Submitted to: Journal of Lipid Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 17, 2000
Publication Date: May 1, 2000
Citation: Pawlosky, R.J., Flanagan, V.P., Novotny Dura, J. 2000. A sensitive procedure for the study of beta-carotene-d8 metabolism in humans using liquid chromatography-mass spectrometry. Journal of Lipid Research. 41:1027-1031. Interpretive Summary: This report describes the development of a robust method for studying the metabolism of (stable hydrogen) deuterium- labeled-Beta-carotene in humans using liquid chromatography and mass spectrometry. Deuterium-labeled-Beta-carotene was extracted from human blood and injected onto a liquid chromatograph. The limit of detection was about 0.3 ng for deuterium-labeled-Beta-carotene. The compound was quantified over a concentration range of two orders of magnitude using an internal standard. The overall coefficient of variance (CV) for determining the concentration of deuterium-labeled-Beta-carotene from blood was 2.4%. Using this method, the concentration of deuterium-labeled-Beta-carotene was determined in the plasma of a subject who had consumed a single 5 mg dose over a thirty day period. The simplified experimental design has both high sensitivity and a high sample through-put. Researchers who study human nutrition especially those interested in how much vitamin-A can be made from Beta- carotene in humans will use this information to help them plan studies in different human populations.
Technical Abstract: This report describes the development of a robust method for studying the metabolism of B-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 solvent was removed under vacuum within the dual-stage particle-beam interface. The de- solvated carotenoids were ionized in the negative-ion mode 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 B-carotene. B-Carotene-d8 was quantified in the plasma over a concentration range of two orders of magnitude using B-carotene-13C40 as an internal standard. The overall coefficient of variance (CV) for determining the concentration of the analytes from 30 ul of plasma was 3.9% for b-carotene and 2.4% for B-carotene-d8. The simplified experimental design having both high sensitivity and a high sample through-put makes large comprehensive studies feasible.