Hplc dad

Lyophilized dry leaf powder (0.1 g) was homogenized with 100% cold acetone (3 mL) using Ultra-Turrax homogenizer for 30 s. Samples were then centrifuged at 11,500× g and 4 °C for 10 min, filtered through Minisart (SRP 15, PTFE) polyamide filters (Sartorius Stedim Biotech, Göttingen, Germany) and transferred to vials. The entire extraction process was performed in dim light.
Chloroplast pigments were analyzed using HPLC-DAD (Thermo Finnigan, San Jose, CA, USA) according to the method described in Vosnjak, et al. [20 (link)]. The content of each compound was calculated using corresponding external standards and expressed as µg g⁻¹ dry weight (DW).
The following sums of compounds identified in our study were expressed as total chlorophylls (sum of chlorophyll a and chlorophyll b), total carotenoids (sum of lutein, β-carotene, neoxanthin, violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z)), xanthophyll cycle pigments (VAZ; sum of V, A and Z). In addition, de-epoxidation state of the xanthophyll cycle (AZ/VAZ; ratio between the sum of A and Z and the sum of V, A and Z) and the chlorophyll a/b ratio (ratio between chlorophyll a and chlorophyll b) were calculated.

Carotenoids were extracted according to the method described by Mikulic-Petkovsek et al. [56 (link)]. Briefly, 0.2 g of the frozen material (pulp with skin, without seeds) was extracted in glass centrifuges with 2 mL of acetone at a temperature of 4 °C using an Ultra-Turrax (IKA-Werke GmbH & Co. KG, Staufen, Germany) homogenizer for 30 s and determined on the Accela HPLC system (Thermo Scientific, San Jose, CA, USA) using the gradient method. Samples were then filtered into labelled vials through a Cromafil A-20/25 polyamide/nylon filter (Macherey-Nagel, Dueren, Germany). The vials containing the extracts were stored at −20 °C until further analysis. The extracts were then analyzed using HPLC-DAD (Thermo Finnigan, San Jose, CA, USA) at 450 nm with a Gemini C18 column (150 × 4.6 mm 3 μm; Phenomenex, Torrance, CA, USA). The first mobile phase was solvent A: acetonitrile/methanol/water (100/10/5, v/v/v) and the second solvent B: acetone/ethyl acetate (2/1, v/v). The flow rate was 1 mL/min with the following gradient: from 10 to 70% B in the first 18 min, then linearly at 70% B up to 22 min and back to the initial conditions until the end of the run.

High-performance liquid chromatographic methods, using HPLC-DAD (Dionex Thermoline Fisher Scientific, Waltham, MA, USA) with gradient elution, were developed to determine anthocyanin 3,5-O-glucosides of pelargonidin. The HPLC gradient method, coupled with the DAD detector, allowed the qualitative and quantitative determination of pelargonidin-3,5-glucoside in extracts from pomegranate flowers. The determinations were carried out using a LiChrospher RP18-5 4.6 mm × 250 mm, 5 µm; the mobile phase contained 5% formic acid in water(A) and 5% formic acid in methanol (B). The gradient developed for the needs of the method assumed changes in the mobile phase according to the scheme: 0–15 min B = 15–55%, 15–20 min B = 55–65%, 20–30 min B = 65–70%, 30–40 min B = 70–75%. The method considers 10 min re-equilibration time to determine the phase equilibrium column relative to the initial injection phase. The phase flow was set to level 1 mL/min, injection for all test samples was 30 μL, and the detection was carried out at 520 nm. The chromatogram of the extract is shown in Figure 5.

After the incubation period, cultures were filtrated with Nalgene™ Rapid-Flow™ Filters of 0.45 μm pore size (Thermofischer Scientific, Waltham, MA, USA) to remove microorganisms. Filtrates were then extracted with 70 mL of ethyl acetate and shaken on a Universal Shaker SM 30 B Control Edmund Bühler^® (Thermofischer Scientific, Waltham, MA, USA) set at 150 rpm overnight. The organic phase was recovered and evaporated until dry under a rotavapor set at 60 °C. Samples were resuspended with 2 mL of acetonitrile/water (30/70, v/v) mixture and filtered through 0.45 µm PTFE syringe filters (Sigma Aldrich, St. Quentin Fallavier, France). Samples were conserved at 4 °C until further analysis. T-2 toxin was analyzed by Gemini C18 columns, 150 mm × 4.6 mm, 3 μm and a pre-column with the same characteristics (Phenomenex). As for PLA, T-2 toxin was detected and quantified using HPLC-DAD (Dionex, Sunnyvale, CA, USA) according to the methodology described by Medina et al. [59 (link)]. T-2 toxin quantification was calculated according to a standard calibration curve with concentrations ranging between 0.2 and 50 μg/mL.