COMPOUND-SPECIFIC CARBON-ISOTOPE RATIO DETERMINATION OF ENRICHED CHOLESTEROL

On-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) was evaluated for use in stable isotope studies of human cholesterol metabolism. Calibration curves were constructed by analyzing underivatized and TMS-cholesterol over a range of C-13 abundances from natural abundance to 0.05 mol of [3,4-C-13]cholesterol per mole of unlabeled cholesterol (delta C-13-24 to 270 parts per thousand). The calibration curves were highly linear (r2 = 0.99) with slopes of 0.98 and 0.88 for underivatized and TMS-cholesterol, respectively. Higher precision of C-13/C-12 ratio determination was obtained using TMS-cholesterol, with a detection limit (2 SD) of 0.3 parts per thousand or 0.004 mol% of [3,4-C-13]cholesterol. By comparison standard GC/MS had a detection limit of 0.06 mol% for (3,4-C-13]cholesterol. A 57-day study of cholesterol elimination was conducted in one human subject with oral administration of 50 mg of [3,4-C-13]cholesterol. Peak enrichment was 9 parts per thousand above baseline (40 times the detection limit) and could be followed for 40-50 days. The results indicate that GC/C/IRMS provides 15-fold lower detection limits for [3,4-C-13]cholesterol than GC/MS and is useful for human studies of cholesterol metabolism.