Determination of methotrexate, several pteridines, and creatinine in human urine, previous oxidation with potassium permanganate, using HPLC with photometric and fluorimetric serial detection

Abstract

A novel HPLC method, using UV and fluorimetric serial detection, for the simultaneous determination of methotrexate (MTX), five disease marker pteridines, and the reference metabolic subproduct creatinine (CREA) in human urine was established. A previous oxidation process using 10 −3 M KMnO 4 (pH 5.0) and 35 min of oxidation time was necessary to transform the analytes in the highly fluorescent pteridinic rings. CREA was not affected by the oxidative medium. Using Tris–HCl/NaCl buffer solution (pH 6.6) as mobile phase, MTX and the assayed pteridines were monitored by fluorescence at λ em = 444 nm and λ ex = 280 nm and creatinine was monitored by absorption measurements at λ abs = 230 nm. All components were well resolved in approximately 7 min. Detection limits, according the criteria of Clayton and co-workers, were 10 ng ml −1 for MTX, less than 1 ng ml −1 for all of the pteridines, and 4 μg ml −1 for CREA.