Development, validation and application of a comprehensive stereoselective LC/MS–MS assay for bupropion and oxidative, reductive, and glucuronide metabolites in human urine

• A stereoselective assay for bupropion and oxidative, reductive, and glucuronide metabolites (16 analytes total) in human urine was developed and validated.
• A trifurcated sample preparation and analysis procedure was used for chiral analysis of (a) free bupropion and primary metabolites, (b) total hydroxybupropion after hydrolysis, and (c) glucuronide metabolites.
• Erythrohydrobupropion and threohydrobupropion glucuronides underwent diastereomer-specific hydrolysis by β-glucuronidase.
• Stereochemistry of glucuronide standards obtained from commercial sources was found to be incorrectly labeled, and the correct stereochemistry was unequivocally reassigned.
• Urine concentration of bupropion and metabolite isomers were determined from a patient who received extended release (R,S)-bupropion.

A stereoselective assay was developed for the quantification of bupropion and oxidative, reductive, and glucuronide metabolites (16 analytes total) in human urine. Initially, authentic glucuronide standards obtained from commercial sources were found to be incorrectly labeled with regard to stereochemistry; the correct stereochemistry was unequivocally reassigned. A trifurcated urine sample preparation and analysis procedure was employed for the stereoselective analysis of bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion enantiomers, and hydroxybupropion, erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers in urine. Method 1 stereoselectively analyzed bupropion (R and S), and unconjugated free hydroxybupropion (R,R and S,S), erythrohydrobupropion (1R,2S and 1S,2R), and threohydrobupropion (1R,2R and 1S,2S) using chiral chromatography with an α1-acid glycoprotein column. Because no hydroxybupropion β-d-glucuronide standards were commercially available, method 2 stereoselectively analyzed total hydroxybupropion aglycones (R,R and S,S-hydroxybupropion) after urine hydrolysis by β-glucuronidase. Hydroxybupropion β-d-glucuronide (R,R and S,S) urine concentrations were calculated as the difference between total and free hydroxybupropion (R,R and S,S) concentrations. Due to incomplete β-glucuronidase hydrolysis of erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers, method 3 stereoselectively analyzed intact erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers using C18 column chromatography. All analytes were quantified by positive ion electrospray tandem mass spectrometry. The assay was fully validated over analyte-specific concentrations. Intra- and inter assay precision were within 15% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), erythrohydrobupropion (1R,2S and 1S,2R) were 10, 50, 100, and 100 ng/mL, respectively. The limits of quantification for (1R,2R)-threohydrobupropion β-d-glucuronide, (1S,2S)-threohydrobupropion β-d-glucuronide, and (1R,2R)-erythrohydrobupropion β-d-glucuronide were each 50 ng/mL. Due to the abundance of bupropion and metabolites in human urine, no efforts were made to optimize sensitivity. All analytes were stable following freeze thaw cycles at −80 °C. This assay was applicable to clinical pharmacokinetic investigations of bupropion in patients and to in vitro metabolism of the primary bupropion metabolites to their glucuronides.