A Novel and High-throughput Method for the Simultaneous Determination of Amoxicillin and Clavulanic Acid in Human Plasma by Liquid Chromatography Coupled with Tandem Mass Spectrometry

A simple, rapid, specific and sensitive liquid chromatography tandem mass spectrometric method has been developed and validated for the simultaneous determination of Amoxicillin and Clavulanic acid from 250μL of human plasma by Solid Phase Extraction. Hydrochlorothiazide was used as an internal standard. Quantified by the transition, 364.060→223.160, 198.061→136.000 for Amoxicillin and Clavulanic acid respectively (Fig 1 and 2) and detected by TSQ Quantum Discovery max triple quadrupole mass spectrometer. Detection was carried out by using ESI source in negative polarity. Chromatographic separation of analyte and internal standard were carried out by reverse phase C18 column at the flow rate of 0.5mL/min with mobile phase of Acetonitrile: 2 mM Ammonium Acetate (70:30) v/v. The assay of Amoxicillin and Clavulanic Acid were linear over the range of 0.103μg/mL to 6.822μg/mL and 0.046μg/mL to 3.026μg/mL respectively with a precision of ≤9.43 % and ≤11.75 % respectively, Mean extraction recovery obtained were 82.04% and 87.14% respectively. Samples were stable at room temperature for 6 hrs and also stable at three freeze-thaw cycle. The method has been used to perform pharmacokinetic and bioequivalence studies in human plasma.