Abstract
Analysis of fatty acids (FA) in food and biological samples such as blood is indispensable in modern life sciences. We developed a rapid, sensitive and comprehensive method for the quantification of 41 saturated and unsaturated fatty acids by means of LC-MS. Optimized chromatographic separation of isobaric analytes was carried out on a C8 reversed phase analytical column (100 × 2.1 mm, 2.6 μm core–shell particle) with a total run time of 15 min with back pressure lower than 300 bar. On an old triple quadrupole instrument (3200, AB Sciex), pseudo selected reaction monitoring mode was used for quantification of the poorly fragmenting FA, yielding limits of detection of 5–100 nM. Sample preparation was carried out by removal of phospholipids and triglycerides by solid-phase extraction (non-esterified fatty acids in oils) or saponification in iso-propanol (fatty acyls). This is not only a rapid strategy for quantification of fatty acyls, but allows the direct combination with the LC-MS-based analysis of fatty acid oxidation products (eicosanoids and other oxylipins) from the same sample. The concentrations of fatty acyls determined by means of LC-MS were consistent with those from GC-FID analysis demonstrating the accuracy of the developed method. Moreover, the method shows high precisions with a low intra-day (≤ 10% for almost all fatty acids in plasma and ≤ 15% in oils) and inter-day as well as inter-operator variability (< 20%). The method was successfully applied on human plasma and edible oils. The possibility to quantify non-esterified fatty acids in samples containing an excess of triacylglycerols and phospholipids is a major strength of the described approach allowing to gain new insights in the composition of biological samples.Graphical abstract
Highlights
Fatty acids play a fundamental role in the biology of living organisms, e.g., by influencing properties of biomembranes, storing and providing energy, or being involved in cell signaling [1,2,3]
Fatty acyls are often analyzed by means of gas chromatography coupled to flame ionization detection (GC-FID) or mass spectrometry (GC-MS) due to the high separation efficiency and good sensitivity of GC [9,10,11]
The method was applied to the analysis of fatty acyls as well as non-esterified fatty acids (NEFA) in plasma and in plant oils
Summary
Fatty acids play a fundamental role in the biology of living organisms, e.g., by influencing properties of biomembranes, storing and providing energy, or being involved in cell signaling [1,2,3]. Long-chain polyunsaturated fatty acids (PUFA) such as arachidonic acid (ARA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) are involved in many (patho)physiological processes, i.a., through their oxidation products These eicosanoids and other oxylipins are highly potent lipid mediators regulating for example inflammation, vasoconstriction or pain [4, 5]. The need for derivatization makes quantifying non-esterified fatty acids (NEFA) in biological samples by GC challenging due to the high amount of fatty acyls occurring in different lipid classes such as triacylglycerols or phospholipids. We developed a new method which (i) covers a comprehensive set of biologically occurring fatty acids, (ii) allows rapid analysis (< 15 min) but separation of positional isomers, and (iii) requires only simple sample preparation by saponification following dilution in organic solvent. The method was successfully applied to analyze human plasma and refined as well as virgin plant oils
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