title: Determination of erythrocyte methotrexate polyglutamates by liquid chromatography/tandem mass spectrometry after low-dose methotrexate therapy in Chinese patients with rheumatoid arthritis.
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 (200 mM) 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 0.5 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.
authors.affiliation: Department of Pharmacy, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Mo
authors.forename: Yuguan
authors.initials: Y
authors.lastname: Wen
authors.forename: Bin
authors.initials: B
authors.lastname: Ren
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Chenshu
authors.initials: C
authors.lastname: Xu
authors.forename: Xueding
authors.initials: X
authors.lastname: Wang
authors.forename: Min
authors.initials: M
authors.lastname: Huang
authors.forename: Xiao
authors.initials: X
authors.lastname: Chen
mesh_terms: D001172:Arthritis, Rheumatoid / Q000097:blood* / Q000188:drug therapy*; D002681:China; D002853:Chromatography, Liquid / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004355:Drug Stability; D004912:Erythrocytes / Q000737:chemistry*; D006801:Humans; D016014:Linear Models; D059625:Liquid-Liquid Extraction; D008727:Methotrexate / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000097:blood / Q000493:pharmacokinetics; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000097:blood; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D008727:Methotrexate