High‐performance liquid chromatography with continuous gradient elution in screening for unknown metabolites of butoprozine directly in dog bile

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Abstract
This paper describes the screening for metabolites of butoprozine, a new anti‐anginal drug, in dog bile by means of reverse‐phase HPLC. Although it does involve a simple clean‐up step to remove a substantial amount of endogenous bile compounds, this screening method nevertheless avoids extraction of metabolites and thus allows all metabolites to be introduced into the chromatographic system. A single run of 100 minutes from 100% water to 100% methanol in a linear gradient effects adequate separation of the great majority of metabolites without interference from remaining endogenous compounds.Two methods of differentiating between metabolite peaks and endogenous peaks have been worked out. The first one makes use of 14C‐labeled butoprozine by measuring the amount of radioactivity in the column effluent while simultaneously recording the UV absorbance. The second method compares continuous gradient chromatograms of bile recorded before and after butoprozine administration under very similar conditions. The latter method can be applied to both radioactive and non‐radioactive materials.