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 -carotene and the bioconversion of -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]--carotene at physiological doses to children between 8 and 11 years of age, blood samples were drawn and serum was prepared. Retinol and -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 -carotene required. APCI LC-MS showed a linear detector response for -carotene over 4 orders of magnitude. Using selected ion monitoring to record the elution profile of protonated circulating -carotene at m/z 537 and [13C10]--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]--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 -carotene were calculated. Combined with standard HPLC measurement of -carotene and retinol concentration and a mathematical model, these results showed that this simple LC-MS method can be used to quantify -carotene bioavailability and its bioconversion to retinol at physiologically relevant doses.
Yan Wang, Xiaoying Xu, van Lieshout, M., West, C. E., Lugtenburg, J., Verhoeven, M. A., & Creemers, A. F. L. (2000). A liquid chromatography-mass spectrometry method for the quantification of bioavailability and bioconversion of beta-carotene to retinol in humans. Analytical Chemistry, 72(20), 4999-5003. https://doi.org/10.1021/ac000454e