A sensitive procedure for the study of beta-carotene-d8 metabolism in humans using high performance liquid...

This report describes the development of a robust method of high sensitivity for studying the metabolism of beta-carotene-d8 in humans using a combination of liquid chromatography/particle beam-mass spectrometry (LC/PB-MS). The utility of the LC/PB-MS method was demonstrated in a pilot study. The carotenoids were extracted from plasma into hexane and separated by reverse phase high performance liquid chromatography (HPLC) using a C-18 column. The HPLC effluent was nebulized using helium and the solvent was removed under vacuum within the dual-stage particle beam interface. The de-solvated carotenoids were ionized in the negative-ion mode (electron capture) using methane chemical ionization and detected using selected ion monitoring. The limit of detection of the method was on the order of 0.3 ng (approximately 0.6 pmol) for beta-carotene. beta-Carotene-d8 was quantified in the plasma over a concentration range of two orders of magnitude using beta-carotene-(13)C(40) as an internal standard. The overall coefficient of variance (CV) for determining the concentration of the analytes from 30 microliter of plasma was 3.9% for beta-carotene and 2.4% for beta-carotene-d8. Using the LC/PB-MS method, the concentration of beta-carotene-d8 was determined in the plasma of a subject who had consumed a single 5-mg dose over a 30-day period. The sensitive semi-automated procedure is capable of high sample throughput and makes large comprehensive studies feasible.