Determination of four omega-3 polyunsaturated fatty acids by UPLC-MS/MS in plasma of hyperlipidemic and normolipidemic subjects

Abstract

BackgroundOmega-3 polyunsaturated fatty acids (PUFAs), including alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA), play critical roles in numerous biochemical reactions. Our aim is to develop a rapid and sensitive method for simultaneous determination of ALA, EPA, DHA and DPA in the plasma of hyperlipidemic and normolipidemic subjects. MethodsAn ultra-high-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method of ALA, EPA, DHA, and DPA was developed with chlorzoxazone as the internal standard (IS). The analytes were separated on an Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm) with gradient elution by acetonitrile and 0.1% ammonia water. ALA, EPA, DHA, DPA, and IS were determined by negative electrospray ionization (ESI-) with multiple reaction monitoring (MRM) at m/z 277.42/259.05, 301.20/257.00, 327.30/283.40, 329.24/285.32, and 168.03/132.02. A total of 80 normolipidemic subjects and 83 hyperlipidemic subjects, who underwent testing for plasma lipids, liver and kidney functions, and blood routine blood test (BRT), were enrolled. ResultsThere was good linearity for ALA within 1–10 μg/mL, and EPA, DHA and DPA were within 0.125–10 μg/mL. The relative standard deviation (RSD) of precision was below 15%. The concentrations of ALA, EPA, DHA and DPA were 3.47 ± 2.58, 0.41 ± 0.26, 2.93 ± 1.39 and 0.25 ± 0.21 μg/mL, respectively, in normolipidemic subjects, increasing to 4.14 ± 3.71, 0.57 ± 0.46, 3.43 ± 2.13, 0.27 ± 0.25 μg/mL, respectively in hyperlipidemic subjects. Among them, only the EPA concentration was significantly different between two groups. There was a high correlation between ALA, EPA, DHA and DPA. ConclusionWe developed a rapid and sensitive method for simultaneously determination of ALA, EPA, DHA and DPA in hyperlipidemic and normolipidemic subjects. In hyperlipidemic and normolipidemic subjects, concentrations of ALA were highest, followed by DHA, EPA and DPA; there were high degrees of correlation between each value.