LC–MS analysis of the plasma metabolome—A novel sample preparation strategy

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 978-979; pp. 83 - 88
• Main Authors: Skov, Kasper, Hadrup, Niels, Smedsgaard, Jørn, Frandsen, Henrik
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 26.01.2015
• Subjects: Animals; Biomarkers - blood; Biomarkers - chemistry; Chromatography; Chromatography, Liquid - methods; Discriminant analysis; Extraction; Feature extraction; LC–MS; Lipids; Male; Metabolome - physiology; Metabolomics; Phospholipids; Plasma samples; Precipitation; Proteins; Rats; Rats, Wistar; Solid phase extraction; Solid Phase Extraction - methods; Solid phases; Spectrometry, Mass, Electrospray Ionization - methods; Metabolomics; Solid phase extraction; PC; LC–MS; MG; PLS-DA; Plasma samples; PFNA
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2014.11.033

•Metabolomics analyses of rat plasma.•New approach using solid phase extraction combined with sub-fractionation.•The three sub-fractions contain phospholipids, lipids and polar compounds.•Comparing SPE with protein precipitation resulted in 2 times more molecular feature.•Possible to detect difference in animals affected by toxic compounds. Blood plasma is a well-known body fluid often analyzed in studies on the effects of toxic compounds as physiological or chemical induced changes in the mammalian body are reflected in the plasma metabolome. Sample preparation prior to LC–MS based analysis of the plasma metabolome is a challenge as plasma contains compounds with very different properties. Besides, proteins, which usually are precipitated with organic solvent, phospholipids, are known to cause ion suppression in electrospray mass spectrometry. We have compared two different sample preparation techniques prior to LC-qTOF analysis of plasma samples: the first is protein precipitation; the second is protein precipitation followed by solid phase extraction with sub-fractionation into three sub-samples: a phospholipid, a lipid and a polar sub-fraction. Molecular feature extraction of the data files from LC-qTOF analysis of the samples revealed 1792 molecular features from the protein precipitation procedure. The protein precipitation followed by solid phase extraction procedure with three sub-samples gave a total of 4234 molecular features. This suggests that sub-sampling into polar, lipid and phospholipid fractions enables extraction of more metabolomic information as compared to protein precipitation alone. Chromatography showed good separation of the metabolites with little retention time drift (<1s) and a mass accuracy below 3ppm was observed. The performance of the method was investigated using plasma samples from rats administered the environmental pollutant perfluorononanoic acid.