Gas chromatography mass spectrometry (GC-MS) has a thermal degradation problem, and it is more pervasive than many scientists might have guessed. True, the heat that GC-MS uses to vaporize samples has been known to alter the structures of specific molecules. But now a metabolomics investigation has shown that GC-MS can skew molecular profiles, altering the structures of as many as 40% of the molecules of interest.
This negative result, unlike many negative results, has been given due prominence. It appeared online October 4 in the journal Analytical Chemistry, in an article entitled, “Thermal Degradation of Small Molecules: A Global Metabolomic Investigation.” According to this article, which was contributed by scientists from The Scripps Research Institute (TSRI), small molecules and metabolites undergo significant time-sensitive alteration when exposed to elevated temperature, especially those conditions consistent with GC-MS experiments.
“We found that even relatively low temperatures used in GC-MS can have a detrimental effect on small molecule analysis,” said study senior author Gary Siuzdak, Ph.D., senior director of TSRI's Scripps Center for Metabolomics and professor of chemistry and molecular and computational biology.
In the TSRI study, a set of small molecule standards and, separately, human plasma metabolites were heated, at different temperatures and for different durations, under GC-MS-like sample conditions. Then the samples were analyzed by liquid chromatography coupled to electron spray ionization mass spectrometry (LC-MS), a technique that does not subject molecules to thermal degradation. Finally, the resulting data were processed by XCMS Online, a metabolomics platform developed in the Siuzdak lab.
It turned out that small molecules transformed and even disappeared during the experiment, throwing into question the nature of the data being generated by GC-MS.
“[Heating] at an elevated temperature of 100°C had an appreciable effect on both the un-derivatized and derivatized molecules, and heating at 250°C created substantial changes in the profile,” wrote the authors of the Analytical Chemistry article. “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.”
These results may generate their own kind of heat, given that GC-MS is such a venerable and familiar technology. For more than 50 years, chemists and biologists have used GC-MS to identify and measure concentrations of small molecules. When a sample is injected in a GC-MS system, it is heated and vaporized. The vapor travels through a gas chromatography column and the molecules separate, allowing the mass spectrometer to measure the individual molecules in the sample. Today, GC-MS is widely used in thousands of laboratories for tasks such as chemical analysis, disease diagnosis, environmental monitoring, and even forensic investigations.
“In retrospect, there was very little to be surprised about: heat degrades molecules,” noted Dr. Siuzdak. “However we've simply taken for granted the extent of thermal degradation.”
The researchers noted that even molecules not typically observed in GC-MS can also be transformed; for example, the energy metabolite adenosine triphosphate (ATP) was readily converted into adenosine monophosphate (AMP). This transformation is relevant for medical research because scientists often use a heating process to look at the ratio of ATP to AMP in cells to estimate the function of cellular components in aging and disease.
Thermal degradation could also explain why many scientists have detected many unknown molecular “peaks” in the past. Based on the new study, the researchers now believe these metabolites may be byproducts of the heating process—the result of reactions between metabolites as they degrade.
The study’s results, though disconcerting, needn’t be demoralizing. If GC-MS has limitations, it may be just as well to know what they are, so that they can be accommodated. “Fortunately, these problems can be overcome with the use of standards in GC-MS as well as using newer, ambient temperature mass spectrometry technologies,” explained Dr. Siuzdak. “[Our] report will likely stimulate more scientists to move to these less destructive alternatives.”