Purospher star

Qualitative and quantitative analyses were performed by HPLC-DAD. Anthocyanin and chlorogenic acids profiles were characterized simultaneously using the Hitachi Chromaster system (Hitachi High-Tech, Tokyo, Japan) with the use of a Purospher STAR (Merck KGaA, Darmstadt, Germany) RP-18e column (5 µm, 4 × 250 mm), according to methods validated and described in our previous publication on the analysis of chokeberry extracts [23 (link)]. A mobile phase gradient system consisted of 4.5% (v/v) formic acid (A) and acetonitrile (B). The gradient conditions were as follows: 0–5 min (5% B), 5–15 min (5–8% B), 15–50 min (8–25% B), 50–55 min (25–50% B), and 55–65 min (5% B). Flow rates were as follows: 1–15 min, 1 mL/min; 15–50 min, 0.8 mL/min; and 50–65 min, 1 mL/min. The CGAs chromatograms were monitored at 330 nm and the anthocyanin at 520 nm. Retention times were as follows: neochlorogenic acid (nCGA)—10.9 min, chlorogenic acid (CGA)—20.5 min, cyanidin 3-galactoside (CyaGal)—26.8 min, and CyaAra—cyanidin 3-arabinoside (CyaAra)—30.7 min. All measurements were performed in triplicate. The concentrations of the compounds were determined using an appropriate calibration curve. The final results were expressed per 1 g of dry weight.

The phenolic profiles of OA extract consisting of cyanidin-3-glucoside (Sigma-Aldrich, USA), gallic acid (Sigma-Aldrich, USA), and quercetin-3-O-rutinoside (Sigma-Aldrich, USA) were determined by high-performance liquid chromatography (HPLC). Chromatography was performed by using a Waters® system equipped with a Waters® 2998 photodiode array detector. Chromatographic separation was performed using Purospher® STAR, C-18 encapped (5 μm), LiChroCART® 250-4.6, and HPLC-Cartridge, Sorbet Lot no. HX255346 (Merck, Germany). The mobile phase (HPLC grade) consisted of 100% methanol (solvent A) (Fisher Scientific, USA) and 2.5% acetic acid (solvent B) (Fisher Scientific, USA) in deionized (DI) water was used to induce gradient elution. The gradient elution was carried out at a flow rate of 1.0 ml/min with the following gradient: 0-17 min, 70% A; 18-20 min, 100% A; and 20.5-25 min, 10% A. The sample was filtered (0.45 μm, Millipore), and a direct injection of the tested sample at the volume of 20 μl on the column was performed. The chromatograms were recorded at 280 nm using the UV detector, and data analysis was performed using Empower™ 3.

High-performance liquid chromatography (HPLC) analysis was used to determine the fingerprint chromatogram. Chromatography was performed by using a Waters® system equipped with a Waters® 2998 photodiode array detector. Chromatographic separation was performed using a Purospher® STAR, C-18 encapped column (5 μm) and LiChroCART® 250-4.6 HPLC cartridge, Sorbet Lot No. HX255346 (Merck, Germany). The mobile phase (HPLC-grade) consisted of 100% methanol (solvent A) (Fisher Scientific, USA), and 2.5% acetic acid (solvent B) (Fisher Scientific, USA) in deionized (DI) water was used to induce gradient elution. The gradient elution was carried out at a flow rate of 1.0 ml/min with the following gradient: 0-17 min, 70% A, 18-20 min, 100% A; 20.5-25 min, 10% A. The sample was filtered (0.45 μm, Millipore), and a direct injection of the tested sample at the volume of 20 μl on the column was performed. Chromatogram detection was performed at 280 nm using a UV detector, and data analysis was performed using EmpowerTM3.

The instrument used for LC-MS/MS was a Waters Acquity UPLC system (Waters, Milford, MA, USA) coupled with a TQD mass spectrometer (Waters Micromass, Manchester, UK). A Purospher Star (Merck, Darmstadt, Germany) RP-18 column (50 × 2.1 mm, 2 μm particle size) was used for the separation of AFB1. The mobile phase was 0.1% acetic acid and methanol (35:65). Isocratic flow was maintained at 0.3 mL/min. Two product ions were monitored (329 > 273 Da and 329 > 259.1 Da). Quantification ion was 273 Da. MassLynx 4.1 software was employed for data acquisition and processing. The detection limit of the method was 0.02 μg/kg, relative standard deviation of reproducibility was 5.4%, and recovery was 65–81%. Linear regression analysis was performed using JMP v.10 software.

The phenolic compounds in the kenaf seed extract were quantified by HPLC, as described by Baydar et al. [20 ] with a slight modification. The HPLC system was equipped with a diode array detector (DAD). Phenolic compound separation was carried out using a reversed-phase HPLC column (Purospher star 5 μm × 250 mm × 4.6 mm, Merck, Germany). The column temperature and detection wavelength were set at 30°C and 210 nm, respectively. A gradient elution system of solvent A (water with 0.1% phosphoric acid) and solvent B (methanol with 0.1% phosphoric acid) was used (5% B (0 min); 50% B (5 min); 55% B (65 min); and 5% B (70 min)). The flow rate was 1 mL/min, and the injected volume was 20 μL. The phenolic compound chromatographic peaks were confirmed by comparing their retention times with those of the reference standards. Gallic acid, tannic acid, catechin hydrate, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, syringic acid, sinapic acid, ferulic acid, naringin, and protocatechuic acid were used as phenolic compound standards.