An optimized UHPLC-MS/MS method for simultaneous determination of erythrocyte methotrexate and polyglutamylated metabolites: Application in pediatric patients with acute lymphoblastic leukemia

Abstract

Abstract Methotrexate (MTX), a widely used chemotherapeutic drug, is critical for achieving long-term complete remission in contemporary maintenance therapy of acute lymphoblastic leukemia (ALL). MTX is intracellularly converted into methotrexate polyglutamates (MTXPGn) by folylpolyglutamate synthase after transporting into the cells. The intracellular levels of active MTXPGn regulate the clinical efficacy and drug-related side effects, but it is still challenged by large inter-individual differences and narrow therapeutic index. Therefore, it is of great significance to develop a sensitive and stable method to monitor MTXPGs concentrations in the erythrocyte. To facilitate clinical application, an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantification of MTX and MTXPGn in erythrocyte. Solid phase extraction was applied to sample clean-up followed by a gradient chromatographic separation. The elution consisting of methanol/water/2 mM ammonium acetate (flow rate: 0.4 mL/min). Linearity of the assay was assured in the range of 1-500 nM (R2 > 0.99). The co-efficient of variation ranged from 2–11% for intraday precision and 0.4–8% for inter day precision. Recovery ranged from 62.51–99.75%, and the matrix-effect varied from 63.24–96.33%. Blood samples from 89 pediatric ALL patients were detected. The concentrations and distribution of MTXPGn in these samples were basically consistent with the current literatures, which confirmed that the optimized method for quantitative detection of MTXPGn in red blood cells had high sensitivity and accuracy. The sensitive method has been fully validated and successfully applied to determine the erythrocyte MTXPGn in pediatric ALL patients during continuation therapy.