HPLC assay for bupropion and its major metabolites in human plasma

Bupropion is used clinically as an antidepressant and in smoking cessation. As it is metabolised to hydroxybupropion specifically by CYP2B6, bupropion has also been used as a probe to assess CYP2B6 activity. A specific and reproducible HPLC assay has been developed to simultaneously quantify bupropion and its major metabolites hydroxybupropion, threohydrobupropion and erythrohydrobupropion in human plasma. The analysis was performed on an Aqua C18 HPLC column, with a mobile phase consisting of 45:55 of methanol:0.05 M phosphate buffer (pH 5.5) and simultaneous UV detection at 214 nm (bupropion metabolites) and 254 nm (bupropion, internal standard timolol maleate). The assay showed a linear response for bupropion (2.5-250 ng/mL), threohydrobupropion (5-250 ng/mL), erythrohydrobupropion (10-250 ng/mL) and hydroxybupropion (10-1000 ng/mL). Extraction recovery was reproducible and greater than 55% for each analyte. The inter- and intra-day assay variability (measured as percent coefficient of variation; %CV) was less than 15% for all analytes. Limit of quantification was 2.5 ng/mL for bupropion, 5 ng/mL for threohydrobupropion and 10 ng/mL for hydroxybupropion and erythrohydrobupropion. This assay is more sensitive than currently published methods using HPLC with UV detection for the simultaneous quantitation of bupropion and metabolites and can be used for assessing CYP2B6 activity in vivo following a single dose of bupropion.