Improving sample preparation to avoid misleading results

Improving sample preparation to avoid misleading results

Source: K. Haslauer

Analysing metabolites to study metabolic pathways and –dysfunctions in human individuals is nowadays a well-known approach. Working with biofluids faces several challenges, which can affect the outcome drastically. The scientific community generally agrees on certain workflows to reach high quality data and therefore reliable results. We recently pointed out the importance of scrutinizing these established procedures by showing the severe decrease in creatinine, one of the frequently used metabolic markers in NMR metabolomics. This work was published in ‘Analytical Chemistry’.

Mass-spectrometry and Nuclear Magnetic Resonance spectroscopy (NMR) are the methods of choice when it comes to metabolomics. In NMR metabolomics, several workflows are established considering sample handling and preprocessing to overcome issues of biofluid dilution effects and degradation of metabolites. In the published work the impact of sample preparation on possible misleading results was shown on the example of D2O concentration on urinary creatinine levels. Deuterated water is used as necessary locking substance in NMR buffer systems and so far not considered as possible impact factor on the acquired spectra. In the case of creatinine, which is a well-described urinary metabolite and due to its stable excretion in healthy individuals frequently used as normalization parameter to correct for urinary dilution, D2O was shown to have a drastic effect on quantification. The underlying mechanism was found to be a proton-deuterium exchange within the creatinine molecule, which results in a significant decrease in quantification of creatinine.

And is there a solution? Yes! We show, that this effect can be minimized by using a maximum of 10% D2O while keeping samples at 4°C prior measurement. Furthermore, we offer a correction factor for existing datasets that allows back-projection of the original creatinine concentration.

For more information see:

Haslauer, K. E., Hemmler, D., Schmitt-Kopplin, P., & Heinzmann, S. S. (2019). Guidelines for the Use of Deuterium Oxide (D2O) in 1H NMR Metabolomics. Analytical Chemistry.

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