Micro HPLC Separation of 3,5-Dinitrobenzoyl Derivatives of Cardiac Glycosides and Their Metabolites

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Abstract
The use of 3,5-dinitrobenzoyl (3,5-DNB) chloride to form an ultraviolet absorbing derivative was attempted in order to improve the detection limit of digitoxin, digoxin, βmethyldigoxin, and their metabolites (bisdigitoxoside, monodigitoxoside, aglycone, and its 3-epimer) by micro high-performance liquid chromatography (MHPLC). The 3,5-DNB derivatives of these cardiac steroids were prepared by standard synthetic procedures and their molar absorptivities were measured at a wavelength of 230 nm. The MHPLC of the derivatives of the digitoxin and digoxin series was performed on an ODS micro column, using an acetonitrile:methanol:water (3:1:1) or acetonitrile:water solvent system and ultraviolet detection (230 nm). The limits of accurate quantitative measurements of digoxin and digoxigenin derivatives were ca. 2 and 0.4 ng, and the detection limits with a signal-to-noise ratio of 3:1 were less than 0.6 and 0.2 ng, respectively.