All samples extracted from fermentation and in vitro assay were preliminary analyzed by Shimadzu HPLC (LC-20AT) with a DAD detector (SPD-M20A) through Phenomenex analytic column (C18, 5 μm, 4.6 × 250 mm). The mobile phase A was 1‰ formic acid in water, and the mobile B was pure acetonitrile (HPLC grade). The flow rate was 1.0 mL min−1. The gradient elution condition was as follows: Initially, the concentration of B was changed from 35 to 65% linearly in 20 min, next increased to 95% gradually from 20 to 28 min. The ratio of mobile phase B was then hold at 95% for 2 min. Finally, it decreased to 35% from 30 to 33 min and hold on for 5 min to make a balance.
Spd m20a
Manufactured by Phenomenex
Sourced in Japan
The SPD-M20A is a diode array detector designed for high-performance liquid chromatography (HPLC) applications. It provides real-time spectral analysis and data acquisition capabilities for the identification and quantification of analytes. The device operates within a wavelength range of 190 to 800 nanometers and can be used with a variety of HPLC systems.
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Lab products found in correlation
Lc 20ad, by Shimadzu (3 mentions)
Lc 20at, by Phenomenex (1 mentions)
Class vp series software, by Shimadzu (1 mentions)
Dgu 14a, by Phenomenex (1 mentions)
Kinetex c18, by Phenomenex (1 mentions)
Hplc system, by Shimadzu (1 mentions)
Lc 20ad system, by Shimadzu (1 mentions)
Synergi 4 m fusion rp 80 column, by Phenomenex (1 mentions)
Luna c18 column, by Phenomenex (1 mentions)
Nmr spectrometer, by Agilent Technologies (1 mentions)
Inova 300, by Agilent Technologies (1 mentions)
Mercury 400, by Agilent Technologies (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Lc 20a, by Shimadzu (1 mentions)
Gemini c18 110a column, by Phenomenex (1 mentions)
Luna silica column, by Phenomenex (1 mentions)
Anhydrous methanol, by Merck Group (1 mentions)
Potassium selenocyanate, by Merck Group (1 mentions)
Synergy polar rp, by Phenomenex (1 mentions)
Lc 20ab prominence liquid, by Shimadzu (1 mentions)
14 protocols using spd m20a
1
Analytical HPLC Characterization of Compounds
2
HPLC Analysis of Colchicine in Plant Extracts
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Each crude extract 0.250 g. (accurately weighted) dissolved in 10 ml HPLC grade methanol in volumetric flask after that 1 ml of stock solution was further diluted in another 10 ml HPLC grade methanol in volumetric flask then subjected to HPLC for qualitative and quantitative analysis of colchicine and other compounds. The HPLC system consists of Shimadzu LC-20AD which was equipped with a photodiode array detector (Shimadzu SPD-M 20 A), Phenomenex Column (RP, Kromasil 5u 100A C-18, 150x4.60 mm), Guard column (Kromasil 5u 100A C-18, 2.1 mm) and data were integrated by Shimadzu Class VP series software. The separation was achieved with a two-pump isocratic program for pump A and B (acetonitrile: H 2 O, 38:62). The flow rate was 1 ml/min, runtime 10 min, and determined at wavelength 350 nm. Results were obtained by comparison of peak areas of the samples with the calibration curve of the referent standard. Every process was repeated 3 times.
3
HPLC Analysis of Compounds
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HPLC analyses were performed on a Shimadzu HPLC system, a LC-10AT liquid chromatograph equipped with four pumps FCV-10AL, a degasser DGU-14A and a photodiode array detector SPD-M20A.
The column used was a C18 Kinetex (150 mm × 4.6 mm, 5 μm particle size), and a guard column, all supplied by Phenomenex (Torrance, CA, USA). The mobile phase was a mixture: 2% acetic acid in water (solvent A) and acetonitrile (solvent B). The eluted compounds were monitored at 280, 320 and 350 nm and the adsorption spectra between 200 and 600 nm.
The column used was a C18 Kinetex (150 mm × 4.6 mm, 5 μm particle size), and a guard column, all supplied by Phenomenex (Torrance, CA, USA). The mobile phase was a mixture: 2% acetic acid in water (solvent A) and acetonitrile (solvent B). The eluted compounds were monitored at 280, 320 and 350 nm and the adsorption spectra between 200 and 600 nm.
4
HPLC-DAD Analysis of Compounds
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HPLC system (FLCXR Shimadzu (Shimadzu corp., Kyoto, Japan)) with DAD detector (SPD-M20A) using Kinetex 2.6u C18 100 A column of 100 mm × 4.60 mm dimension (Phenomenex, Torrance, CA, USA) was applied. Two mobile phases were used, 0.1% solution of phosphoric acid in water (solvent A) and in methanol (solvent B). The separation started with using only the solvent A as a mobile phase and then gradually increasing the percentage of the solvent B in the mobile phase.
5
Controlled Release of Vancomycin from Modified Collagen Sheets
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Collagen sheets were loaded with 1 mg of vancomycin in PBS containing 0.01% (w/v) RB and modified by RGX. Unmodified collagen sheets loaded with 1 mg of vancomycin in PBS were used as control. To investigate the release of vancomycin, collagen sheets were used directly after treatment loading and incubated in a 24-well plate in 1 mL of PBS at either RT or 37 °C. Samples were taken after 30 min, 1 h, 2 h, 4 h, 8 h and 24 h by agitating and withdrawing the entire supernatant and adding 1 mL of fresh PBS to the collagen sample. The samples were analyzed by reversed-phase HPLC on a Shimadzu LC20-AD system, SIL-20AC autosampler, SPD-M20A photodiode array detector and a C18 250 × 4.6 mm Synergi™ 4 µm Fusion-RP 80 Å column (Phenomenex, Aschaffenburg, Germany). Then, 100 µL of the supernatant was injected onto the column by autosampler. After washing for 20 min with eluent A (95% water, 5% acetonitrile, 0.1% TFA), the concentration of eluent B (5% water, 95% acetonitrile, 0.1% TFA) was steadily increased from 0% to 90% over 50 min at a flow rate of 0.5 mL/min. Absorption was monitored at 280 nm for vancomycin and at 220 nm for collagen. Vancomycin eluted after a retention time of 41 min. The amount of released vancomycin was calculated from a calibration curve of vancomycin from 0 to 1 mg/mL.
6
Docetaxel-loaded Nanoparticle Entrapment Assay
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For estimation of the percentage of drug entrapped in DTX-NPs, reported HPLC method was used with minor modifications. In brief, supernatant obtained after centrifugation of formulation suspension was discarded. The pellet obtained as sediment was taken and solubilized in acetonitrile. Analysis was carried out by HPLC (Shimadzu, Kyoto, Japan) consisting of SPD-M20A diode array detector and Luna C18 column (Phenomenex Inc., Torrance, CA, USA).
Acetonitrile and orthophosphoric acid (OPA) (58:42) were the mobile phase pumped at a flow rate of 1.0 mL/min with constant column temperature of 37 °C. Ten µL was the injection volume with docetaxel eluting at 7.4 min with λ-max 230 nm.
Acetonitrile and orthophosphoric acid (OPA) (58:42) were the mobile phase pumped at a flow rate of 1.0 mL/min with constant column temperature of 37 °C. Ten µL was the injection volume with docetaxel eluting at 7.4 min with λ-max 230 nm.
7
Characterization of DNA-Conjugated Agents
8
Phenolic Extraction and Quantification
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The free and bound phenolics were extracted following the method of Ma et al. (2019) (link). The total phenolic content was assayed according to the Folin-Ciocalteu colorimetric method (Ma et al., 2019 (link)).
The content of phenolic acids was measured as described by Ma et al. (2019) (link) using high performance liquid chromatography (HPLC, Shimadzu LC-20A, Japan) fitted with a SPD-M20A diode array detector and an Gemini C18 110A column (4.6 mm × 150 mm, 5 µm; Phenomenex, Torrance, California, USA).
The content of phenolic acids was measured as described by Ma et al. (2019) (link) using high performance liquid chromatography (HPLC, Shimadzu LC-20A, Japan) fitted with a SPD-M20A diode array detector and an Gemini C18 110A column (4.6 mm × 150 mm, 5 µm; Phenomenex, Torrance, California, USA).
9
Fingerprinting of H-Ei Extract
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The fingerprinting of the H-Ei extract was performed on Shimadzu Prominence Series coupled with photodiode array detector SPD-M20A using Phenomenex Luna Silica column (250 mm × 4.6 mm, 100 Å, 5 μm). The mobile phase consisted of solvent A, hexane, and solvent B, 2-propanol, with an injection volume of 20 µL of 1 mg/mL H-Ei. The gradient program was as follows: 100% A (0-5 min) and 100-0% A (5-25 min) at a constant flow rate of 1 mL/min.
10
Synthesis of Selenium-Substituted 2'-Deoxyuridine
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The synthesis of SeCNdU was already summarized before 30 and thus only a brief overview is provided here. For the synthesis of SeCNdU, a modified procedure described by Agenäs 32 was used. A solution of Br2 (142 µL, 2.76 mmol) in methanol (800 µL) was added to the suspension of KSeCN (100 mg, 0.69 mmol) in anhydrous methanol (166 µL). After 1 h of stirring in a dry ice bath (-80°C), a suspension of 2'-deoxyuridine (75 mg, 0.34 mmol) in anhydrous methanol (3 mL) was added. After that, the mixture was brought up to -20°C and stirred for another hour.
In the next step, reaction mixture was evaporated and the crude product was purified with semipreparative HPLC (Shimadzu, LC 20AD) equipped with a UV detector (SPD M20A) and a Synergy Polar-RP (Phenomenex) reversed-phase column (10 × 250 mm, 4 µm in particle size and 100 Å in pore size). The linear gradient of 10-25% phase B in 20 min was used (mobile phase A: 0.2% formic acid and B: 80% ACN). The resulting product was obtained as a white solid (20 mg) in a 6.3% yield.
Potassium selenocyanate, bromine and anhydrous methanol were purchased from Sigma-Aldrich. 1
In the next step, reaction mixture was evaporated and the crude product was purified with semipreparative HPLC (Shimadzu, LC 20AD) equipped with a UV detector (SPD M20A) and a Synergy Polar-RP (Phenomenex) reversed-phase column (10 × 250 mm, 4 µm in particle size and 100 Å in pore size). The linear gradient of 10-25% phase B in 20 min was used (mobile phase A: 0.2% formic acid and B: 80% ACN). The resulting product was obtained as a white solid (20 mg) in a 6.3% yield.
Potassium selenocyanate, bromine and anhydrous methanol were purchased from Sigma-Aldrich. 1
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