A high-performance liquid chromatographic method was developed for the sensitive determination of methotrexate (MTX) in monkey plasma using direct injection and on-line solid-phase extraction. After application of a 100-microliters aliquot of plasma to a pre-treatment column, the column was washed with 0.02 M phosphate buffer (pH 7) to eliminate plasma proteins and endogenous substances, and subsequently the adsorbed MTX was eluted. The MTX fraction was transferred to an analytical column by a column-switching (heart-cutting) technique, and MTX was analyzed using ion-pair chromatography with tetrabutylammonium bromide. More than 50 injections could be performed onto one pretreatment column. The accuracy, precision, reproducibility and linearity were satisfactory over a wide range of MTX concentrations (5-1000 ng/ml). The quantitation limit was 5 ng/ml with a signal-to-noise ratio of 5. The method was suitable for the pharmacokinetic study of MTX in monkey.
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