Simultaneous determination of four anticoagulant rodenticides in human urine using high performance liquid chromatography cloud point extraction

A new method was developed for the simultaneous determination of four rodenticides including warfarin, coumatetralyl, bromadiolone and brodifacoum in human urine samples by reverse-phase high performance liquid chromatography after clound point extraction(CPE) by Triton X-114. The chromatographic conditions and extraction parameters were optimized. Under the optimum CPE conditions: 0.030 g NaCl, 30 mu L formic acid and 5% (w/V) Triton X-114 400 mu L for 5 mL unine sample with the extraction temperature of 50 degrees C and 15 min of equilibration time, the extraction efficiencies were more than 85%. Then the extracts were seperated and eluted by an ODS-C-18 column (150 mm x 4.6 min i.d., 5 mu m) and the mobile phase comprising of methanol-1% formic acid (90: 10, V/V) at 1.0 mL/min under 40 degrees C. The dectecion was done at wavelength of 306 nm and completed within 10 minutes. The linear ranges of four rodenticides were in the range of 0.02-5.0 mg/L except for coumatetralyl 0.01-5.0 mg/L with the correlation coefficients more than 0.997. The recoveries of the four rodenticides from spiked urine samples ranged from 71.2% to 114.5% and the relative standard deviations were less than 7.0%. The concentration factor(Fc) could be changed with the final diluted volume of surfactant-rich phase, and the theory concentration factor could reach to 28 times when 50 mu L methanol was added after CPE of 5 mL sample. The limits of quantification were 0.016 mg/L, 0.001 mg/L, 0.005 mg/L, 0.003 mg/L for warfarin, coumatetralyl, bromadiolone and brodifacoum, respectively. The proposed method is simple, rapid, sensitive, accurate and friendly to environment. It can meet the requirements for simultaneous determination of four rodenticides for poisoning patients.