LC-MS Method for Studying the Pharmacokinetics and Bioequivalence of Clonidine Hydrochloride in Healthy Male Volunteers

A simple and sensitive high performance liquid chromatography-electrospray ionization mass spectrometry method has been evaluated for the assignment of clonidine hydrochloride in human plasma. Methods: The mobile phase composed of acetonitrile-water 60:40 (v/v) and 0.2% formic acid 20 μl of sample was chromatographically analyzed using a repacked ZORBAX-XDB-ODS C18 column (2.1 mm×30 mm, 3.5 μ). Detection of analytes was achieved by tandem mass spectrometry with Electrospray Ionization (ESI) interface in positive ion mode operated under the multiple-reaction monitoring mode (m/z 230.0 →213). Sample pretreatment consisted of a one-step Protein Precipitation (PPT) with methanol and perchloric acid (HClO4) of 0.10 ml plasma. Results: Standard curve was linear (r=0.998) over the concentration range of 0.01–10.0 ng/ml and showed suitable accuracy and precision. The Limit of Quantification (LOQ) was 0.01 ng/ml. The mean (SD) Cmax, Tmax, AUC0–t and AUC0–∞ values after administration of the test and reference formulations, respectively, were in this manner: 6.16 (0.32) versus 6.21 (0.07) ng/ml, 30.12 (0.86) versus 30.13 (0.73) hr, 290.37 (1.13) versus 293.39 (1.22) ng/ml/hr, and 350.17 (1.98) versus 352.96 (1.67) ng/ml/hr. The mean (SD) t1/2 was 120.12 (1.90) hr for the test formulation and 120.96 (1.54) hr for the reference formulation. No statistical differences were showed for Cmax and the area under the plasma concentration-time curve for test and reference tablets. Conclusion: The method is rapid, simple, very steady and precise for the separation, assignment, pharmacokinetic and bioavailability evaluation of clonidine in healthy Iranian adult male volunteers.