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Analysis of lipids using hydrophilic interaction chromatography coupled to mass spectrometry: optimization of sample preparation and validation

Autoři: Chocholoušková Michaela | Wolrab Denise | Holčapek Michal

Rok: 2019

Druh publikace: ostatní - přednáška nebo poster

Strana od-do: nestránkováno

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Jazyk	Název	Abstrakt	Klíčová slova
eng	Analysis of lipids using hydrophilic interaction chromatography coupled to mass spectrometry: optimization of sample preparation and validation	Hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry is one of the methods suitable for the quantitative analysis of lipids in biological samples. The main advantage of HILIC is that separation is based on the interaction of the polar stationary phase with the polar head group of the lipid species, leading to the lipid class separation. Consequently, exogenous internal standards and analytes within the same lipid class coelute. Reliable quantitation is achievable as the same suppression effects and matrix effects for the IS and the analytes occur. Another critical point for comprehensive quantitation of various lipid classes in human plasma or serum is the employed sample preparation protocol, whereby liquid-liquid extraction is the most commonly used approach. In this work, we aimed to optimize the sample preparation. We evaluated the influence of various extraction protocols, the influence of different solvents for dissolving the extracts (mixtures of MeOH, MTBE, CHCl3, IPA), the influence of pH during the extraction and the influence of freeze/thawing cycles. In total, 6 extraction protocols varying in the choice of solvent (Folch, MTBE, BUME, etc.) were employed and compared to each other with regard to the matrix effect, selectivity, and recovery rate. The pH during the extraction was modified using additives, such as ammonium acetate, ammonium carbonate, sodium hydrogen phosphate, acetic acid, etc. The best conditions for sample preparation were chosen according to the performance parameters, such as recovery rate and reproducibility as well as practicability, sensitivity, and the lowest amount of impurities.

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