New sample preparation approach for mass spectrometry-based profiling of plasma results in improved coverage of metabolome

Abstract

Sample preparation remains a challenge in untargeted metabolomics studies and no method currently results in complete extraction of all metabolite classes in human plasma. Because a large variety of molecules, with vast differences in dynamic range, could be involved in human disease, there is an urgent need to develop analytical techniques that result in comprehensive coverage of metabolites. Furthermore, analysis of more focused molecular classes could be necessary to more fully interrogate markers of human disease. However, such techniques, which generally involve multiple steps, often result in high variability. We have optimized a combined liquid-liquid and solid phase extraction method for plasma and have compared that to traditional methanol precipitation using spiked internal standards as controls. This method, based largely on previously published methods, results in 5 separate fractions enriched for aqueous species, phospholipids, fatty acids, neutral lipids, and hydrophobic lipids. Using liquid chromatography mass spectrometry as the analytical method, we detect over 3,806 metabolites using the new method versus 1,851 metabolites using methanol alone. Qualitative analysis and quantitative analysis of both internal standards (ISTDs) and endogenous metabolites demonstrate excellent reproducibility with CV’s below 15% for the combined method compared to 30% using the methanol method. While both methods have applications for clinical metabolomics, fractionation resulted in greater overall coverage and can be used for initial classification of molecular species.

High-Resolution Metabolomics Assessment of Military Personnel

“…We noted in our study that different HRM strategies resulted in differential detection of representative chemicals. Overall, our study results are in agreement with previous studies and others (20, 23,29,38,47–50), as we conclude that employing dual HRM with positive and negative ionization and orthogonal chromatography increases chemical coverage of human serum metabolites and that the quality and quantification of detected chemicals may vary depending on HRM conditions. Increased chemical coverage facilitates the use of non-targeted metabolomics pathway enrichment software i.e.…”

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

“…Although the list of compounds was greatly reduced, the results were more reliable as they were not based on multiple adducts from the same compound. The full method is comprehensive and allows isolation of hydrophobic metabolites such as neutral lipids, phospholipids, fatty acids, triglycerides, and steroids, while also isolating hydrophilic classes in the aqueous fraction, of which eicosanoids, sugars, flavonoids, and amino acids have been identified 12,26 .…”

Comprehensive analysis of lipids in biological systems by liquid chromatography-mass spectrometry

“…Specifically, Yang et al [16] used C18 and hydrophilic interaction LC (HILIC) columns for separation of lipid and polar fractions, respectively, while Whiley et al [9] and Godzien et al [8] analyzed those two fractions on C8 and C18 columns. In these last two studies [8,9] the authors streamlined the procedure even further, performing the plasma extraction within an HPLC vial with a fixed 0.3-mL glass insert and using the same vial for analysis.…”