Elsevier

Analytical Biochemistry

Volume 393, Issue 2, 15 October 2009, Pages 163-175
Analytical Biochemistry

Metabolite analysis of human fecal water by gas chromatography/mass spectrometry with ethyl chloroformate derivatization

https://doi.org/10.1016/j.ab.2009.06.036Get rights and content

Abstract

Fecal water is a complex mixture of various metabolites with a wide range of physicochemical properties and boiling points. The analytical method developed here provides a qualitative and quantitative gas chromatography/mass spectrometry (GC/MS) analysis, with high sensitivity and efficiency, coupled with derivatization of ethyl chloroformate in aqueous medium. The water/ethanol/pyridine ratio was optimized to 12:6:1, and a two-step derivatization with an initial pH regulation of 0.1 M sodium bicarbonate was developed. The deionized water exhibited better extraction efficiency for fecal water compounds than did acidified and alkalized water. Furthermore, more amino acids were extracted from frozen fecal samples than from fresh samples based on multivariate statistical analysis and univariate statistical validation on GC/MS data. Method validation by 34 reference standards and fecal water samples showed a correlation coefficient higher than 0.99 for each of the standards, and the limit of detection (LOD) was from 10 to 500 pg on-column for most of the standards. The analytical equipment exhibited excellent repeatability, with the relative standard deviation (RSD) lower than 4% for standards and lower than 7% for fecal water. The derivatization method also demonstrated good repeatability, with the RSD lower than 6.4% for standards (except 3,4-dihydroxyphenylacetic acid) and lower than 10% for fecal water (except dicarboxylic acids). The qualitative means by searching the electron impact (EI) mass spectral database, chemical ionization (CI) mass spectra validation, and reference standards comparison totally identified and structurally confirmed 73 compounds, and the fecal water compounds of healthy humans were also quantified. This protocol shows a promising application in metabolome analysis based on human fecal water samples.

Section snippets

Chemicals and materials

ECF, pyridine, and sodium hydroxide (NaOH) from Fluka (Saint Quentin en Yvelines, France) and anhydrous ethanol and n-hexane from Riedel-de Haën (Seelze, Germany) were used for derivatization reagents. l-2-Chloro-phenylalanine (a Sigma product) was used as an internal standard (IS) for batch quality control. Sodium bicarbonate and sodium sulfate were analytical reagent grade. All standards (Table 1), which were commercially obtained from Sigma–Aldrich (St. Louis, MO, USA), were prepared in

Chromatographic separation

Fecal water is a complex mixture of various metabolites, including fatty acids, amino acids, amines, and phenolic compounds, with a wide range of boiling points. The application of ECF derivatization in fecal water is beneficial not only to decrease the boiling points of metabolites but also to narrow the boiling point window of the derivatives. Consequently, capillary gas chromatography separation can cover as many compounds as possible. When the initial oven temperature was decreased from 80

Conclusions

Fecal water is a complex mixture of varieties of metabolites with a wide range of physicochemical properties and boiling points. The analytical method developed here provides a qualitative and quantitative GC/MS, with high sensitivity and efficiency, coupled with derivatization of ECF in aqueous medium. The parameters affecting derivatization efficiency and GC chromatographic separation were discussed and optimized. The pH of extraction solvent and storage conditions of fecal samples were

Acknowledgment

Xianfu Gao is thankful to INRA, France, for providing a bursary award.

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