Gas Chromatography–Mass Spectrometry Measurements of Butyrate

Samples were processed as described before (Krautkramer et al., 2016). Briefly, an aliquot of 50 μl of cultures incubated for 12 h with starch and quercetin and E. ramulus monocultures with glucose and quercetin (control) were mixed with 20 mM of a butyric-d7 acid, 99.5 atom % D, CDN isotopes #D-171 as internal standard, acidified with 5 μl of 33% HCl, extracted twice with Diethyl Ether, then 60 μl of each sample was mixed with 2 μl of derivatizing reagent (N-Methyl-N-tert-butyldimethylsilyltrifluoroacetamide, MTBSTFA) and incubated at RT for 2 h. For detection, 1 μl of each sample was injected in a gas chromatography–mass spectrometry (GC-MS) instrument (Agilent 7890B/5977A GC/MSD), and an Agilent DB-1 ms column was used. Oven program was: initial temperature, 40°C for 2.25 min; then 20°C min^-1 to 200°C; next 100°C min^-1 to 300°C, maintained for 7 min.