Waters micro mass zq 4000

Samples were derivatized with the AMP + MaxSpec Kit (Cayman Chemical, Ann Arbor, USA, 710000) according to the manufacturer's instructions. Briefly, the samples were derivatized by adding 20 l of acetonitrile/dimethylformamide, 20 l of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide, 10 l of 1-hydroxybenzotriazole (Sigma-Aldrich), and 30 l of the AMP + reagent. The reaction mixture was incubated for 30 min at 60°C. Samples were stored at -20°C before analysis. AMP + -derivatized SCFAs were separated on a Hypercarb column (3 m, 100 × 2.1 mm; Thermo Fisher Scientific, 35003-031030) using a gradient of solvent A (5% acetonitrile and 0.1% formic acid) and solvent B (100% acetonitrile). After 5 min of 100% solvent A, the proportion of solvent B was increased to 100% within 15 min at a flow rate of 200 l/min at 30°C. Quantification of SCFA was performed by LC-ESI-MS (Alliance HT Waters 2790 with Waters Micromass ZQ 4000, Waters, Eschborn, Germany, 176004001) using the selected ion recording mode for the AMP + signals 227 (acetic acid), 241 (propionic acid), 255 (butyric acid), and 269 (valeric acid) (Sigma-Aldrich). For absolute quantification, an SCFA standard calibration mixture (WSFA-2; Merck, Darmstadt, Germany, 46975-U) was used.