Development and validation of a subcritical fluid extraction and high performance liquid chromatography assay for...

A simple, rapid and sensitive method was developed for the determination of medroxyprogesterone in aquatic products by extraction with subcritical 1,1,1,2-tetrafluoroethane (R134a) and high performance liquid chromatography (HPLC). A response surface methodology (RSM) was adopted to optimise extraction pressure, temperature and co-solvent volume. The optimum extraction conditions predicted within the experimental ranges were as follows: pressure, 3 MPa; temperature, 25 °C; and co-solvent volume, 6 ml. The analysis was carried out on Zorbax SB-C₁₈ column (4.6 mm × 150 mm, 5 μm) with the mobile phase acetonitrile-water (55:45, v/v), flow rate 1.0 ml/min, temperature 30 °C and wavelength 240 nm. Good linearity of detection was obtained for medroxyprogesterone between concentrations of 50-250 ng/ml, r²=0.999. The method was validated using samples fortified with medroxyprogesterone at levels of 10, 30 and 50 ng/g, the mean recovery exceeds 90%, and the RSD values were less than 10%.