Assessment of critical extraction and chromatographic parameters for the determination of bupropion and its three primary metabolites in human plasma by LC-MS/MS

Optimization of extraction and chromatographic conditions is vital for the analysis of drugs and their metabolites in biological samples, especially for in vivo applications. Herein, we report a sensitive, specific and high throughput bioanalytical assay for reliable concurrent estimation of bupropion (BUP) and its major metabolites, hydroxybupropion (HBUP), erythrohydrobupropion (EHBUP) and threohydrobupropion (THBUP) in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample clean-up was optimized using solid-phase extraction after prior acidification with ortho-phosphoric acid. Critical parameters like washing and elution solvents, which play a major role in extraction efficiency, were systematically evaluated to obtain consistent and quantitative recovery for all the analytes. The extraction recoveries obtained were in the range of 95.4-99.8% for all the analytes. Chromatographic conditions were tested on columns with different polarities under isocratic and gradient conditions. Ascentis RP-Amide (100 × 4.6 mm, 5 μm) column afforded complete separation of the analytes within 5.0 min under gradient elution using acetonitrile and 5 mM ammonium formate (pH 3.5) as the mobile phase. The resolution factor between BUP & HBUP and EHBUP & THBUP was 2.7 and 3.1 respectively. Calibration curves were linear in the concentration range of 0.10-250, 0.10-300, 0.10-100 and 0.25-500 ng mL− 1 for BUP, HBUP, EHBUP and THBUP, respectively using 100 μL plasma. The method was validated for sensitivity, selectivity, accuracy, precision, matrix effect, recovery, stability, ruggedness and was used to analyze subject samples obtained after oral administration of BUP.