Abstract
High-performance liquid chromatography (HPLC) is significantly used for the rapid detection and preparative separation of several pteridines, folic acid derivatives, pterins and pteroyloligoglutamates. This chapter focuses on separation of methotrexate (MTX) analogs containing terminal lysine or ornithine and their dansyl derivatives by HPLC. The emphasis of most of these separation studies is on improving the analytical sensitivity, ease of sample preparation, and rapid quantitation of the folate antagonist MTX and its metabolites in biological fluids such as plasma, serum, saliva, and urine. Sample identification has been achieved by monitoring the absorption at 303, 305, and 315 nm and by detecting the fluorescence of MTX derivatives obtained by permanganate oxidation or dithionite reduction. HPLC is employed in the analysis of amides, esters, and poly(γ-glutamyl) derivatives of MTX and to demonstrate the cellular uptake of radioactive impurities found in commercial [ 3 H]MTX. Preparative and analytical-scale HPLC are performed using a Waters Associates system consisting of a Model 720 system controller, an M45 solvent delivery system, and a Model 450 variable wavelength detector.
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