Spd m20a

HPLC was conducted using Shimadzu liquid chromatography system (LC-2AD) with a diode array detector (SPD-M20A) and a Shim-pack Synergi^TM 4 μm Polar-RP 80 Å (250 mm × 4.6 mm I.D.) at 35 °C. The mobile phase consisted of two positions (solvents A and B). Solvent A was distilled water with 0.1 M ammonium acetate (pH 5.0 adjusted for acetic acid), and solvent B was acetonitrile. The gradient was 0–5.0 min, 10% B; 5.0–10.0 min, 10–20% B; 10.0–65.0 min, 20% B; 65.0–65.1 min, 20–95% B; 65.1–70.0 min, 95% B; 70.0–70.1 min, 95–10% B; 70.1–77.0 min, 10% B. The run time was 77 min, with a flow rate of 1.0 mL/min. The UV detection wavelength was 245 nm, and peaks were identified by comparing their relative retention times with those of authentic standards analyzed under the same conditions. The monosaccharide standards were derivatized by the same method as the sample. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The concentrations of phenolic compounds were analyzed using high-performance chromatography (HPLC) (LC-20AB, SPD- M20A photodiode array detector (PDA), Shimazu, Kyoto Japan) equipped with an InfinityLab Poroshell 120 EC-C18 chromatography column, 150 mm length, 4.6 mm width, and particle size 2.7 μm at column oven temperature 26 °C. The binary gradient method was used in HPLC analysis incorporated 2% acetic acid dissolved in water (A) and a mixture of concentrated acetic acid, water, and acetonitrile (1:9:40 v/v/v) (B). The total runtime of the analysis was 93 min referring to the method described elsewhere^{14 (link)} as follows: (a) initially 0–25 min, 10–30% B; (b) 25–50 min, 30–40% B; (c) 50–75 min, 40–90% B; (d) 75–93 min, 10% B. An amount of 20 μL of the samples was injected onto the column, and three wavelengths 280, 360 and 520 nm were chosen for analysis in this investigation using HPLC–DAD. For quantitative purposes, a calibration curve was constructed by analysis of known concentrations of different standard compounds.

Analytical and semi-prep high-performance liquid chromatography (HPLC) were performed on a Shimazu HPLC system equipped with a SPD-M20A prominence diode array detector. A C18 reversed-phase column (Jupiter 5 µm C18 300 Å, 250 × 4.6 mm) was used for analytical analysis. A C18 reversed-phase column (Jupiter 5 µm C18 300 Å, 250 × 10 mm) was used for semi-prep purification. The analytes were eluted using a gradient mixture of two solvents: solvent A was distilled deionized water containing 0.05% trifluoroacetic acid (TFA) and solvent B was 90% acetonitrile (ACN) in distilled water containing 0.05% TFA. The mobile phase flow rate was 1 ml min⁻¹ for analytical analysis and 2.5 ml min⁻¹ for semi-prep purification. The separation temperature was room temperature.

The main polyphenols in ROE were determined using high-pressure liquid chromatography (HPLC, SHIMAZU LC-20AT liquid chromatography, Japan) with a photodiode array (PDA, SHIMAZU SPD-M20A) detector. Separation and identification were based on our previous report^{16 (link)} and performed with a COSMOSIL 5C18-MS-II (4.6 mm × 250 mm, 5 μm) at 30 °C with a gradient elution solution A, composed of a formic acid–water solution (0.1% formic acid), and solution B, comprising an acetonitrile and formic acid–water solution (0.1% formic acid) (6 : 4; v/v), which were delivered at a flow rate of 0.7 mL min⁻¹ as follows: 0 min, 96% (A); 180 min, 70% (A); 205 min, 96% (A); and 210 min, 0% (A). The UV spectra of every identified peak were recorded at 280 nm. The tested standards, including procyanidin B3 (1), EGC (2), catechin (3), epicatechin (4), (−)-fisetinidol-(4α,8)-(−)-catechin (5), (4α,8)-(−)-fisetinidol-(−)-epicatechins (6), and (+)-guibourtinidol-(4β,8)-epicatechin (7), were made by our laboratory. These compounds were isolated and identified from ROE by our group and the purity of each standard was higher than 95%.

Four phytochemicals (chlorogenic acid, ellagic acid, gallic acid, and quercetin) were quantified using an HPLC analysis system (Shimadzu chromatographic system, Shimadzu, Japan). The HPLC system consists of an LC pump (LC-20AD), autosampler (SIL-20AC HT), HPLC column oven (CTO-20A), a photodiode array detector (SPD-M20A), system controller (CBM-20A) and a Shimazu LCsolution^® software. An Inertsil^® ODS-3 analytical column 5020-01732 (reversed-phase C18 column) was used for the analysis (22 (link)). The HPLC-grade chemical standards and fruit extract samples were prepared in 1 mL of Milli-Q^® type 1 ultrapure water (Merck Millipore, USA) followed by sonication for 30 sec, filtered through a 0.45 μm nylon filter, and kept on ice throughout the process to protect the biomolecules from degradation. The mobile phases and detection wavelengths are shown in Table 1. Several solvent combinations were developed and optimized to obtain good separation of peaks and symmetry of peak shapes referring to Sawant et. al. (23 ). The determination of each of the phytochemical contents was extrapolated from HPLC standard curves ranging between 0 and 100 μg/mL. Each sample was injected at least three times and the means of data points were presented.

A Shimadzu chromatographic system (Model no. LC-20AD) with a dual λ UV absorbance diode array detector (SPD-M20A) was used for the RP-UFLC analysis. The data processing was performed using a built-in LC-Solution software system. The separation of the compounds was achieved on a Qualisil BDS 250 × 4.6 mm (5 µm) C18 column for phenolic compounds and a Hibar 250 × 4.6 Lichrospher 60 RP-select B (5 µm) column for the piperine. Acetonitrile, water, and glacial acetic acid (with a ratio of: 12:85:3) was used as a solvent system for the separation of the phenolics, whereas piperine was separated using water and methanol (30:70) in an isocratic mode with a common injection volume (20 µL). The pressures were maintained by adjusting the flow rates (0.7 and 1.4 mL/min) at a λmax of 280 nm and 343 nm in order to detect the phenolic compounds and piperine, respectively.
The calibration, linearity, LOD, LOQ, retention time, and the tailing factor parameters according to the ICH guidelines for the phenolic compounds and piperine are provided in Table 2. The standard signal/noise methods and parameters were used in order to perform the detection and quantification limits (LOD and LOQ) [11 (link),41 (link)].