TECHNICAL NOTE: Analysis of volatile fatty acids in rumen fluid by gas chromatography mass spectrometry using a dimethyl carbonate extraction

Analysis of rumen fluid volatile fatty acids (VFA) is typically conducted by injecting acidified aqueous rumen fluid into a gas chromatograph (GC) with a flame ionization detector (FID). Aqueous samples are highly problematic because of the large vapor volume that can lead to poor peak shape and contamination of inlets, potentially causing sample carryover. Methods using aqueous samples are not well suited for use in a mass spectrometer (MS) detector system. The objective of this project was to validate a dimethyl carbonate (DMC) extraction process and GCMS method for rumen VFA analysis. To perform the extraction, 100 mu L of sample, KHSO4 (500 g/L), and 2-ethylbutyrate (internal standard; 8.5 mM) were added to a microcentrifuge tube (in order) followed by 1 mL of DMC. The mixture was thoroughly vortexed and centrifuged. The organic layer (top) was removed and placed in a GC vial. The DMC extract was injected (0.5 mu L) into an Agilent 5977B GCMS (8:1 split injection) with a polar DB-FFAP column. The column was held at 105 degrees C for 5 min, increased at 10 degrees C/min to 150 degrees C, then 65 degrees C/min to 240 degrees C, and held constant for 10 min. The peak area of acetate relative to the internal standard is linear from approximately 2 mM to at least 130 mM and encompasses the expected values of rumen concentrations for the other VFA. Recovery of VFA from spiked rumen fluid was tested at three concentrations in rumen fluid from steers fed a finishing diet or grazing wheat pasture. Recovery was not affected by the diet of the animals (P > 0.10) or the amount of VFA spiked (P > 0.19) for acetate, propionate, isobutyrate, or butyrate. There was an interaction of amount of VFA spiked and the diet of the animal (P = 0.021) for valerate and a tendency for an interaction (P = 0.051) for isovalerate, due to the recovery of the VFA being lower in the medium spike amount in rumen fluid from cattle on wheat pasture. Overall, recovery was greatest for propionate (101.9 +/- 1.67%) and lowest for valerate (95.7 +/- 1.95%). Including the 10-min hold at 240 degrees C at the end of each run prevented carryover from sample to sample. This method appears to perform well in a GCMS system and accurately and precisely quantifies rumen fluid VFA. Using dimethyl carbonate to extract volatile fatty acids from rumen fluid provides a method that can be used to analyze volatile fatty acids using gas chromatography with a mass spectrometer detector. This method avoids hazardous chemicals commonly used in other analytical procedures. Lay Summary Scientists need to measure the end-products of microbial digestion called volatile fatty acids in the gastrointestinal tract of ruminants for many reasons. The methods currently available for the analysis of volatile fatty acids have evolved over time along with new technology. Many of the methods available are either not compatible with a mass spectrometer detector or require hazardous chemicals to prepare the samples. The method described here uses a less-hazardous chemical to prepare the samples for analysis with a mass spectrometer detector. The method tested in this manuscript was adequate in preparing samples for volatile fatty acid analysis.

Automated summary

This article describes a method for extracting volatile fatty acids from rumen fluid using dimethyl carbonate and analyzing them with a gas chromatography-mass spectrometer detector. The method performs well in the GCMS system and accurately quantifies rumen fluid VFA.