No abstract

No abstract

Development of a straightforward strategy for simultaneous quantitative analysis of nonesterified fatty acids (NEFA) species in biofluids is a challenging task because of the extreme complexity of fatty acid distribution in biological matrices. In this study, we present a direct immersion solid phase microextraction method coupled to liquid chromatography--mass spectrometry platform (DI--SPME--HPLC--ESI --MS ) for determination of unconjugated fatty acids (FA) in fish and human plasma. The proposed method was fully validated according to bioanalytical method validation guidelines. The LOD and LOQ were in the range of 0.5--2 and 5--12 ng/ml, respectively, with a linear dynamic range of 100 fold for each compound. Absolute and relative matrix effects were comprehensively evaluated and found to be in the acceptable range of 91--116%. The affinity constant (K a ) of individual FAs to protein albumin was determined to be 9.2×10 4 to 4.3×10 5 M -1 . The plasma protein binding (PPB%) was calculated, and found to be in the range of 98.0--99.7% for different polyunsaturated fatty acids (PUFAs). The PUFAs under study were found at a high concentration range in fish plasma, whereas only a few were within quantification range in control human plasma. The method was successfully applied for monitoring PUFA changes during the operation in plasma samples obtained from patients undergoing cardiac surgery with the use of cardiopulmonary bypass (CPB). The most significant contribution induced by surgery was noticed in the concentration level of α--linolenic acid (18:3, ALA), arachidonic acid (20:4, AA), and docosahexanoic acid (22:6, DHA) soon after administration of CPB in all cases.

A new SPME method for untargeted lipidomic study of cell line cultures was proposed for the first time. In this study the feasibility to monitor changes in lipid profile after external stimuli was demonstrated and compared to the conventional Bligh & Dyer method. The human hepatocellular carcinoma (HCC) cell line was used as a model. The obtained results provided a list of up-regulated and down-regulated lipids through a comparison between control (non-stimulated) cells versus the group of cells treated with polyunsaturated fatty acid (20:5). Use of the SPME technique yielded a list of 77 lipid species whose concentrations were recognized to be significantly different between control and treated cells, from which 63 lipids were up-regulated in treated cells. In general, the list was comparable to the peer list obtained by the Bligh & Dyer method. However, more diversity of lipid classes and subclasses such as LPC, sphingomyelins, ceramides, and prenol lipids were observed with the application of the SPME method. Method precision for the SPME approach was within the acceptable analytical range (5-18% RSD) for all detected lipids, which was advantageous over solvent extraction applied. The evaluation of ionization efficiency indicated no matrix effect for the SPME technique, while Bligh & Dyer presented significant ionization suppression for low abundant species such as LysoPC, PG, ceramides, and sphingomyelins, and ionization enhancement for high abundant phospholipids such as PE.

No abstract