Determination of biogenic amines in ileal and colonic digesta with high performance liquid chromatography (HPLC)

The concentrations of methylamine, spermidine, cadaverine, putrescine, and histamine in chyme were detected by HPLC with a method adapted from the previous report (Fan et al., 2017b) with modifications. Briefly, 0.2 g ileal or colonic chyme was put into a 2 mL screw-capped tube along with 1 mL trichloroacetic acid solution, then homogenized for 10 min and centrifuged at 3,600 × g at 4°C for 10 min. An equal volume of n-hexane was mixed with pooled supernatant for 5 min. The organic phase was abandoned and the same protocols were used to re-extract the water phase. The pretreated sample was mixed with 20 mL internal standard saturated sodium bicarbonate solution, 1 mL dansyl chloride, and 1 mL NaOH, then set at 60°C for 45 min with several soft reversing. The reaction was then stopped by adding 100 μL ammonia. The solution was maintained in the 40°C water bath and the acetone was vaporized with nitrogen flow. The solution was finally extracted twice with diethyl ether. The extract was dried with nitrogen flow and the residue for injection was dissolved in acetonitrile. HPLC analysis was performed with elution of ammonium acetate-acetonitrile gradient and an Agilent 1200 series system with a dual low-pressure gradient pump, as well as an auto sampler and a column compartment and variable wavelength detector (VWD). The column was a reversed-phase ZORBAX 80A Extend-C18 (4.6 mm × 250 mm; 5 μm) (Agilent, Santa Clara, CA, USA). The two solvent reservoirs contained acetonitrile and 0.2 mol/L ammonium acetate. The flow rate was 1.0 mL/min and the temperature was 30°C. The wavelength was 260 nm for VWD. Each sample was measured three times.