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

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 et al. J Lipid Res. 2012 Apr.

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.

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Figures

Fig. 1.

Fig. 1.

Chemical structure, formula, and formula weights of the stable isotope-labeled and native β-carotene and retinol analyzed in this study.

Fig. 2.

Fig. 2.

HPLC-MS standard curves for d8-β-carotene (m/z 543.5 + 544.5; top) and d4-retinyl acetate (273 > 94,217; bottom). Insets show linearity of curves at the lowest three concentrations used. The y axis represents the ratio of peak area of the analyte to the internal standard. Amounts of internal standards added were 2.6 × 10−11 moles of 13C-β-carotene for the d8-β-carotene analysis and 7.3 × 10−11 moles of 12C- retinyl acetate for the d4-retinyl acetate analysis.

Fig. 3.

Fig. 3.

Representative chromatograms of the CM fraction sample at h 6 of one of the subjects analyzed for β-carotene. The four chromatograms, from top to bottom, are the absorbance at 452 nm, m/z 576.5 for 13C40-β-carotene stable isotope internal standard, m/z 543.5 +544.5 for deuterium labeled β-carotene, and m/z 536.5 for native β-carotene and lycopene isomers.

Fig. 4.

Fig. 4.

Representative chromatograms for retinyl linoleate (RL), retinyl oleate (RO), retinyl palmitate (RP), and retinyl stearate (RS) in the CM fraction. The top chromatogram shows the absorbance at 325 nm, the middle chromatogram shows the native retinyl esters as 269 > 93,107 MS/MS transitions, and the bottom chromatogram shows the d4-retinyl esters as 273 > 94,217 transitions.

Fig. 5.

Fig. 5.

Quantitative HPLC-MS of retinoids in a CM fraction. The top panel shows the total d4-retinyl esters (d4 Total) and d8-βC (d8bcar) whereas the bottom panel contains the individual d4-retinyl esters (RL, retinyl linoleate; RO, retinyl oleate; RP, retinyl palmitate; RS, retinyl stearate) and d8-βC for the same subject. Individual retinyl esters followed the same trend as the total retinyl esters and β-carotene with the peak concentrations of both d4-RE and d8-βC at 5–6 h postdose.

Fig. 6.

Fig. 6.

Conversion of d8-β-catotene to d4-retinyl esters as quantified by HPLC-MS. The upper panel shows an extract of human whole plasma and the bottom plot shows the corresponding CM-rich fraction. Both are monitored for d-8-β-carotene (d8bcar), d-4-retinyl palmitate (d4RP), and d4-retinol (d4Rol) by m/z 543.5 + 544.5, and 273 > 94,217 MS/MS transitions.

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