Multiple inverse isotope dilution assay for cadralazine and four metabolites in biological fluids.

An inverse isotope dilution assay was developed for the specific determination of 14C-labelled cadralazine and four of its metabolites in biological samples. After addition of unlabelled carrier substances to the sample, metabolite IV was derivatized. The derivative and the unaltered compounds (I, II, III, V) were extracted and separated by high-performance liquid chromatography on silica gel. Quantitation was performed by on-line ultraviolet detection at 274 nm and off-line radiometry by liquid scintillation counting. Endogenous compounds and unknown metabolites did not interfere in the assay. The analysis of water, plasma and urine samples spiked with [14C]cadralazine showed mean recoveries between 98.4 and 101.3%. The lower limit of detection was 10 nmol/1 (3 ng/ml) for any of the compounds I-V. The method was used for the analysis of plasma and urine samples of rats dosed with [14C]cadralazine.