Application of Solid Phase Microextraction for Quantitation of Polyunsaturated Fatty Acids in Biological Fluids

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 possible link between polyunsaturated fatty acids and uremic toxins from the gut microbiota in hemodialysis patients: A hypothesis

“…37 Furthermore, fatty acids have an association constant (Ka) with albumin at 25 C (9.2 × 10 4 to 4.3 × 10 5 M −1 ) that is higher than that of uremic toxins (0.98 × 10 5 M −1 ), allowing them to have a stronger connection with HSA. 38,39 Indeed, de Loor et al 40 observed that when sodium octanoate (a medium chain fatty acid characterized as a binding competitor) was added to normal and uremic serum, p-CS, and IS were inhibited to bind HSA. Moreover, Tao et al 32 revealed that 0.6 μmol/min of DHA increased IS removal by 25% in an in vitro HD model.…”

Solid-Phase Microextraction in Binding Studies

“…Birjandi et al evaluated the effect of varying concentration of human serum albumin in the range from 5 to 100 g/L on the extraction efficiency of polyunsaturated fatty acids by SPME. They found constant extraction efficiencies in the normal range of human serum albumin, but significant differences at very low (<20 g/L) and very high (>70 g/L) albumin concentrations [23]. This should be considered in experimental design to ensure accuracy of total concentrations if they are of interest in a given study.…”

“…This is in agreement with other studies where both kinetic and equilibrium SPME were used for accurate determination of binding constants such as presented in Table 10.1. In another study, SPME was applied to measure human serum albumin binding constants and % PPB of polyunsaturated fatty acids [23]. The affinity constants (K a ) of individual polyunsaturated fatty acids to human serum albumin ranged from 9.2 × 10 4 to 4.3 × 10 5 M −1 , while % PPB ranged from 98.0 to 99.7%.…”

Mass‐spectrometry‐based lipidomics

“…Birjandi et al. developed a direct immersion SPME method that was coupled to an LC–MS platform (DI‐SPME–HPLC–ESI‐MS) to quantify free FAs in fish and human plasma . SFE, which is based on an increase in the solvation power of a supercritical fluid, is used to effectively extract and separate different lipid species in a sample .…”