High‐resolution isotope‐dilution mass spectrometry using metabolism of isotope‐labeled compounds: Application to drug metabolites

RATIONALE: Herein we describe a generic quantitative method using high-resolution, isotope-dilution (HRID) metabolism of isotope-labeled compounds and apply it to the analysis of drug metabolites (DMs) in human plasma. Metabolites (drug) in Safety Testing (MIST) application was one goal. METHODS: Testosterone (T) and diclofenac (D) were chosen for mass defect characteristics. T, [(14)C]T, [(13)C(3)]T, D, [(14)C]D, and [(13)C(6)]D were metabolized separately in vitro to produce test metabolites. Liquid chromatography/radioactivity monitoring (LC/RAM) analysis was used to determine the concentration of the test metabolites in the incubates. The incubates containing 6β-hydroxy-T (6βHT), [(13)C(3)]6βHT, 4'-hydroxy-D (4'HD) and [(13)C(6)]4'HD were used to make standard curves. Plasma samples were prepared by 'dilute-and-shoot' and analyzed by LC/MS using SCIEX 5000 and Thermo Orbitrap instrumentation. RESULTS: Human hepatic microsomes and the S9 fraction produced between 2-6 μM β-hydroxy-T and 4'-hydroxy-D at 60 min starting with 10 μM parent drug as determined by LC/RAM. It was assumed that the amounts of [(13)C(3)]6βHT and [(13)C(6)]4'HD produced were similar. Dilutions and standard curves were prepared in human plasma. Analysis of the DMs by LC/MS/MS and LC/HRMS exhibited linear responses over a useable range. CONCLUSIONS: HRID with metabolism of an isotope-labeled compound reduces the number of analytical variables considerably. Metabolism of the parent drug to DMs represents a simpler alternative quantitative method compared with traditional approaches. The method will have useful applications for evaluating MIST situations.