MSACL 2023 Abstract

Mass Spectrometry Quantitation of Immunosuppressive Drugs in Clinical Specimens Using Accurate-Mass Full Scan-Single Ion Monitoring Priscilla S.-W. Yeung (1, 2) Paige Miller (2), Tran Bao Lai-Nyugen (2), Phil Cheng (2), Amira Ibrahim (2), Run-Zhang Shi (1, 2), Raffick A.R. Bowen (1, 2), Ruben Yiqi Luo (1, 2) (1) Department of Pathology, Stanford University, Stanford, CA, USA (2) Clinical Laboratories, Stanford Health Care, Palo Alto, CA, USA Priscilla Yeung, MD, PhD (Presenter) Stanford University Department of Pathology

Presenter Bio: Priscilla Yeung is a Clinical Pathology Resident at Stanford University interested in using mass spectrometry to discover improved biomarkers for clinical testing. Her current research with Dr. Ruben Luo is focused on applying top-down mass spectrometry to the diagnosis of monoclonal gammopathies. Prior to residency, she completed her MD/PhD training at Northwestern University, where she studied the biophysical mechanisms of calcium channel gating with Dr. Murali Prakriya, and her undergraduate studies at University of Pennsylvania, where she studied amyloid-beta protein misfolding with Dr. Paul Axelsen.

INTRODUCTION: Therapeutic drug monitoring (TDM) of immunosuppressive drugs including tacrolimus, sirolimus, everolimus, and cyclosporine A is a critical part of optimizing clinical outcomes for solid organ transplant patients. The most commonly used methodologies for TDM are immunoassays and liquid chromatography-mass spectrometry (LC-MS). Although immunoassays have shorter turnaround times and fewer trained personnel requirements, they can suffer from interference from heterophile antibodies and similar compounds such as other drugs from the same class or metabolites. Current mass spectrometry assays based on multiple reaction monitoring (MRM) can be tedious to optimize and are limited in the number of compounds that can be detected in each run.

OBJECTIVES: The purpose of this study was to implement a mass spectrometry-based test for immunosuppressant TDM using accurate-mass full scan-single ion monitoring (FS-SIM) and online solid phase extraction (SPE).

METHODS: LC-MS analysis was performed on a TLX-2 multi-channel HPLC with a Q-Exactive Plus mass spectrometer. Mobile phase A contained 10 mM ammonium formate in water with 0.1% formic acid and mobile phase B was methanol. A TurboFlow online SPE column was used for sample clean up prior to loading onto the analytical C18 column. The accurate-mass MS was set to positive electrospray ionization mode with FS-SIM for quantification of tacrolimus, sirolimus, everolimus, and cyclosporine A. During the LC-MS analysis, Fragmentation of each analyte was performed using high-energy C-trap dissociation (HCD) for compound confirmation.

RESULTS: The method was validated according to the guidelines for laboratory-developed tests. Within-run and between-run imprecision, analytical bias, limit of quantitation, analytical measurement range, and linearity were all within acceptable limits. Quantification of tacrolimus, sirolimus, everolimus, and cyclosporine A correlated closely with the results from Mayo Clinic Laboratories with R2 = 0.90-0.96. Tacrolimus results also had excellent correlation with Roche immunoassay with R2 = 0.97. Several major metabolites of the four drugs were able to be observed within the LC-MS method.

CONCLUSIONS: Accurate-mass FS-SIM can be utilized for immunosuppressant TDM with tight correlation with results generated by standard methods. Turboflow online SPE allows for a simple “protein crash and shoot” sample preparation protocol. Compared with traditional MRM, analyte quantification by FS-SIM has a more streamlined assay optimization process and allows for detection of more drug metabolites within the same run.