Quantification of Medium-Chain Fatty Acids

The MCFA levels in the plasma and intestinal samples were determined following a previously described protocol with modifications (17 (link)). Briefly, the samples containing an internal control (C19:0) were homogenized in methanol and mixed with chloroform and water to extract the lipids. The samples were centrifuged at 2,000 × g at 17 °C for 10 min. The supernatant containing MCFAs was collected and dried. The samples were resuspended in chloroform:methanol (1:3, v/v) and subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis using an ultra-performance LC system (UPLC, Waters, Milford, MA, USA) equipped with an Acquity UPLC system coupled to a Waters Xevo TQD mass spectrometer (Waters). The samples were separated on an ACQUITY UPLC BEH C18 column (2.1 × 150 mm, 1.7 μm; Waters) using a methanol gradient in 10 mM ammonium formate aqueous solution.