STABLE-ISOTOPE METHODS FOR THE STUDY OF BETA-CAROTENE-D(8) METABOLISM IN HUMANS UTILIZING TANDEM MASS-SPECTROMETRY AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

This report presents analytical methods for the isolation and quantification of all-trans-beta-carotene-d(8) in human plasma following a 73 mu mol oral dose. Retinol-d(4) derived from beta-carotene-d(8) was also determined in the same plasma. Plasma samples drawn over a 24 day period were analyzed. beta-Carotene and retinol were isolated and purified for analysis using a solid phase extraction protocol with aminopropyl-bonded silica sorbent. Ratios of beta-carotene-d(8)/beta-carotene were determined using reversed-phase HPLC with spectrophotometric detection, which fully resolved the isotopomers, and by tandem mass spectrometry (MS/MS) with electron ionization. Results obtained from MS/MS and HPLC analysis showed close agreement and demonstrated improved selectivity relative to analysis using a single mass analyzer. Retinol-d(4) was converted to its tert-butyldimethylsilyl ether and analyzed by gas chromatography/mass spectrometry using selected ion monitoring. The ability to resolve the beta-carotene isotopomers by HPLC makes it feasible for investigators without mass spectrometers to investigate the dynamics of absorption and metabolism of beta-carotene-d(8) in humans.