Specific and sensitive high-performance liquid chromatographic method with fluorescence detection for measurement of...

A reversed-phase high-performance liquid chromatographic (HPLC) assay is described for the quantitative determination of lometrexol in biological samples; the assay is rapid, simple, specific, and highly sensitive. The method requires the dissociation of lometrexol from folate-binding proteins present in blood and formation of a fluorescent oxidized derivative of the compound. The dissociation of lometrexol from folate-binding proteins was achieved by acidification to pH 3.5 using ammonium formate, followed by serum protein precipitation with perchloric acid. The protein-free lometrexol was subsequently oxidized by MnO2 at 90 degrees C for 10 min. Chromatographic separation of lometrexol without interference was achieved on a C18 reversed-phase column with a convex gradient, using acetonitrile-0.1% ammonium formate, pH 7.0, as the mobile phase. In human serum and urine the calibration curve was linear between 5 and 300 nM. The lower limit of quantification was 5 nM. The method has been applied successfully to measure serum and urinary levels of lometrexol in patients.