 Methionine MET M 208 277 101 203 129 0 9 0 2 3 Hydroxyproline 3HYP 172 259 130 4 Hydroxyproline 4 OHPro 172 86 172 86 2 0 2 Phenyl glycine PHE GLY Seleno methionine Se MET Glutamic acid GLU E 230 170 84 142 2 0 2 Phenylalanine PHE F 206 190 147 128 91 0 5 0 2 a Aminoadipic acid AAA 244 98 98 125 1 0 2 Cysteine cys C 248 162 206 4 Aminobenzoic acid PABA 265 206 163 Homophenylalanine HPHE a Aminopimelic acid APA 198 258 286 198 138 112 0 5 04 Chloro phenylalanine CI PHE Histamine HA
9. 180 168 223 Glutamine GLN Q 84 187 84 112 8 10 Theanine THE 112 215 Bicine 290 260 2 4 Diamino n butyric acid DABA 203 142 245 Glycyl glycine dipeptide GLY GLY 117 144 201 Homocysteine HCYS 142 203 Methionine sulfone Methionine sulfoxide 229 182 138 S Carboxymethyl cysteine 144 203 262 Ornithine ORN 0 156 70 156 139 114 1 0 2 Glycyl proline dipeptide GPR G P 70 300 153 114 1 5 Tyramine 120 107 162 Lysine LYS K 170 128 153 170 128 1 0 2 Threonine aspartic acid dipeptide THR ASP 218 360 130 Histidine HIS H 282 168 267 222 136 1 0 2 Naphthyl alanine Seleno cystine Se C C Hydroxylysine 2 isomers HLY OHLys 129 169 87 129 2 10 TYR Y 206 107 164 107 0 4 0 2 Diaminopimelic DAPA 256 168 Proline hydroxyproline dipeptide 156 186 156 114 0 9 10 Tryptophan TRP 130 130 04 0 1 Lysine alanine dipeptide LYS ALA 170 224 153 Dopamine DA 179 136 123 3 Nitrotyrosine 152 209 EZ faast User s Manual Table 1 continued Aspartame 302 Cystathionine CTH 203 272 146 114 4 10 3 4 Dihydroxyphenylalanine DOPA 222 123 Cystine c c Cys 2 248 216 114 173 4 10 Serotonin SRO 146 288 348 Homocystine HC CH Hcys 2 230 188 128 Arginino succinic acid ARG SUC 441 326 Ethionine ETH 203 291 143 LODs were determined for amino acids included in standard mixtures provided with the kit Several amino acids coelute under the chromatographic conditio
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11. amples prepared for GC MS analysis following the procedure outlined in this manual may be stored for several days in a freezer before analysis For longer storage we recommend that samples be desiccated with anhydrous sodium sulfate vials be capped and placed in the freezer Since sample preparation is expeditious with this procedure we recommend analyzing samples prepared freshly Samples prepared during the day may be left on the autosampler tray at room temperature to be analyzed during the night or the next day 7 0 CLEANING AND or SUPPLIES The Drummond Dialamatic Microdispenser should be flushed with isopropanol acetone approx 1 1 at the end of the day Please review the Drummond Microdispenser users manual for further care and use notes The same organic mix is recommended as wash for both manual syringes and autosamplers Plastic syringes used for sample clean up by SPE can be cleaned by flushing with propa nol water 1 2 v v mixture Always tightly cap the reagent bottles when not in use in order to avoid solvent evapora tion and alteration of reagent composition Cover the racks holding sorbent tips when not in use to prevent contamination FACCHI User s Manual 8 0 QuaLiTY AsSURANCE All components of the EZ faast Gas Chromatographic Amino Acid Analysis kit are subjected to rigorous quality control testing These measures help to ensure the best results If poor results occur please contact your Phenom
12. e Physiological Amino Acid Analysis Kit KG0 7166 ea GC FID Protein Hydrolysate Kit KG0 7167 ea GC MS Protein Hydrolysate Kit KG0 7168 ea LC MS Free Physiological Amino Acid Analysis Kit with 250 x 2 0mm column KH0 7337 ea LC MS Free Physiological Amino Acid Analysis Kit with 250 x 3 0mm column KH0 7338 ea LC MS Protein Hydrolysate Kit with 250 x 2 0mm column KH0 7339 ea LC MS Protein Hydrolysate Kit with 250 x 3 0mm column KH0 7340 ea GC Free Physiological Amino Acid Standards SD1 SD2 amp SD3 2mL vial x 2 AGO 7184 ea GC Protein Hydrolysate Standard SD 2mL vial x 2 0 7263 LC MS Free Physiological Amino Acid Standards for LC 501 502 amp 03 2mL vial x 2 ALO 7500 ea LC MS Protein Hydrolysate Standard SD 2mL vial x 2 ALO 7501 ea www phenomenex com d amp 1 A e y Sa E products are available worldwide For the distributor in your coi k milk E phenomenex E contact Phenomenex USA International Department by telephone fax or e iternationalOphenomenex com breaking with tradition p USA Puerto Rico Canada France United Kingdom Germany New Zealand Australia mail 411 Madrid Ave 273 Sierra Morena 411 Madrid Ave Parc des Grillons Bat3 Queens Avenue Queens Avenue Zeppelinstr 5 P 0 Box 31601 PO Box 4084 Torrance CA Suite 104 Torrance CA 60 route de Sartrouville Hurdsfield Ind Est Hurdsfield Ind Est 63741 Aschaffenburg Milford Lane Cove
13. enex technical consultant or distributor 9 0 Pnopucr Limitations Phenomenex Analyte Specific Reagent products are not intended for clinical use Because they are not intended for clinical use no claim or representation is made or intended for their clinical use including but not limited to diagnostic prognostic therapeutic or blood banking It is the user s responsibility to validate the performance of Phenomenex products for any par ticular use since the performance characteristics are not established Phenomenex products may be used in clinical diagnostic laboratory systems after the laboratory has validated their complete system as required by the Clinical Laboratory Improvements Amendments of 1988 CLIA 88 regulation in the U S or equivalent in other countries Trademarks EZ faast Sorbent Tips are patented Phenomenex Inc U S Patent 6 770 246 EZ faast is a trademark of Phenomenex Inc Phenex is a trademark of Phenomenex Inc FocusLiner and Auto Sep T are trademarks of SGE SoftGrip is a trademark of Hamilton Drummond is a registered trademark of the Drummond Corp Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by Law 92005 2006 Phenomenex Inc All rights reserved EZ faast Kit Each kit includes a ZB AAA GC column or EZ faast LC column GC liners with GC kits sample prep and derivatization reagents sample pre
14. enomenex P N AH0 5967 or equivalent e 10 100yL pipette SoftGrip pipette Phenomenex P N AH0 5966 or equivalent e Pipette tips Phenex Phenomenex P N AH0 5917 200pL and AH0 5920 1mL or equivalent e Vortex e Vials of an appropriate volume with caps see section 3 2 e Pasteur pipettes for sample transfer see section 3 3 step 13 Container for proper waste disposal Septa Auto Sep T 11mm SGE P N 041883 fits Agilent or Carlo Erba instruments or equivalent EZ faast User s Manual 2 0 Overview 2 1 Overview The EZ faast amino acid analysis procedure consists of a solid phase extraction step followed by a derivatization and a liquid liquid extraction derivatized samples are quickly analyzed by gas chromatography mass spectrometry The solid phase extraction is performed via a sorbent packed tip that binds amino acids while allowing interfering compounds to flow through Amino acids on sorbent are then extruded into the sample vial and quickly derivatized with reagent at room temperature in aqueous solution Derivatized amino acids concomitantly migrate to the organic layer for additional separation from interfering compounds Organic layer is then removed evaporated and re suspended in re dissolution solvent and analyzed on a GC MS system Total sample preparation time takes around 8 minutes and analysis is performed in around 7 minutes for a total start to finish time of around 15 minutes A
15. epta come in one convenient kit pack Description Clear wide mouth vial cap and septa kit pack with Rubber PTFE septa Silicone PTFE septa PTFE Silicone PTFE septa Amber wide mouth vial cap and septa kit pack with Rubber PTFE septa Silicone PTFE septa Clear wide mouth vial cap with pre slit septa Silicone PTFE septa SGE FocusLiners Description GC Model No Thermo Electron 8000 series Carlo Erba Single Taper Gooseneck Liner Agilent 5880 5890 Technologies 6890 Single Taper Gooseneck Liner PerkinElmer Autosystem Single Taper Gooseneck Liner Shimadzu 17B Single Taper Gooseneck Liner Varian 1075 77 Single Taper Gooseneck Liner Varian 1078 79 Double Taper Gooseneck Liner Dimensions ID Material IDxLxOD mm deactivated 5 x 105 x 8 0 y 4 78 5 x 6 3 y 4x92x6 2 3 4 95 5 4x72x6 3 y 3 4x54x5 y B Borosilicate Deactivated Yes y or No n Quartz Wool Y N Y Order No AH0 4610 AH0 4613 AH0 4616 AH0 4619 4622 7507 Mfr P N 092046 092003 092095 092068 092025 092036 Unit 1000 pk 1000 pk 1000 pk 1000 pk 1000 pk 1000 pk Order No AG0 4679 AGO 4680 AGO 4681 AGO 4683 AGO 4684 AGO 4685 Unit 5 pk 5 pk 5 pk 5 pk 5 pk 5 pk Detach Here N eo eo e FACCHI User s Manual EZ faast Free Physiological Ami
16. even while dispensing reagents In case the sorbent tip cannot reach to the bottom of the vial tilt the vial to about 45 push the tip into the vial gently and proceed with the SPE step 4 Pipette 2001 Reagent 2 Washing Solution into the same sample preparation vial Pass the solution SLOWLY through the sorbent tip and into the syringe barrel Drain the liquid from the sorbent bed by pulling air through the sorbent tip Detach the sorbent tip and leave it in the sample preparation vial then discard the liquid accumulated in the syringe Note save the syringe as it can be reused with many other samples For convenience place it into the pipette rack 5 Pipette 200uL Eluting Medium prepared fresh each day section 3 2 into the same sample preparation vial FACCHI User s Manual Keep THE SORBENT TIP IN THE VIAL FicurE 3 Keep the sorbent tip in the sample preparation vial through steps 3 8 even while dispens ing reagents Figure 3 6 Pull back the piston of a 0 6 mL syringe halfway up the barrel and attach the sorbent tip used in steps 3 8 7 Wet the sorbent with Eluting Medium watch as the liquid rises through the sorbent particles and stop when the liquid reaches the filter plug in the sorbent tip 8 Eject the liquid and sorbent particles out of the tip and into the sample preparation vial Repeat step 7 and 8 until the sorbent particles in the tip are expelled into the sample preparation vial Only the
17. filter disk should remain in the empty tip see figure 4 Keep the syringe as it can be reused with many other samples 9 Using the adjustable Drummond Dialamatic Microdispenser included transfer 50yL Reagent 4 into the sample preparation vial Caution Avoid cross contamination by not touching the inner wall of the sample vial with the tip of the Microdispenser The piston will ensure proper transfer of liquids into the vial without the need of touching the vial wall Use the same Microdispenser with both Reagents 4 and 5 There is no need to change Microdispenser tips or to wash between uses Change Microdispenser tips only when broken Warning Do not use regular pipettes and tips with Reagents 4 and 5 as they will contaminate the sample Use the included Microdispenser for Reagents 4 and 5 ONLY Note for all subsequent sample preparation steps use a vortex mixer set in the touch pulse mode to about 80 of max speed for any mixing operations 10 Emulsify the liquid in the vial by repeatedly vortexing for about 5 8 seconds During vortexing hold the sample vial firmly between fingers and keep it straight as you push it onto the vortex plate Do not let vial wobble otherwise liquid may come out of the vial Allow reactions to proceed 1 minute or more The emulsion will gradually separate into two layers Note a longer reaction time than 1 minute at step 10 and at step 11 or later at step 12 does not affect results 11 Re emulsif
18. holds a reagent tray a vial rack a pipette rack and a section for sorbent tips and vials To speed up sample preparation it is recommend that the workstation be arranged as shown in figure 1a By following directions and markings on the reagent box by breaking it along perforations it can be transformed into a reagent tray When the kit is not in use for several days the reagent tray figure 1b may be conveniently removed and placed in the refrigerator 3 2 Preparing the Eluting Medium The volume of prepared Eluting Medium depends upon the number of samples to be ana lyzed during the day 200uL sample The eluting medium should be prepared fresh each day 1 Use capped vials of appropriate size not included for preparation of the Eluting Medium EZ faast User s Manual 2 Combine 3 parts Reagent Eluting Medium Component 1 with 2 parts Reagent Eluting Medium Component Il in an appropriate sized vial see Table 2 page 5 for reagent volumes based on number of samples Mix briefly 3 Store prepared eluting medium during the day at room temperature Discard any unused mixture at the end of the day e WorksTATION ARRANGEMENT Ficun 1 To speed up sample preparation it is recommended that the workstation be arranged as shown below Figure 1a Figure 1b Table 2 For your convenience check the table below to determine the volume of Eluting Medium components needed depending on your number of samples
19. ired calibration Calibration Solution 1 25uL of SD1 solution plus 25uL of 502 and or SD3 depending on your application plus 100uL Reagent 1 calibration level 1 50 nmoles mL ll 50uL 501 50uL SD2 and or 03 100pL Reagent 1 calibration level 11 100 nmoles mL 100uL 501 100 02 and or SD3 100 Reagent 1 calibration level Ill 200 nmoles mL The concentration of the internal standard 15 Norvaline in the sample prepared for gas chromatographic analysis is 200 nmoles mL serum urine etc Note Disregard the first 3 5 injections when performing method calibration or after replacing column or liner These injections act as primers and mask active sites inside the liner and chromatographic column Use subse quent duplicate runs for calibration Remember the SD1 SD2 and SD3 vials should be placed in the freezer after use Allow standards to reach room temperature before use 4 8 Calibration of analytical results Calculations are performed by the Data Analysis portion of the software controlling the analytical instrument gas chromatograph Calculations and calibration are based on an in ternal standard Results are reported in the units entered for the internal standard and analyte levels in calibration mixtures Note nmols mL are equivalent to pmols L EZ faast User s Manual 5 0 TROUBLESHOOTING Problem Decrease in peak height for some amino acids components of amino acid standard
20. mixtures SD2 or SD3 Possibly related to improper sample storage See sample standard storage sec tion 6 0 Decrease in peak height for basic amino acids Old eluting medium Prepare eluting medium daily based on the number of samples to be analyzed on that day Decrease in peak height or missing peaks for late eluting HIS C C HC CH or polar amino acids SER HYP GLN Improper liner Use deactivated liners see section 4 4 Analyze samples only after mak ing priming injections Decrease in peak height for early eluting amino acids Sample too concentrated the capacity of the SPE tip exceeded Use less sample for preparation see section 3 3 step 2 Constantly moni tor the area for the IS peak Volatile derivatives evaporated during removal of residual reagents with nitrogen gas Adjust nitrogen flow to minimal stop evaporation before sample gets completely dry Low peak height for late eluting derivatives Carrier gas leak Check instrument for leaks reinstall the column and check o ring on liner Peak height for IS Norvaline lower compared to other early eluting amino acids in the standard mix Pipetting error Recalibrate pipette used for dispens ing Reagent 1 Constantly monitor area for IS Ghost peaks Pipette tips used for dispensing reagents or for transferring prepared samples may be a source of con taminant peaks Use polypropylene pipette tips of appr
21. ncen trations recorded as a result of the GC analysis will be half one quarter of the actual concentrations for these samples Conversely when low concentrations of amino acids have to be quantified the volume of sample to be prepared should be 200uL or more The total amount of amino acids present in the sample to be loaded onto the SPE tip should not exceed 1 2 umols 3 Attach a sorbent tip to a 1 5mL syringe and loosen the syringe piston immerse the tip and let the solution in the sample preparation vial pass through the sorbent tip by SLOWLY pulling back the syringe piston in SMALL steps Caution Do not quickly pull back the piston Try to take at least one minute to pass low viscosity sample such as urine or standard through the sorbent tip For very viscous samples like concen trated plasma up to 200uL of water can be added to ease the sample transfer through the sorbent The syringe should be capable of drawing all sample and subsequent wash reagent into the barrel Watch as the liquid accumulates inside the syringe barrel and move the piston only as the accumu lation slows down Urine passes relatively fast through the sorbent bed while serum and plasma pass much slower If you run out of piston range detach the sorbent tip expel the solution from the Syringe barrel then reattach the sorbent tip and proceed with sample preparation Note the sorbent tip should stay in the sample preparation vial through steps 3 8 see figure 3
22. no Acid Analysis by GC MS Quick GuipE Summary of Procedure 1 For each sample line up one glass sample preparation vial in the vial rack 2 Pipette 100uL sample serum plasma urine or other see section 3 3 2 and 100 Reagent 1 into each sample preparation vial 3 Attach a sorbent tip to a 1 5mL syringe pass the solution in the sample preparation vial through the sorbent tip by slowly pulling back the syringe piston Pipette 200 Reagent 2 Washing Solution into the sample preparation vial Slowly pass the solution through the sorbent tip and into the syringe barrel Detach the sorbent tip and discard the liquid accumulated in the syringe Pipette 200yL Eluting Medium prepared fresh each day section 3 2 into the sample preparation vial Pull back the piston of a 0 6mL syringe halfway up the barrel and attach the sorbent tip Wetthe sorbent with Eluting Medium stop when the liquid reaches the filter plug in the sorbent tip CO do N cc P Eject the liquid and sorbent out of the tip and into the sample preparation vial Repeat until all sorbent particles in the tip are expelled into the sample preparation vial Discard the empty tip 11 Using the Drummond Dialamatic Microdispenser transfer 50uL Reagent 4 12 Emulsify by repeatedly vortexing the solution for about 5 seconds Allow reaction to proceed for 1 minute or more 13 Vortex the solution again for a few seconds to re em
23. ns specified in the user manual e g GABA amp SER 2 3 Storage and Stability Store Reagents 1 3B and 4 at 4 C do not freeze Store amino acid standard solutions in the freezer All other components may be stored at room temperature For your convenience the bottom of the reagent box has been designed as a tray that can be easily lifted from the work station and placed in the refrigerator when the kit is not in use for an extended period of time All components are guaranteed for 12 months or more see label on bottle vial from the date of purchase when stored at recommended temperatures and used as described in this manual Please review the Instruction Manual included with the Drummond Dialamatic Microdispenser for recommended usage and warranty information Please observe recommendations for solvent bottle handling and syringe cleaning in Section 7 0 of this manual 2 4 Safety Although the concentration of all toxic components in any of the reagent bottles is low for safety reasons the sample preparation station should be placed in an exhaust hood and protective gloves and goggles should be worn When working with biological fluids please take any necessary precautions to prevent infection with blood borne pathogens Appropriate bio safety precautions and disposal of bio hazardous wastes should be followed 3 0 SawPLE PREPARATION PROCEDURE 3 1 Setup The EZ faast kit packaging has been designed as an efficient workstation It
24. opriate quality see order ing info on page 14 Always use the Microdispenser for dispensing Reagents 4 5 and 6 Use Pasteur pipettes to transfer the reconstituted sample into a glass insert Interfering peaks may result from extracted contaminants in plastic sample preparation vials Use the vials provided in the kit For autosampler vials order Phenomenex P N AHO 4610 and for inserts 4604 Early deterioration of column performance Residual sample preparation reagents degrade column stationary phase Evaporate residual reagents at step 13 Do not use samples prepared as described in the EZ faast kit for the analysis of free amino acids by GC FID Interfering peaks drug metabolites Physiological sample anticoagulants like citrate or citric acid may inter fere in the amino acid profile Samples collected with EDTA and heparin anticoagulants are recom mended Confirm peak identity based on mass spectrum EZ faast User s Manual 6 0 SAMPLE STORAGE AND STABILITY Some amino acids are chemically unstable in physiological fluids e g progressive decline of plasma glutamine and cystine in time and also in standard mixtures Keep samples and standard mixtures in the freezer Old amino acid standard mixtures and mixtures which have not been stored properly should not be used for instrument calibration Order fresh mixtures from Phenomenex see ordering info in Phenomenex catalog S
25. p vials AA standard mixtures SPE sorbent tips vial rack autosampler vials with inserts come with MS kits Microdis penser for Reagents 4 and 5 and demo video Description Order No Unit GC FID Free Physiological Amino Acid Analysis Kit KG0 7165 ea GC MS Free Physiological Amino Acid Analysis Kit KG0 7166 ea GC FID Protein Hydrolysate Kit KG0 7167 ea GC MS Protein Hydrolysate Kit KG0 7168 ea LC MS Free Physiological Amino Acid Analysis Kit with 250 x 2 0mm column KH0 7337 ea LC MS Free Physiological Amino Acid Analysis Kit with 250 x 3 0mm column KH0 7338 ea LC MS Protein Hydrolysate Kit with 250 x 2 0mm column KH0 7339 ea LC MS Protein Hydrolysate Kit with 250 x 3 0mm column KH0 7340 ea GC Free Physiological Amino Acid Standards SD1 SD2 amp SD3 2mL vial x 2 AGO 7184 ea GC Protein Hydrolysate Standard SD 2mL vial x 2 0 7263 LC MS Free Physiological Amino Acid Standards for LC 501 502 amp 03 2mL vial x 2 ALO 7500 ea LC MS Protein Hydrolysate Standard SD 2mL vial x 2 ALO 7501 ea Phenex Vials This universal vial can be used in any autosampler that utilizes a 12 x 32mm vial It may be used in place of crimp top and snap ring top vials Eliminates the need of stocking many different style vials The top screws down in 1 3 turn and eliminates the chore of crimping de crimping and snapping caps on Cap comes with a bonded in septa that eliminates septa slipping into vials Vials and caps with bonded in s
26. ulsify the content of the vial Allow the reaction to proceed for at least one additional minute 14 Using the Microdispenser transfer 100pL Reagent 5 and re emulsify by vortexing for about 5 seconds Let the reaction proceed for 1 minute 15 Transfer part of the upper organic layer 50 100pL using a Pasteur pipette into an autosampler vial Avoid transferring aqueous layer along with the organic layer Evaporate the solvent SLOWLY to almost dry under a gentle stream of nitrogen max 10 min Re dissolve amino acid derivatives in 100pL or less of Reagent 6 using a Pasteur pipette Transfer the reconstituted sample into an insert and place the insert in the same autosampler vial The reconstituted sample is ready for GC MS analysis Gas Chromatographic Analysis GC Settings Injection Split 1 15 250 C 2 0uL Carrier Gas Helium 1 1mL min Oven Program 30 C min from 110 to 320 C MS Settings MS Source 240 C MS Quad 180 C Auxiliary 310 C Scan Range 45 450 m z Sampling Rate 2 3 5 scans s C EZ faast Kit Each kit includes a ZB AAA GC column or EZ faast LC column GC liners with GC kits sample prep and derivatization reagents sample prep vials AA standard mixtures SPE sorbent tips vial rack autosampler vials with inserts come with MS kits Microdis penser for Reagents 4 and 5 and demo video Description Order No Unit GC FID Free Physiological Amino Acid Analysis Kit KG0 7165 ea GC MS Fre
27. y the liquids in the vial by vortexing again for about 5 seconds Allow the reaction to proceed for one additional minute or more 12 Transfer with the Microdispenser 100uL Reagent 5 50uL twice for convenience and mix for about 5 seconds Let the reaction proceed for one more minute 13 Transfer part of the upper organic layer about 50 1001 using a Pasteur pipette into an autosampler vial included Avoid transferring aqueous layer along with the organic layer Evaporate the solvent SLOWLY to almost dry under a gentle stream of nitrogen Do not leave samples under the nitrogen stream for more than 10 minutes Re dissolve amino acid derivatives in 100uL or less of Reagent 6 Transfer the reconstituted sample into an insert included using a Pasteur pipette and place the insert in the same autosampler vial The reconstituted sample is ready for GC MS analysis see GC MS set up and calibration in section 4 EZ aast User s Manual 3 4 Optimizing Sample Preparation Time For experienced users sample preparation proceeds in 7 8 minutes per sample This process can be further improved by preparing up to ten samples at a time For example at step 2 dis pense Reagent 1 and at later steps all other reagents in ten vials successively using the same pipette tip At step 9 after dispensing Reagent 4 vortex 2 3 vials simultaneously During each one minute wait at steps 10 12 prepare autosampler vials for sample transfer
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