Abstract
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 (200mM) at 37°C for 3h 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 0.5ng/mL and 1ng/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.
Published Version
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