Simultaneous determination of erythrocyte methotrexate polyglutamates by a novel and simple HPLC‐MS/MS method with stable isotope‐labeled internal standards

Abstract Low‐dose methotrexate is the first‐line therapy for juvenile idiopathic arthritis. In vivo, methotrexate is converted into a series of methotrexate polyglutamates whose intracellular levels contribute significantly to its efficacy and toxicity. In this study, a novel high‐performance liquid chromatography–tandem mass spectrometry method was developed and validated to simultaneously determine erythrocyte methotrexate polyglutamates using stable isotope‐labeled internal standards. Erythrocyte samples were precipitated by perchloric acid and then determined on an XBridge BEH C18 column with an XP vanguard precolumn in 12 min. The mobile phase consisted of 10 nM ammonium acetate (pH 10) and methanol under gradient elution. The detection was carried out in multiple reaction monitoring mode via an electrospray ionization source in positive ionization mode. The calibration curve for each metabolite was linear from 2.0 to 500.0 nmol/L ( r 2 > 0.99). The intraday and interday accuracies were between 93.0 and 107.0%, and the corresponding precisions were between 0.8 and 5.2%. The relative recovery ranged from 82.7 to 105.1%, and the relative matrix effect varied from 96.5 to 104.4%. The erythrocyte metabolites were stable for 30 days at −80°C. This simple and accurate method is applicable to routine monitoring of the concentration of erythrocyte methotrexate polyglutamates in patients to achieve individualized treatment.