An LC/MS method for d8-β-carotene and d4-retinyl esters: β-carotene absorption and its conversion to vitamin A in humans

Matthew K Fleshman,Ken M Riedl,Janet A Novotny,Steven J Schwartz,Earl H Harrison

doi:10.1194/jlr.d021139

Abstract

The intestinal absorption and metabolism of β-carotene is of vital importance in humans, especially in populations that obtain the majority of their vitamin A from provitamin A carotenoids. MS has provided a better understanding of the absorption of β-carotene, the most potent provitamin A carotenoid, through the use of stable isotopes of β-carotene. We report here an HPLC-MS method that eliminates the need for complicated sample preparation and allows us to detect and quantify newly absorbed d8-β-carotene as well as its d4-retinyl ester metabolites in human plasma and chylomicron fractions. Both retinoids and β-carotene were recovered in a single simple extraction that did not involve saponification, thus allowing subsequent quantitation of individual fatty acyl esters of retinol. Separation of d8-β-carotene and its d4-retinyl ester metabolites was achieved using the same C30 reversed-phase liquid chromatography followed by mass spectrometry in selected ion monitoring and negative atmospheric pressure chemical ionization modes, respectively. Total time for the two successive runs was 30 min. This HPLC-MS method allowed us to quantify the absorption of intact d8-β-carotene as well as its extent of conversion to d4-retinyl esters in humans after consumption of a single 5 mg dose of d8-β-carotene.

Highlights

The intestinal absorption and metabolism of ␤carotene is of vital importance in humans, especially in populations that obtain the majority of their vitamin A from provitamin A carotenoids
Blood was collected hourly for 9 h and enrichment of chylomicrons with d8-␤-carotene and d4-retinyl esters was determined by HPLC-MS
We optimized the method for ␤-carotene and were able to detect lycopene as the same mass ion in selected ion monitoring (SIM) but it is not optimized for lycopene detection and other MS modes are more sensitive for lycopene quantification

Summary

Introduction

The intestinal absorption and metabolism of ␤carotene is of vital importance in humans, especially in populations that obtain the majority of their vitamin A from provitamin A carotenoids. We report here an HPLC-MS method that eliminates the need for complicated sample preparation and allows us to detect and quantify newly absorbed d8-␤carotene as well as its d4-retinyl ester metabolites in human plasma and chylomicron fractions. Both retinoids and ␤carotene were recovered in a single simple extraction that did not involve saponification, allowing subsequent quantitation of individual fatty acyl esters of retinol. Total time for the two successive runs was 30 min This HPLC-MS method allowed us to quantify the absorption of intact d8-␤-carotene as well as its extent of conversion to d4-retinyl esters in humans after consumption of a single 5 mg dose of d8-␤-carotene.— Fleshman, M.

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Conclusion