Lichrocart 250 4

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.

A commercially available mangosteen-based functional juice (Lord Duke Biotechnology Corporation, Taipei, Taiwan) was employed as a supplemental treatment in the present study. The mangosteen-based juice is a liquid beverage containing 50 % whole-fruit mangosteen, 7 % pomegranate juice concentrate, 7 % acerola juice concentrate, 12 % white grape juice concentrate, 6 % strawberry juice concentrate, 10 % passion fruit juice concentrate and 8 % tamarind jams blend. High-performance liquid chromatography (HPLC) was used to determine the major components of the mangosteen-based juice. HPLC-UV analysis was conducted using an HPLC system (Hitachi, Tokyo, Japan) consisting of a L-2420 UV–Vis detector, a L-2200 autosampler, and a L-2130 pump. LiChroCART^® 250–4,6 (Merck KGaA, Darmstadt, Germany) was used as an analytical column. The injection volume was 20 μL. For determination of the content of α-mangostin, the mobile phase, composed of solvent A (methanol) and solvent B (0.15 % formic acid) in a ratio of 2:98, was delivered at a flow rate of 1 mL · min⁻¹. The detection UV wavelengths were set at 254 nm. For determination of the HCA content, the mobile phase, composed of solvent A (methanol) and solvent B (0.1 % H₃PO₄) in a ratio of 98:2, was delivered at a flow rate of 1 mL · min⁻¹. The detection UV wavelengths were set at 220 nm. All of the analyses were performed in triplicate.

To optimize and study the effect of each parameter on cotinine binding and extraction, the HPLC method was used for the whole experiment. Cotinine determination was performed using HPLC with 260 nm UV detection at 40 °C. A reverse phase column, Purospher® STAR RP-18 endcapped (5 µm) LiChroCART® 250-4.6 (250 mm × 4.6 mm id, particle size 5 µm, Merck), was used for separation. The mobile phase consisted of 71% ammonium acetate buffer (13 mM) and 29% acetonitrile. The pH of the mobile phase solvent was adjusted to 5.0 by glacial acetic acid. Samples were filtered through a 0.45 µm syringe filter before injection. The injection volume was 20 µL and the flow rate was set at 0.8 mL/min. Cotinine standards in a concentration range of 62.5–500 µg/mL with internal standard (2-phenylimidazole) were run as the mentioned condition. The chromatogram of standard mixture is shown in the Supplementary Data (Supplementary Figure S2 and Table S1).

The fingerprint chromatogram of AZC was established through high-performance liquid chromatography (HPLC) employing a Waters® system equipped with a Waters® 2998 photodiode array detector. Separation was achieved using Purospher® STAR, C-18 encapped (5µM), LiChro-CART® 250 − 4.6, and HPLC-Cartridge, Sorbet Lot No. HX255346 (Merck, Germany).
In this study, the mobile phase gradient comprised 100% methanol (solvent A) (Fisher Scientific, USA) and 2.5% acetic acid (solvent B) (Fisher Scientific, USA) in deionized (DI) water. The gradient elution was conducted at a flow rate of 1.0 mL/min, with the following gradient profile: 0–17 min, 70% A; 18–20 min, 100% A; 20.5–25 min, 10% A. Before administration, the sample underwent filtration (0.45 μm, Millipore), and a 20 µL aliquot of the sample was directly utilized. The chromatogram was assessed at 280 nm using a UV detector, and data analysis was carried out employing EmpowerTM3 [22 (link)].

The fingerprint chromatogram of PMG was determined by using the high-performance liquid chromatography (HPLC) analysis. Chromatography was performed by using a Waters® system equipped with a Waters® 2998 photodiode array detector. The separation of chromatogram was performed using Purospher® STAR, C-18 encapped (5 μm), LiChroCART® 250-4.6, and HPLC-Cartridge, Sorbet Lot No. HX255346 (Merck, Germany). According to this study, 100% methanol (solvent A) (Fisher Scientific, USA) and 2.5% acetic acid (solvent B) (Fisher Scientific, USA) in deionized (DI) water were used to produce the gradient of mobile phase. The gradient elution of mobile phase 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 an aliquot of sample at the volume of 20 μl was directly administered. The chromatogram assessment was performed at 280 nm using a UV detector, and data analysis was performed using EmpowerTM3.

The concentrations of main active compounds (aloin A and aloenin A) were determined by using the HPLC-Diode-Array Detection method (equipment LC system, Dionex Thermoline Fisher Scientific) with Chromeleon software version 7.0) described previously [20 (link)]. Briefly, separations were performed on a LiChrospher RP-18 column, 5 μm particle size, 250 mm × 4 mm LiChroCART^® 250-4 (Merck, Darmstadt, Germany). The detection was performed using a diode array detector at a wavelength maximum (λ_max) of 295 nm. The following mobile phase was composed of water (A) and methanol (B) with a gradient elution: 0–35 min, 35–95% B; 35–40 min, 95% B; 40–45 min, 35 B with a flow rate of the mobile phase set at 1.0 mL/min and the column temperature was maintained at 30 °C.
The presence of aloin A and aloenin A in the lyophilizate and extract was confirmed by comparison of the retention time and UV spectra of analyzed substances with their reference standards.

The sugar content of carob honey was determined by a method described by Liviu et al. [16 ]. Five grams of honey samples were dissolved in water and transferred to a 100 mL volumetric flask containing 25 mL of methanol; the volume was adjusted to 100 mL with water. The solution was passed through a 0.45-mm filter and the sugar content was determined in a high-performance liquid chromatography system equipped with a refractive index detector (Hitachi model L-2490, Japan). Sugar separation was performed in a Merck amino-bonded high purity silica column (LiChroCART 250-4; particle size diameter of 5 mm). The mobile phase was acetonitrile/water (75:25 v/v) at a flow rate of 1.3 mL/min and it was filtered through a membrane filter (0.45 µm) from Technochroma before the elution. The injection volume of the samples was 10 mL, with a flow rate of 1.3 mL/min. Sample peaks were identified by comparing their retention times with the standards. The samples were spiked with standards to verify the identity of the chromatographic peaks. The average peak areas of triplicate injections were used for peak quantification. A calibration curve was made for each sugar using standard solutions of different concentrations (0.5-80 mg/mL). The linear regression factor of the calibration curves was higher than 0.9982 for all sugars. The samples in crystallized form were liquefied in a 40°C water bath.