Evaluation of Fumaric Acid and Maleic Acid as Internal Standards for NMR Analysis of Protein Precipitated Plasma, Serum, and Whole Blood
2021
Nagana Gowda, G. A. | Hong, Natalie N. | Raftery, Daniel
Significant advances have been made in unknown metabolite identification and expansion of the number of quantifiable metabolites in human plasma, serum, and whole blood using NMR spectroscopy. However, reliable quantitation of metabolites is still a challenge. A major bottleneck is the lack of a suitable internal standard that does not interact with the complex blood sample matrix and also does not overlap with metabolite peaks apart from exhibiting other favorable characteristics. With the goal of addressing this challenge, a comprehensive investigation of fumaric and maleic acids as potential internal standards was made along with a comparison with the conventional standards, TSP (trimethylsilylpropionic acid) and DSS (trimethylsilylpropanesulfonic acid). Both fumaric acid and maleic acid exhibited a surprisingly high performance with a quantitation error <1%, while the TSP and DSS caused an average error of up to 35% in plasma, serum, and whole blood. Further, the results indicate that while fumaric acid is a robust standard for all three biospecimens, maleic acid is suitable for only plasma and serum. Maleic acid is not suited for the analysis of whole blood due to its overlap with coenzyme peaks. These findings provide new opportunities for improved and accurate quantitation of metabolites in human plasma, serum, and whole blood using NMR spectroscopy. Moreover, the use of protein precipitation prior to NMR analysis mirrors the sample preparation commonly used for mass spectrometry based metabolomics, such that these findings further strengthen efforts to combine and compare NMR and MS based metabolite data of human plasma, serum, and whole blood for metabolomics based research.
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