Spd m30a

The phenolic compounds were analyzed using a ultra-high performance liquid chromatography (UPLC) system (CBM-20A, Shimadzu) with two gradient pump systems (LC-30AD, Shimadzu), a UV-detector (SPD-M30A, Shimadzu), an auto sample injector (SIL-30AC, Shimadzu), and a column oven (CTO-30A, Shimadzu). Separation was achieved on an XR-ODS column (3.0 × 100 mm, 1.8 μm, Shimadzu) using a linear gradient elution program with a mobile phase containing solvent A (0.1%, v/v, trifluoroacetic acid in distilled deionized water) and solvent B (0.1%, v/v, trifluoroacetic acid in acetonitrile). The phenolic compounds were separated using the following gradient: 0–5 min, 10–15% B; 5–10 min, 15–20% B; 10–15 min, 20–30% B; 15–25 min, 30–50% B; 25–30 min, 50–75% B; 30–35 min, 75–100% B; 35–40 min, 100–5% B; and 40–45 min, 5–0% B. The phenolic compounds and anthocyanins were detected at 280 nm and 520 nm, respectively. The chlorogenic acid (CGA), caffeic acid (CA), delphinidin-3-sambubioside (Dp3-Sam), delphinidin-3-glucoside (Dp3-Glu), cyanidin-3-sambubioside (Cy3-Sam), all obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) were identified based on the retention times of commercial standards (UV spectrum). Gallocatechin (GC) and gallic acid (GAL) were identified as described in a previous study [16 (link)] and by their UV-visible spectral characteristics.

The HPLC analysis of crude methanolic L. stavium seed extract was performed on a Shimadu i-series of liquid chromatograph comprising of a solvent delivery pump Model LC-20AT, controller Model LC-2030, UV visible detector with variable wavelengths Model UV-2600, and autosampler equipped with a photodiode array detector (SPD-M30A, Shimadzu). Separation of flavonoids was performed on Inertsil ODS III analytical column with 250 mm × 4.6 mm dimensions and 5 μm particle size. The analytical column was externally protected by C18 Guard‐Pak cartridge (Waters, Milford). Injection volume was used to be 10 µL. The mobile used were: 0.2% of acetic acid in 45% of Millipore water and 0.2% of acetic acid in methanol and an isocratic program was applied. All the separations were carried out at 20°C temperatures with a 0.8 mL/min flow rate. The peaks were identified on the chromatogram by comparing their retention times and spectra with those of commercial and authentic standard solution. Identification of flavonoid compounds was based on the appearance of the absorption maxima between 275 and 320 nm in the spectrum of the individual peaks.

Quantification and carotenoid profile of permeated PEC-DMSO solution into receptor cells were determined by HPLC-PDA-MS/MS [27 (link)]. The mobile phases and gradient conditions used for HPLC carotenoid analysis are shown in Table S1, while Table S2 summarizes the MS parameter settings for optimized analysis, performed in a Shimadzu LCMS-QP (Kyoto, Japan), equipped with binary pumps (LC-40D X3) and connected, in series, to a photodiode array (PDA) detector (SPD-M30A) and triple quadrupole mass spectrometer (LCMS-8050TM). The UV-visible spectra were obtained between 250 and 700 nm, and the chromatograms were processed at 450 nm; 1 µL volume of each of the withdrawn aliquots of permeated PEC-DMSO solution was subjected to carotenoid analysis.
Calibration curves for lutein, β-cryptoxanthin, β-carotene, and lycopene with a minimum of five concentration (0.01–0.50 mg/mL) levels were built. Retention times of carotenoid molecular species were determined using standard compounds. UV-visible (λmax) and mass spectra were compared with data available in literature [36 (link),37 (link),38 (link),39 (link),40 (link)].

The PC content analysis of pale ale craft beer microparticles was performed according to the adaptations made previously (18 (link)) using soluble and conjugate phenolic extraction methods. Conjugated PCs were extracted via alkaline and acid hydrolysis. All extracts were filtered through 0.45 μm cellulose ester membranes (Merck Millipore Co., Darmstadt, Germany) prior to high-performance liquid chromatography (HPLC). The HPLC system was equipped with a 5 μm C18 guard column (10 × 3.0 mm, I.D., Ascentis^®), 5 μm reverse-phase C18 column (250 × 4.6 mm, I.D., Ascentis^®, Los Angeles, CA, United States) and SPD-M30A (Shimadzu, Kyoto, Japan) diode-array detector (DAD). The DAD wavelength was monitored from 190 to 370 nm. The temperature of column was set at 40°C and the volume of injection was 20 μL. The mobile phase (1.0 mL·min⁻¹) was 0.3% formic acid (in H₂O-DD), methanol (100%), and acetonitrile (100%) with gradient elution. The quantification of PC was performed using a calibration curve (1–50 ppm) with a minimum of five standards concentrations.

The method was developed with a Nexera X2 UHPLC system comprising two LC-30AD pumps with degassers, SIL-30AC autosampler, CTO-30AD oven and SPD-M30A photo diode-array detector, controlled by LabSolutions software (Shimadzu Corp., Kyoto, Japan).
The stationary phase was a Symmetry C18 (2.1 × 100 mm, 3.5 µm) analytical column (Waters, Milford, USA) preceded by a Symmetry C18 guard column (2.1 × 10 mm, 3.5 µm) (Waters). Mobile phase was 20 mM sodium phosphate buffer at pH 3.0 in 12.5% acetonitrile delivered isocratically at 0.25 mL/min. The autosampler was held at 4°C, and injection volume was 0.5 µL. The photodiode-array detector scanned from 200 to 500 nm and ceftazidime, avibactam and pyridine were quantified at wavelengths of 260, 230 and 254 nm, respectively.

The molecular weight analysis of melanoidin EAM was determined using high-performance gel permeation chromatography with photodiode array detection (HPGPC-PDA) at liquid chromatograph LC-20 Prominence coupled with photodiode array detector SPD-M30A (wavelength 270 nm; all Shimadzu) and TSK-Gel G4000 SWXL column (7.8 × 300 mm; Shimadzu, Tosoh Bioscience LLC, Tokyo, Japan) at the column temperature of 35 °C. Isocratic elution with 20 mM KH₂PO₄ was used for the separation. The injection volume was 5 μL, and the flow rate was 500 μL/min. To calibrate the column, the dextrans with 10, 400, and 400 kDa were used (all Sigma-Aldrich).