High-pressure liquid chromatography (HPLC) was developed and validated for probucol analyses in plasma, stomach, small intestine, large intestine, pancreas, liver, spleen, heart, skeletal muscles, brain, white adipose tissues, kidney, urine, and feces, based on our well-established methods [11 ]. In brief, standard concentrations of probucol in mobile phase acetonitrile in water were prepared for the range of 0.4 to 1000 μg/ml. An autosampler injection volume of pooled samples used was 10 μL per injection. A 250mmx4.6mm Phenomenex Luna C-18 column (5 μm ID) was used, and the UV detector was set at wavelengths of 241nm. The HPLC used was a Shimadzu Prominence consisting of a Shimadzu DGU-20A5 degasser, LC-20AT liquid chromatographer, SIL-20A autosampler, and SPD-20A UV/Vis detector (Japan). The mobile phase consisted of acetonitrile: water as 96:4% v/v ratio. The flow rate was 1.5mL/min with an autosampler injection volume of 10 μL, and the HPLC setup was an isocratic Shimadzu Prominence HPLC system consisting of a Shimadzu DGU-20A5 degasser, LC-20AT liquid chromatographer, SIL-20A autosampler, and SPD-20A UV/Vis detector (Shimadzu, Japan).
Spd 20a uv vis detector
Manufactured by Shimadzu
Sourced in Japan, United States, Australia
The SPD-20A UV/VIS detector is a laboratory instrument used for the detection and quantification of compounds in liquid chromatography (LC) applications. It measures the absorbance of light by samples at specific wavelengths, providing data on the concentration and identity of the detected compounds.
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Lab products found in correlation
Sil 20a ht autosampler, by Shimadzu (11 mentions)
Lc 20at pump, by Shimadzu (10 mentions)
Lc 20ad pump, by Shimadzu (9 mentions)
Hplc system, by Shimadzu (9 mentions)
Luna c18 column, by Phenomenex (6 mentions)
Prominence uflc system, by Shimadzu (6 mentions)
Sil 20a autosampler, by Shimadzu (5 mentions)
Cto 20ac, by Shimadzu (4 mentions)
Cbm 20a, by Shimadzu (4 mentions)
Lc solution software, by Shimadzu (3 mentions)
Lc 20a hplc system, by Shimadzu (3 mentions)
Dgu 20a5r, by Shimadzu (3 mentions)
Lc 10at, by Shimadzu (3 mentions)
Silica gel, by Qingdao Marine Chemical (3 mentions)
Sil 20ac autosampler, by Shimadzu (3 mentions)
Av 600 nmr spectrometers, by Bruker (2 mentions)
Arx 400, by Bruker (2 mentions)
Sephadex lh 20, by GE Healthcare (2 mentions)
Sil 20a ht, by Shimadzu (2 mentions)
Lc 20ab, by Shimadzu (2 mentions)
81 protocols using spd 20a uv vis detector
1
Validated HPLC Method for Probucol Analysis
2
Quantification of MRN by HPLC
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The quantification of MRN was performed via reverse-phase HPLC (HPLC Prominence LC-20AB pump, Shimadzu North America, Columbia, MD, USA), following a method reported in the literature [58 (link)], with slight modification. Briefly, 20 μL of each sample was injected into a C-18 column (Discovery C18 column, 250 mm × 4.6 mm inner diameter (i.d.), 5 μm, Supelco) and the analysis was performed at a flow rate of 1 mL/min using a mobile phase consisting of 0.5% acetic acid in water/acetonitrile (80:20, v/v). MRN was detected at 355 nm using a UV–Vis detector (Shimadzu UV–Vis detector SPD-20A). A chromatogram of MRN is reported in the Supplementary Materials (Figure S1) , as an example.
3
Quantification of Tetracycline by HPLC
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The amount of tetracycline present in the fractions collected at the outlet of the chip chamber was quantified by HPLC. The instrument employed was a Shimadzu composed of the following modules: detector Shimadzu prominence UV/Vis Detector SPD-20A, Shimadzu prominence Communications Bus Module CBM-20A, Shimadzu Liquid Chromatograph LC-20AB. A degasser (Waters™ In-Line Degasser) was also mounted to improve HPLC performances, while fractions were manually injected through a Rheodyne® model 7725i injector connected with a 50 µL loop. Data were acquired with the LCSolution software (Shimadzu). An inverse phase column was used for separations (Luna® C18 by Phenomenex®, 5 µm, 100 Å, 250 mm × 3 mm connected with the pre-column Security Guard™ by Phenomenex®). All fractions were centrifuged at 21,000× g for 10 min at room temperature, before injection in HPLC, to possibly present pellet beads. A mobile phase was composed of solvent A (oxalic acid 0.01 M pH 2.7) and solvent B (methanol and acetonitrile in a 1.5:1 ratio), pumped with a 0.35 mL/min flux. The separation method was the following: 10% solvent B for 5 min, gradient up to 58% of solvent B in 20 min.
4
Structural and Surface Characterization of UiO-66@MIP
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X-ray diffraction (XRD) measurements were carried out on a Bruker D8 Advance Scattering system (Bruker, Germany). Fourier transform infrared spectra (FT-IR) (4000–400 cm−1) in KBr were recorded on a Varian DRX-400 Fourier Transform Spectrophotometer (Agilent Technologies, USA). Thermogravimetric analysis (TGA) was performed using a TG 209 F3 thermal analyzer (Netzsch, Germany) in a N2 atmosphere at a heating rate of 5 °C min−1. The morphology of UiO-66@MIP was characterized by transmission electron microscopy (TEM) on a JEM-2010HR at 120 kV (JEOL, Japan). The pore size of the materials and their N2 adsorption–desorption measurements were analysed with an ASAP-2460 surface area and pore size analyzer (Micromeritics Instrument Corp, USA). HPLC was performed using an LC-20AT LC system coupled with an SPD-20A UV-Vis detector (Shimadzu, Japan).
5
HPLC Analysis of Silymarin
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The HPLC was used on a Shimadzu LC-20AVP system with two LC-20AT solvent delivery units, an SPD-20A UV-vis detector, a CTO-10ASVP column oven (Shimadzu, Kyoto, Japan), a T2000P workstation (Shenyang, China) and a reversed-phase C18 column (250 × 4.6mm, 5μm, DiamodsilTM). The conditions for the HPLC detection of silymarin were as follows: solvent A, methanol; solvent B, water (1 ‰ formic acid); gradient (A%), initial 4 min 43 %, 4-25 min 43-70 %, 25-30 min 70 %, 30.01 min 43 %, 40 min, stop; flow rate, 1mL/min; injection volume, 10μL, wavelength, 288 nm; column temperature,40°C. A sample chromatogram from the extraction of silymarin is shown in Figure 1(Fig. 1) .
6
HPLC Analysis of Allium cepa Extracts
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The analysis of the A. cepa extracts was carried out by high-performance liquid chromatography (HPLC), using a Prominence LC-20A HPLC system coupled with a SPD-20A UV-VIS detector (Shimadzu, Kyoto, Japan). The column used was an Ascentis Express C18-PCP 15 cm × 4.6 mm, particle size 2.7 µm (Supelco Co. Bellefonte, PA, USA). Ultrapure water with 0.05% formic acid (v/v) as solvent A and acetonitrile with 0,02% formic acid (v/v), solvent B, was the mobile phase, on the following gradient condition: 1 min 5% B; 5–60% B in 20 min; 60–100% B in 25 min; returning to initial condition (5% B) in 28 min, held for 5 min for system re-equilibration. Separations were developed with the column temperature at 40 °C and a flow rate of 0.5 mL/min. Samples were prepared at 2 mg/mL in methanol–water (1:1, v/v) and injected at a volume of 2 µL.
7
Analytical Techniques for Compound Characterization
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Melting points were determined using an MP-S3 apparatus (Yanaco, Tokyo, Japan). UV spectra were recorded at room temperature on a U-0080-D spectrophotometer (Hitachi, Tokyo, Japan). IR spectra were obtained with a FT-IR Spectrum RX I spectrophotometer (PerkinElmer, Waltham, MA, USA). Optical rotations were measured using a P-2000 digital polarimeter (JASCO, Tokyo, Japan). 1H- and 13C-NMR spectra were recorded on Avance III HD 700 and Avance III 400 NMR spectrometers (Bruker, Billerica, MA, USA). Chemical shifts are shown in δ values (ppm) with tetramethylsilane as an internal standard. The ESIMS and HRESIMS were taken on a Bruker Daltonics APEX II 30e spectrometer (positive-ion mode). Column chromatography (CC) was performed on silica (70–230 mesh and 230–400 mesh, Merck, Darmstadt, Germany), Diaion HP-20 (Mitsubishi, Tokyo, Japan), and C18 (Sigma-Aldrich, St. Louis, MO, USA) gels, respectively, and preparative TLC (thin-layer chromatography) was conducted on Merck precoated silica gel 60 F254 plates, using UV light to visualize the spots. High-performance liquid chromatography (HPLC) was performed on an LC-20AT series pumping system (Shimadzu, Kyoto, Japan) equipped with a Shimadzu SPD-20A UV-vis detector, and a SIL-10AF auto-sampling system at ambient temperature.
8
SEC-MALS Analysis of PRDX5 Redox Forms
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SEC-MALS experiments were performed using an LC-20 Prominence BioInert HPLC system (Shimadzu, Kyoto, Japan), coupled to a miniDAWN TREOS-II MALS instrument (Wyatt Technology, Santa Barbara, CA, USA), a RID-20A refractive index detector (Shimadzu) and an SPD-20A UV-VIS detector (Shimadzu). For chromatographic separation, 50 µL of sample were loaded alternately onto a Superdex 200 Increase 10/300 GL (Cytiva, Marlborough, MA, USA) and a Superdex 75 Increase 10/300 GL (Cytiva) (data not shown) equilibrated in the PBS running buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, NaCl 137 mM, KCl 2.7 mM pH 7.4). Data were analyzed with ASTRA 7.3 software (Wyatt Technology) and molecular mass was calculated using a Zimm fit model.
Relative quantification of the absolute molar masses of PRDX5 redox form species was performed by taking the ratio of the calculated mass of the selected peak over the total calculated mass of all peaks of interest, expressed as a percentage.
Relative quantification of the absolute molar masses of PRDX5 redox form species was performed by taking the ratio of the calculated mass of the selected peak over the total calculated mass of all peaks of interest, expressed as a percentage.
9
HPLC Analysis of Monosaccharides
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Glucose, fructose, and mannose were determined at room temperature using an HPLC system with an SPD-20A UV-Vis detector (Shimadzu) at 190 nm and an LC-20AD pump (Shimadzu) connected to a COSMOSIL Sugar-D packed column (3.0 mm I.D. × 250 mm, Nacalai Tesque). The eluent was 80 % acetonitrile with a flow rate of 0.5 mL/min.
10
Monosaccharide Composition Analysis of PPp-W
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The monosaccharide compositions and their mass ratios of PPp-W were determined by PMP–HPLC based on a previous report (Ma et al., 2020 (link)) with some modifications. PPp-W (6 mg) was hydrolyzed with 4 mol/L trifluoroacetic acid (TFA) at 110°C for 4 h. The hydrolysates were mixed with 240 μl water, 600 μl Lyx (0.1 mg/ml), 600 μl PMP methanol solution (0.5 M), and 240 μl NaOH (0.23 M); reacted at 70°C for 100 min; and neutralized with 400 μl HCl (0.15 M). The mixture was extracted with CH3Cl and filtered through 0.22 μm microporous membrane for HPLC. The standard monosaccharides were derivatized in the same way. The monosaccharide compositions were determined by Shimadzu LC-20A HPLC system (Shimadzu co. Ltd.), fitted with a SPD-20A UV/Vis detector and a TC-C18 column (250 mm × 4.6 mm, 5 μm). Mobile phase A and B were acetonitrile and sodium phosphate buffer (pH = 6.8), respectively, and flow rate was 1.0 ml/min, column temperature was 35°C, and injection volume was 10 μl. The monosaccharide content was calculated by internal standard method, and Lyx was used as the internal standard.
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