Fast LC-ESI-MS/MS analysis and influence of sampling conditions for gut metabolites in plasma and serum

Abstract

In the past few years, the gut microbiome has been shown to play an important role in various disorders including in particular cardiovascular diseases. Especially the metabolite trimethylamine-N-oxide (TMAO), which is produced by gut microbial metabolism, has repeatedly been associated with an increased risk for cardiovascular events. Here we report a fast liquid chromatography tandem mass spectrometry (LC-MS/MS) method that can analyze the five most important gut metabolites with regards to TMAO in three minutes. Fast liquid chromatography is unconventionally used in this method as an on-line cleanup step to remove the most important ion suppressors leaving the gut metabolites in a cleaned flow through fraction, also known as negative chromatography. We compared different blood matrix types to recommend best sampling practices and found citrated plasma samples demonstrated lower concentrations for all analytes and choline concentrations were significantly higher in serum samples. We demonstrated the applicability of our method by investigating the effect of a standardized liquid meal (SLM) after overnight fasting of 25 healthy individuals on the gut metabolite levels. The SLM did not significantly change the levels of gut metabolites in serum.

High-Throughput Fractionation Coupled to Mass Spectrometry for Improved Quantitation in Metabolomics

“…We have previously reported the effects of salts and phospholipids on the ESI. 11 The fractionation method provides a fast solution to minimize ion suppression caused by these matrix interferences. The use of three orthogonal columns allocated phospholipids, negative and positive salts into three different fractions.…”

“…The amines (quaternary amines, creatinine, urea, and uric acid) did not reveal a significant difference over a period of 24 h. This is in accordance with our prior work, in which we demonstrated that gut metabolites (quaternary amines) were not affected by the fasting state of an individual. 11 The amino acid levels started to rise after wake time. The levels remained high throughout the morning/afternoon and decreased again toward baseline levels just before dinner.…”

“…Phospholipids and salts are held responsible for a majority of signal suppression during electrospray ionization of plasma samples. 11 By using a fractionation approach based on polarity and charge, phospholipids are retained on the reversed phase column, whereas positive and negative salt ions are trapped on and eluted from the cation and anion exchange, respectively. Another benefit of serially coupled columns is the flow-through fraction, which is cleaned by three sorbent chemistries instead of one in conventional single-column methods.…”

Electromembrane Extraction of Highly Polar Compounds: Analysis of Cardiovascular Biomarkers in Plasma

“…A previously developed fast LC-MS/MS method for analysis of metabolites linked to gut metabolism was used in this study [10]. Briefly, a 1290 Infinity II ultra-high pressure liquid chromatography (UHPLC) system from Agilent Technologies (Waldbronn, Germany) was used for fast LC-MS/MS experiments.…”

“…The most common practice in metabolomics is to remove proteins by protein precipitation [7] or ultrafiltration [8]. However, these two approaches cannot separate the compounds of interest from interferences such as salts or phospholipids [9,10]. To do this, more complex sample preparation approaches, e.g., liquid-liquid extraction or reversed phase solid phase extraction are often considered.…”