Immunosuppressants in Whole Blood – On
Contents
1. 4 4 2 MS MS parameters The MS MS parameters indicated in the tables of sections 4 4 2 1 to 4 4 2 4 are recommended values only This particularly applies to the mass transition specific parameters The values should be regarded as starting points for optimisation The optima vary between different MS MS systems and therefore should be optimised for the system to be used compound optimisation see section 5 3 1 For analyte internal standard assignments see the table below Table 4 Analyte internal standard assignments and corresponding LC retention times RT Analyte RT min Internal Standard IS order no MS1212 RT min Cyclosporine A 1 45 Cyclosporine D 1 50 Tacrolimus 1 26 Ascomycine 1 25 Sirolimus 1 34 d Everolimus 1 35 Everolimus 1 35 d Everolimus 1 35 D Immunosuppressants in Whole Blood Page 13 4 4 2 1 Agilent The following parameters refer to the MS MS system Agilent 6430 6410 with Hotbox and Agilent 6460 Table 5 MS MS parameters Agilent 6430 6410 w Hotbox Agilent 6460 lon Source ESI positive ESI Agilent Jet Stream positive Resolution MS1 and MS2 unit 0 7 amu unit 0 7 amu Gas Temperature 275 C 225 Gas Flow 9 9 min Nebulizer 35 psi 35 psi Sheath Gas Temperature 325 Sheath Gas Flow 12 l min Capillary Voltage 4000 V 4000 V2. Vial septum contaminated Mobile phase contaminated Use another septum Change the mobile phases and flush the system Column s guard analytical column contaminated Change the guard analytical column Mass resolution too low Optimise mass resolution System not correctly configured Check all connections No signals Injector defect Check injector Defective HPLC pump Check the pumps MS MS system not ready for operation Check the MS MS system D 28 Immunosuppressants in Whole Blood Problem Decrease of sensitivity Possible cause lon source contaminated Corrective measure Clean the ion source Mass spectrometer contaminated Clean the mass spectrometer Leakage of injection valve Check the injector Shift of mass calibration Recalibrate MS MS system MRM transitions not optimal Optimise MRM transitions lonisation conditions not optimal Optimise the parameters of the ion source Mass resolution too high Optimise the mass resolution High fluctuations of signals Spray instable Check the spray needle capillary and clean or exchange if necessary Fluctuation of the flow rate Gas flow rate instable Check the HPLC pumps Check the gas pipes No vacuum Defective vacuum pumps Check the pre and high vacuum pumps Leakage within the vacuum Check the vacuum
3. Analyte Retention Time min i Ascomycine 1 25 58e Tacrolimus 1 26 i Sirolimus 1 34 d Everolimus 1 35 4804 Everolimus 1 35 Mh Cyclosporine D 1 50 40e4 3 8e4 3 6e4 3 404 Intensity 32e4 3 0e4 28e4 2 604 24 4 2224 20 4 18 4 A 1 664 10e4 8000 0 6000 0 bus 20000 LY a 00 0 1 0 2 03 0 4 05 0 6 07 08 0 9 1 0 14 12 13 14 15 16 17 18 19 20 43 25 37 62 74 86 98 110 122 134 148 158 171 123 195 207 218 231 243 Figure 4 Chromatogram of the ClinCal Whole Blood Calibrator level 4 D Immunosuppressants in Whole Blood Page 23 6 Evaluation The analyte detection is achieved using compound specific mass transitions see section 4 4 2 The evaluation of the analyte concentration is performed with the internal standard method using the peak areas Calibration curves are achieved for the calibrators by plotting the ratio analyte peak area internal standard peak area against the ratio analyte concentration internal standard concentration The analyte concentrations for samples and controls are calculated from the calibration curve Please consult the software user manual of the MS MS manufacturer in order to ensure correct evaluation of the results For the calculation of mass concentrations ug l into molar concentrations and vice versa the analytical results have to be multi
4. _ 5013 Whole Blood Calibrator lyophil Wi Store at 2 8 C Whole Blood Calibrator additional 8 9028 level for order nos 9033 and 9933 T Store at 2 8 lyophil Whole Blood Calibrator Set Level 0 3 9033 7 Store at 2 8 lyophil 2c Whole Blood Calibrator Set Level 0 6 9933 Store at 2 8 lyophil 2v MS1030 Analytical Column A Store at 15 30 C 30 C MS1031 SPE Column 4 Store at 15 30 C MS1032 ac Guard Column 4 Store at 15 30 MS1033 7400 Inline Filter Store at ambient temperature Switching valve Sykam 6 port FK1102 Uae ont Store at ambient temperature 3 channel FK7330 Endfittings Store at ambient temperature FK7340 Sealings and sieves Store at ambient temperature FK7350 Endstoppers Store at ambient temperature Whole Blood Controls Level III 8 8830 8833 Store 2 8 lyophil 2 C Whole Blood Controls Level IV V 8 i 8903 Store at 2 8 lyophil 2 Refers to the lyophilised product For storage conditions after reconstitution please refer to the product data sheet 2 1 3 Disposal of laboratory waste For disposal laboratory waste should be collected separately with regard to its different chemical properties Recommendations for the disposal of the product and of the packaging are given in section 13 of the appropriate Material Safety Data Sheet MSDS D WAG VARA Immunosuppressants in Whole
5. 2 The use of liquid chromatography with tandem mass spectrometry LC MS MS allows a highly selective quantification of the main drug independently from its metabolites see section 1 3 D Immunosuppressants in Whole Blood Page 3 1 3 General description of the analytical procedure In this analytical method Cyclosporine A Tacrolimus Sirolimus and Everolimus are determined from human whole blood by on line SPE HPLC with electrospray tandem mass spectrometry Prior to the on line analysis a short sample clean up is performed in order to remove the sample matrix and to spike with the internal standards sample pretreatment see section 5 2 Conventional HPLC methods require manual sample preparation performed prior to the sample injection With on line HPLC methods the sample preparation is performed within the LC MS MS configuration by column switching using a multiple port valve and a SPE column on line sample preparation After chromatographic separation on the second the analytical column within the HPLC system the analytes are ionised by electrospray ionisation ESI and detected by the tandem mass spectrometer MS MS In electrospray ionization the sample components are ionized and then transferred to the gas phase where they subsequently pass into the MS MS which is composed of two quadrupoles connected through a collision cell In the present analytical method the MS MS measurement of the analyte
6. are used on the product labels and in this user manual For in vitro diagnostic use Order number Manufacturer Lot number Upper temperature limit C Temperature limits C to C Expiry date See instructions for use 5 Ed gt 1 2 Clinical background Cyclosporine A Tacrolimus Sirolimus and Everolimus see figure 1 are immunosuppressive drugs used after organ transplantation The goal of the therapy is to prevent an acute allograft rejection by inhibition of the immunological defence of the recipient with as far as possible minimal effect on the immunological resistance towards infections 1 3 Immunosuppressive drugs function through various mechanisms Cyclosporine A and Tacrolimus are calcineurin inhibitors and block the interleukin 2 production leading to a decrease in T lymphocyte proliferation Sirolimus and Everolimus act at a later stage than the calicineurin inhibitors by inhibiting the interleukin 2 stimulated cell cycle progression 4 Due to the complementary mechanisms of action these two classes of agents are often combined in patient treatment whereby taking advantage of the synergistic effects 5 D 2 Immunosuppressants in Whole Blood Cyclosporine A Tacrolimus OMe CH CH 3 Sirolimus Everolimus Figure 1 Structural formulas of Cyclosporine A Tacrolimus Sirolimus and Everolimus The administration of immuno
7. 35 177 16 7 Quantifier CyclosporineD 1234 1198 8 35 177 28 7 Qualifier Tacrolimus u 821 6 768 4 35 140 17 7 Quantifier Tacrolimus A 821 6 786 4 35 140 12 7 Qualifier 809 5 756 4 35 154 16 7 me 809 5 774 4 35 154 12 7 Qualifier Sirolimus n 931 7 864 5 35 160 13 7 Quantifier Sirolimus 931 7 846 5 35 160 21 7 Qualifier Everolimus 975 6 908 6 35 190 13 7 Everolimus e 975 6 858 5 35 190 24 7 Qualifier d Everolimus 979 6 912 5 35 154 12 7 d Everolimus e 979 6 894 5 35 154 20 7 Qualifier DAE RA Immunosuppressants in Whole Blood Page 15 4 4 2 2 Applied Biosystems The following parameters refer to the MS MS systems API 3000 and API 4000 Table 8 MS MS parameters 3000 API 4000 lon Source Turbolonspray ESI positive Turbolonspray ESI positive Resolution Q1 and Q3 unit 0 7 amu unit 0 7 amu Nebuliser Gas GS 1 14 65 652 60 Curtain Gas 14 30 Collision Gas 10 6 lon Spray Voltage 5000 V 5000 V Source Temperature 500 C 350 Interface Heater Mass transitions see table 9 see table 10 Table 9 Mass transitions 3000 Substance Precursor Product Dwell time DP FP CE amu amu ms V V V V V CyclosporineA 4154199 12032 35 41 260 23 54 1
8. Blood Page 7 3 Required instruments Using this test kit requires a LC system with tandem mass spectrometer LC MS MS and evaluation software Requirements for the tandem mass spectrometer The tandem mass spectrometer should be of comparable or higher sensitivity as the instruments described in section 4 4 2 Required LC modules e Autosampler with cooling function 4 C e socratic HPLC pump 1 SPE buffer e lsocratic HPLC pump 2 mobile phase e 6 port 3 channel switching valve e g RECIPE order no FK1102 e Column heater 60 C Degasser optional For sample pretreatment the following laboratory instruments are required e Pipettes pipette tips e Tabletop centrifuge e Vortex mixer D 8 Immunosuppressants in Whole Blood 4 Operation of the analytical system 41 Configuration of the LC system Connect the LC modules P1 P2 AS and the automatic switching valve ASV as shown in the figure below with exception of the columns SPE GC AC Put the outlet capillaries from ASV to waste into a safe waste container waste Abbreviations P1 HPLC pump 1 P2 HPLC pump 2 AS autosampler PF inline filter order no FK7400 ASV automatic switching valve high pressure switching valve 6 port 3 channel order no FK1102 SPE SPE column GC guard column AC analytical column CH column heater Figure 2 Configuration of the LC system 4 1 1 Reagent MP For operat
9. Nozzle Voltage 300 V Mass Transitions see table 6 see table 7 Table 6 Mass transitions Agilent 6430 6410 with Hotbox Substance Precursor Product Dwelltime Fragmentor CE Cell Accelerator amu amu ms V V Voltage V Cyclosporine A 1219 9 1202 9 35 200 13 7 Quantifier Cyclosporine A 1219 9 1184 9 35 200 33 7 Qualifier Cyclosporine D 1233 8 1216 6 35 220 16 7 Cyclosporine 0 x 1233 8 1198 8 35 220 28 7 Qualifier Tacrolimus an 821 5 768 4 35 110 17 7 Quantifier Tacrolimus PN 821 4 786 4 35 110 12 7 Qualifier Ascomycine 809 6 756 6 35 120 15 7 Ascomycine 809 6 774 4 35 120 12 7 Qualifier Sirolimus 931 5 864 4 35 160 13 7 Quantifier D 14 Immunosuppressants in Whole Blood Sirolimus m 931 5 846 5 35 160 21 7 Qualifier Everolimus u 975 5 908 4 35 180 13 7 Quantifier Everolimus m 975 5 858 5 35 180 24 7 Qualifier d Everolimus 5i 979 6 912 5 35 154 12 7 Quantifier d Everolimus 979 6 894 5 35 154 20 7 Qualifier Table 7 Mass transitions Agilent 6460 Substance Precursor Product Dwelltime Fragmentor CE Cell Accelerator amu amu ms V V Voltage V Cyclosporine A M 1219 9 1202 9 35 200 13 7 Quantifier Cyclosporine A n 1219 9 1184 9 35 200 33 7 Qualifier CyclosporineD 1234 1216 8
10. labels and table 1 Unused components stored under appropriate conditions can be used until the expiry date given on the product label After use of ClinMass Reagents and ClinMass Mobile Phases the bottles must be closed tightly and stored immediately under the required conditions Provided proper use and storage procedures are followed the lifetime of the reagents is the same as for the unused products For storage conditions and life times of ClinMass Internal Standard and Optimisation Mixes as well as for ClinCal Calibrators and ClinChek Controls lyophilised after reconstitution please also refer to the appropriate product data sheets Table 1 Storage conditions of kit components Order no Product description Storage conditions utosampler Washing Solution Store at 15 30 MS1005 A pler Washing Sol eagent E Store at 15 30 C MS1006 Reagent MP Buffer di Store at 15 30 C MS1009 SPE Buff MS1010 Mobile Phase Y dd Store at 15 30 C MS1014 Optimisation Mix 1 lyophil W Store below 18 C MS1015 Optimisation Mix 2 lyophil I ore below 18C MS1020 Sample Pretreatment Vials Store at ambient temperature MS1021 P Precipitant Store at 15 30 C D 6 Immunosuppressants in Whole Blood MS1212 IS Internal Standard lyophil W store below 18 C 8 C
11. tubes and system fittings No gas supply Defective of nitrogen Check the nitrogen generator generator Defective compressor Check the compressor Gas bottle is empty Replace the gas bottle Inlet gas pressures are not within the specified range Regulate the inlet gas pressures D WAG VARA Immunosuppressants in Whole Blood Page 29 10 Appendix EC Declaration of Conformity Declaration of Conformity for in vitro diagnostic medical devices acc to article 9 1 of the directive 98 79 EC The company RECIPE Chemicals Instruments GmbH Dessauerstraf e 3 80992 Munich Germany declares that the CE labelled product ClinMass Complete Kit for Immunosuppressants order no MS1000 meets all applicable provisions of the directive on in vitro diagnostic medical devices 98 79 EC The conformity assessment was performed according to annex III The technical documentation is held according to annex III no 3 Munich 15 03 2013 Z Alfred Bauer General Manager w h CHEMICALS INSTRUMENTS GmbH DessauerstraBe 3 D 80992 M nchen Tel 49 89 54 70 81 0 Fax 11 info Zertifiziert nach Certified acc to ISO 9001 ISO 13485 www recipe de VAL VAAN AL
12. 0 Quantifier Cyclosporine A 1219 9 1184 6 35 41 310 45 30 10 Qualifier CyclosporineD 45339 12172 35 46 300 49 30 10 Quantifier Tacrolimus 821 6 768 4 35 51 280 29 20 10 Quantifier Ascomycine 809 5 756 6 35 51 330 29 18 10 Quantifier Sirolimus 931 7 864 4 40 46 290 23 22 10 Everolimus 975 6 908 4 40 46 260 23 24 10 Quantifier d Everolimus 979 6 912 6 40 46 280 25 24 10 Quantifier D DAE A RA 16 Immunosuppressants in Whole Blood Table 10 Mass transitions 4000 Substance Precursor Product Dwell time DP CE CXP EP amu amu ms IV IV Cyclosporine A 1219 7 1202 8 25 66 27 42 10 Quantifier Cyclosporine A 1219 7 1184 8 25 66 49 40 10 Qualifier Cyclosporine D 1233 8 1216 9 25 66 37 22 10 Quantifier Cyclosporine D 1233 8 1198 7 25 66 49 22 10 Qualifier Tacrolimus 821 5 768 4 25 61 31 26 10 Quantifier Tacrolimus 821 5 786 3 25 61 25 26 10 Qualifier Ascomycine 809 4 756 3 25 66 39 26 10 Quantifier Ascomycine 809 4 774 4 25 61 25 26 10 Qualifier Sirolimus 931 5 864 5 30 66 25 16 10 Quantifier Sirolimus 931 5 882 5 30 66 19 30 10 Qualifier Everolimus 975 6 908 4 30 66 33 16 10 Quantifier Everolimus 975 6 926 7 30 66 20 16 10 Qualifier d Evero
13. 3 analytical series each by 6 fold determination n 18 n number of values per sample The following coefficients of variation CV were obtained mean values Cyclosporine A Tacrolimus Sirolimus Everolimus CV 96 CV 96 CV 96 Sample 1 3 98 6 57 6 01 6 95 Sample 2 1 51 3 40 4 49 3 59 Sample 3 1 47 4 34 3 84 4 37 D Immunosuppressants in Whole Blood Page 25 7 1 3 2 Interassay 7 2 For the determination of the interassay precision the samples were measured in 8 analytical series each by 2 fold determination n 16 n number of values per sample The following coefficients of variation CV were obtained Cyclosporine A Tacrolimus Sirolimus Everolimus CV 96 CV 96 Sample 1 8 88 4 28 5 65 4 48 Sample 2 7 82 4 78 4 00 3 85 Sample 3 6 26 1 63 3 54 3 83 Reference ranges The therapeutical ranges depend on several factors such as the type of transplantation time after graft and co medication with other immunosuppressive agents For this reason general therapeutical ranges cannot be given but must be established individually for each patient 26 Immunosuppressants in Whole Blood 8 References 1 Thomas Ed Labor und Diagnose Indikation und Bewertung von Laborbefunden 7 Auflage TH Books Verlagsgesellschaft Frankfurt Main 2008 p 1214 1225 2 K M Re
14. 31 Whole Blood Control lyophil Level 8832 Whole Blood Control lyophil Level III 8833 Whole Blood Control lyophil Level I Il 8903 Whole Blood Control lyophil Level IV V Quantity 1 pce 1 x MS1005 2 x MS1009 1 x MS1010 3 x MS1212 4 x 51020 1 x MS1021 1000 ml 8ml 1000 ml 800 ml 2ml 3 ml 100 pcs 80 ml 3 ml 5x2ml 2x2ml 4x1x2ml 7x1x2ml 1 pce 1 pce 1 pce 5 pcs 1 pce 1 pce 2 pcs 4 pcs each 2 pcs 5x2ml 5x2ml 5x2ml 3x2x2ml 2x2x2ml Immunosuppressants in Whole Blood 5 2 1 1 Safety information Several of the kit components e g mobile phases and reagents are chemical preparations and thus may contain hazardous substances For safety information please consult the appropriate Material Safety Data Sheet MSDS for each component The calibrator and control materials are prepared from human whole blood Although the products are tested for the absence of common infection markers they should still be considered as potentially infectious For this reason we recommend the product to be handled with the same precautions as patient samples Detailed safety information is given in the appropriate Material Safety Data Sheet MSDS 2 1 2 Storage conditions and lifetime of kit components Please unpack the kit components from the transport packaging immediately upon receipt and follow the instructions for the storage conditions given on the product
15. PROCEDURE 5 1 Collection and storage of whole blood samples 5 2 Sample pretreatment 5 2 1 Reconstitution of the lyophilised whole blood calibrators controls 5 2 2 Work flow 5 2 2 1 Precipitation 5 2 2 2 LC MS MS analysis 5 2 2 3 Stability of the pretreated samples 5 3 LC MS MS analysis 5 3 1 Compound optimisation MS MS 5 3 2 Equilibration of the analytical system and test run 5 3 3 Calibration run 5 3 4 Accuracy control 5 3 5 Example chromatogram C N ounn A w 11 12 13 15 17 18 18 19 19 19 19 19 20 20 20 20 20 21 21 21 22 Contents 6 EVALUATION 23 7 TEST DATA 24 7 1 Test performance 24 7 1 1 Linearity quantitation limit detection limit 24 7 1 2 Recovery 24 7 1 3 Precision 24 7 1 3 1 Intraassay 24 7 1 3 2 Interassay 25 7 2 Reference ranges 25 8 REFERENCES 26 9 TROUBLESHOOTING 27 10 APPENDIX EC DECLARATION OF CONFORMITY 29 D Immunosuppressants in Whole Blood Page 1 1 Introduction 1 1 Intended use This ClinMass Complete Kit is intended for the determination of Cyclosporine A Tacrolimus Sirolimus and Everolimus from human whole blood with LC MS MS The kit components have to be used in accordance with this user manual The kit is not designed for combination with components by other manufacturers 1 1 1 IVD symbols Symbols according to EU directive 98 79 EC for in vitro diagnostic medical devices IVDD which
16. ble in five different concentrations see section 5 3 4 D 4 Immunosuppressants in Whole Blood 2 Components of the complete kit and accessories 2 1 Ordering information Order No Description 51000 ClinMass Complete Kit for Immunosuppressants in Whole Blood for 400 assays Contents Autosampler Washing Solution SPE Buffer Mobile Phase IS Internal Standard lyophil Sample Pretreatment Vials P Precipitant Manual Separately available components MS1005 Autosampler Washing Solution MS1006 Reagent MP 51009 SPE Buffer MS1010 Mobile Phase MS1014 Optimisation Mix 1 lyophil MS1015 Optimisation Mix 2 lyophil 51020 Sample Pretreatment Vials MS1021 P Precipitant MS1212 IS Internal Standard lyophil 5013 Whole Blood Calibrator lyophil single point 9028 Whole Blood Calibrator lyophil additional level for order nos 9033 and 9933 9033 Whole Blood Calibrator Set lyophil Level 0 3 9933 Whole Blood Calibrator Set lyophil Level 0 6 Start Accessories MS1030 Analytical Column with test chromatogram MS1031 SPE Column MS1032 Guard Column Holder incl 1 Guard Column MS1033 Guard Column FK7400 Inline Filter stainless steel sieve free of dead volume Accessories FK1102 Switching valve Sykam 6 port 3 channel incl electr drive PEEK FK7330 Endfittings FK7340 Sealings and sieves FK7350 Endstoppers ClinChek Controls 8830 Whole Blood Control lyophil Level 88
17. e f 1 j TIT 1 5 n 288 15 ge N P 4 4 PN j 4 1 Instruction Manual ClinMass LC MS MS Complete Kit Immunosuppressants in Whole Blood On Line Analysis 51000 For in vitro diagnostic use C IVDD 98 79 EC RECIPE D D DAG VARA RECIPE Chemicals Instruments GmbH Dessauerstrafse 3 80992 Munich Germany Phone 49 89 54 70 81 0 Fax 449 89 54 7081 11 e mail info recipe de MS1000 IVD Forin vitro diagnostic use Document version 3 0 Date of revision 15 03 2013 File name MS1000_m_e doc D D DAG VARA Contents 1 INTRODUCTION 1 1 Intended use 1 1 1 IVD symbols 1 2 Clinical background 1 3 General description of the analytical procedure COMPONENTS OF THE COMPLETE KIT AND ACCESSORIES 2 1 Ordering information 2 1 1 Safety information 2 1 2 Storage conditions and lifetime of kit components 2 1 3 Disposal of laboratory waste REQUIRED INSTRUMENTS OPERATION OF THE ANALYTICAL SYSTEM 4 1 Configuration of the LC system 4 1 1 Reagent MP 4 2 Flushing of the LC system 4 3 Equilibration of the LC system 4 4 Starting the analytical system 4 4 1 LC parameters 4 4 1 1 Automatic switching valve on line analysis 4 4 2 MS MS parameters 4 4 2 1 Agilent 4 4 2 2 Applied Biosystems 4 4 2 3 Thermo 4 4 2 4 Shimadzu 4 5 Standby mode IMPLEMENTATION OF THE ANALYTICAL
18. e and d Everolimus Optimisation Mix 1 and 2 are lyophilised and thus have to be reconstituted before use Information regarding the reconstitution is given in the appropriate product data sheets If necessary Optimisation Mix 1 and 2 should be diluted with mobile phase according to the sensitivity of the MS MS system in use The compound optimisation procedure for the MS MS system in use should then be followed in order to optimise the ionisation source parameters and the compound specific mass transition parameters D Immunosuppressants in Whole Blood Page 21 5 3 2 Equilibration of the analytical system and test run Equilibrate the entire analytical system for at least 30 min before injecting samples In order to confirm the performance of the analytical system repeatedly inject a mixture of the Optimisation Mix 1 and 2 see preparation below until two consecutive chromatograms comparable in retention times and peak areas are obtained The mixture is prepared from e 50 Optimisation Mix 1 order no MS1014 e 100 ul Optimisation Mix 2 order no MS1015 e 850 ul Mobile Phase order no MS1010 A further dilution of the mixture with mobile phase may be required depending on the sensitivity of the MS MS system in use 5 3 3 Calibration run For calibration a ClinCal 4 Level Whole Blood Calibrator Set level O 3 order no 9033 and a ClinCal 7 Level Whole Blood Calibrator Set level O 6 order no 9933 a
19. eds 10 bar at a flow rate of 2 5 ml min or if that of the guard column exceeds 10 bar at a flow rate of 0 5 ml min 60 C Autosampler Set the autosampler cooling function to 4 C The autosampler washing solution order no MS1005 is available for the flushing of the injection system Please also refer to the user manual of the autosampler manufacturer Injection volume 50 ul Injection interval 2 min Automatic switching valve See section 4 4 1 1 Immunosuppressants in Whole Blood Page 11 4 4 1 1 Automatic switching valve on line analysis The SPE sample clean up is performed on line by use of a 6 port 3 channel automatic switching valve A description of the working principle is given in figure 3 ASV position as ASV position inject PF CH CH exhaust exhaust Abbreviations P1 HPLC pump 1 P2 HPLC pump 2 AS autosampler PF1 inline filter order no FK7400 ASV automatic switching valve high pressure switching valve 6 port 3 channel order no FK1102 SPE SPE column GC guard column AC analytical column CH column heater MS tandem mass spectrometer ASV position load switching interval 0 0 50 min The sample is injected into the LC system and is transferred to the SPE column which extracts the analytes selectively from the sample matrix The matrix components are washed through the SPE column and
20. he samples may be stored for up to 7 days If the samples shall be stored for a longer period of time the samples must be stored at temperatures below 18 C multiple freeze thaw cycles should be avoided 5 2 Sample pretreatment 5 2 1 Reconstitution of the lyophilised whole blood calibrators controls ClinCal Whole Blood Calibrators and ClinChek Whole Blood Controls see section 2 1 lyophilised and thus must be reconstituted before use Information regarding reconstitution along with analyte concentrations and information about storage and stability is given in the appropriate product data sheets 5 2 2 Work flow Sample pretreatment Precipitation 200 ul 20 ul 100 ul P Precipitant IS Internal Standard whole blood calibrator control patient mix for 30 sec vortex mixer incubate 5 min room temp mix for 10 sec vortex mixer centrifuge 5 min 10000 x g LC MS MS analysis inject 50 ul of the supernatant 20 Immunosuppressants in Whole Blood 5 2 2 1 Precipitation Pipette 200 ul Precipitant P 20 ul Internal Standard IS and 100 ul of the whole blood sample calibrator control patient into a sample pretreatment vial Mix for 30 sec on a vortex mixer and afterwards incubate for 5 min at room temperature 15 30 C After this mix for 10 sec again vortex mixer and centrifuge for 5 min with a rotation speed of 10000 x g N b P
21. ion with an Agilent MS MS system Reagent MP order no MS1006 must be added to the mobile phase Add the whole amount of Reagent MP 8 ml to a freshly opened bottle of mobile phase D Immunosuppressants in Whole Blood Page 9 42 Flushing of the LC system Set both HPLC pumps P1 P2 at a flow rate of 1 ml min and flush the LC system with 10 ml SPE buffer and mobile phase respectively Afterwards connect the SPE column SPE the guard column GC and the analytical column AC as shown in figure 2 The guard column and the analytical column have to be installed within the column heater CH When connecting the SPE column and the analytical column please take care that the flow direction follows the arrow marking on the columns 4 3 Equilibration of the LC system After flushing the system see section 4 2 the equilibration is performed as follows e Set both HPLC pumps P1 P2 to a flow rate of 0 5 ml min set the column heater to 60 C and allow approximately 10 ml SPE buffer and mobile phase respectively to flow through the columns e After this stop the HPLC pumps and connect the outlet capillary of the analytical column AC with the tandem mass spectrometer 4 4 Starting the analytical system The following sections provide the parameters for the LC system see section 4 4 1 and the tandem mass spectrometer see section 4 4 2 For optimisation equilibration and testing as well as for calibratio
22. limus 979 6 912 5 30 61 37 16 10 Quantifier d Everolimus 979 6 930 5 30 66 21 16 10 Qualifier D Immunosuppressants in Whole Blood Page 17 4 4 2 3 Thermo The following parameters refer to the MS MS system TSQ Quantum Ultra Table 11 MS MS parameters TSQ Quantum Ultra lon Source H ESI II positive Resolution Q1 and Q3 0 7 amu Spray Voltage 3000 V Vaporiser Temperature 300 C Sheath Gas Pressure 40 lon Sweep Gas Pressure 2 0 Aux Gas Pressure 15 Capillary Temperature 200 C Skimmer Offset bV Collision Pressure Probe Position 1 5 mTorr argon C Mass Transitions see table 12 Table 12 Mass transitions TSQ Quantum Ultra Substance Precursor Product Dwell time Collision Tube Lens amu amu ms Energy Offset V Cyclosporine A 1220 0 1203 3 50 17 190 Cyclosporine D 1234 0 1217 0 50 17 190 Tacrolimus 821 6 768 4 50 18 190 Ascomycine 809 5 756 6 50 18 190 Sirolimus 931 7 864 6 75 15 190 Everolimus 975 7 908 8 75 16 190 d Everolimus 979 7 912 6 75 16 190 D DAE aA RA 18 Immunosuppressants in Whole Blood 4 4 2 4 Shimadzu 4 5 The following parameters refer to the MS MS system LCMS 8030 Table 13 MS MS parameters LCMS 8030 lon Source ESI positive Resolution Q1 and Q3 0 7 amu Interface Voltage 2500 V Conversion D
23. n of the LC MS MS system please refer to section 5 3 Please consult the user manual of the tandem mass spectrometer to ensure appropriate usage User trainings provided by the instrument manufacturer may also be advisable Immunosuppressants in Whole Blood LC parameters Table 2 LC parameters Isocratic HPLC pump 1 SPE buffer Flow rates 0 1 ml min 2 5 ml min see section 4 4 1 1 table 3 Isocratic HPLC pump 2 mobile phase Flow rates 0 5 ml min 1 0 ml min see section 4 4 1 1 table 3 SPE buffer mobile phase Reagent MP Columns Column heater Make sure that the bottles are closed well to avoid alteration of the retention times through evaporation of components of the SPE buffer and mobile phase For operation with an Agilent MS MS system Reagent MP must be added to the mobile phase see section 4 1 1 The analytical column AC and the guard column GC are installed within the column heater 60 C At a flow rate of 0 5 ml min the backpressure of the analytical column should not exceed 80 bar At a flow rate of 2 5 ml min the backpressure of the SPE column should not exceed 150 bar For the complete HPLC system the backpressure should not exceed 250 bar The sealings and frits of the inline filter PF and the guard column should be replaced after every 500 injections These parts should also be replaced if the backpressure of the inline filter exce
24. ntsch Monitoring von Immunsuppressiva Therapeutische Umschau 2008 65 545 550 3 D B Kaufman R Shapiro M R Lucey W S Cherikh R T Bustami D B Dyke Immunosuppression practice and trends American Journal of Transplantation 2004 4 Suppl 9 38 53 4 Taylor CJ Watson J A Bradley Immunosuppressive agents in solid organ transplantation mechanism of action and therapeutic efficacy Critical Reviews in Oncology Hematology 2005 56 23 46 5 B Nashan Maximizing the clinical outcome with mTOR inhibitors in the renal transplant recipient defining the role of calcineurin inhibitors Transplant International 2004 17 279 285 6 Holt Therapeutic drug monitoring of immunosuppressive drugs in kidney transplantation Current Opinion in Nephrology and Hypertension 2002 11 657 663 D Immunosuppressants in Whole Blood Page 27 9 Troubleshooting Problem Possible cause Corrective measure Gradient profile cannot be generated Defective HPLC pump Air within the system Check the pumps Degas the mobile phases and flush and purge the HPLC system thoroughly Fluctuation of the flow rate Check the pumps Interference signals Injection system contaminated Sample vials contaminated e Rinse needle with methanol or inject 10 x mobile phase e Check flushport solvent level e Clean exchange needle seat assembly and or injection valve Use new vials
25. plied with the factors shown in table 15 Table 15 Conversion factors Analyte Molecular weight Conversion factor Conversion factor g mol gt ug l ug l gt umol l Cyclosporine 1202 63 1202 63 8 315 x 10 Tacrolimus 804 15 804 15 1 244 x 10 Sirolimus 914 17 914 17 1 094 x 10 Everolimus 958 24 958 24 1 044 x 10 D 24 Immunosuppressants in Whole Blood 7 Test data 7 1 Test performance 7 1 3 1 The results were obtained with the MS MS system Agilent 6460 Linearity quantitation limit detection limit Cyclosporine Tacrolimus Sirolimus Everolimus 45 1 0 011 0 004 0 073 0 126 LLOQ yg I 0 037 0 014 0 244 0 420 Linearity ug l 0 037 2000 0 014 80 0 244 80 0 420 80 LLOD Lower limit of detection S N 3 LLOQ Lower limit of quantitation 5 10 S Signal N Noise Recovery For Cyclosporine A Tacrolimus Sirolimus and Everolimus mean recovery rates between 90 100 were obtained Precision For the evaluation of the intra concentrations were used and interassay precision 3 samples with the following Cyclosporine A Tacrolimus Sirolimus Everolimus ug l ug l ug l ug l Sample 1 62 5 3 28 3 64 3 34 Sample 2 132 6 67 11 2 10 6 Sample 3 258 13 3 18 9 18 2 Intraassay For the determination of the intraassay precision the samples were measured in
26. re available For an extended calibration range with an additional high calibration point level 7 the whole blood calibrator with order no 9028 is optionally available After reconstitution see section 5 2 1 the calibrators must be pretreated as described for the patient samples see section 5 2 For each analytical series freshly prepared calibrators are required 5 3 4 Accuracy control For the quality control of the analytical measurements ClinChek Whole Blood Controls are available in five different concentrations level order no 8830 level Il order no 8831 level III order no 8832 level Ill order no 8833 as well as level IV V order no 8903 Please note The usage of control levels IV and V order no 8903 requires an extension of the calibration range with level 7 of the whole blood calibrator with order no 9028 see section 5 3 3 These controls are lyophilised and subsequently to reconstitution must be pretreated as described for the patient samples see section 5 2 For each analytical series freshly pretreated controls must be used In case of large analytical series we recommend to inject these controls additionally at the end of the series 22 Immunosuppressants in Whole Blood 5 3 5 Example chromatogram Example chromatogram of the ClinCal Whole Blood Calibrator order no 9933 level 4 recorded with the MS MS system API3000 5 524 5 824
27. recipitant and internal standard may be pre mixed mixing ratio 200420 in accordance to the actual daily amount required 220 of this mixture is then mixed with 100 ul whole blood sample Multipettes with some multipettes the setting of 220 ul may not be possible In these cases a volume between 200 240 ul can be alternatively set 5 2 2 2 LC MS MS analysis Transfer the centrifuge supernatant to a sample vial which is suitable for the autosampler in use brown glass vials are recommended Inject 50 ul of the supernatant into the LC MS MS system 5 2 2 3 Stability of the pretreated samples Stored in the dark at temperatures between 2 8 C pretreated samples are stable for at least 12 hours 5 3 LC MS MS analysis Independent from the analytical method the mass accuracy of the tandem mass spectrometer MS MS should be checked at regular intervals A mass calibration may be required For information regarding the check up of the MS MS system please refer to the documentation provided by the instrument manufacturer 5 3 1 Compound optimisation MS MS For the optimisation of the MS MS system parameters Optimisation Mix 1 and 2 order nos MS1014 and MS1015 are provided compound optimisation Optimisation Mix 1 contains a preparation of the analytes i e Cyclosporine A Tacrolimus Sirolimus and Everolimus Optimisation Mix 2 contains a preparation of the internal standards i e Cyclosporine D Ascomycin
28. s is performed in the MRM Multiple Reaction Monitoring mode In this mode only selected ions known as the precursor ions with a defined mass charge ratio m z are isolated in the first quadrupole and subsequently are transferred into the collision cell These ions are then fragmented by impact with an inert gas argon or nitrogen at selectively appropriate voltage settings Among the fragments generated known as the product ions only those with a defined m z ratio are isolated in the final quadrupole for subsequent detection Thus measurement in MRM mode ensures identification and quantification with high selectivity and sensitivity with the analyte identification based on highly characteristic mass transitions for the compound of interest ClinMass Optimisation Mixes are provided for the optimisation of the MS MS parameters see section 5 3 1 and for the test run of the analytical system see section 5 3 2 The calibration of the analytical system is performed by use of ClinCal Multi Level Calibrators see section 5 3 3 For this purpose a 4 level calibrator set level 0 3 order no 9033 as well as a 7 level calibrator set level 0 6 order no 9933 are available For an extended calibration range with an additional high calibration point level 7 the whole blood calibrator with order no 9028 is optionally available Quality control is performed by use of ClinChek Whole Blood Controls These controls availa
29. suppressants requires accurate therapeutic drug monitoring TDM within a narrow therapeutic concentration range Overdose of these drugs increases the risks of severe side effects whilst underdose can result in immunological rejection and organ loss or damage Both can significantly reduce the lifespan of the organ recipients The pharmacokinetics of immunosuppressants in general characteristically shows poor bioavailability and a large intra and inter individual variation Consequently the correlation between drug dosage and blood concentration is poor and results in a need to individualise the dose regimen for different recipients To avoid over or under administration dosage regimens are usually adjusted according to the whole blood concentration For the dose adjustment the trough concentrations termed as C 0 are often used i e the values which are measured before the next medication For Cyclosporine A the C 2 level blood level measured 2 hours after dose administration is preferred due to a better correlation with the pharmacological effect 2 6 For the therapeutic drug monitoring liquid chromatography LC based methods are considered the methodology of choice Immunological methods although widely used in clinical laboratories lack in analytical specificity Cross reactions between drug and drug metabolites can result in an overestimation of the measured drug with unacceptable biases in some clinical situations see e g
30. thus are eluted to the waste Meanwhile the analytical column is reequilibrated from the previous injection cycle ASV position inject switching interval 0 50 1 65 min The automatic switching valve switches to the inject position The extracted analytes are eluted from the SPE column with mobile phase under backflush mode They are transferred to the analytical column on which they are chromatographically separated ASV position load switching interval 1 65 2 min The automatic switching valve switches back to the load position Both columns SPE and analytical column are reequilibrated for the next injection Figure 3 Automatic switching valve and configuration of the LC system 12 Immunosuppressants in Whole Blood The switching times and positions of the automatic switching valve ASV and the flow rates of the HPLC pumps P1 and P2 are programmed according to the following table Table 3 Switching times and positions ASV flow rates of the HPLC pumps P1 P2 Time ASV Pump P1 Event Pump P2 Event min position flow rate SPE column flow rate analytical column ml min ml min 0 00 load 0 1 0 5 0 01 2 5 loading equilibration 0 50 inject 2 5 elution loading 0 51 0 1 separation 1 30 0 5 1 35 1 0 1 50 0 1 1 51 2 5 1 55 1 0 1 65 load equilibration 0 5 equilibration 1 99 2 5 2 00 0 1 0 5
31. ynode 10000 V Desolvation Line 220 C Heat Block 400 C Nebulizer Gas 3 L min Drying Gas 15 L min Mass Transitions see table 14 Table 14 Mass transitions LCMS 8030 Substance Precursor Product Dwell time Collision amu amu ms Energy Cyclosporine A 1219 9 1203 0 30 21 Cyclosporine D 1233 9 1216 9 30 20 Tacrolimus 821 5 768 6 30 22 Ascomycine 809 3 756 4 30 20 Sirolimus 931 6 864 7 30 18 Everolimus 975 8 908 7 30 19 d Everolimus 979 5 912 6 30 20 Standby mode When the analytical system is not in use the pumps have to be switched off The SPE buffer and the mobile phase can be left within the LC system The vacuum pumps of the tandem mass spectrometer MS MS system should be in permanent operation In order to protect the ion source and multiplier the MS MS system should be switched into the standby mode For a longer operation pause the SPE Column and the analytical column should be disconnected and stored tightly closed The LC system should then be flushed with a water methanol mixture 1 1 D Immunosuppressants in Whole Blood Page 19 5 Implementation of the analytical procedure 51 Collection and storage of whole blood samples The analysis is performed from EDTA whole blood If the determination is performed within the same day the samples may be stored at room temperature 15 30 C At temperatures between 2 8 C t
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