[Highly sensitive analytical procedure for methotrexate and its main metabolite 7-hydroxymethotrexate in serum by high-performance liquid chromatography].

Recently, methotrexate (MTX) low dose therapy (5-10 mg/m2) has been used for the treatment of patients with rheumatoid arthritis. Hence a practical and sensitive high-performance liquid chromatographic method for the determination of MTX and its main metabolite 7-hydroxymethotrexate (7-OH-MTX) in human serum has been studied. After deproteinization with perchloric acid followed by the addition of pH 5.0 acetate buffer, the serum sample was purified by solid-phase extraction on a Sep-Pak C18 cartridge. The analyte was chromatographed on a reversed-phase Inertsil ODS-2 column using a phosphate buffer-acetonitrile at pH 3.0 system as the mobile phase, and the effluent from the column was monitored at 303 nm. Gradient elution was employed to increase the sensitivity for 7-OH-MTX. A good linear relationship between peak height and concentration was found for the two compounds in the range 2.5 to 100 ng/ml of the human serum, and the detection limits were about 1 ng/ml for the two compounds. The day-to-day coefficients of variation assay were 2.3% (20 ng/ml) and 4.4% (100 ng/ml) for MTX and 4.6% (20 ng/ml) for 7-OH-MTX. The present method was successfully applied to the analysis of the serum after a single oral administration of MTX 2.5 mg tablet to male dogs. MTX was rapidly absorbed, reached to the maximal level at about 1.4 h and thereafter decreased monoexponentially with a half-life of about 1.4 h. A metabolite, 7-OH-MTX was not detected in the serum up to 24 h post dose.