Determination of methotrexate and its main metabolite 7-hydroxymethotrexate in human urine by high-performance liquid chromatography with normal solid-phase extraction

A practical and sensitive high-performance liquid chromatographic method using normal solid-phase extraction has been developed for the determination of methotrexate (MTX) and its main metalbolite 7-hydroxymethotrexate (7-OH-MTX) in human urine. A urine specimen followed by the addition of pH 5.0 acetate buffer was purified by solid-phase extraction on a Sep-Pak silica cartridge. The analyte was chromatographed on a reversed-phase Inertsil ODS-2 column using phosphate buffer-acetonitrile at pH 5.3 as the mobile phase, and the effluent from the column was monitored at 303 nm. A good linear relationship between peak height and concentration was found for both of MTX and 7-OH-MTX in the range 5 to 1000 ng/ml of human urine. The inter-day coefficients of variation for the assay (n=5) were 8.8% (5 ng/ml), 3.4% (50 ng/ml) and 2.0% (500 ng/ml) for MTX, and 7.2, 2.7 and 2.3% for 7-OH-MTX in urine, respectively. The present method should prove useful for the evaluation of urinary drug excretion in patients undergoing MTX low-dose therapy.