1260 infinity high performance liquid chromatograph

HPLC chromatograph, 1260 Infinity high performance liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) was used to determine polyphenolic compounds in the experimentally produced edible packaging. The method of Gómez-Estac et al. [27 (link)] with slight modifications was used. The mobile phase consisted of 1% phosphoric acid (A) and acetonitrile (B) in the following composition: 80% of A and 20% of B for 20th min, 70% of A and 30% of B from 20th to 25th min, 60% of A and 40% of B from 25th to 40th min. The separation was performed on a Zorbax SB-C18 4.6 × 250 mm column (Agilent Technologies, Santa Clara, CA, USA) (the temperature was 25 °C) and detection was performed on a DAD array detector (detection wavelength was 324.5 nm) The injection volume was 10 µL. Each sample was measured in triplicate. The software used for HPLC chromatohraph was Agilent ChemStation.

A Hypersil HILIC hydrophilic column (specification 150 × 2.1 mm, 1.7 μm, ThermoFisher Scientific, Waltham, MA, USA) was constructed as follows: mobile phase A: water (5 mM ammonium acetate); mobile phase B: acetonitrile. Conditions for the gradient elution using gradient program were: 0–5 min, 50% A; 5–10 min, 50% A-80%, 10–15 min, 80% A, total run time 15 min; flow rate: 0.3 mL/min; amount: 3 μL; column temperature: 30 °C. The electrospray ion source was in positive ion mode with high purity nitrogen (purity 99.999%) as dry gas, drying gas temperature 325 °C, flow rate 10 L/min. The scanning method was in multiple reaction monitoring mode, quantitative ion pair: TMAO m/z 76 → 58, d9-TMAO m/z 85 → 66; qualitative ion: TMAO m/z 42, d9-TMAO m/z 46; and collision energy: TMAO 20 eV, d9-TMAO 25 eV. Analyses were conducted using 1260 Infinity High Performance Liquid Chromatograph (Agilent, Santa Clara, CA, USA) and 6420 Triple Quad Triple Quadrupole Mass Spectrometer (Agilent, Santa Clara, CA, USA). Circulating interleukin-6 (IL-6) levels were measured using commercial ELISA kits (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions.