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Abstract
A paired-ion high pressure liquid chromatography procedure (HPLC) is described for the separation of methotrexate and its major metabolites (7-hydroxymethotrexate, 4-amino-4-deoxy-NlO-methylpteroic acid, methotrexate diglutamate, methotrexate triglutamate), and therapy-related compounds (Diamox and 5-methyltetrahydrofolate). The mobile phase consisted of a 70% solution of 5 mM hexanesulfonic acid, pH 3.75, and 30% methanol eluted on a reverse phase column or columns and monitored at 305 nm or 280 nm UV absorption. The lower limit of sensitivity for methotrexate and 7-hydroxymethotrexate in plasma was 2 ng/ml.