Determination of osthole in rat plasma by high-performance liquid chromatograph using cloud-point extraction.

A new straightforward method based on cloud-point extraction (CPE) was developed to determine osthole in rat plasma by reversed phase high-performance liquid chromatography with ultraviolet detection using a photodiode array detector. The non-ionic surfactant Triton X-114 was chosen as the extract solvent. Variable parameters affecting the CPE efficiency were evaluated and optimized. A Zorbax SB-C(18) column was used for elution separation at 25 degrees C with detection wavelength at 322 nm. Under the optimum conditions, the method was shown to be reproducible and reliable with intra-day precision below 7.62%, inter-day precision below 6.37%, and accuracy within +/-5.02% and mean extraction recovery more than 90.4%, which were all calculated using a range of spiked samples at three concentrations of 0.5, 5.0 and 15.0 microgmL(-1) for osthole in plasma. The calibration curve for the analyte was linear in the range from 0.1 to 20 microgmL(-1) with the correlation coefficients greater than 0.9981. Limit of detection (S/N=3) was less than 0.03 microgmL(-1)and limit of quantification (S/N=10) was less than 0.1 microgmL(-1). After strict validation, the method was successfully applied to the pharmacokinetic study of osthole in rats after oral and intravenous administration, respectively.