title: Simple and sensitive HPLC method for the fluorometric determination of methotrexate and its major metabolites in human plasma by post-column photochemical reaction.
abstract: A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 2,4-diamino-N(10-) methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7-OH-MTX, DAMPA and 2,4-diaminopteroic acid (DAPA) as an internal standard were separated on a reversed-phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7-OH-MTX and DAMPA from plasma at 100 pmol/mL were 81.5±5.4, 82.5±5.3 and 56.2±7.0%, respectively. The limits of quantification for MTX, 7-OH-MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within- and between-day variations for MTX, 7-OH-MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy.
authors: Department of Pharmacy, Fukuoka University Hospital, Fukuoka, Japan.
authors: Masanobu
authors: M
authors: Uchiyama
authors: Takanori
authors: T
authors: Matsumoto
authors: Taichi
authors: T
authors: Matsumoto
authors: Shiro
authors: S
authors: Jimi
authors: Yasushi
authors: Y
authors: Takamatsu
authors: Kazuo
authors: K
authors: Tamura
authors: Shuuji
authors: S
authors: Hara
mesh_terms: D000368:Aged; D000964:Antimetabolites, Antineoplastic / Q000097:blood / Q000493:pharmacokinetics / Q000627:therapeutic use; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D016403:Lymphoma, Large B-Cell, Diffuse / Q000097:blood / Q000188:drug therapy; D008297:Male; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000097:blood* / Q000493:pharmacokinetics / Q000627:therapeutic use; D009952:Ornithine; D010782:Photolysis; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D000964:Antimetabolites, Antineoplastic; C000203:deoxyaminopteroic acid; D009952:Ornithine; C011864:7-hydroxymethotrexate; D008727:Methotrexate