A UHPLC-MS/MS method for the simultaneous quantification of BTK inhibitors and their active metabolite in human plasma and cerebrospinal fluid

BACKGROUND: Bruton's tyrosine kinase inhibitors (BTKis, ibrutinib, zanubrutinib, orelabrutinib) are key targeted therapies for B-cell lymphomas, but interindividual variability and adverse reactions limit their use. Metabolized by CYP3A, co-medications or hepatic dysfunction cause plasma fluctuations, necessitating therapeutic drug monitoring (TDM). Cerebrospinal fluid (CSF) distribution is also critical for CNS lymphoma treatment. OBJECTIVE: To develop and validate a UHPLC-MS/MS method for simultaneous quantification of three BTKis and one metabolite in human plasma and CSF to support TDM. METHODS: After protein precipitation with methanol, samples were analyzed on an ACQUITY HSS T3 column with a 3-min gradient at 0.3 mL/min and ESI (+) MRM. Mobile phase: water-acetonitrile-formic acid-ammonium acetate (1 M) (950:50:1:1, A) and (50:950:1:1, B). Recoveries were 89.4-103.8%, matrix factors 0.93-1.06, and precision CV ≤8.5%. Stability was confirmed. The method was applied to 34 clinical samples (25 plasma, 9 CSF) from 20 patients. CONCLUSION: This method detects four BTK-related analytes in plasma/CSF with a 3-min run time, evaluation of hemolytic/lipemic matrices, and clinical validation, increasing throughput, reducing costs, and broadening applicability, providing preliminary feasibility data for clinical TDM.