Simultanous determination of dacarbazine, its photolytic degradation product, 2-azahypoxanthine, and the metabolite 5-aminoimidazole-4-carboxamide in plasma and urine by high-pressure liquid chromatography

A high-pressure liquid chromatographic method has been developed that allows for the simultaneous analysis of dacarbazine (DTIC), 5-aminoimidazole-4-carboxamide (AIC), and 2-azahypoxanthine (2-AZA) in plasma or urine. Plasma samples were prepared by ultrafiltration, whereas urine samples were filtered and diluted for analysis. Chromatography was done with a C₁₈ μBondapak column along with gradient elution of the drugs. The mobile phase consisted of 100% 0.5 M sodium acetate (pH 7.0) and 25% acetonitrile in 0.05 M sodium acetate (pH 5.5) with detection at 280 nm. Linearity was observed up to 500 μg/ml for DTIC and up to 53 μg/ml for AIC and 2-AZA. The assay methodology was reproducible, with a lower limit detection of 5.0, 0.5, and 0.5 μg/ml for DTIC, AIC, and 2-AZA, respectively. Interday and intraday coefficients of variation ranged between 4 to 14% and 2 to 16%, respectively. The analytical method was applied to the analysis of plasma and urine samples resulting from the isolation perfusion chemotherapy of an extremity with 57 mg of DTIC per kg in a patient with melanoma.