Abstract
Alpha-linolenic acid (ALA), an omega-3 fatty acid essential for humans, must be supplied by diet, mainly from plants. It's a precursor of omega-3 long-chain fatty acids, eicosapentaenoic and docosahexaenoic acids which are essential for human cardiovascular health. ALA consumption may also reduce the risk of heart disease. The geometric isomers of ALA in humans are derived from consumption of industrially produced partially hydrogenated and deodorized vegetable oils. The presence of trans-isomers of ALA in humans may affect the levels of regular ALA, potentially impacting the beneficial effects of ALA. Some studies have reported geometric isomers of ALA in human milk and serum but demonstrate incomplete chromatographic separation. The objective of this work is to develop a method to separate geometric isomers of ALA in human plasma using silver ion high-performance liquid chromatography (Ag-HPLC) and detect them using gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS). Sample preparation involves acidic and alkaline hydrolysis of samples, followed by extraction of free fatty acids with hexane. Ag-HPLC is then employed for isomer separation, HPLC fractions containing each isomer are collected, and finally the samples are analyzed by GC-NCI-MS. Preliminary data shows all eight geometric isomers of ALA (n-3t6t9t, n-3t6t9c, n-3c6t9t, n-3t6c9t, n-3t6c9c, n-3c6t9c, n-3c6c9t, and n-3c6c9c) in plasma can be separated using this approach. Ag-HPLC separation of ALA isomers in human plasma resulted in the same elution times as the isomers in a standard mixture. The identities of the isomers were confirmed by comparing their retention times and mass-to-charge ratios with those of ALA isomer standards via GC-NCI-MS. This approach can potentially be used to investigate the distribution of ALA geometric isomers in human plasma. The full resolution of ALA geometric isomers can ensure accurate identification of these isomers in human plasma, which may aid in understanding their impact on human health.
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