Solid phase microextraction: a versatile technique for lipid analysis

Abstract

Extensive multidisciplinary research and improvements to instrumentation and workflows are continuously enabling new insights into the role of lipids in health and disease. As with other metabolites, the accurate analysis of lipids in biological samples requires robust and efficient analytical methodologies that enable reliable quantitation. Since its introduction in the 1990s, solid phase microextraction (SPME) has proven well-suited for numerous applications in various scientific fields (environmental chemistry, forensics, clinical, and others) thanks to its many advantages and compatibility with different instruments. Despite being present on the bioanalytical landscape for a decade, SPME research aimed at lipids remains unusual and often qualitative. This thesis documents the development of three approaches for the analysis of lipids based on SPME coupled with different instrumental platforms, namely: liquid chromatography-mass spectrometry (LC-MS) (Chapter 2); mass spectrometry (MS) (Chapter 3), and Raman spectroscopy (Chapter 4). Chapter 2 details the development of an LC-MS platform, the analysis of sampling parameters crucial to SPME, the steps taken to determine the total concentration of glycerophospholipids in human plasma and strategies for the analysis of the free concentration of model lipids. The reversed-phase liquid chromatography (RPLC) method was applied to analyze exogenous lipids in human blood plasma. It was validated with respect to instrumental linearity and dynamic linear range, inter– and intra–day accuracy and precision, and limits of quantitation. The SPME method was optimized by investigating several crucial parameters, including coating chemistry, extraction time, and desorption conditions. This proposed SPME-RPLC-MS/MS platform was utilized to construct matrix-matched calibration curves for the total concentration in four plasma lots, including NIST™ SRM® 1950 human blood plasma. Moreover, SPME probes were used to quantitate endogenous lipid compounds present in the NIST™ SRM® 1950 plasma using a standard addition approach, which resulted in good agreement with previous studies on this sample. Furthermore, the lipidome of NIST™ SRM® 1950 was qualitatively assessed via an untargeted approach using high-resolution mass spectrometry. The lipidome obtained using SPME, an unconventional technique, is in good agreement with previous studies. This chapter also proposes a simple approach for the calibration of free concentration of lipid metabolites using SPME devices and external calibration curves free of binding matrix components. To this end, this section evaluated a ‘standard generating vial’ to produce and maintain a stable free concentration in aqueous media. The standard generating vial employs a lipid laden PDMS film coated onto a carbon mesh acting as an analyte reservoir. These vials were evaluated regarding their equilibration time, short-term stability, and linearity attained at various spiking concentrations. Finally, this chapter briefly explores the binding between human serum albumin and two long-chain fatty acids using experimental and in silico approaches. This study took advantage of SPME’s extraction mechanism from the pool of unbound analytes; thus, the free concentration of fatty acids was measured with SPME fibres and used to construct Scatchard plots to estimate the binding affinities with human serum albumin. The mass uptake of fatty acids by the SPME probes under different kinetic conditions (static extraction, agitated extraction, and agitated extraction with a binding matrix component) was validated by mathematical modelling using the COMSOL Multiphysics® software package. In Chapter 3, solid-phase microextraction is directly coupled to mass spectrometry (SPME–MS) for the analysis of glycerophospholipids in plasma while bypassing chromatographic separation. Coated blade spray (CBS), the initial SPME–MS technology surveyed, is a sword-like device that provides the sampling/pre-concentration capabilities of SPME devices while also serving as an ion source requiring minimal additional instrumentation. While lipid detection was easily achieved through the proper selection of modifiers, CBS’s geometry and ambient nature posed a challenge regarding lipids' fast and reproducible desorption. As an alternative, the flow-isolated desorption chamber of an open microfluidic interface (MOI)—the second SPME–MS technology assessed—was employed to study the desorption of lipids with virtually no evaporation. Substantial ionization/absolute matrix effects were detected for SPME–MS technologies, mirroring the phenomena observed in other MS-based lipid analyses. Chapter 4 presents a simple, proof-of-concept approach for the on-fibre detection of unsaturated lipids based on the direct coupling of SPME probes and Raman spectroscopy. The SPME protocol was optimized by investigating various parameters influencing extraction efficiency, including coating chemistry, extraction time, extraction temperature, and washing solvent. Our findings show that the developed SPME–Raman method is suitable for detecting lipids enriched on the coating. A clear dependence is observed between the number of double bonds and the ratio of the Raman bands at 1655/1445 cm–1. Although additional studies are needed to establish how these double bonds contribute to the observed Raman bands, the proposed platform has great potential for fast profiling applications.