HPLC Analysis of Merbarone (NSC 336628) in Plasma Using MG(II) Asamobile Phase Modifier

Abstract

Merbarone is a thiobarbituric acid derivative which has been shown to be curative in murine leukemias and is currently under evaluation in the treatment of cancer patients. A reversed-phase HPLC method was developed for the quantitation of plasma levels of merbarone. The protein of plasma (50 p. μl) was precipitated with 250 μl of methanol-DMSO (82:18, v/v) containing the internal standard, 3′-F-merbarone. After centrifugation, the supernatant (200 μl) was diluted with the mobile phase less methanol (300 μl) and 350 μl was injected on column. Chromatography was carried out on a 4 μm Nova-Pak C-¹⁸column (3.9 mm × 15 cm) using an isocratic mobile phase and UV detection at 306 nm. The mobile phase was methanol-ammonium acetate (0.1 M)-acetic acid (0.4 M)-MgSO₄·7H₂O (2.08 M) (30:60:8:2, by volume) containing 1 mM sodium dodecyl sulfate. Employing 50 μl of plasma, concentrations of merbarone in the range 0.05–5.00 μg/ml were quantitated. Very low levels of one metabolite, tentatively identified as 2-oxo-desthiomerbarone, were observed in chromatograms of patient plasma.