Methanol Generates Numerous Artifacts during Sample Extraction and Storage of Extracts in Metabolomics Research

Abstract

Abstract:Many metabolomics studies use mixtures of (acidified) methanol and water for sample extraction. In the present study, we investigated if the extraction with methanol can result in artifacts. To this end, wheat leaves were extracted with mixtures of native and deuterium-labeled methanol and water, with or without 0.1% formic acid. Subsequently, the extracts were analyzed immediately or after storage at 10 • C, −20 • C or −80 • C with an HPLC-HESI-QExactive HF-Orbitrap instrument. Our results showed that 88 (8%) of the >1100 detected compounds were derived from the reaction with methanol and either formed during sample extraction or short-term storage. Artifacts were found for various substance classes such as flavonoids, carotenoids, tetrapyrrols, fatty acids and other carboxylic acids that are typically investigated in metabolomics studies. 58 of 88 artifacts were common between the two tested extraction variants. Remarkably, 34 of 73 (acidified extraction solvent) and 33 of 73 (non-acidified extraction solvent) artifacts were formed de novo as none of these meth(ox)ylated metabolites were found after extraction of native leaf samples with CD 3 OH/H 2 O. Moreover, sample extracts stored at 10 • C for several days, as can typically be the case during longer measurement sequences, led to an increase in both the number and abundance of methylated artifacts. In contrast, frozen sample extracts were relatively stable during a storage period of one week. Our study shows that caution has to be exercised if methanol is used as the extraction solvent as the detected metabolites might be artifacts rather than natural constituents of the biological system. In addition, we recommend storing sample extracts in deep freezers immediately after extraction until measurement.

Comprehensive investigation on synthetic cannabinoids: Metabolic behavior and potency testing, using 5F‐APP‐PICA and AMB‐FUBINACA as model compounds

“…No information about the N ‐methylation of related drugs has been found. N ‐methylation of amines are common artefacts when working with methanolic solutions, particularly at elevated temperatures. In the present study, the compounds were dissolved in methanol and later incubated at a low final methanolic concentration level of 0.3%.…”

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“…The reported that extraction of plant material by methanol can led to the artifacts containing methoxyl groups [12]. Since compound 1 contains a methoxyl group, we therefore change the solvent extraction of the plant material by using EtOAc.…”

NMR Metabolomics Applied on the Discrimination of Variables Influencing Tomato (Solanum lycopersicum)

“…In tomato, several extraction protocols using methanol-water (for polar/semi-polar metabolites, such as organic acids, carbohydrates, amino acids, quaternary ammonium compounds, and hydroxycinnamic acids), and/or chloroform (for a polar/nonpolar metabolites, such as carotenoids and fatty acids) [ 34 ], with variations in the relative amount of each solvent, in one- or multistep processes, and possibly at different temperatures, are described in the literature [ 35 ]. In untargeted plant metabolomics, methanol–water mixtures with or without acidification (e.g., formic or acetic acid) have become popular extraction solvents, since they allow extracting a wide range of metabolites [ 36 ]. For lycopene extraction, a mixture of methanol and chloroform (80:20, v / v ) was also reported [ 37 ].…”