A fast LC-MS/MS assay for methotrexate monitoring in plasma: validation, comparison to FPIA and application in the setting of carboxypeptidase therapy

Abstract

High-dose methotrexate remains a mainstay in the treatment of acute lymphoblastic leukaemia, osteosarcoma and non-Hodgkin lymphoma. Therapeutic drug monitoring of plasma MTX is important to monitor efficacy and adverse events. The authors aimed at developing a liquid chromatography tandem mass spectrometry (LC-MS/MS) method with online extraction to determine MTX and 7-OH-MTX in plasma for therapeutic drug monitoring. The analysis combined straightforward sample preparation, consisting of protein precipitation with methanol/ZnSO4, with a 4-minute run time consisting of an on-line enrichment by a flush/back-flush cycle (Poros column, R1/20, 2.1 mm × 30 mm) before the second dimension chromatography (Phenomenex Luna 5 μm Phenyl Hexyl, 2 mm × 50 mm column). Samples were analysed using an HPLC Agilent 1200 Series and ABSciex API 3200. The electrospray was operated in positive ionization mode monitoring the following mass transitions: m/z 455.11 → 308.3 for MTX, 471.14 → 324.3 for 7-OH-MTX and m/z 459.1 → 312.3 for internal standard (MTX¹³C²H₃). The method was linear up to 50 μmol L⁻¹, and intra-day and inter-day quality control CVs were below 8.3% for MTX and 11.71% for 7-OH-MTX. Average recovery was 24% for MTX and 57% for 7-OH-MTX. The lower limit of quantitation was 25 nmol L⁻¹ for the 2 analytes. For MTX and 7-OH-MTX the standard line slope CV percentage was <3% and the slope difference <6%, indicating that our analytical method is almost free from a significant relative matrix effect. Method comparison with the Abbott TDx fluorescent polarization immunoassay (FPIA) showed excellent agreement: LC-MS/MS = 0.0011 + 1.0334 (FPIA). Because of antibody cross-reactivity between DAMPA and MTX, none of the immunoassays can be used after carboxypeptidase administration. The goal of our work was to develop a specific LC-MS/MS method to monitor both MTX and 7-OH-MTX plasma concentrations within the clinically relevant range. It's expected that the LC-MS/MS method for MTX monitoring after carboxypeptidase administration will be very rarely used since it concerns only exceptional cases. Therefore, the geographically balanced distribution of University Hospital able to ensure follow-up of these patients, within 24 hours of collection, can draw a reliable solution for the security of the patients. We develop a fast and reliable LC-MS/MS method for both routine TDM of MTX as in the setting of carboxypeptidase therapy.