High Performance Liquid Chromatography (HPLC) for Short Chain Fatty Acid (SCFA) Analysis

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Overview

SCFAs are an emerging biomarker in metabolic diseases. A high concentration of SCFAs in fecal matter is associated with central obesity, hypertension, low-grade chronic inflammation, and dyslipidemia. The underlying mechanism begins with gut microbiota dysbiosis, which is caused by both an imbalance in SCFA production and gut permeability alteration. To further understand the distribution and function of SCFAs in organs and tissues, it is vital to accurately measure blood levels. SCFAs including pyruvate, lactic acid, formic acid, acetic acid, propionic acid, isobutyric acid, and butyric acid were analyzed by high-performance liquid chromatography (HPLC). Over the past few years, HPLC coupled to-tandem mass spectrometry (LC-MS/MS) has been increasingly used to quantify SCFAs. It reduced the analysis time of 45-60 min required from GCMS to as short as 15 min or less.

HPLC for SCFAs Analysis at Creative Biolabs

HPLC is another method for analyzing SCFAs and is a good alternative to GC. The most commonly used technique is a reverse phase HPLC (RP-HPLC), where the stationary solid phase (column) is hydrophobic (non-polar) and the mobile liquid phase is hydrophilic (polar, watery). In addition to GC, HPLC has also been applied to the analysis of fecal SCFAs in humans and rats. The detection limit of fecal SCFAs by HPLC is affected by the sensitivity of the detector. The most remarkable method for HPLC analysis of SCFA in biological samples is the direct conversion of acids to their non-volatile chromophobe compounds without the need for complex steps to separate the acids. As with GC, the sample pretreatment process plays an important role in HPLC. The derivatization of non-chromogenic SCFAs improves the sensitivity and selectivity of HPLC analysis.

Fig.1 Production of SCFA in every bioreactor, quantified by HPLC. (Gutiérrez, 2019)

Samples

• Fecal Sample
• Serum Sample
• Others

The most widespread pretreatment method for fecal SCFAs analysis is simple acidification (deproteinization) with perchloric acid or sulfuric acid.

Advantages

The greatest advantage of the HPLC over the GC technique is the lower running temperatures. Similar to GC, specific combinations of pretreatments, columns, running conditions, and detectors have to be optimized for a successful SCFAs analysis. The determination of SCFAs by HPLC is convenient and time-consuming. In addition, it does not require any preprocessing steps compared to GC.

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Reference

1. Gutiérrez, N.; Garrido, D. Species deletions from microbiome consortia reveal key metabolic interactions between gut microbes. MSystems. 2019, 4(4): e00185-19.

For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.