Simultaneous quantification of bupropion and hydroxybupropion in human plasma by UA-DLLME-LC-MS/MS with central...

A sensitive and selective LC-MS/MS bioanalytical method was developed, optimized, and validated for the simultaneous quantification of bupropion and hydroxybupropion in human plasma using ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) as the sample preparation technique. Four critical UA-DLLME parameters - extraction solvent volume, disperser solvent volume, sample pH, and ultrasonication time - were simultaneously optimized using Central Composite Design and response surface methodology, yielding extraction recoveries of 93.8 ± 1.2% and 95.6 ± 0.9% for bupropion and hydroxybupropion, respectively. Chromatographic separation of both analytes and the internal standard carbamazepine was achieved on an Agilent Poroshell 120 EC-C18 column (50 × 4.6 mm, 2.7 μm) using an isocratic mobile phase of 10 mM ammonium formate (pH 4.0) and acetonitrile (35:65, v/v) at 0.4 mL/min, with a total runtime of 5 min. Detection was performed by triple quadrupole mass spectrometry in positive electrospray ionization mode using multiple reaction monitoring transitions of m/z 240.1 → 184.1 for bupropion and m/z 256.1 → 238.1 for hydroxybupropion. Comprehensive validation following ICH M10 guideline recommendations demonstrated linearity across 2-1000 ng/mL, LLOQ of 2 ng/mL, intraday and interday precision within 14.13% CV, accuracy within ±8.92% RE, and stability under all evaluated conditions for both analytes. The method was successfully applied to a single-dose pharmacokinetic study in five healthy volunteers following bupropion SR 150 mg administration. The assay measures total (non-stereoselective) hydroxybupropion, and the hydroxybupropion/bupropion AUC metabolic ratios of 6.55-11.68 serve as surrogate indices of CYP2B6 hydroxylation activity, classifying all subjects as CYP2B6 extensive metabolizers and demonstrating the method's utility for CYP2B6 phenotyping and precision pharmacotherapy applications.