Abstract
A sensitive and reliable HPLC method consisting of a solid-phase extraction, post-column photoreaction of the analytes in a polyethylene tubing by UV lamp, and fluorimetric detection for the simultaneous determination of methotrexate (MTX) and 7-OH-methotrexate (7-OH-MTX) in plasma was developed. The linear relationships between the peak area and MTX (0.1 ng/ml-1000 ng/ml, r=0.9997) or 7-OH-MTX (6.25 ng/ml-400 ng/ml, r=0.9995) concentrations were obtained. The average coefficients of variation for the intraday and interday replications were less than 11% and the absolute recoveries were 96.8%-101.2% for MTX and 76.4%-86.7% for 7-OH-MTX for the calibration ranges used. The limit of detection for MTX in plasma was 0.05 ng/ml. This most sensitive HPLC method reported so far was used for the trace assay of plasma MTX concentrations in dogs following a topical application of MTX in gel.

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