2.3. SCFA Derivatisation

All derivatisation reagents and SCFA standards were prepared in acetonitrile/water (2:1, v/v). Raw milk and serum samples were allowed to thaw thoroughly at room temperature, and SCFAs in raw milk and serum were extracted by adding two volumes of pure acetonitrile in one volume of milk/serum, followed by vortex for 2 min and centrifugation for 15 min (13,000× g); the supernatant was used for SCFA measurement. SCFAs were derivatised based on the protocol for carboxylic acid analysis described by Han et al. [19] with modifications. Briefly, the derivatisation reaction was conducted in a 2-millilitre Eppendorf tube, placed in a water bath (30 °C) for 30 min with frequent shaking after sequentially adding 50 µL of 50 mM 3-NPH·HCl, 50 mM EDC and 7% pyridine to 100 µL of standards or samples. When the reaction was completed, 250 µL of Milli-Q water was added to each reaction mixture before LC-MS analysis.