B-372 Development and Validation of a Quantitative Ultra-Performance Liquid Chromatography-Mass Spectrometry (LC-MS/MS) for Quantitative Determination of Tacrolimus, Sirolimus, Everolimus, and Cyclosporine A in Whole Blood

Abstract

Waters provides a comprehensive immunosuppressant kit, which includes the MassTrak™ Immunosuppressant Calibrator, Quality Control, and Internal Standard sets, along with a validated analytical method. To optimize performance for our specific clinical needs, the manufacturer’s protocol was modified to reduce matrix effects and improve precision. This optimized LC-MS/MS method was fully validated to support the assessment of tacrolimus, sirolimus, everolimus, and cyclosporine A in whole blood, which is critical for preventing toxicity while achieving therapeutic efficacy. 50 µL of whole blood specimen, calibrator, or quality control (QC) material was combined with 200 µL of 0.1 M aqueous zinc sulfate and vortexed for 5 seconds. Following the addition of the 500 µL of internal standard, the mixture was vortexed for 2 minutes and then centrifuged for 10 minutes. A 10 µL aliquot of the supernatant was analyzed by a Waters Xevo TQ Absolute system with a Waters Acquity UPLC HSS C18 SB column using positive electrospray ionization (ESI+) mode. LC methods of 2, 3, and 4 minutes were developed and studied. Sirolimus was evaluated using both ascomycin and sirolimus-D3 as internal standards. Repeatability, reproducibility, and carryover were assessed using the QC materials. Analytical Measurement Range (AMR) was established using the Chromsystems multilevel whole blood calibrator set and spiking negative whole blood pool with cyclosporin A. Accuracy was evaluated by analyzing patient, spiked, and previously tested proficiency testing samples against a 5-minute validated in-house LC-MS/MS method. Matrix effect was assessed by matrix dilution study. The 3-minute method was ultimately selected as it provided a significant reduction in matrix effect compared to the vendor provided 2-minute method, while extending the runtime to 4 minutes yielded no further analytical improvement. Sirolimus-D3 was ultimately selected for the validated method because longitudinal data demonstrated that ascomycin did not consistently mirror the analytical behavior of sirolimus, leading to decreased precision. Repeatability and reproducibility were <=8% CV for all compounds at all QC levels and no carryover was observed. The AMR range was from 1.0 to 35.0 (ng/mL) for tacrolimus, sirolimus, and everolimus, and from 25.0 to1800.0 (ng/mL) for cyclosporine A, and correlation coefficients of all compounds were R^2 = 0.999. Method comparison using Passing-Bablok regression yielded a slope of 0.92 and intercept of 0.18 ng/mL for tacrolimus (N=62), a slope of 1.02 and intercept of –0.09 ng/mL for everolimus (N=26), and a slope of 1.10 and intercept of -5.97 ng/mL for cyclosporine A (N=32). A bias was seen in sirolimus (N=75) with a Passing-Bablok regression yielding a slope of 1.20 and intercept of -0.10 ng/mL. Minimal matrix effect was observed. We have developed and validated a comprehensive and rapid LC-MS/MS method for the simultaneous measurement of tacrolimus, and cyclosporine A for clinical use. The bias observed in sirolimus is currently being investigated.