Analysis of Phytostanyl Fatty Acid Esters in Enriched Foods via UHPLC-APCI-MS

Abstract

A method for the analysis of phytostanyl fatty acid esters, the functional ingredients of cholesterol-lowering enriched foods, was developed. The procedure is based on (i) separation of the intact esters via reversed-phase ultrahigh-performance liquid chromatography; (ii) detection by atmospheric pressure chemical ionization-mass spectrometry; and (iii) quantitation using selected ion monitoring (SIM) mode. Employing a C8 column, phytostanyl fatty acid esters sharing the same stanol nucleus could be separated according to the esterified fatty acids while esters with different stanol moieties could be distinguished via SIM based on the formation of an intense fragment ion [M - fatty acid + H](+). The suitability of the approach was demonstrated using different types of enriched foods reflecting the diversity in potential matrices (skimmed milk drinking yogurt, margarine, and soft-cheese-style spread). The developed methodology extends the analytical basis for authenticity and quality assessments of functional foods enriched with phytostanyl fatty acid esters.

Heating Two Types of Enriched Margarine: Complementary Analysis of Phytosteryl/Phytostanyl Fatty Acid Esters and Phytosterol/Phytostanol Oxidation Products

“…The compositions of the two mixtures have been previously described. 26,27 Reference Compounds of Sitostanol Oxidation Products. Reference compounds of 6α-hydroxy-and 7-hydroxysitostanol were obtained via semipreparative HPLC separation of a thermo-oxidized sitostanol standard.…”

“…27 Identification of the individual esters was based on relative retention times and mass spectrometric data and quantitation on the generation of calibration functions. 26,27 All analyses were performed in triplicate.…”

“…18 The lack of further data on the changing contents of individual intact phytosteryl and/or phytostanyl fatty acid esters might be due to a long-time unavailability of analytical methodologies allowing their quantitation in complex mixtures. Recently, novel LC-APCI-MS-based approaches enabling for the first time the quantitative analysis of mixtures of phytostanyl fatty acid esters 26 and of phytosteryl and phytostanyl fatty acid esters 27 on an individual basis in their intact forms in enriched foods have been described. In addition, an online LC-GC-based methodology for the analysis of POPs has been developed.…”

Mass Spectrometric Approaches for the Analysis of Phytosterols in Biological Samples

“…However, acid hydrolysis is required when steryl glycosides (SG) and acylated steryl glucosides (ASG) are present as the acetal bond between the sterol hydroxyl and the sugar moiety is resistant to base hydrolysis 118,119 . Both free phytosterols (FS) and conjugated phytosterols (SG, ASG, fatty acid steryl esters-SE) have been isolated and quantified in functional foods (spreads, beverages) and cereals (spelt, wheat) 108,112,120 . The quantification was achieved in the SIM mode by monitoring [M+H-H2O] + , [M-FA+H] + , [M-FA-sugar+H] + , and [M-sugar+H] + for FS, SE, ASG, and SG respectively, where FA represents a fatty acid moiety.…”

Direct Analysis Mass Spectrometry

“…In the wider area of LC-APCI-MS, a large number of investigations have focused on identifying particular contaminants [27][28][29] or quantification of ingredients [30,31].…”