Lipid-Soluble Inhibitors of Dihydrofolate Reductase III: Quantitative Thin-Layer and High-Performance Liquid Chromatographic Methods for Measuring Plasma Concentrations of the Antifolate, 2,4-Diamino-6-(2,5- dimethoxybenzyl)-5-methylpyrido-[2,3-d]pyrimidine

Abstract Specific methods using high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) for the analysis of the potent antifolate, 2,4-diamino-6-(2,5-dimethoxybenzyl)-5- methylpyrido-[2,3-dJpyrimidine (I) in plasma were developed. The HPLC system employed paired-ion chromatography using a mobile phase of water-acetonitrile (65:35, v/v) in conjunction with a reversed-phase C-l column and detection by UV absorbance measurement. The TLC system utilized a scanning densitometer operating in the reflectance mode with detection of I by fluorescence measurement. The lower limits of detection for the HPLC and TLC methods were ~ 0.005 μg/ml. The coefficients of variation for the measurement of drug concentrations over the range of 0.04-1.0 μg/ml of plasma were 5 and 6%, respectively.