Quantification of short-chain fatty acids

SCFA levels were measured in a subset of fecal samples by gas chromatography – mass spectroscopy (GC-MS) at the McMaster Regional Centre of Mass Spectrometry. A weight equivalent amount of 3.7% HCl, 10 µL of internal standard, and 500 µL of diethyl ether was added to each fecal sample and vortexed for 15 min. After vortexing, 400 µL of diethyl ether fecal extract was transferred to a clean 1.5 mL Eppendorf tube. In a chromatographic vial containing an insert, 20 µL of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide was added, after which 60 µL of diethyl ether fecal extract was added. The mixture was incubated at room temperature for one hour and analyzed using GC-MS (6890N GC, coupled to 5873N Mass Selective Detector; Agilent Technologies, Santa Clara, CA, USA). Statistical significance was assessed by linear mixed model with pBMI, GWG category, and parity as fixed effects and participant ID as a random effect, and multiple comparisons by pBMI and GWG category were performed within primiparous and multiparous participants (Kenward-Rogers degrees of freedom with Tukey adjustment for multiple comparisons).