Microdosing assessment to evaluate pharmacokinetics and drug metabolism in rats using liquid chromatography-tandem mass spectrometry

Purpose. To evaluate the sensitivity requirement for LC-MS/MS as an analytical tool to support human microdosing study with sub-pharmacological dose, investigate proportionality of pharmacokinetics from the microdose to therapeutic human equivalent doses in rats and characterize circulating metabolites in rats administered with the microdose. Materials and Methods. Five drugs of antipyrine, metoprolol, carbamazepine, digoxin and atenolol were administered orally to male Sprague-Dawley rats at 0.167, 1.67, 16.7, 167 and 1,670 mu g/kg doses. Plasma samples were extracted using either solid phase extraction or liquid-liquid extraction, and analyzed using LC-MS/MS. Results. Using 100 mu l of plasma sample, the lower limit of quantitation for antipyrine (10 pg/ml), carbamazepine (1 pg/ml), metoprolol (5 pg/ml), atenolol (20 pg/ml), and digoxin (5 pg/ml) were achieved using an API 5000 (TM). Proportional pharmacokinetics were observed from 0.167 mu g/kg to 1,670 mu g/kg for antipyrine and carbamazepine and from 1.67 to 1,670 mu g/kg for atenolol and digoxin, while metoprolol exhibited a non-proportional pharmacokinetics relationship. Several metabolites of carbamazepine were characterized in plasma from rats dosed at 1.67 mu g/kg using LC-MS/MS. Conclusions. This study has shown the promise of sensitive LC-MS/MS method to support microdose pharmacokinetics and drug metabolism studies in human.