Improvement And Application Of The Method For Determination Of Clopidogrel In Human Plasma By LC-MS/MS

Background Clopidogrel is a thienopyridine antiplatelet agent,and is mainly used in the prevention of heart or brain and other arterial circulation disorders caused by high platelet aggregation.Objective Improvements have been made to the method for determining clopidogrel in human plasma to establish an accurate and sensitive liquid chromatography-tandem mass spectrometry?LC-MS/MS?method for the determination of clopidogrel in human plasma.The method was then successfully applied to the bioequivalence of two tablet formulations of clopidogrel in healthy Chinese Volunteers.Method Two plasma sample pretreatment methods?liquid-liquid extraction and protein precipitation?were used to test the stability of clopidogrel in human plasma.Mixed 0.5 h(T_max)and 8 h?elimination phase?of plasma that collected from the healthy volunteers after oral administration of clopidogrel hydrogen sulfate tablets,respectively,so that we can get actual plasma samples to test the stability of clopidogrel.After storage at room temperature for 6 h,after storage at 0?C for 6 h.The post-preparative stability was measured after exposure of the processed samples at 4?C for 44 h and 120 h,respectively.Stability of plasma samples were evaluated after three freeze-thaw cycles from-70?C to 0?C.The results showed that the plasma samples were stable under the above storage conditions use the LLE method.However,in the low concentrations of clopidogrel sample,clopidogrel increased by32.4%and 70.8%at 4°C for 44 h and 120 h storage conditions after the plasma sample was handled by PPT method,respectively.Based on the above experimental results,the experimental data obtained by the liquid-liquid extraction method is more accurate and reliable.LC-MS/MS method was established for the determination of clopidogrel in human plasma by liquid-liquid extraction.The analytes and internal standards?clopidogrel-d₄?were extracted from plasma by MTBE,and separated on a Venusil C₁₈?4.6×50 mm,3.0?m?column using a isocratic elution procedure.The mobile phase consisted of acetonitrile?0.2%formic acid?and 5 mmol·L^-1 ammonium acetate?0.1%formic acid?at a flow rate of 0.65 m L·min^-1.The total run time was 3.0 min.Positive electrospray ionization was performed using multiple reaction monitoring?MRM?with transitions of m/z 322.3?m/z 212.1 for clopidogrel and m/z 326.3?m/z 216.1 for its internal standard clopidogrel-d₄.The standard curves were linear in the range of 0.02-5.00 ng/m L for clopidogrel.Results The standard curves were linear in the range of 0.0200 to 5.00 ng/mL for clopidogrel.The precision and accuracy values were within the acceptable rang.The matrix effect of clopidogrel were 103%and 98.6%at the low and high concentration,and the RSD was below 2.3%.These results showed that the method is not affected by variations in the sample matrix.Extraction recoveries were generally high and consistent for clopidogrel at the test concentrations,ranging from 72.8%to77.4%.The method was validated and successfully applied to the bioequivalence of two tablet formulations of clopidogrel in healthy Chinese Volunteers.Conclusion An accurate and sensitive liquid chromatography-tandem mass spectrometry?LC-MS/MS?method was developed and validated for the determination of clopidogrel in human plasma.The human plasma samples was handled by liquid-liquid extraction method could avoid the conversion of clopidogrel glucuronide conjugates to clopidogrel,and make sure that the experimental data was reliable.The method was then successfully applied to the bioequivalence of two tablet formulations of clopidogrel in healthy Chinese volunteers.