Immunosuppressants by LCMSMS. Grant Moore Toxicology

Transcription

1 Immunosuppressants by LCMSMS Grant Moore Toxicology CHLabs

2 Past experience of Immunosuppressant TDM Azathioprine metabolites been tested for >5 years Initially two HPLC methods, TGN and MMP Two different λ for detection Labour intensive Weekly runs of samples Converted to LCMSMS method 2-3 years ago

3 Past Experience of Immunosuppressant TDM Checked out LCMSMS systems in lab for sensitivity TGN is critical analyte for sensitivity considerations ABI 3200 system not sensitive enough ABI 4000 system was Single run now with all samples on one assay Ability to report MMP results s now without having to reanalyse Now looking at providing back-up on new system (Agilent 6460)

4 Background to Cyc/Tacr/Siro Currently performed by immunoassay Various immunoassays have been used for around the last 20 years All these methods are antibody based immunological methods Cross reactivity occurs due to antibody binding to metabolites Differing metabolite patterns for different individuals and different transplant types Desire to become less dependent d on kit suppliers! Cost savings

5 Background LC-MSMS methodologies have been available for a few years now Allows simultaneous analysis of a number of immunosuppressants ABI 3200 system evaluated 5 years ago Cyclosporin and Tacrolimus set up and validated Logistical issues occurred

6 Why change methodology? LC-MSMS provides specific mass analysis for the immunosuppressants Negligible cross reactivity to metabolites or other drugs/foreign species Heterophilic antibodies (rare cases) will not interfere as they can in immunoassays It is far more specific!

7 How do we sell the idea? Business case prepared Trade-off reagent kit cost against new technology Bundled up a number of analyses to make required savings Cost benefits to lab in the change Sirolimus for eg, $1600 for 100 test kit, ~25% patient results, ie about $65 per patient result LCMSMS analysis considerably less per patient result

8 Agilent 6460 LC-MSMS purchased

9 Changing g scenery!

10 LCMSMS Tender Requirements Tender brief was to provide solutions, not instruments! Wanted functioning methods to be supplied Didn t quite work out that way Ensure the hardware provided d can perform the supplied method, i.e pumps, switching valves! Know what the capabilities of the quoted system are

11 Immunosuppressant Methodology Involves a 2-D column set-up Agilent Zorbax Eclipse Plus-C18 used as a loading column Agilent Poroshell EC-C18 column used as the analytical column Two pump system employed Agilent 1290 pump is used as the loading pump with a solvent ramp Agilent 1260 pump is used as the analytical pump with a simple flow rate increase during the run

12 Methodology Sample preparation similar to immunoassay systems Internal standard/precipitation d/ it ti reagent prepared fresh daily Well mixed whole blood is aliquoted into microfuge tube Precipitation reagent added Vortexed Centrifuged rpm Supernatant decanted into autosampler vial 20 μl injected into LCMSMS

13 Methodology 2 minutes for loading column 2 minutes for analytical column Total run time less than 5 minutes per sample Samples batched and run as a single run in early afternoon Reported same day

14 Methodology Recipe Clincal standards, Recipe Clinmass internal standards and UTAK quality control material supplied by PM Separations used in the method Aliquots are stored frozen and thawed on the day of use

15 Pitfalls and speed bumps Cyclosporin D didn t work because metabolite of Cyclosporin A co-eluting with patients Cyclosporin D was set up in supplied method perhaps they hadn t tested on real samples! Timing around 2-D setup is crucial especially since supplied pump couldn t achieve required flow rate! Need switching valve for 2-D setup because not enough positions to leave it permanently plumbed. Need to buy suppliers version of switching valve to get it programmed into the method not many staff want to sit around all night hitting the manual switch!

16 Pitfalls and speed bumps Deuterated Tacrolimus gave non-linear standard curve and not consistent.no obvious reason why. Not a cross-talk effect or carryover effect.? Suppressing itself! Using sirolimus internal standard corrects problem Sirolimus assay straightforward set-up QC ranges can be challenging depending on what methodology they are designed for. Comparison with IA results not always helpful Extraction process straightforward, very similar to IA extraction method Needs to be prepared fresh or ZnSO4 falls out of solution.

17 Pitfalls and speed bumps Column life seems to be exceedingly good Using a Poroshell column from Agilent Will it be robust enough? So far so good Using an in-line frit filter and guard pre-column improves column longevity Frits replaced regularly and are reused after ultrasonic cleaning Pre-column and frit performance monitored by system pressure and peak shape Always cheaper to clean and replace these than analytical column

18 Ciclosporin comparison 1000 Ciclosporin LCMSMS vs mean of peers LCMS Mean Peers LCMS Linear (Mean Peers LCMS) y = x R² = Mean peers LCMS

19 Ciclosporin 1000 Ciclosporin LCMSMS vs Architect, Patient samples y = x R² = LCMS Cyc Architect Linear (Cyc Architect) Architect

20 Tacrolimus Tacrolimus LCMSMS vs mean of peers LCMS y = x R² = Mean peers

21 Tacrolimus 30 Tacrolimus LCMSMS vs Architect Patient samples y = x R² = LCMS Tacr LCMS Siro Int std Linear (Tacr LCMS Siro Int std) Linear (Tacr LCMS Siro Int std) Architect

22 Sirolimus Sirolimus LCMSMS vs mean of peers y = x R² = LCMS 8 6 Mean Peers LCMS Linear (Mean Peers LCMS) Mean peers LCMS

23 Sirolimus Sirolimus LCMSMS vs Architect Patient samples y = x R² = LCMS Siro Architect Linear (Siro Architect) Architect

24 Method statistics - Interday Ciclosporin QC1 QC3 QC4 Nominal Conc (ug/l) Mean Accuracy (%) C.V. (%) Sirolimus QC1 QC3 QC4 Nominal Conc (ug/l) Mean Accuracy y( (%) C.V. (%) Tacrolimus QC1 QC3 QC4 Nominal Conc (ug/l) Mean Accuracy (%) C.V. (%)

25 Reference ranges and Daily timing May need to be adjusted to fit the observed negative biases May be performed as a single batched run each day Meetings held with key stakeholders to ensure they are engaged Renal Specialists individualise reference range for their patients Communications sent out to referring laboratories and specialists Potential ti problem where patients t are tested t across different laboratory sites This still to be resolved!

26 Calculated Costings Description, incl. size, brand, etc. Unit of purchase, eg 250ml bottle or50/pk Price/Unit of purchase, eg $ per bottle or pack Qty used per test $ per Test Internal Std MS1312 Recipe 5mL $ $ 0.38 Immuno Calibrators 9933 Recipe 7 x 2mL $ $ 0.91 PP autosampler vial 100/pk $ $ 0.42 Autosamplervial cap 100/pk $ $ Microcentrifuge tube 1.5mL 1000/pk $ $ uL pipette tip 960/pk $ $ 0.05 Poroshell 120 EC-C18 column AGI $ 1, $ 0.29 Eclipse plus Guard C18 AGI /pk $ $ 0.06 Utak IMControl L UTAK 6mL $ $ 0.10 Utak IM Control L UTAK 6mL $ $ 0.10 Utak IM Control L UTAK 6mL $ $ 0.10 Methanol 2.5L $ $ 0.07 Zinc Sulfate Z g 100g $ $ 0.00 Formic Acid id89 91% 91% 1L $ $ Ammonium Formate 100g $ $ 0.01 Isopropanol 2.5L $ $ 0.03 Acetonitrile 4L $ $ 0.02 Total direct costs $ 2.93 Cluster Overhead 8% $0.23 General Admin Overhead 10% $0.29 TOTAL COST $ 3.46

27 Service Roll-out and Future directions Due to kick-off Monday! Watch this space Everolimus monitoring System already monitoring this as it s in the calibrator mix Mycophenolate monitoring Currently run total and unbound by HPLC

28 Acknowledgements Mark Lewis Berit Packart Jensen AACB CHLabs Questions?