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Heated Dispute Over Analytical Method

Study finds that GC-MS changes or destroys sample compounds

by Stu Borman
October 21, 2015 | APPEARED IN VOLUME 93, ISSUE 42

Credit: Anal. Chem.
LC-MS peaks from analyses of a small-molecule sample maintained at room temperature (bottom) and heated for five minutes at 250 °C (top) differ considerably.

If you’ve used gas chromatography-mass spectrometry (GC-MS) to analyze unknown compounds from cells and biological tissue, a new study suggests you may want to throw away most—if not all—of your data. But some researchers believe it might be better to keep the data and throw away the new study instead.

Gary Siuzdak of Scripps Research Institute California and coworkers heated standard samples of known small molecules and samples of unknown metabolites from cells to mimic the GC stage of a GC-MS instrument. This initial stage uses heating to volatilize and separate samples. The researchers then used liquid chromatography-MS (LC-MS), which does not use heat in its initial LC separation stage, to analyze the heated samples as well as unheated, but identical, samples (Anal. Chem. 2015, DOI: 10.1021/acs.analchem.5b03003). The result: Up to 40% of the heated compounds were modified or destroyed, compared with the unheated ones—even in samples whose components were derivatized by trimethylsilylation, a method widely used to protect compounds from heating.

The results question what we are really analyzing when GC-MS experiments are used to identify unknown compounds in a sample, says Siuzdak, a specialist in MS and metabolomics. “The injected sample, or its thermal degradation products?” he asks.

“We found that even relatively low temperatures used in GC-MS can have a detrimental effect on small-molecule analysis,” he continues. GC and GC-MS have been used to identify and measure small molecules for more than 50 years. So the new results might trigger an “uh-oh moment” for analytical chemists.

Using standard laboratory procedures, Siuzdak and coworkers used LC-MS to analyze small-molecule standards and human plasma metabolites stored at room temperature (25 °C). Then they compared the results with LC-MS analyses of the same samples heated to 60 °C, 100 °C, or 250 °C, to mimic a variety of common heating conditions used in GC-MS. The heating changed the identities of or destroyed roughly 5%, 15%, and 30% of compounds in both types of samples, respectively.

Siuzdak suggests that work-arounds would involve using GC-MS primarily to measure sample concentrations by using reference standards rather than trying to identify unknowns, or switching to techniques such as LC-electrospray ionization MS, which does not use heating. “In fact, that is the direction things are going,” he says. “However, tens of thousands of GC-MS instruments are still being used on a daily basis” to analyze unknowns in nutrition, forensics, clinical and environmental analysis, and similar fields.

The findings have sparked some strong opinions, both heated and unheated. For example, “I am unclear why a scientific journal would publish work that is such clear nonsense,” says metabolomics expert Oliver Fiehn of the University of California, Davis. “The publication of this paper is, in my opinion, a major embarrassment for Analytical Chemistry. There was a peer review process involved, but perhaps not all peer reviewers understood the study design the Siuzdak group used.”

When analytical chemists “develop, validate, and implement an analytical method for GC-MS, they carefully control for important method parameters,” such as the temperatures used and the conditions used to derivatize compounds to protect them from heating, he says, noting that the Siuzdak group sidestepped such controls.

In some experiments, Siuzdak’s team “used underivatized metabolites and heated them intensely,” Fiehn says. “That is called cooking—like in a kitchen. Primary metabolites have lots and lots of hydroxyl and amino groups, and blood plasma has a lot of sugars,” groups that should be protected to prevent breakdown before heating them for analysis, he adds.

Fiehn believes the Scripps researchers made other methodological mistakes, such as analyzing trimethylsilyl-derivatized samples in a water-containing LC-MS solvent, because water cleaves trimethylsilyl groups. “You cannot inject trimethylsilylated compounds in an aqueous solvent into an LC-MS system and expect that peaks survive,” he says. “That’s why their MS spectra could not identify compounds: The authors destroyed them.”

And metabolomics specialist Warwick Dunn of the University of Birmingham, in England, pointed out that “the published study heated dry samples, whereas GC heats samples in a liquid solution for a few seconds during injection and then in the gas phase. Heat transfer in a solid can be expected to be higher and could therefore provide a higher level of degradation than in the gas phase.” Hence, “further validation of the degradation of many metabolites is required before we should worry about the terabytes of data already collected.”

Other scientists who spoke with C&EN were less critical. For example, Stephen Barnes, director of the Targeted Metabolomics & Proteomics Laboratory at the University of Alabama, Birmingham, says that whole-metabolome analysis typically identifies fewer than 20% of cellular metabolites, and the new study could help explain why.

At this year’s International Conference of the Metabolomics Society, Barnes says, “a group reported incubating a series of pure metabolites at 70 °C and analyzing them to see if new compounds appeared without added enzymes. The answer was yes.” He notes that compounds in samples can react with each other, albeit slowly, and that any elevated temperatures to which they are exposed can also alter their composition.

“I don’t think the problem is confined to GC-MS,” Barnes adds. “Those doing LC-MS analyses need to think hard about this too.” For example, samples can get modified during the extraction process used to prepare them, in which heated solvents are sometimes used. Or they can even get modified at the source from which they are obtained—for example, the human body is about 37 °C. GC-MS and LC-MS analyses are used as evidence in criminal cases, Barnes says, and “it seems like one could defend guilty people on the basis that prosecutors have no way of knowing whether the data are valid. The forensic science community needs to develop a more rigorous understanding of the pitfalls of analysis.”

Liang Li of the University of Alberta, whose group develops MS methods for proteomics and metabolomics applications, says the new study is important because it reminds people, particularly new practitioners in the metabolomics field, not to use high temperatures or other conditions that are more extreme than necessary for processing complex metabolomic samples.

Siuzdak says his team’s study wasn’t intended to disparage “thousands of papers’ worth of research.” But molecular transformation from sample heating “has been a fundamental yet unrecognized problem with GC-MS technology since its inception,” he says. “I remember asking someone about it many years ago during the question period after his talk, and he simply didn’t know how to respond. For me, it has always been the elephant in the room.”



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CH Lam (October 21, 2015 2:20 PM)
If this study is conducted by experts that mastered GC/LC combined with low and high resolution mass spectrometry, with or without fragmentation, for example Prof Fiehn, the finding would be more precise.
Ming Yeh (October 21, 2015 3:27 PM)
I believe the risk of using GC-MS due to high temperature concerns is very much a common sense in chemistry lab. This report surely recorded such.
Simon Maher (October 21, 2015 3:52 PM)
Very interesting paper which, despite some control issues, raises a fundamental question regarding heating of samples in mass spec.
Paul C. Goodley (October 21, 2015 4:31 PM)
The original analytical application of GC and GC/MS was in the petreolium indrustry for complex hydrocarbons mixtures. Thermal degratation was a well known phenomonon for compounds materials other than hydrocarbon. The derivitation methods which Pierce Chemical help to develop was a direct result and recognition that many chemical compounds would be thermally degraded without modifiying their chemical thermal stability.
Are we re-discovering thermodynamics and differential thermal analysis in the twenty-first century?
Stephen C. Brown, PhD (October 21, 2015 11:32 PM)
Indeed, this seems another unfortunate example of a peer-review failure. I'm not aware of anyone in Prof. Siuzdak's Lab that has any training or long experience in GCMS, a technology practiced for some 50 years. I suggest he buy a copy of ISBN: 978-0-444-87158-9, for starters, and do a year sabbatical under some long-standing practitioners, like Cedric Shackleton. PubMed is not likely to retrieve much of the relevant literature on GCMS artifacts, most of which was published pre-1980 before funding, patience, and glamour dried up for these types of investigations. Ephraim Racker's aphorism still stands: "Don't waste clean thoughts on dirty enzymes (or cooked specimens)".
M.Gnana sekar (October 22, 2015 3:24 AM)
It is quite understandable that GC-MS experiments of cells and biological tissues should be done at the human body's temperature to rely on the data. A best way to know the stability of the cell or biological tissue could be, to do Bio-NMR at the GC-MS experimental temperature. This would clearly reveal the reality and the reliability of GC-MS data. Human metabolism could change with temperature and that would change the molecular structure too.
Nathan (October 22, 2015 9:52 AM)

First of all, I'll be more outraged by the figure presented here - why the bottom one appears inverted? unless it is the difference between the two chromatograms.
Dan Harring (October 24, 2015 9:04 PM)
For the most part, GC does not work for carbohydrates, peptides, polymers, metals. But tens of thousands of other small molecules have been demonstrated to consistently survive GC and GC-MS separation without decomposition or artifact formation. The confines/limitations of GC methods have been thoroughly examined since it was invented in the 1950s; this information is readily available to experienced analysts and beginners alike. Countless journal articles, book chapters and application notes have reported the specifics. One example that pre-dates modern analytical chemistry - perfume makers have known for centuries that distillation has adverse effects on a few thermally labile components of certain botanical essential oils, so alternate extraction methods must be used for those particular materials. Despite these exceptions, distillation is successfully used for many many essential oils, aroma compounds, fuels, solvents, liquors, etc.
Paul Begley (November 2, 2015 4:27 AM)
Three points:
1/ TMS derivatives are notoriously prone to hydrolysis, so analysing them by HPLC using high water content mobile phases is unlikely to work well.
2/ Derivatisation for GC is designed to increase volatility, not to improve thermal stability.
3/ GC-MS stationary phases were explicitly developed for high chemical inertness. A molecule in a solid phase mixture of metabolites (ie in the conditions the authors used) has a much wider range of reaction possibilities than the same molecule isolated in a GC stationary phase at the same temperature.
Pat Perkins (November 2, 2015 3:18 PM)
I find it incredulous that a study suggesting problems with GC/MS analysis contains no experiment run on a GC/MS to prove/disprove the hypothesis, and that the reviewers of the submission did not request this information.
Mekibib Dawit (November 12, 2015 2:23 PM)
It looks to me a simple misunderstanding of what GC-MS can and can't analyse. By definition GC should only be used for compounds that are 'GC amenable' i.e. for those compounds that don't degrade or react at the GC conditions (column, mobile and stationary phase combination, temperature of inlet and so on) to be used. This works well for known samples. Unknowns by GC-MS can only be analysed after a thorough understanding of the sample matrix, possible analytes and after such precautions such as appropriate devitalisation of potential sensitive groups have been taken care of. Even then comparative study of other analytical techniques such as LC-MS should be attempted. Otherwise the results will not be defendable under any proper regulatory scrutiny.

In my experience a greater confidence of results for any analytical procedure can only be achieved if one can confidently answer the following;

1) Has the appropriate sampling, sample pretreatment (clean up, extraction,
concentration...) been used?
2) Has the appropriate instrumental technique/s (validated, for known samples)
been used?
3) Have appropriate QC/QA procedures been followed?
4) Has appropriate risk analysis been done including characterisation of errors?

Mike (December 13, 2015 8:05 AM)
You want to select the best method for your molecules of interest and GC is not the best for heat labile compounds. Just like reversed phase is not great for TMS derivatives but normal phase would work fine. The important part of any experiment is to think about the resuts that you are getting. Does the result make sense for the type of molecule and sample matrix that you are working with. In some cases testing the same sample with two different methods can be very illuminating.

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