Development and Validation of a LC⁻MS/MS-Based Assay for Quantification of Free and Total Omega 3 and 6 Fatty Acids from Human Plasma

Abstract

Few high-performance liquid chromatography⁻tandem mass spectrometry (LC-MS/MS) methods have been developed for the full quantitation of fatty acids from human plasma without derivatization. Therefore, we propose a method that requires fewer sample preparation steps, which can be used for the quantitation of several polyunsaturated fatty acids in human plasma. The method offers rapid, accurate, sensitive, and simultaneous quantification of omega 3 (α-linolenic, eicosapentaenoic, and docosahexaenoic acids) and omega 6 fatty acids (arachidonic and linoleic acids) using high-performance LC-MS/MS. The selected fatty acids were analysed in lipid extracts from both free and total forms. Chromatographic separation was achieved using a reversed phase C18 column with isocratic flow using ammonium acetate for improving negative electrospray ionization (ESI) response. Mass detection was performed in multiple reaction monitoring (MRM) mode, and deuterated internal standards were used for each target compound. The limits of quantification were situated in the low nanomolar range, excepting linoleic acid, for which the limit was in the high nanomolar range. The method was validated according to the U.S. Department of Health and Human Services guidelines, and offers a fast, sensitive, and reliable quantification of selected omega 3 and 6 fatty acids in human plasma.

Keywords: C18 column; MRM; PUFA; high-performance liquid chromatography; human plasma; metabolomics; negative electrospray ionization; polyunsaturated fatty acids; tandem mass spectrometry.

Figure 1

Multiple reaction monitoring (MRM) chromatograms of ALA, ALA-d14, ARA, ARA-d8, DHA, DHA-d5, EPA, EPA-d5, LA, and LA-d4 standards at concentration of 0.016, 0.2, 0.08, 0.1, 0.016, 0.1, 0.016, 0.1, 0.08, and 0.2 µg/mL respectively. Legend: ALA—α-linolenic acid, ARA—arachidonic acid, DHA—docosahexaenoic acid, EPA—eicosapentaenoic acid, LA—linoleic acid.

Figure 2

The overlaid total ion chromatograms of the targeted fatty acids. Data was obtained following analysis of a plasma sample (total FAs content). Legend: ALA—α-linolenic acid, ARA—arachidonic acid, DHA—docosahexaenoic acid, EPA—eicosapentaenoic acid, LA—linoleic acid.