Stable isotope- and mass spectrometry-based metabolomics as tools in drug metabolism: A study expanding tempol pharmacology

The application of mass spectrometry-based metabolomics in the field of drug metabolism has yielded important insights not only into the metabolic routes of drugs but has provided unbiased, global perspectives of the endogenous metabolome that can be useful for identifying biomarkers associated with mechanism of action, efficacy, and toxicity. In this report, a stable isotope- and mass spectrometry-based metabolomics approach that captures both drug metabolism and changes in the endogenous metabolome in a single experiment is described. Here the antioxidant drug tempol (4-hydroxy-2,2,6,6- tetramethylpiperidine-N-oxyl) was chosen because its mechanism of action is not completely understood and its metabolic fate has not been studied extensively. Furthermore, its small size (MW = 172.2) and chemical composition (C ₉H₁₈NO₂) make it challenging to distinguish from endogenous metabolites. In this study, mice were dosed with tempol or deuterated tempol (C₉D₁₇HNO₂) and their urine was profiled using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Principal component analysis of the urinary metabolomics data generated a Y-shaped scatter plot containing drug metabolites (protonated and deuterated) that were clearly distinct from the endogenous metabolites. Ten tempol drug metabolites, including eight novel metabolites, were identified. Phase II metabolism was the major metabolic pathway of tempol in vivo, including glucuronidation and glucosidation. Urinary endogenous metabolites significantly elevated by tempol treatment included 2,8-dihydroxyquinoline (8.0-fold, P < 0.05) and 2,8-dihydroxyquinoline- β-d-glucuronide (6.8-fold, P < 0.05). Urinary endogenous metabolites significantly attenuated by tempol treatment including pantothenic acid (1.3-fold, P < 0.05) and isobutrylcarnitine (5.3-fold, P < 0.01). This study underscores the power of a stable isotope- and mass spectrometry-based metabolomics in expanding the view of drug pharmacology.