HPLC determination of methotrexate and its metabolite in serum.

Determination of methotrexate (MTX) residues in biological samples (serum, cerebrospinal fluid) was worked out using liquid-liquid and solid-phase extractions and high performance liquid chromatography (HPLC) with UV detection. Clinical samples from patients treated by low and high doses of MTX were analyzed using reversed-phase and ion-pair liquid chromatography, and buffer-methanolic mobile phases. Chromatographic conditions were optimized for the simultaneous determination of MTX and its metabolite and the minimal analysis time was recommended. The results were statistically evaluated, elimination curves and chromatograms have been demonstrated. Solid-phase extraction recovery was 93.1 +/- 1.5% and the determination limit for methotrexate in serum samples was 5.10(-7) mol/l and after the preconcentration of samples 5.10(-8) mol/l.