Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS

Abstract

The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34–80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics.

Characterization of rapid extraction protocols for high‐throughput metabolomics

“…Historically, well‐established extraction methods such as Bligh‐Dyer and Folch methods and more recent variants have been used to recover water‐soluble hydrophilic and hydrophobic metabolites through the sequential exposure of the matrix of interest to progressively less polar and more organic solvents (e.g. from water to methanol and chloroform).…”

Evaluating the performance of sample preparation methods for ultra‐performance liquid chromatography/mass spectrometry based serum metabonomics

“…It is worth noting that though the different extraction and deproteination efficiencies of some solvent-based protein precipitation methods for serum extraction were investigated in previous works, [29][30][31][32][33][34][35][36][37] the impact of different extraction methods on multivariate analysis, biomarker identification in particular, is obscure and controversial. The aim of this study was to investigate the efficiency of different sample pre-treatment methods on serum metabonomics and to develop a protocol for accurate and precise biomarker identification.…”

The analysis of advanced glycation endproducts

“…Besides the use of a state-of-the-art technique, such as UPLC-MS/MS, to quantify AGEs and other biomolecules, derivatization of the compounds of interest could increase sensitivity and selectivity. A broad range of derivatization reagents have therefore been used to detect biomolecules with MS techniques, including 1-butanol to derivatize amino acids 122 , diacetyl-L-tartaric anhydride (DATAN) to derivatize glutaric acids 123 and O-phenlylenediamine (oPD) to derivatize -oxoaldehydes 124 . These three reagents have been used in this thesis to separate and quantify butylated AGEs, enantiomeric L-and D-lactate and -oxoaldehydes as quinoxaline derivatives with ESI-UPLC-MS/MS.…”

“…After collecting the sample it has to be prepared for analysis. Many sample preparation methods have been described, including various solvent precipitation techniques, ultrafiltration, liquid-liquid extraction and solid-phase extraction methods 25,36,122,123 . For the measurement of circulating molecules, such as free AGEs, D-lactate or -oxoaldehydes in plasma, it is of utmost important to remove proteins by precipitation or ultra-filtration before analysis.…”

“…On the contrary, protein-bound AGEs are determined after acid hydrolysis of the protein pellet and subsequently detected after derivatization. However, these sample preparation techniques can influence quantitative results by low metabolite coverage, low recovery and matrix effects 122 .…”