An HPLC-MS/MS method for the separation of α-retinyl esters from retinyl esters

Enzymatic cleavage of the nonsymmetric provitamin A carotenoid a.-carotene results in one molecule of retinal (vitamin A), and one molecule of alpha-retinal, a biologically inactive analog of true vitamin A. Due to structural similarities, alpha-retinyl esters and vitamin A esters typically coelute, resulting in the overestimation of vitamin A originating from alpha-carotene. Herein, we present a set of tools to identify and separate alpha-retinol products from vitamin A. alpha-Retinyl palmitate (alpha RP) standard was synthesized from alpha-ionone following a Wittig-Horner approach. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method employing a C30 column was then developed to separate the species. Authentic standards of retinyl esters and the synthesized alpha-RP confirmed respective identities, while other alpha-retinyl esters (i.e. myristate, linoleate, oleate, and stearate) were evidenced by their pseudo molecular ions observed in electrospray ionization (ESI) mode, fragmentation, and elution order. For quantitation, an atmospheric pressure chemical ionization (APCI) source operated in positive ion mode was used, and retinol, the predominant in-source parent ion was selected and fragmented. The application of this method to a chylomicron-rich fraction of human plasma is demonstrated. This method can be used to better determine the quantity of vitamin A derived from foods containing alpha-carotene. (C) 2016 Elsevier B.V. All rights reserved.