PSVII-17 - Use of a dimethyl carbonate extraction for rumen fluid volatile fatty acid quantification by gas chromatograph mass spectrometry

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 ignition detector (FID). Water in GC samples can lead to poor peak shape and to contamination of inlets, potentially causing sample carryover. Aqueous methods are not well suited for use in mass spectrometer (MS) detector systems. 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 µL of sample, KHSO₄ (50 g/L), and 2-ethylbutyrate (internal standard; 0.86 mM) are added to a microcentrifuge tube (in order) followed by 1 mL of DMC. The mixture is thoroughly vortexed and centrifuged. The organic layer (top) is removed and placed in a GC vial. The DMC extract is injected (0.5 µL) into an Agilent 5977B GCMS (8:1 split injection) with a polar DB-FFAP column. The column was held at 105°C for 5 min, increased at 10°C/min to 150°C, then 65°C/min to 240°C, and held constant for 10 min. The assay is linear for acetate from approximately 2 mM to at least 130 mM and covers 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 > 0.19) or the amount of VFA spiked (P > 0.27) and averaged 99.9% for all VFA, with valerate being the lowest (95.9%). Including the 10 min hold at 240°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 quantify rumen fluid VFA.