A Liquid Chromatography−Mass Spectrometry Method for the Quantification of Bioavailability and Bioconversion of β-Carotene to Retinol in Humans

A method based on high-performance liquid chromatography−atmospheric pressure chemical ionization mass spectrometry (APCI LC−MS) was developed for the quantification of the bioavailability of retinyl palmitate and β-carotene and the bioconversion of β-carotene to retinol in humans. Following oral administration of [8,9,10,11,12,13,14,15,19,20-13C10]-retinyl palmitate and [12,13,14,15,20,12‘,13‘,14‘,15‘,20‘-13C10]-β-carotene at physiological doses to children between 8 and 11 years of age, blood samples were drawn and serum was prepared. Retinol and β-carotene were extracted from 0.2- and 1.0-mL serum samples, respectively, and analyzed using reversed-phase HPLC with a C30 column interfaced to an APCI mass spectrometer. Unlike other LC−MS assays for carotenoids, no additional purification steps were necessary, nor was any derivatization of retinol or β-carotene required. APCI LC−MS showed a linear detector response for β-carotene over 4 orders of magnitude. Using selected ion monitoring to record the elution profile of protonated circulating β-carotene at m/z 537 and [13C10]-β-carotene at m/z 547, the limit of detection was determined to be 0.5 pmol injected on-column. To assess the ratio of labeled to unlabeled retinol, selected ion monitoring was carried out at m/z 269, 274, and 279. These abundant fragment ions corresponded to the loss of water from the protonated molecule of circulating retinol, [13C5]-retinol (metabolically formed from orally administered [13C10]-β-carotene), and [13C10]-retinol (formed by hydrolysis of [13C10]-retinyl palmitate). The ratios of labeled to unlabeled retinol and the ratio of labeled to unlabeled β-carotene were calculated. Combined with standard HPLC measurement of β-carotene and retinol concentration and a mathematical model, these results showed that this simple LC−MS method can be used to quantify β-carotene bioavailability and its bioconversion to retinol at physiologically relevant doses.