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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.