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).

Results

The method showed excellent selectivity and linearity (r > 0.997) over 1-200 ng/mL (ibrutinib, zanubrutinib, ibrutinib metabolite) and 5-1000 ng/mL (orelabrutinib) in both matrices. 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.