Application of Solid Phase Microextraction for Lipid Analysis and Lipidomics

Although the latest advancements in lipid analysis technology provide an excellent opportunity for the comprehensive study of lipidome, fulfilling the analytical requirements at the desired quality is still a major challenge. In lipidomic workflow, sample treatment and extraction for simultaneous analysis of endogenous lipids is of a great interest, especially when a number of undesirable circumstances such as ionization suppression are the matter of concern. In this thesis, I describe several approaches for both in vivo and high throughput in vitro sample preparation for molecular lipidomics based on solid phase microextraction (SPME) in the direct extraction mode. The initial research presented in this thesis focused on the analysis of fatty acids (FA) as the major building block and most fundamental category of entire lipid family. In this study, we present a direct immersion solid phase microextraction method coupled to a 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 affinity constant (Ka) of individual FAs to protein albumin was determined to be 9.2 × 104 to 4.3 × 105 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 in plasma samples obtained during operation of a group of patients undergoing cardiac surgery with the use of cardiopulmonary bypass (CPB). The most significant v alteration 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 inception of CPB in all cases. The quantitative results obtained for fatty acid measurement have encouraged me to expand my study for analyzing a broad spectrum of complex lipids as the next step. Therefore, in Chapter 3 biocompatible thin film SPME technique is introduced for quantitative analysis of lipids from human plasma in a high throughput manner. Robotic assisted 96-blade thin-film SPME was employed for the extraction of a group of non-endogenous lipids from plasma, and satisfactory results for recovery and reproducibility were achieved. In addition, the new extraction method eliminated the need for multi-step sample handling including centrifugation, filtering, solvent evaporation, and reconstitution. The total preparation time was reduced to less than 2 min per sample, while artifacts and sample loss were minimized due to the simplified method. Absolute recoveries were typically 1-2%, thereby avoiding sample disturbance while providing proper sensitivity. Furthermore, endogenous and non endogenous groups of target lipids were quantified, and excellent linearity and method precision were obtained. The regression calibrations obtained from five different plasma batches represents RSD less than 5% in all cases, clearly indicating the absence of matrix effects and lot-to-lot variations of the proposed method. The linearity (R2 > 0.99), inter and intra-day reproducibility (2-7%), and precision (1-12%) provided verification of method validity. Finally, the evaluated SPME method was compared to the conventional Bligh & Dyer technique for the untargeted lipidomic study of human hepatocellular carcinoma (HCC) cell vi lines. Cells were cultured under standard conditions in two individual groups; one cultivated normally as a control group and another supplemented by eicosapantanoic acid (20:5), as a highly polyunsaturated fatty acid, to study the effect of treatment on the lipidome pattern of cancerous cells. The obtained results provided a list of up-regulated and down-regulated lipids through a comparison between control versus treated cells. Method precision for the SPME approach was excellent (5-18% RSD) for all detected lipids. The relative LOD range was 0.5-1 ng/ml for the proposed SPME method, while for Bligh & Dyer, it was found to range between 0.05-0.1 ng/ml. Automated 96-blade sample handling enhanced extraction rates by approximately 2 min per sample, while generating comparable or enhanced product yields in comparison to conventional methods This study highlights important advantages of both SPME approaches using the rod fiber and thin-film geometries to capture fatty acids and lipids from different biological media including human and fish plasma as well as cell culture. The experimental results confirm the suitability of SPME for both in vitro and in vivo study in clinical samples as a new tool for lipidomic analysis

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

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/ac502627wDevelopment 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 a 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 (Ka) of individual FAs to protein albumin was determined to be 9.2 × 104 to 4.3 × 105 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.Natural Sciences and Engineering Research Council of Canada (NSERC) Supelc

High throughput solid phase microextraction: A new alternative for analysis of cellular lipidome?

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.jchromb.2016.09.034 © 2017. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/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.Natural Sciences and Engineering Research Council of Canada (NSERC