Quantification of nicotine, chlorpyrifos and their metabolites in rat plasma and urine using high-performance liquid chromatography

This study describes a high-performance liquid chromatographic method for the separation and quantification of nicotine, its metabolites nornicotine and cotinine, the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl]phosphorothioate), and its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl]phosphate), and TCP (3,5,6-trichloro-2-pyridinol) in rat plasma and urine. The compounds were separated using gradient mobile phase of methanol, acetonitrile and water (pH 3.20) at a flow-rate of 0.8 ml/min in a period of 17 min, and gradient UV detection ranging between 260 and 280 nm. The retention times ranged from 3.4 to 16.7 min. The limits of detection were ranged between 20 and 150 ng/ml, while limits of quantitation were 50–200 ng/ml. Average percentage recovery of five spiked plasma samples were 84.7±8.3, 78.2±7.6, 80.1±7.6, 79.0±6.4, 74.0±7.4, 87.6±7.5, and from urine 85.1±5.2, 75.9±7.0, 82.1±6.1, 79.5±6.1, 71.3±7.4 and 81.3±6.9 for nicotine, nornicotine, cotinine chlorpyrifos, chlorpyrifos-oxon and TCP, respectively. Intra-day accuracy and precision for this method were ranged between 2.2–3.6 and 2.1–2.8%, respectively. The relationship between peak areas and concentration was linear over range between 200 and 2000 ng/ml. This method was applied to analyze the above chemicals and metabolites following combined oral administration in rats.