Ruminal organic acid analysis by gas chromatography/mass spectrometry

Abstract

Rumen fluid was analyzed by the gas chromatography/mass spectrometry (GC/MS) technique in order to identify the organic acids contained. The gas chromatogram obtained showed more than 200 peaks, and 60 organic acids were identified from their mass spectra obtained under electron impact conditions from the relative chromatographic peaks. Keto acids, polycarboxylic acids, hydroxy acids, aromatic acids, and saturated and unsaturated fatty acids were present, which were subdivided into three main groups: (i) short- and long-chain fatty acids; (ii) polyfunctional organic acids such as intermediate metabolic products; (iii) phenolic acids mainly from lignin and tannin degradation. It was concluded that GC/MS is a very specific and sensitive technique to detect the presence of fermentation products in biological fluids and that it could allow for the simpler and cheaper GC technique to be used for routine quantitative analyses of the identified compounds. Research on ruminal fermentation of feedstuffs is mainly based on measurements of pH, volatile fatty acids concentration, and turnover of markers. A more analytical approach is limited by difficult laboratory procedures regarding the identification and quantitation of the different biochemical pathways. We have recently used mass spectrometry/mass spectrometry (MS/MS) techniques to analyze the gas produced during the fermentation in the rumen (Bonsembiante et al., 1987a,b), and we have also adapted the MS/MS (McLafferty, 1983) and the gas chromatography/mass spectrometry (GC/MS) (McFadden, 1973) techniques to cope with the mixture analysis in order to study the chemical changes of ensiled grass (Bonsembiante et al., 1985; Daolio et al., 1986). The present work extends the method to analyze the nonvolatile organic acid profile of rumen fluid and to obtain the mass spectra of each component of the mixture. This basic research is a preliminary but essential step in order to carry out a complete compound screening and to perform routine quantitative analyses with a less sophisticated technique such as gas chromatography.