Determination of erythrocyte methotrexate polyglutamates by liquid chromatography/tandem mass spectrometry after low-dose methotrexate therapy in Chinese patients with rheumatoid arthritis

Methotrexate (MTX) is currently one of the most widely used drugs for treatment of rheumatoid arthritis (RA) through polyglutamation of methotrexate polyglutamates (MTXPGs), a process attaching sequential γ-linked glutamic residues to MTX. A new and sensitive LC/MS/MS method was developed and validated for determination of whole-blood MTX and total MTX (MTX + MTXPGs), and then concentration of MTXPGs was calculated. To determine whole-blood MTX, whole blood was precipitated with 50% trifluoroacetic acid, and extraction was performed using ethyl acetoacetate. Analytes were subjected to LC/MS/MS analysis using positive electrospray ionization. To determine whole-blood total MTX, whole blood was incubated with ascorbic acid (200 mM) at 37 °C for 3 h to enzymatically convert the MTXPGs to MTX, and then processed with the same method mentioned above. Recoveries of spiked MTX at ppb (ng/mL) level were between 26.2% and 37.8% with intra- and inter-day precision less than 15.8% and 11.8%, respectively. The lower limit of detection (LLOD) and lower limit of quantitation (LLOQ) were 0.5 ng/mL and 1 ng/mL, respectively. The sensitive LC/MS/MS method was fully validated with high selectivity and acceptable accuracy and precision, which was successfully applied to determine the erythrocyte methotrexate polyglutamates in patients with RA.

► An LC/MS/MS method was developed for determination of MTXPGs in erythrocyte.
► It's the first study using LC/MS/MS with liquid–liquid extraction to detect MTXPGs.
► The levels of MTXPGs are generated indirectly.
► The method is sensitive, simple, low cost and can be widely applied in many labs.
► The method has been applied to determine the concentration of MTX in patients with RA.