New study shows GC-MS can alter samples during analysis

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  • Published: Oct 7, 2015
  • Author: Jon Evans
  • Source: TSRI
  • Channels: Gas Chromatography / Base Peak

A new study led by scientists at The Scripps Research Institute (TSRI) at La Jolla, California, US, has revealed that gas chromatography-mass spectrometry (GC-MS) can fundamentally alter the samples it analyzes. The study appears in Analytical Chemistry.

‘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, senior director of TSRI's Scripps Center for Metabolomics and professor of chemistry, molecular and computational biology.

Using new capabilities within XCMS, a web-based data analysis tool developed in the Siuzdak lab, the researchers observed small molecules transforming – and even disappearing – during an experiment meant to mimic the GC-MS process. According to Siuzdak, this finding throws into question the nature of the data being generated by GC-MS.

The new experiments were initiated when Siuzdak was preparing a short course for students at the American Society for Mass Spectrometry annual meeting. The question arose of how heat from the vaporization process during GC-MS could affect results, so Siuzdak and TSRI research associate Mingliang Fang ran a series of experiments to investigate how a set of 64 small molecules responded to thermal stress.

To their surprise, the molecular profiles of as many as 40% of the molecules were altered, suggesting that heat from the GC-MS process could dramatically change the chemical composition of the samples. ‘The results were quite astounding – as this is a technology that has been used for decades,’ said Siuzdak.

The finding led the researchers to take a closer look at how molecules degrade and transform during GC-MS. They analyzed small molecule metabolites heated at 60°C, 100°C and 250°C, mimicking sample preparation and analysis conditions in GC-MS. The team then used XCMS with a low-temperature liquid chromatography-mass spectrometry (LC-MS) technique that has been previously shown not to degrade molecules thermally to assess the extent of the thermal effects.

The researchers observed significant degradation even at the lower temperatures. At the higher temperatures, almost half of the molecules were degraded or completely transformed.

‘In retrospect, there was very little to be surprised about: heat degrades molecules,’ said Siuzdak. ‘However we've simply taken for granted the extent of thermal degradation. While this is a negative result and scientists rarely publish them, I felt compelled especially for the students just getting started in their careers to report the limitation of such a ubiquitous technology.’

The researchers noted that even molecules not typically observed in GC-MS can 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, in order to estimate the role of cellular components in aging and disease.

‘People use this ratio to detect disease, but if the ratio can be changed by the heating process, the results will not be accurate,’ said Fang. ‘It is known that ATP is thermally sensitive, but not how it changed under these conditions.’

Thermal degradation could also explain why scientists have detected many unknown molecular peaks in the past. Based on the new study, the researchers now believe these peaks may be by-products of the heating process – the result of reactions between metabolites as they degrade.

So why haven't scientists figured out the effect of heating before now? Siuzdak explained that while some scientists had noticed changes in specific metabolites, it was difficult to see changes in overall molecular profiles comprising thousands of molecules.

This study was made possible by new capabilities within the XCMS tool. ‘With XCMS, we could expand our study to obtain a global profile of how the metabolites were altered – not just a few compounds,’ said Fang.

‘Fortunately these problems can be overcome with the use of standards in GC-MS as well as using newer, ambient temperature mass spectrometry technologies, and this report will likely stimulate more scientists to move to these less destructive alternatives,’ said Siuzdak.

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