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

作者：Teitelbaum Aaron M; Flaker Alicia M; Kharasch Evan D^*

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2016, 1027: 239-253.

DOI：10.1016/j.jchromb.2016.05.036

• Bupropion
• Urine
• LC-MS/MS
• Analytical method
• Stereoselectivity

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 beta-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 alpha(1)-acid glycoprotein column. Because no hydroxybupropion beta-D-glucuronide standards were commercially available, method 2 stereoselectively analyzed total hydroxybupropion aglycones (R,R and S,S-hydroxybupropion) after urine hydrolysis by beta-glucuronidase. Hydroxybupropion beta-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 beta-glucuronidase hydrolysis of erythrohydrobupropion and threohydrobupropion beta-D-glucuronide diastereomers, method 3 stereoselectively analyzed intact erythrohydrobupropion and threohydrobupropion beta-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 beta-D-glucuronide, (1S,2S)-threohydrobupropion beta-D-glucuronide, and (1R,2R)-erythrohydrobupropion (beta-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 degrees 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.

• 出版日期2016-8-1