Mass Spectrometry Quantitation of Immunosuppressive Drugs in Clinical Specimens Using Online Solid-Phase Extraction and Accurate-Mass Full Scan-Single Ion Monitoring

Introduction: Therapeutic drug monitoring (TDM) of immunosuppressants is essential for optimal care of trans-plant patients. Immunoassays and liquid chromatography-mass spectrometry (LC-MS) are the most commonly used methods for TDM. However, immunoassays can suffer from interference from heterophile antibodies and structurally similar drugs and metabolites. Additionally, nominal-mass LC-MS assays can be difficult to optimize and are limited in the number of detectable compounds.Objectives: The aim of this study was to implement a mass spectrometry-based test for immunosuppressant TDM using online solid-phase extraction (SPE) and accurate-mass full scan-single ion monitoring (FS-SIM) data acquisition mode.Methods: LC-MS analysis was performed on a TLX-2 multi-channel HPLC with a Q-Exactive Plus mass spec-trometer. TurboFlow online SPE was used for sample clean up. The accurate-mass MS was set to positive elec-trospray ionization mode with FS-SIM for quantitation of tacrolimus, sirolimus, everolimus, and cyclosporine A. MS2 fragmentation pattern was used for compound confirmation.Results: The method was validated in terms of precision, analytical bias, limit of quantitation, linearity, carry-over, sample stability, and interference. Quantitation of tacrolimus, sirolimus, everolimus, and cyclosporine A correlated well with results from an independent reference laboratory (r = 0.926-0.984).Conclusions: Accurate-mass FS-SIM can be successfully utilized for immunosuppressant TDM with good corre-lation with results generated by standard methods. TurboFlow online SPE allows for a simple "protein crash and shoot" sample preparation protocol. Compared to traditional MRM, analyte quantitation by FS-SIM facilitates a streamlined assay optimization process.