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Techniques & Tools Liquid Chromatography, Mass Spectrometry, Data Analysis

Is Your Biomarker Analysis Accurate?

Supercritical fluid chromatography (SFC) finally appears ready for prime time with both Agilent and Waters selling viable machines. But, in the bioanalytical field at least, there has been surprisingly little interest shown to date. Might this be due, in part at least, to the fact that chromatography in general has taken a back seat to the selectivity provided by triple sector mass spectrometry(MS) and highly selective antibodies in the case of ligand-binding assays?

Not only can we not demonstrate specificity, we are also much less likely to achieve it.

Indeed, why buy an expensive new chromatographic platform if there is no ‘apparent’ need? Apparent being the operative word. There is an easy trap for us to fall in to here. All of our confidence in MS/MS has grown while, for several decades, we have been measuring drugs and their metabolites – typically xenobiotics. For analytes not normally found in the matrix of interest, demonstrating the specificity of a method is straightforward. In the absence of the drug, if the matrix generates no signal, it is highly likely that the method is specific for the analyte of interest. And we can often achieve that specificity with little or even no chromatography.  However, we are now becoming more and more involved with the analysis of biomarkers.

Our move to biomarkers introduces a very important but not always completely obvious challenge – the fact that a blank matrix is often not available, so it is not possible to demonstrate specificity. Nevertheless, we have developed so much confidence in the selectivity of MS/MS and our antibodies while quantifying xenobiotics, that we are not inclined to be concerned.

Unfortunately, there is a very real reason to be concerned! Biomarkers are created by complex biosynthetic processes that also produce a number of other structurally very similar substances. Not only can we not demonstrate specificity, we are also much less likely to achieve it. Chromatography suddenly becomes a critically important part of our analytical methodology. And we cannot know how much chromatography is required because we can never be sure that our analyte signal is not partly produced by another similar molecule.  

I am not saying that every biomarker method should include an extensive and time-consuming chromatographic step – but if accuracy is important, we could benefit by testing our method against one that does utilize the best chromatography available – better still, more than one type of chromatography, which is why SFC can be a very important tool. Not only is SFC comparable to liquid chromatography (LC) in ease of use, but it also tends to produce very different retention times for a given analyte compared with LC. In addition, SFC yields very different retention times on different stationary phases. The orthogonality gained can be as useful as brute force chromatography, benefitting from high numbers of theoretical plates. And it requires only a few minutes to test each stationary phase using SFC. If the intended method (LC-MS/MS or ligand-binding assay, for example) has been used to analyze the amount of the endogenous biomarker in a given lot of matrix, and if we test the same lot using various SFC stationary phases, we will either discover that the intended method is not specific – or we will be as confident as possible that it is.

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About the Author
James Settlage

James Settlage is Senior Research Investigator at inventive Health, Princeton, New Jersey. Dr Settlage received his bachelor’s degree in chemistry from the University of California at Berkeley and his PhD from the University of California at Davis under the tutelage of the late Professor Walter Jennings. Jimbegan his bioanalytical career in 1981 in bioanalysis at LAB, in Neu Ulm, Germany in 1981, where he was among the first people to introduce GC-MS to the field of bioanalysis. He has since served as the Director of R&D at PPD in Richmond, Virginia, USA, and Vice President of Lab Operations at Taylor Technology, now inventive Health Clinical in Princeton, New Jersey, USA. He has served on FDA advisory panels as an expert bioanalytical scientist and he was also a presenter and participant at the 2001 Crystal City conference on bioanalytical method validation, which was a pivotal point in the history of bioanalysis.

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