HPLC-UV method development and validation for 16-dehydropregnenolone, a novel oral hypolipidaemic agent, in rat...

An accurate and precise HPLC assay has been developed and validated for determination of dehydropregnenolone (DHP) in rat plasma, bile, urine and feces. Separation was achieved using a C-18 reversed phase column with a mobile phase comprising of acetonitrile and deionized water (55:45% v/v) using a UV detector, set at a wavelength of 248 nm. The method, applicable to 200-microl plasma, bile and urine, involved double extraction of the samples with n-hexane. The sample clean up for feces involved single extraction of 50 mg of sample with 3 ml of acetonitrile. The method was sensitive with a limit of quantitation of 20 ng/ml in all the matrices and absolute recovery >92%. Precision and accuracy were within the acceptable limits, as indicated by relative standard deviation varying from 4.7 to 11.2% and bias values ranging from 1.8 to 8.8%. Moreover, DHP was stable in plasma, bile and urine up to 90 days of storage at -60 degrees C and after being subjected to three freeze-thaw cycles. The method was applied to generate the pharmacokinetics of DHP in rats after oral and intravenous administration.