Simultaneous quantitation of hydrochlorothiazide and metoprolol in human plasma by liquid chromatography-tandem mass...

A rapid and sensitive method for the simultaneous quantitation of hydrochlorothiazide (HCT) and metoprolol (MET) in human plasma based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. MS/MS detection involved switching the electrospray ionization (ESI) mode during chromatography from negative to detect HCT and its internal standard (I.S.) 5-bromouracil to positive to detect MET and its I.S. tramadol. Sample preparation by liquid-liquid extraction with diethyl ether-dichloromethane (60:40, v/v) was followed by chromatography on a Venusil MP-C18 column using methanol-ammonium acetate (10mM)-formic acid (pH 3.4) (50:50:0.05, v/v/v) at a flow rate of 0.8mL/min. The method was linear in the concentration range 3-1000ng/mL for both HCT and MET using 100microL human plasma. Intra- and inter-day precisions (as relative standard deviation) for HCT were 2.9-3.9% and 3.9-4.7%, respectively and for MET were 2.4-4.1% and 4.7-6.2%, respectively. Accuracies (as relative error) were +/-3.8% and +/-2.6% for HCT and MET, respectively. The assay was successfully applied to a pharmacokinetic study involving a single oral dose of a combination tablet (25mg HCT, 50mg MET) in healthy volunteers.