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 beta-carotene and the bioconversion of beta-carotene to retinol in humans. Following oral administration of [8,9,10,11,12,13,14,15,19,20-C-13(10)]-retinyl palmitate and [12,13,14,15,20,12',13',14',15',20'-C-13(10)]-beta-carotene at physiological doses to children between 8 and 11 years of age, blood samples were drawn and serum was prepared. Retinol and beta-carotene were extracted from 0.2- and 1.0-mL serum samples, respectively, and analyzed using reversed-phase HPLC with a C-30 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 beta-carotene required. APCI LC-MS showed a linear detector response for beta-carotene over 4 orders of magnitude. Using selected ion monitoring to record the elution profile of protonated circulating beta-carotene at m/z 537 and [C-13(10)]-beta-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, [C-13(5)]-retinol (metabolically formed from orally administered [C-13(10)]-beta-carotene), and [C-13(10)]-retinol (formed by hydrolysis of [C-13(10)]-retinyl palmitate); The ratios of labeled to unlabeled retinol and the ratio of labeled to unlabeled beta-carotene were calculated. Combined with standard HPLC measurement of beta-carotene and retinol concentration and a mathematical model, these results showed that this simple LC-MS method can be used to quantify beta-carotene bioavailability and its bioconversion to retinol at physiologically relevant doses.