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. 2015 Dec;148(2):531-43.
doi: 10.1093/toxsci/kfv198. Epub 2015 Sep 9.

Reference Standardization for Mass Spectrometry and High-resolution Metabolomics Applications to Exposome Research

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Free PMC article

Reference Standardization for Mass Spectrometry and High-resolution Metabolomics Applications to Exposome Research

Young-Mi Go et al. Toxicol Sci. .
Free PMC article

Abstract

The exposome is the cumulative measure of environmental influences and associated biological responses throughout the lifespan, including exposures from the environment, diet, behavior, and endogenous processes. A major challenge for exposome research lies in the development of robust and affordable analytic procedures to measure the broad range of exposures and associated biologic impacts occurring over a lifetime. Biomonitoring is an established approach to evaluate internal body burden of environmental exposures, but use of biomonitoring for exposome research is often limited by the high costs associated with quantification of individual chemicals. High-resolution metabolomics (HRM) uses ultra-high resolution mass spectrometry with minimal sample preparation to support high-throughput relative quantification of thousands of environmental, dietary, and microbial chemicals. HRM also measures metabolites in most endogenous metabolic pathways, thereby providing simultaneous measurement of biologic responses to environmental exposures. The present research examined quantification strategies to enhance the usefulness of HRM data for cumulative exposome research. The results provide a simple reference standardization protocol in which individual chemical concentrations in unknown samples are estimated by comparison to a concurrently analyzed, pooled reference sample with known chemical concentrations. The approach was tested using blinded analyses of amino acids in human samples and was found to be comparable to independent laboratory results based on surrogate standardization or internal standardization. Quantification was reproducible over a 13-month period and extrapolated to thousands of chemicals. The results show that reference standardization protocol provides an effective strategy that will enhance data collection for cumulative exposome research. In principle, the approach can be extended to other types of mass spectrometry and other analytical methods.

Keywords: amino acids; analytical chemistry; environment; mass spectrometry; metabolomics.

Figures

FIG. 1.
FIG. 1.
Cross-laboratory comparison of amino acid (AA) quantification. Randomized and blinded analyses of 117 human plasma samples were performed in independent laboratories by HRM and automated AA analysis. Pearson correlation coefficients (r) are provided in Table 2; all correlations were significant at P < .05.
FIG. 2.
FIG. 2.
Comparison of absolute concentration measurements for methionine (Met) and tyrosine (Tyr) by HRM and amino acid (AA) analysis. Met (A) and Tyr (B) concentrations were obtained by HRM using stable isotope dilution and directly compared to corresponding measurements by conventional AA analysis.
FIG. 3.
FIG. 3.
Comparison of signal intensities for amino acids (AAs) in NIST SRM1950 and Qstd with repeated analyses over 13 months. To evaluate reproducibility of reference standardization over time, AA signals were compared for analyses performed during different time periods. The results confirm that long-term referencing is robust. The results also showed that variability for different AAs occurred with triplicate analyses, providing a basis to change standard operating procedures to perform 6 technical replicates of the reference standards after each batch of 20 samples.
FIG. 4.
FIG. 4.
Histograms of selected metabolite concentrations in 157 healthy adults. Samples were analyzed in triplicate by HRM and quantification was performed using reference standardization.
FIG. 5.
FIG. 5.
Histograms of selected environmental and dietary chemicals in 157 healthy adults. Samples were analyzed in triplicate by HRM and quantification was performed using reference standardization.
FIG. 6.
FIG. 6.
Representative ultra-high resolution MS1 scans of human plasma show that many low abundance ions are present within a 0.5 atomic mass unit (AMU) window. Four examples of mass spectra are given and are representative of nominal mass <1000.

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