A pre‐column derivatization high‐performance liquid chromatography method for simultaneous determination of short‐chain and medium‐chain fatty acids in a fecal sample

Abstract

Short‐chain and medium‐chain fatty acids have plentiful biological functions, which play a crucial role in the diagnosis and therapy of many diseases. Herein, a new method for simultaneous quantifying 17 short‐chain and medium‐chain fatty acids with high‐performance liquid chromatography coupled with an ultraviolet detector was developed and the pre‐column derivatization by indole‐3‐acetic acid hydrazide was performed to improve the separation and detection. The conditions of the derivatization reaction were systematically investigated. Subsequently, the method was validated and the results showed a satisfactory linearity (linear regression coefficients > 0.9969), the limit of detection (4.0×10−3–1.9×10−2 μmol/L), precision (0.9%–7.3% for intra‐day and 2.0%–9.8% for inter‐day), recovery (90.0%–109.1% with relative standard deviation <7.7%) and stability (0.1%–3.3% for standard solution and 0.2%–3.9% for fecal sample). Finally, the established method was successfully applied to quantify short‐chain and medium‐chain fatty acids in the feces of healthy control and diabetic rats. Eleven kinds of short‐chain and medium‐chain fatty acids were detected and six of them showed a significant difference between the control group and the model group.

Simultaneous quantification of fatty acids in serum with dual derivatization by liquid chromatography‐tandem mass spectrometry

“…Nevertheless, it is impractical to use stable isotope labeling with high purity which was usually expensive and difficult to synthesize as the IS in the quantification of FAs. In our previous study [38], SCFAs and MCFAs in the fecal sample were derivatized using IAH and determined on HPLC, and the derivant of IAH was proved to be stable. In this study, commercially available ICH and IAH were selected as the double derivatization reagents for FAs.…”

“…To obtain satisfactory sensitivity, accuracy, and high throughput of FAs quantification, two different parameters of the derivative conditions were optimized: (1) the concentration of EDC, HOBt, and ICH, and (2) reaction temperature and time. In our previous study [38], we investigated the effect of the reaction solvent and found that the derivatization would be efficient in the methanol solvent system. Thus, all the essential derivative solvents and standard solutions were prepared with methanol.…”

In vitro simulation of oral gastric intestinal digestion and fecal fermentation of Cordyceps militaris polysaccharides reveal its digestion profile and effect on the modulation of the gut microbiota

“…The monosaccharide composition of IPS was analyzed using pre-column derivatization of 1-phenyl-3methyl-5-pyrazolone (PMP) integrated with high-performance liquid chromatography (HPLC) because of the optical properties of the monosaccharides [29] .…”

Determination of six short‐chain fatty acids in rat feces using headspace solid‐phase dynamic extraction coupled with GC‐MS

“…Besides, a variety of methods can be used for quantitative determination, such as gas chromatography (GC) or combined mass spectrometry (MS) [22], high-performance liquid chromatography (HPLC), ion exclusion chromatography, and capillary electrophoresis [23]. Due to the weak UV absorption, it is necessary to conduct pre-column or post-column derivatization on SCFAs before HPLC [11,[24][25][26], which is more cumbersome. When analyzing SCFAs with GC, it is necessary to carry out derivation to increase signal response and improve chromatographic peak shape [27], which is also time consuming.…”