Liquid chromatography coupled with multi-channel electrochemical detection for the determination of daidzin in rat blood sampled by an automated blood sampling system

Daidzin, a soy-derived biologically active natural product, has been reported to inhibit mitochondrial aldehyde dehydrogenase and suppress ethanol intake. This paper describes a method for the determination of daidzin in rat blood. After administration of daidzin, blood samples were periodically collected from awake, freely moving animals by a Culex automated blood sampler. Daidzin was extracted from 50 μl of diluted blood (blood and saline at a ratio of 1:1) with ethyl acetate. Chromatographic separation was achieved within 12 min using a microbore C18 (100×1.0 mm) 3 μm column with a mobile phase containing 20 mM sodium acetate, 0.25 mM EDTA, pH 4.3, 4% methanol and 11% acetonitrile at a flow-rate of 90 μl/min. Detection was attained using a four-channel electrochemical detector with glassy carbon electrodes using oxidation potentials of +1100, 950, 850, 750 mV vs. Ag/AgCl. The limit of detection for daidzin in rat plasma was 5 ng/ml at a signal-to-noise ratio of 3:1. The extraction recovery of daidzin from rat plasma was over 74%. Linearity was obtained for the range of 25–1000 ng/ml. The intra- and inter-assay precisions were in the ranges of 2.7–6.6 and 1.9–3.7%, respectively. This method is suitable to routine in vivo monitoring of daidzin in rat plasma.