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Anal Chem. 2019 Mar 19;91(6):4055-4062. doi: 10.1021/acs.analchem.8b05611. Epub 2019 Mar 11.

Combining NMR and MS with Chemical Derivatization for Absolute Quantification with Reduced Matrix Effects.

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College of Chemistry , Jilin University , Changchun 130021 , P. R. China.
Northwest Metabolomics Research Center, Department of Anesthesiology and Pain Medicine , University of Washington , Seattle , Washington 98109 , United States.
Chongqing Blood Center , Chongqing 400015 , P. R. China.
Arizona Metabolomics Laboratory, College of Health Solutions , Arizona State University , Scottsdale , Arizona 85259 , United States.
College of Plant Protection , Southwest University , Chongqing 400715 , P. R. China.
Department of Chemistry , University of Washington , Seattle , Washington 98109 , United States.
Fred Hutchinson Cancer Research Center , Seattle , Washington 98109 , United States.


Absolute quantitation is a major challenge in metabolomics. Previously, we [ Nagana Gowda et al. Anal. Chem. 2018 , 90 , 2001 - 2009 ] showed that nuclear magnetic resonance (NMR) spectroscopy can guide absolute quantitation using mass spectrometry (MS); however, this method does not account for the matrix effect in MS measurements. To surmount this challenge, we have developed a novel method, qNMR-MS, for the absolute quantitation of metabolites using MS by combining it with NMR and chemical derivatization. Metabolite concentrations are first obtained using NMR for a reference sample. Subsequently, both reference and study samples are chemically derivatized with isotope-labeled and unlabeled reagents, respectively. The derivatized reference sample is then mixed with study samples and measured using MS. Comparison of paired isotope unlabeled and labeled MS peaks enables absolute quantitation with virtually no matrix effects. As a proof of concept, we applied the qNMR-MS method for absolute quantitation of amino acids using propyl-chloroformate (PCF) derivatization. For standards, the observed coefficients of determination ( R2) of most amino acids were greater than 0.99 across concentrations of 0.2 to 20 uM. For human serum, the results of the qNMR-MS method are comparable to the conventional isotope-labeled internal standard (iSTD) method ( R2 ≥ 0.99), with an average median coefficient of variation (CV) of 5.45%. The qNMR-MS method is relatively simple, highly quantitative, has high cost-efficiency (no iSTD required), and offers new avenues for the routine quantitation of amino acids in blood samples; it can, in principle, be extended to a wide variety of metabolites in different biological samples.

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