High Throughput and Quantitative Measurement of Microbial Metabolome by Gas Chromatography/Mass Spectrometry Using Automated Alkyl Chloroformate Derivatization

Abstract

The ability to identify and quantify small molecule metabolites derived from gut microbial-mammalian co-metabolism is essential for the understanding of the distinct metabolic functions of the microbiome. To date, analytical protocols that quantitatively measure a complete panel of microbial metabolites in biological samples have not been established, but urgently needed by the microbiome research community. Here, we report an automated high-throughput quantitative method using a gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) platform to simultaneously measure over one hundred microbial metabolites in human serum, urine, feces and Escherichia coli cell samples within 15 minutes per sample. A reference library was developed consisting of 145 methyl and ethyl chloroformate (MCF and ECF) derivatized compounds with their mass spectral and retention index information for metabolite identification. These compounds encompass different chemical classes including fatty acids, amino acids, carboxylic acids, hydroxylic acids and phenolic acids, as well as, benzoyl and phenyl derivatives, indoles, etc., that are involved in a number of important metabolic pathways. Within an optimized range of concentrations and sample volumes, most derivatives of both reference standards and endogenous metabolites in biological samples exhibited satisfactory linearity (R2 > 0.99), good intra-batch reproducibility and acceptable stability within 6 days (RSD<20%). This method was further validated by examination of the analytical variability of 76 paired human serum, urine, and fecal samples as well as quality control samples. Our method involved using high-throughput sample preparation, measurement with automated derivatization and rapid GC/TOFMS analysis. Both techniques are well suited for microbiome metabolomics studies.

Effects of dietary fat on gut microbiota and faecal metabolites, and their relationship with cardiometabolic risk factors: a 6-month randomised controlled-feeding trial

“…Raw sequencing data have been deposited in the National Center for Biotechnology Information Sequence Read Archive database with accession number PRJNA480547. Targeted identification and measurement of faecal metabolomics were performed based on a validated method 18. Details of the 16S rRNA sequence data preparation and analysis as well as the quantitative measurement of faecal metabolomics are available in online supplementary materials.…”

Vegan and Animal Meal Composition and Timing Influence Glucose and Lipid Related Postprandial Metabolic Profiles

“…For bile acids analysis, a method providing a broader coverage of targeted biochemical species was employed, differing from the quantification of 18 major bile acids used for the fasting plasma sample analyses. Plasma samples were extracted and prepared according to previously published methods . Briefly, all standards were obtained from Steraloids Inc. (Newport, RI, USA) and TRC Chemicals (Toronto, ON, Canada), and nine stable isotope‐labeled bile acid standards were used as internal standards.…”

Metabotypes Related to Meat and Vegetable Intake Reflect Microbial, Lipid and Amino Acid Metabolism in Healthy People

“…Plasma and urine samples were analyzed according to previously published methods, (Tables S1 and S2, Supporting Information).…”

“…We have developed a novel targeted and quantitative metabolomic method, which covers major metabolic pathways including bile acids, fatty acids, amino acids, carboxylic acids, hydroxylic acids, and aromatic derivatives and applied this to the study of a 48‐h vegan diet challenge in healthy women and men and children with and without inflammatory bowel disease . The objective of this present study was to use a new methodological approach to assess if dietary patterns could be linked to particular metabotypes in the urine or the blood plasma of healthy people, even in the case of a diverse population and uncontrolled dietary conditions.…”