Abstract
A novel High Performance Liquid Chromatography β electrospray mass spectrometric method has been developed for the estimation of Ursodiol (Ursodeoxycholic acid)βa bile acid, in human plasma using Ornidazole as internal standard. The methodology involved solid phase extraction of the analyte from human plasma matrix. The chromatographic separation was achieved within seven minutes by an isocratic mobile phase containing 1.0 mM ammonium acetate and Acetonitrile (65: 35, v/v), flowing through XTerra MS C18, 100 Γ 2.1, 3.5 ΞΌm analytical column, at a flow rate of 0.2 ml/min. Ion signals were measured in negative mode for Ursodiol and internal standard at m/z 391.3 and 278.1, respectively. A detailed validation of the method was performed as per USFDA guidelines and the standard curves were found to be linear in the range 50.0 ng/ml to 3000.0 ng/ml with the mean correlation coefficient more than 0.99. The absolute recovery was more than 54.90 % for Ursodiol and 76.51 % for internal standard. Ursodiol was stable for sixtyβnine days at β70 Β°C and for eight hours at ambient temperature. After extraction from plasma, the reconstituted samples of Ursodiol were stable in autosampler at 10 Β°C for fortyβeight hours. Upon subjecting to three freeze thaw cycles, there was no change in the recovery of the analyte. The integrity of the plasma samples remained unaffected even upon fourβfold dilution with drug free human plasma. The method was simple, specific, sensitive, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. It was successfully applied to the pilot bioequivalence study of Ursodiol in male human subjects.