Extraction, identification, and quantification of volatile fatty acids (VFA) in rumen fluid samples using Reverse Phase High-Performance Liquid Chromatography with Diode Array Detector (RP HPLC-DAD)
In ruminant animals, volatile fatty acids (VFA) or short-chain fatty acids (SCFAs) are derived from the protein and carbohydrate fermentation by rumen microorganism. Hence, the VFA determination in rumen fluid allows the evaluation of the nutritional quality of a diet, as well as its potential impact on the chemical composition of ruminant milk and meat. Thus, we developed a protocol to extract, identify, and quantify acetic, propionic, butyric, valeric, and caproic acids in ruminal fluid samples using RP-HPLC-DAD. Despite literature findings had shown that the most suitable column for VFA chromatographic separation under HPLC-DAD is an ion-exchange column, our protocol showed that a C18 column also allows an efficient VFA separation of the aforementioned acid, except for butyric and iso-butyric acids. This condition may constitute a limitation of the Hypersil GOLD C18 column use for VFA determination. However, considering that the concentration of iso-butyric acid is significantly lower than that of butyric acid, a good estimation of butyric acid can be obtained.
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Table 1. Quantities of reagents to be used to prepare the calibration curve
Table. 2. Gradient used in the chromatographic run.
Posted 05 Sep, 2020
Extraction, identification, and quantification of volatile fatty acids (VFA) in rumen fluid samples using Reverse Phase High-Performance Liquid Chromatography with Diode Array Detector (RP HPLC-DAD)
Posted 05 Sep, 2020
In ruminant animals, volatile fatty acids (VFA) or short-chain fatty acids (SCFAs) are derived from the protein and carbohydrate fermentation by rumen microorganism. Hence, the VFA determination in rumen fluid allows the evaluation of the nutritional quality of a diet, as well as its potential impact on the chemical composition of ruminant milk and meat. Thus, we developed a protocol to extract, identify, and quantify acetic, propionic, butyric, valeric, and caproic acids in ruminal fluid samples using RP-HPLC-DAD. Despite literature findings had shown that the most suitable column for VFA chromatographic separation under HPLC-DAD is an ion-exchange column, our protocol showed that a C18 column also allows an efficient VFA separation of the aforementioned acid, except for butyric and iso-butyric acids. This condition may constitute a limitation of the Hypersil GOLD C18 column use for VFA determination. However, considering that the concentration of iso-butyric acid is significantly lower than that of butyric acid, a good estimation of butyric acid can be obtained.
Figure 1
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