A Rapid imethod TM Test for Analysis of Four Immunosuppressants

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1 imethod Test A Rapid imethod TM Test for Analysis of Four Immunosuppressants The following description outlines the instrument requirements and expected results obtainable from the Spark Holland imethod TM Test for the quantification of Cyclosporine A, Everolimus, Serolimus, and Tacrolimus when using a Spark Holland Symbiosis PICO integrated online SPE and HPLC system with a mistral column oven, an Applied Biosystems/ MDS Analytical Technologies 3200 series (API 3200 or 3200 QTRAP ) LC/MS/MS instrument and Chromsystems calibrators and controls. Sample preparation is based upon precipitation of whole blood followed by centrifugation and automated clean up of the supernatant using the on-line sample preparation capabilities of the Spark PICO system when using an SPE cartridge packed with Applied Biosystems POROS R1 sorbent. More in depth sample preparation, and instrument parameter information is included as part of the standard operating procedure provided with the method, as is the required analytical columns. Solvents, standards and any supplies required for sample preparation are not. The mobile phase consists of the use of methanol, ammonium acetate and acetic acid with separation on a Phenomenex Luna 5 u Phenyl-Hexyl 50 x 2.1 mm HPLC column. An example chromatogram of the separation achieved is shown below in figure 1. Figure 1:Chromatogram of the level 1 Chromsystems calibrator run on an API 3200 TM LC/MS/MS System

2 Results The following calibration curves are representative of the performance obtained on the instrument using the method described here, and may not be representative of performance on any other instrument. Cyclosporine A Everolimus Sirolimus Tacrolimus Table 1: Representative calibration curves for the four immunosuppressants included in the method are shown above. The concentration for each calibrator level is summarized in the table below. Analyte Analyte Concentration in Calibrator (ng/ml) Level Level 2 Level 3 Level 4 Level 5 Level 6 1 Cyclosporine A Tacrolimus Sirolimus Everolimus Table 2: Chromsystems Whole Blood Calibrator Concentrations (batch: Lot No. 2907)

3 A level 0, blank, containing zero concentration of each of the four analytes was also used. Concentrations of Calibrators will vary slightly from lot-to-lot. Analyte S/N* Level 1 Calibrator Cyclosporine A Everolimus Sirolimus Tacrolimus Table 3: Representative Signal to Noise Ratios * Signal to Noise (S/N) is the peak height divided by the noise measured at three standard deviations of the noise. Please note that the results presented above were obtained using a single instrument and single set of standards and samples. Prior to production use, the method should be fully validated with real samples, and the results here may not be typical for all instruments. Variations in LC column properties, chemicals, environment, instrument performance and sample preparation procedures will impact performance, thus these results should be considered as informative rather than representative. System Requirements In order to run this method as outlined above, the following equipment and reagents are required: An Applied Biosystems/ MDS Analytical Technologies 3200 Series LC/MS/MS System Spark Holland Symbiosis PICO Online SPE HPLC system with a Mistral column oven Immunosuppressant calibrators and controls ( Immunosuppressant internal standards ( LC/MS grade methanol, and ammonium acetate A Phenomenex Luna 5u Phenyl-Hexyl 50 x 2.1 mm HPLC column Spark SPE cartridges filled with Applied Biosystems POROS R1 sorbent Pipettes and standard laboratory glassware Please note that the Phenomenex HPLC is required but not included with this imethod Test.

4 Important Note The purchase and use of certain of the chemicals listed above may require the end user to possess any necessary licenses, permits or approvals, if such are required in accordance with local laws and regulations. It is the responsibility of the end user to purchase these chemicals from a licensed supplier, if required in accordance with local laws and regulations. The suppliers and part numbers listed below are for illustrative purposes only and may or may not meet the aforementioned local requirements. Applied Biosystems is not responsible for user s compliance with any statute or regulation, or for any permit or approval required for user to implement any imethod procedure. Legal Acknowledgements / Disclaimers The imethod Test described above has been designed by Applied Biosystems to provide the sample prep and instrument parameters required to accelerate the adoption of this method for routine testing. This method is provided for information purposes only. The performance of this method is not guaranteed due to many different potential variations, including instrument performance, tuning, and maintenance, chemical variability and procedures used, technical experience, sample matrices, and environmental conditions. It us up to the end user to make adjustments to this method to account for slight differences in equipment and/or materials from lab to lab as well as to determine and validate the performance of this method for a given instrument and sample type. Please note that a working knowledge of Analyst Software may be required to do so. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of either Life Technologies Corporation, MDS Inc., Applied Biosystems/MDS Analytical Technologies or otherwise, their respective owners Applied Biosystems LLC and MDS Inc. Joint Owners. All Rights Reserved. Publication no. 114AP104-01

5 Multidimensional SPE and on-line POPLC- MS/MS for the analysis of immunosuppressants Rosa Morello Karl-Siegfried Boos in whole blood Institute of Clinical Chemistry Medical Center of the University of Munich Munich, Germany Spark Holland Symbiosis User Group Meeting 18 th September 2009, Utrecht

6 2 Processing of Whole Blood Manual (off-line) / Robotic (at-line) Blood Spots LC-MS/MS

7 Processing of Whole Blood Manual (off-line) / Robotic (at-line) Blood Spots Dialysis Liquid-Liquid- Extraction Precipitation (Hemo)Lysis Membrane Filtration ± Anticoagulation Centrifugation Drying Dialysate Evaporation (Hemo)Lysate Plasma / Serum Filtrate Plasma / Serum Punching Precipitation Precipitation Precipitation Extraction Centrifugation Derivatization Supernatant Matrix-depleted (preprocessed) Samples 3 LC-MS/MS off-line at-line on-line SPE

8 On-line SPE of secondary blood specimens Processing of Whole Blood Manual (off-line) / Robotic (at-line) Blood Spots Dialysis Liquid-Liquid- Extraction Precipitation (Hemo)Lysis Membrane Filtration Anticoagulation Centrifugation Drying Dialysate Evaporation (Hemo) Lysate Plasma / Serum Filtrate Plasma / Serum Punching Precipitation Precipitation Precipitation Extraction Centrifugation Derivatization Supernatant Matrix-depleted (preprocessed) Samples Integrated 4 LC-MS/MS off-line at-line on-line SPE Matrix-containing (native) Samples

9 Processing of Whole Blood Manual (off-line) / Robotic (at-line) Blood Spots Dialysis Liquid-Liquid- Extraction Precipitation (Hemo)Lysis Membrane Filtration Anticoagulation Centrifugation Drying Dialysate Evaporation (Hemo)Lysate Plasma / Serum Filtrate Plasma / Serum Punching Precipitation Precipitation Precipitation Extraction Centrifugation Derivatization Supernatant Matrix-depleted (preprocessed) Samples Integrated 5 LC-MS/MS off-line at-line on-line SPE Matrix-containing (native) Samples

10 Processing of Whole Blood in-line Manual (off-line) / Robotic (at-line) Celldisintegrated Blood (CDB) Blood Spots Dialysis Liquid-Liquid- Extraction Precipitation (Hemo)Lysis Membrane Filtration Anticoagulation Centrifugation Drying Dialysate Evaporation (Hemo)Lysate Plasma / Serum Filtrate Plasma / Serum Punching Precipitation Precipitation Precipitation Extraction Centrifugation Derivatization Supernatant Matrix-depleted (preprocessed) Samples Integrated 6 LC-MS/MS off-line at-line on-line SPE Matrix-containing (native) Samples

11 Conversion of Whole Blood into Cell-Disintegrated Blood (CDB) CDB : Homogenous, red-coloured blood specimen containing the complete matrix but no cellular components which sediment In-line Processing : Whole blood Sedimented Whole blood CDB Heat shock-treatment of an anticoagulated whole blood sample (e.g. 25 µl) under defined conditions (heating time and temperature, e.g. 16 sec at 75 C) while being pumped through a stainless-steel capillary (e.g. 300 x 0.5 mm I.D.) 7

12 Target analytes : Immunosuppressants / Internal standards Sirolimus Tacrolimus Cyclosporine A Everolimus Ascomycin Cyclosporine D 8 Desmethoxysirolimus

13 Total Analysis System (TAS) for fully automated determination of pharmaceuticals Mixing and Injection Processing Fractionation Separation Detection Dataprocessing Direct Injection Heat-shock Solid Phase High performance of whole blood treatment Extraction liquid chromatography SPE HPLC MS/MS (Single cartridge mode) Whole blood 9 Cell disintegrated blood (CDB)

14 10 Goals for the on-line SPE LC MS/MS analysis of immunosuppressants in whole blood Isocratic elution 1 Analyte B Flow-rate 3 Organic modifier Gradient elution System-Peak(s) Analyte A Internal standard 5 Ionisation yield Ion suppression 2 Fractionation Transfer Separation Re-equilibration SPE (RAM) SPE LC Time

15 Total Analysis System (TAS) for fully automated determination of pharmaceuticals Mixing and Injection Processing Fractionation Separation Detection Dataprocessing Direct Injection Heat-shock Solid Phase High performance of whole blood treatment Extraction liquid chromatography SPE POPLC MS/MS (Single cartridge mode) Whole blood 11 Cell disintegrated blood (CDB)

16 POPLC Theory k total = Ф A k A + Ф B k B + Ф C k C + = ΣФ i k i k total = total retention factor for an analyte of interest k i Ф i = corresponding retention factors concerning the individual stationary phases = fraction of length of each column segment relative to the total column system ΣФ i = total column system = Ф A + Ф B + Ф c = 1 k total A x= x 12

17 POPLC Theory k total = Ф A k A + Ф B k B + Ф C k C + = ΣФ i k i k total = total retention factor for an analyte of interest k i Ф i = corresponding retention factors concerning the individual stationary phases = fraction of length of each column segment relative to the total column system ΣФ i = total column system = Ф A + Ф B + Ф c = 1 13

18 Determination of R t of target analytes : Operational procedure 1. Determination of R t for : 2. Different predicted chromatograms : ProntoSIL C18 SH 2 (red) % ProntoSIL C18 EPS (green) MRM of 7 Channels ES+ TIC 3.05e minutes too long MRM of 7 Channels ES+ TIC 2.75e5 % 1 % ProntoSIL C30 (yellow) MRM of 7 Channels ES+ TIC 3.26e5 3 minutes short, but no separation of Cyclosporine A and Cyclosporine D ProntoSIL CN 2 (blue) 1.84 MRM of 7 Channels ES+ TIC 4.95e5 % 1 % ProntoSIL Phenyl 2 (white) Time MRM of 7 Channels ES+ TIC 3.00e5 Compromise : 6 minutes base-line separation of Cyclosporine A and Cyclosporine D POPLC column : C18 (20 mm x 3 mm ID) + C30 (20 mm x 3 mm ID) + CN (30 mm x 3 mm ID) + Phenyl (10 mm x 3 mm ID), dp 5 µm

19 On-line SPE POPLC MS/MS : Ion Suppression Ascomycin / Tacrolimus 100 Original POPLC column Final POPLC column Sirolimus / Everolimus / Desmethoxysirolimus Cyclosporine A 9 Cyclosporine D % Infusion chromatogram Infusion : Solution of immunosuppressants and internal standards, each 10 ng/ml in MeOH / H 2 O (80/20), v/v) Injection : 25 µl CDB Transfer and In-line dilution Ion Suppression Sample : Solution of immunosuppressants and internal standards, (each 100 ng/ml), MeOH / H 2 O (80/20, v/v) Injection volume : 5 µl Fractionation SPE cartridge : Oasis HLB (10 x 2 mm ID), dp µm Mobile Phase : H 2 O / ACN (95/5, v/v) ; Flow-rate : 270 µl/min ; 1.85 min Transfer In-line dilution with water ; Flow-rate 100 μl/min ; 1.85 min Step-gradient : MeOH / 2mM NH 4 Ac (85/15, v/v) ; Flow-rate : 250 µl/min ; min Separation Original POPLC column : C18 (20 mm x 3 mm ID) + C30 (20 mm x 3 mm ID) + CN (30 mm x 3 mm ID) + Phenyl (10 mm x 3 mm ID), dp 5 µm Mobile phase : MeOH / 2 mm NH 4 Ac (80/20, v/v) ; Final POPLC column : C30 (200 mm x 3 mm ID) + CN (90 mm x 3 mm ID), dp 5 µm Step gradient : min ; MeOH / 2 mm NH 4 Ac (80/20, v/v) ; min ; MeOH / 2 mm NH 4 Ac (90/10, v/v) ; Flow-rate : 250 µl/min ; 5 Temperature : 60 C Detection Quattro Micro, Waters, USA Mode : ESI Time 2

20 16 In-line dilution during transfer step In-line dilution water (100 vol.%) ; min at 100 µl/min Symbiosis Pharma (Spark Holland) Peak compression on head of POPLC column Step gradient and flow-rate of mobile phase : Methanol / water (61/39, v/v) min at 350 µl/min SPE POPLC Methanol / water (57/43, v/v) min at 350 µl/min Elution of analytes from SPE-cartridge Step gradient and flow-rate of mobile phase : Methanol / water (85/15, v/v) min at 250 µl/min Methanol / water (80/20, v/v) min at 250 µl/min

21 Total Analysis System (TAS) for fully automated determination of pharmaceuticals Mixing and Injection Processing Fractionation Separation Detection Dataprocessing Direct Injection Heat-shock Solid Phase High performance of whole blood treatment Extraction liquid chromatography MD - SPE (Dual cartridge mode) POPLC MS/MS Whole blood 17 Cell disintegrated blood (CDB)

22 On-line MD SPE POPLC MS/MS platform : Dual cartridge mode Injection Processing Heated Capillary HPD 2 HPD 1 A MS/MS W W POPLC column B 2 C D W E 5 F HPLC Pumps Waste Internal T-piece Internal T-piece Waste 1. SPE-cartridge 2. SPE-cartridge 18

23 19 Elimination of ion suppression A) Early eluting (hydrophilic) matrix components Three-dimensional (3D) SPE : RP + SEC + hydrophobic IEX LiChrospher ADS RP4 (Dual cartridge mode) + Oasis MCX

24 On-line MD-SPE POPLC MS/MS platform: Dual cartridge mode % 9 8 Infusion chromatogram Infusion : Solution of immunosuppressants and internal standards, each 100 ng/ml in MeOH / H 2 O (80/20), v/v) Injection : 25 µl CDB Time 20 Sample : Solution of immunosuppressants and internal standards, (each 100 ng/ml), MeOH / H 2 O (80/20, v/v) Injection volume : 25 µl Fractionation 1. SPE cartridge : Oasis HLB (10 x 2 mm ID), dp µm Mobile Phase : H 2 O / ACN (95/5, v/v) ; Flow-rate : 270 µl/min ; 1.00 min, followed by H 2 O / ACN (95/5, v/v) ; Flow-rate : 3 ml/min ; 3.00 min Transfer (SPE SPE) MeOH ; Flow-rate : 450 µl/min ; 2.00 min, In-line dilution with 10mM NH 4 formiat, ph = 2,8 ; Flow-rate 2 ml/min ; 2.00 min 2. SPE cartridge : Oasis MCX (10 x 1 mm ID), dp µm Mobile Phase : MeOH / 10mM NH 4 formiat (18/82, v/v) ; Flow-rate : 2.45 ml/min ; 2.00 min Transfer (SPE AC) Mobile Phase : MeOH / 2mM NH 4 Ac (78/22, v/v) ; Flow-rate : 250 µl/min ; 2.00 min Separation Original POPLC column : C18 (20 mm x 3 mm ID) + C30 (20 mm x 3 mm ID) + CN (30 mm x 3 mm ID) + Phenyl (10 mm x 3 mm ID), dp 5 µm Mobile Phase : MeOH / 2mM NH 4 Ac (78/22, v/v) ; Flow-rate : 500 µl/min ; min Temperature : 60 C Detection Quattro Micro, Waters, USA Mode : ESI +

25 21 On-line MD-SPE POPLC MS/MS platform: Comparison of matrix effects SPE : Single cartridge mode (1D) MRM of 7 Channels ES+ TIC 6.31 e 6 MD SPE : Dual cartridge mode (3D) MRM of 7 Channels ES+ TIC 6.80 e 6 Asco Tacro Eve Sir Desm CyA CyD Asco Tacro Eve Sir CyA (Phospho-)lipids? Desm CyD HySphere C8 EC-SE HySphere C8 EC-SE / Oasis MCX

26 22 On-line MD SPE POPLC MS/MS platform : Detection of Phospholipids (m/z )

27 23 Goals for the On-line MD SPE POPLC MS/MS analysis of immunosuppressants in whole blood Isocratic elution 1 Analyte B Flow-rate 3 Organic modifier Gradient elution 9 System-Peak(s) Analyte A Internal standard 5 Ionisation yield Ion suppression 8 Ion suppression 10 2 Fractionation Transfer Separation Re-equilibration SPE (RAM) SPE LC Time

28 Total Analysis System (TAS) for fully automated determination of pharmaceuticals Mixing and Injection Processing Fractionation Separation Detection Dataprocessing Direct Injection Heat-shock Solid Phase High performance of whole blood treatment Extraction liquid chromatography MD - SPE (Triple cartridge mode) POPLC MS/MS Whole blood 24 Cell disintegrated blood (CDB)

29 25 Elimination of ion suppression A) Early eluting (hydrophilic) matrix components Three-dimensional (3D) SPE : RP + SEC + hydrophobic IEX LiChrospher ADS RP4 + Oasis MCX (Dual cartridge mode) B) Late eluting (hydrophobic) matrix components Four-dimensional (4D) SPE : RP + SEC + shape selectivity + hydrophobic IEX LiChrospher ADS RP4 + PLR Bischoff Chromatography + Oasis MCX (Triple cartridge mode)

30 26 Shape Selectivity

31 Shape selectivity : Retention of phospholipids Standard solution of immunosuppressants Phospholipids in CDB 27 Sample : Solution of immunosuppressants and internal standards, (each 100 ng/ml), MeOH / H 2 O (80/20, v/v), CDB (off-line) Injection volume : 25 µl Fractionation 1. SPE cartridge : HySphere C8 EC-SE (10 x 2 mm ID), dp 10 µm Mobile Phase : H 2 O / ACN (95/5, v/v) ; Flow-rate : 270 µl/min ; 1.00 min, followed by H 2 O / ACN (95/5, v/v) ; Flow-rate : 3 ml/min ; 3.00 min Transfer (SPE SPE) MeOH ; Flow-rate : 450 µl/min ; 2.00 min, In-line dilution with 10mM NH 4 formiat, ph = 2,8 ; Flow-rate 2 ml/min ; 2.00 min 2. SPE cartridge : PLR Bischoff Chromatography (20 x 3 mm ID), dp 5 µm Mobile Phase : MeOH / 10mM NH 4 formiat (18/82, v/v) ; Flow-rate : 2.45 ml/min ; 2.00 min Transfer (SPE AC) Mobile Phase : MeOH / 2mM NH 4 Ac (78/22, v/v) ; Flow-rate : 500 µl/min ; min Detection Quattro Micro, Waters, USA Mode : ESI + MRM of all immunosuppressants and internal standards ; MRM m/z

32 28 Depletion of phospholipids from CDB Fractionation (depletion) of phospholipids on PLR-cartridge by valve-switching (Front-cut) Efficacy : approx. 90 %

33 On-line MD SPE POPLC MS/MS platform: Triple cartridge mode 10 Infusion chromatogram Infusion : Solution of immunosuppressants and internal standards, each 100 ng/ml in MeOH / H 2 O (80/20), v/v) Injection : 25 µl CDB 9 29 Sample : Solution of immunosuppressants and internal standards, (each 100 ng/ml), MeOH / H 2 O (80/20, v/v) Injection volume : 25 µl Fractionation 1. SPE cartridge : LiChrospher ADS RP 4 (10 x 2 mm ID), dp 25 µm Mobile Phase : H 2 O / ACN (95/5, v/v) ; Flow-rate : 270 µl/min ; 1.00 min, followed by H 2 O / ACN (95/5, v/v) ; Flow-rate : 3 ml/min ; 3.00 min Transfer (1.SPE 2.SPE) MeOH ; Flow-rate : 450 µl/min ; 3.00 m In-line dilution with 10mM NH 4 formiat, ph = 2,8 ; Flow-rate 2 ml/min ; 3.00 min 2. SPE cartridge : PLR Bischoff Chromatography (10 x 2 mm ID), dp 25 µm Transfer (2.SPE 3.SPE) MeOH/10mM NH 4 formiat, ph = 2,8 (75/25, v/v) ; Flow-rate : 500 µl/min ; 3.00 min, In-line dilution with 10mM NH 4 formiat, ph = 2,8 ; Flow-rate 1.5 ml/min ; 3.00 min 3. SPE cartridge : Oasis MCX (10 x 1 mm ID), dp 30 µm Mobile Phase : MeOH / 10mM NH 4 formiat (18/82, v/v) ; Flow-rate : 2.0 ml/min ; 2.00 min Transfer (SPE AC) Mobile Phase : MeOH / 2mM NH 4 Ac (75/25, v/v) ; Flow-rate : 500 µl/min ; 2.00 min Separation Original POPLC column : C18 (30 mm x 3 mm ID) + C30 (20 mm x 3 mm ID) + CN (30 mm x 3 mm ID) + Phenyl (10 mm x 3 mm ID), dp 5 µm Mobile Phase : MeOH / 2mM NH 4 Ac (75/22, v/v) ; Flow-rate : 500 µl/min ; min Temperature : 60 C Detection Quattro Micro, Waters, USA Mode : ESI +

34 On-line MD SPE POPLC MS/MS platform : Triple cartridge mode Injection Processing Heated Capillary HPD 2 HPD 1 A MS/MS W W POPLC column B 2 C D W E 5 F HPLC pumps Waste Internal T-piece Internal T-piece 30 Waste 1. SPE-cartridge 2. SPE-cartridge

35 On-line MD SPE POPLC MS/MS platform : Triple cartridge mode Injection Processing Heated Capillary HPD 2 HPD 1 A MS/MS W W POPLC column B 2 C D W E 5 F HPLC pumps Waste Internal T-piece Internal T-piece 31 Waste 2. SPE-cartridge 3. SPE-cartridge

36 32 Dr. Jelena Milojković Melita Fleischmann Team from Spark Holland B.V. EUREKA PROJECT E!4112 An intergovernmental initiative supporting European innovation Waters Corporation Bischoff Chromatography

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