OPEN Analytical chemistry | 6 Oct 2015
M Fang, J Ivanisevic, CH Johnson, ME Kurczy, GJ Patti, LT Hoang, W Uritboonthai, HP Benton and G Siuzdak
Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization especially in gas chromatography mass spectrometry (GC/MS). In this study the effect of heating was examined on a set of 64 small molecule standards and, separately, on human plasma metabolites. The samples, either derivatized or underivatized, were heated at three different temperatures (60, 100, and 250°C) at different exposure times (30s, 60s, and 300s). All the samples were analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry (LC/MS) and the data processed by XCMS Online (xcmsonline.scripps.edu). The results showed that heating at an elevated temperature of 100°C had an appreciable effect on both the underivatized and derivatized molecules, and heating at 250°C created substantial changes in the profile. For example, over 40% of the molecular peaks were altered in the plasma metabolite analysis after heating (250°C, 300s) with a significant formation of upregulated, degradation and transformation products. Derivatized samples were similarly affected by thermal degradation. The analysis of the 64 small molecule standards validated the temperature-induced changes observed on the plasma metabolites, where most of the small molecules degraded at elevated temperatures even after minimal exposure times (30s). For example, tri- and di-organophosphates (e.g., adenosine triphosphate and adenosine diphosphate) were readily degraded into a mono-organophosphate (e.g., adenosine monophosphate) during heating. Nucleosides and nucleotides (e.g., inosine and inosine monophosphate) were also found to be transformed into purine derivatives (e.g., hypoxanthine). A newly formed transformation product, oleoyl ethyl amide, was also identified in both the underivatized and derivatized of the plasma metabolites and small molecule standard mixture, and was likely generated from reaction(s) with oleic acid. Overall these analyses show that small molecules and metabolites undergo significant time-sensitive alterations when exposed to elevated temperatures, especially those conditions consistent with GC/MS experiments.
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