The HPLC system consisted of a Shimadzu (Kyoto, Japan) model LC-10AD pump, a Shimadzu degasser model DGU-12A, and a Corona Veo detector (Thermo Fisher Scientific, Inc., Waltham, MA, USA). To analyze the oligosaccharide fraction, an Asahipak NH2P-50 4E column (5 µm, 4.6 mm internal diameter ×250 mm, Showa Denko K.K., Tokyo, Japan) was used, and the mobile phase was acetonitrile/water (3:1; v/v). Elution was carried out at a flow rate of 1 mL/min at room temperature (~23 °C). The injection volume was 20 µL. To purify the target oligosaccharide, an Asahipak NH2P-50 10E column (5 µm, 10.0 mm internal diameter ×250 mm; Showa Denko K.K.) was used with a flow rate of 2 mL/min. As a post-column type splitter, an Adjustable Flow Splitter (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used. The split ratio was 1:20, with the low-flow outlet directed to CAD. The remaining (~95%) volume was then collected from the high-flow outlet of the splitter. The other conditions were as described above.
Lc 10ad pump
Manufactured by Shimadzu
Sourced in Japan
The LC-10AD pump is a high-performance liquid chromatography (HPLC) pump designed for reliable and stable liquid delivery. It features a piston-type design and delivers a flow rate range of 0.0001 to 10.0 mL/min. The pump is capable of operating at a maximum pressure of 40 MPa (400 bar, 5,800 psi).
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Lab products found in correlation
Hplc system, by Shimadzu (3 mentions)
Rid 6a refractive index detector, by Shimadzu (2 mentions)
C r7a integrator, by Shimadzu (2 mentions)
Polyspher chpb column, by Merck Group (2 mentions)
Lc 10a system, by Shimadzu (2 mentions)
V 530 uv vis spectrophotometer, by Jasco (2 mentions)
P 1020 digital polarimeter, by Jasco (2 mentions)
Spd m10a pda detector, by Shimadzu (2 mentions)
Apex 2 spectrometer, by Bruker (2 mentions)
Scl 10a system controller, by Shimadzu (2 mentions)
Spd 10a uv vis detector, by Shimadzu (2 mentions)
Lc solution software, by Shimadzu (2 mentions)
Spd 10av, by Shimadzu (2 mentions)
Spd m10a vp pda detector, by Shimadzu (2 mentions)
Cto 10a column oven, by Shimadzu (2 mentions)
Lichrospher 100 rp 18, by Merck Group (2 mentions)
Class vp software, by Shimadzu (2 mentions)
Koh tris, by Merck Group (2 mentions)
Spd 20 a av uv vis wavelength detector, by Shimadzu (2 mentions)
Lichrocart 4 4 guard columns, by Merck Group (2 mentions)
50 protocols using lc 10ad pump
1
Oligosaccharide Analysis and Purification
2
HPLC Analysis of Carbohydrates in Food
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The contents of lactose, lactulose, and galactose were determined by HPLC using a Shimadzu LC-10A system equipped with an LC-10AD pump (Shimadzu Corporation Co., Ltd., Kyoto, Japan), an RID-6A refractive index detector (Shimadzu Corporation), and a C-R7A integrator (Shimadzu Corporation) under the following conditions: column, Polyspher CHPB column (7.9 mm φ × 300 mm; Merck KGaA, Darmstadt, Germany); guard column, Polyspher CHPB column (4 mm φ × 50 mm; Merck KGaA); column temperature, 80 °C; mobile phase, distilled water; and flow rate, 0.4 mL/min. The sample volume applied was 10 μL. Degradation products formed in the reaction were analyzed by HPLC using a JASCO LC system equipped with an 880-PU pump (JASCO Corporation Co., Ltd., Tokyo, Japan), a UV-970 ultraviolet/visible detector, an RID-6A refractive index detector (Shimadzu Corporation), and an 807-IT integrator (JASCO) under the following conditions: column, Polyspher OAKC (7.8 mm φ × 300 mm; Merck KGaA, Darmstadt, Germany); guard column, Polyspher OAKC column (4 mm φ × 50 mm; Merck KGaA); column temperature, 40 °C; mobile phase, 1 mM phosphoric acid; flow rate, 0.4 mL/min; and UV wavelength, 210 nm. The sample volume applied was 50 μL.
3
Quantification of Flavonoids from Skin and Mucosa
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QCT, LUT, and 3-O-MQ content into crude extract, nanoemulsions, and skin/mucosa after permeation studies were determined using a liquid chromatography equipment Shimadzu LC-10A, equipped with LC-10AD pump, CBM-10A system controller, SIL-10A autosampler, SPD-20AV UV/vis detector (set at 362 nm), and LC Solution software. The chromatographic system was composed by a Synergi Polar-RP 150 × 4.6 mm i.d., 4 μm (Phenomenex, Torrance, CA) column protected by precolumn packed with silica C18 Phenomenex (150 μm, 140 Å), temperature system of 30 ± 1°C, isocratic flux of 0.8 mL/min, and 20 μL as injection volume. The mobile phase consisted of methanol : 0.16 M phosphoric acid : acetonitrile (46 : 44 : 10, v/v/v) and the samples were diluted in methanol : phosphoric acid 16 mM (50 : 50, v/v) before analyses. The analytical method was previously validated for the determination of QCT, LUT, and 3-O-MQ in ethanolic extract and nanoemulsion [8 (link)], demonstrating to be specific, linear (0.25 to 10 µg/mL), precise, and accurate. In this paper, the revalidation of the analytical method was performed in terms of specificity and recovery of QCT, LUT, and 3-O-MQ from porcine ear skin and porcine esophageal mucosa. The limits considered acceptable for the evaluated parameters are in accordance with the “The Guidance for Industry: Bioanalytical Method Validation” (FDA).
4
Liquid Chromatography-Mass Spectrometry Analysis
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Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis was performed with an LC-10AD pump (Shimadzu, Kyoto, Japan) with Sunfire C18 column (100 Å, 5 μm, 4.6 × 250, Waters Corporation) and API2000 mass spectrometer (AB SCIEX, Foster City, CA, United States). The purified compounds (1 mg) were separately dissolved in 200 μL MeOH to attain a final concentration of 5 mg mL–1. An aliquot (10 μL) of each solution was then injected into the instrument.
The 1H-NMR and 13C-NMR data of the purified compounds were recorded using Bruker Avance III HD 500 MHz instrument (Bruker Biospin GmbH, Rheinstetten, Germany) in chloroform-d (Cambridge Isotope Laboratories, Inc., MA, United States), and tetramethylsilane (TMS) was used as an internal standard in the NMR analysis (Nguyen et al., 2020 (link)).
The 1H-NMR and 13C-NMR data of the purified compounds were recorded using Bruker Avance III HD 500 MHz instrument (Bruker Biospin GmbH, Rheinstetten, Germany) in chloroform-d (Cambridge Isotope Laboratories, Inc., MA, United States), and tetramethylsilane (TMS) was used as an internal standard in the NMR analysis (Nguyen et al., 2020 (link)).
5
HPLC-EC Analysis of Extracellular Dopamine
6
Comprehensive Analytical Characterization
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Optical rotation was measured on a JASCO P-1020 digital polarimeter (Tokyo, Japan). UV data were recorded on a JASCO V-530 UV/VIS Spectrophotometer (Tokyo, Japan). High-resolution ESIMS data were obtained on a Bruker APEX II spectrometer (Billerica, MA, USA)). The IR spectrum was measured on a Perkin Elmer system 2000 FT-IR spectrophotometer (Waltham, MA, USA). The NMR spectra were obtained by JEOL JNM-ECS 400 MHz NMR (Akishima, Japan). Merck (Darmstadt, Germany) silica gel 60 and GE Healthcare (Chicago, IL, USA) Sephadex LH-20 were used for column chromatography. The instrumentation for HPLC was composed of a Shimadzu LC-10AD pump (Kyoto, Japan) and a Shimadzu SPD-M10A PDA detector.
7
AMCA-PLGA Polymer Characterization Protocols
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The AMCA-PLGA preparation was analyzed by size exclusion chromatography (SEC) using a Shimadzu system equipped with an LC-10AD pump, a degasser DGU-14A, a CTO-10A column oven, a RID-10A refractive index detector, and a SPD-10AD VP ultraviolet detector. A 5% lithium chloride solution (ACROS Organic, Morris Plains, NJ, USA) in N,N-dimethylacetamide (Sigma-Aldrich) was used as eluent at a flow rate of 1 mL/min and a column oven temperature of 40°C. Polystyrene (Polymer kit) was used to calibrate the GRAM guard/1000/30 Å column (10 μm particle size) (PPS Polymer Standards Service GmbH, Mainz, Germany). The absorbance of AMCA-PLGA was determined at 350 nm.
For thin layer chromatography (TLC), AMCA (0.1 mg/mL) and AMCA-PLGA (2 mg/mL) were diluted in methanol (Merck KGaA, Darmstadt, Germany) and 1 μL aliquots were spotted on silica thin-layer plates (TLC Silica gel 60 F254, Merck KGaA). A methanol/chloroform (both Merck KGaA) 1:9 (v/v) mixture was used as the mobile phase. After drying the plates at room temperature, spots were identified by exposure to ultraviolet light at 366 nm.
For thin layer chromatography (TLC), AMCA (0.1 mg/mL) and AMCA-PLGA (2 mg/mL) were diluted in methanol (Merck KGaA, Darmstadt, Germany) and 1 μL aliquots were spotted on silica thin-layer plates (TLC Silica gel 60 F254, Merck KGaA). A methanol/chloroform (both Merck KGaA) 1:9 (v/v) mixture was used as the mobile phase. After drying the plates at room temperature, spots were identified by exposure to ultraviolet light at 366 nm.
8
Isocratic HPLC Analysis of Compounds
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An LC–10 AD pump by Shimadzu (Kyoto, Japan) was used for the delivery of the mobile phase to the analytical column at a flow rate of 1.0 mL/min. Chromatographic separation was achieved isocratically in a PerfectSil Target ODS-3 (250 mm × 4.6 mm, 5 µm) analytical column (MZ AnalysenTechnik, Mainz, Germany) at room temperature, with CH3CN/H2O, 58/42% v/v as mobile phase. The samples were injected via a Rheodyne 7125 injection valve, with a loop of 20 μL volume (Rheodyne, Cotati, CA, USA). Detection was achieved at a wavelength of 215 nm and a sensitivity setting of 0.002 AUFS using an SSI 500 UV–vis detector (SSI, State College, PA, USA).
9
Quantitative HPLC-UVD Analysis of S-Allyl Cysteine
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The levels of S‐allyl‐L‐cysteine were analyzed by the Korean Food Research Institute (Chonbuk). Briefly, the extracts were filtered through a 0.45‐m syringe filter (Merck KGaA) and the filtrate was analyzed using S‐allyl‐L‐cysteine (≥98%; Sigma‐Aldrich) as a standard. A HPLC‐UVD system (Shimadzu, Shimadzu Corporation) fixed with a LC‐10AD pump, a SPD‐10A UV/Vis detector, a CTO‐10AC column thermostat, and a manual sample injector was used to analyze S‐allyl cysteine content in extracts. The mobile phase consisted of 0.1% H3PO4 solution and acetonitrile (Sigma‐Aldrich) with isocratic elution. A flow rate of 0.5 ml/min and injection volume of 10 µl were applied. The analyte was separated using a LiChroCART® column (250 × 4 mm, 5 m, Merck KGaA) at room temperature, and SAC was detected at 210 nm.
10
Cecum Content Organic Acid Analysis
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Cecum and cecum contents were isolated and weighed. Each 0.3 g sample of cecum content was transferred into 0.6 mL of distilled water, and stood on ice for 10 min after adding 0.09 mL of 12% peroxide acid. The supernatant was filtered after centrifugation with 15,000× g at 4 °C for 10 min, and then used for organic acid analysis using ion-exclusion high-performance liquid chromatography with LC-10AD pump (Shimadzu, Kyoto, Japan) and electrical conductivity meter (Waters431, Kyoto, Japan). Component identification was performed by CBM-20A data module (Shimadzu, Kyoto, Japan) [30 (link)].
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