Performance of Human Mass Balance/Metabolite Identification Studies Using Stable Isotope (13C, 15N) Labeling and Continuous-Flow Isotope-Ratio Mass Spectrometry as an Alternative to Radioactive Labeling Methods


Neurology Service (127), V.A. Medical Center, 150 S. Huntington Avenue, Boston, MA 02130.


Stable isotope labeling in therapeutic and subtherapeutic quantities of drug (15N213C-phenobarbital) can be quantitated in biological matrices (urine) and high performance liquid chromatography (HPLC) peaks from urine using continuous-flow isotope-ratio mass spectrometry (CF-IRMS). Standard curves for 15N213C-phenobarbital were reproducible and linear (R2 > 0.985) over the ranges of 3–100 μg/mL for whole urine (1SN2 or 13C labeling) and 0.1–8.0 μg/mL for HPLC peaks derived from urine (15N2 labeling). The lower limit of quantitation values for urine drug concentration was 0.46–2.62 μg/mL in whole urine and 0.10–0.70 μg/mL in HPLC peaks. Validation samples quantitated with these standard curves yielded close to expected values. These data suggest stable isotope labeling and CF-IRMS may be used as an alternative to 14C labeling and radioactivity counting methods in mass balance/metabolite identification and other biomedical studies.