Dionex ultimate 3000 rs u hplc liquid chromatograph system

The AAS enzyme assays were performed in 100 μL of reaction buffer (50 mM sodium phosphate, pH 6.8, 200 μM PLP) containing 7 μg of recombinant OeAAS. Reactions were carried out with a range of amino acid substrate concentrations (0.05–5 mM) at 30 °C for 20 min prior quenching with 100 μL of 0.8 M formic acid. The reaction mixture was centrifuged and the supernatant was analyzed by high-performance liquid chromatography (HPLC). Analysis of substrates and reaction products was performed in a Beckman Coulter HPLC system equipped with a DAD System Gold 168 detector and a Superspher RP 18 column (4.6 × 250mm, particle size 4 μm: Dr. Maisch GmbH, Ammerbuch, Germany). The amount of product in reaction mixtures containing different concentrations of substrates were quantitated based on a standard curve generated using authentic 2,4-DHPAA, 4-HPAA, or phenyl acetaldehyde (PAA) standards. Identification of compounds was confirmed by HPLC/ESI-qTOF-HRMS on a liquid chromatograph Dionex Ultimate 3000 RS U-HPLC liquid chromatograph system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a similar column and elution program.

VOO phenolic compounds were isolated by solid phase extraction (SPE) on a diol-bonded phase cartridge (Supelco, Bellefonte, PA, USA) based on the method by Mateos et al. [25 (link)] using p-hydroxyphenylacetic and o-coumaric acids as internal standards. Phenolic compounds were analyzed by HPLC on a Beckman Coulter liquid chromatography system equipped with a System Gold 168 detector, a solvent module 126 and a Waters column heater module following a previously described methodology [26 (link)]. A Superspher RP 18 column (4.6 mm i.d. × 250 mm, particle size 4 µm: Dr Maisch GmbH, Ammerbuch, Germany) at flow rate 1 mL min⁻¹ and a temperature of 35 °C was used. Tentative identification of compounds was conducted with their UV-Vis spectra and later confirmed with HPLC/ESI-qTOF-HRMS on a liquid chromatograph Dionex Ultimate 3000 RS U-HPLC liquid chromatograph system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a similar column and elution program. Mass spectra were acquired in MS fullscan mode and data were processed using TargetAnalysis 1.2 software (Bruker Daltonics, Bremen, Germany).