As described in our previous article46 (link), all reagents and starting materials were purchased from commercial chemical suppliers (Sigma-Aldrich, TCI and Across Organics) and used as received. All the anhydrous organic solvents of purity greater than 99.9% were purchased from Aldrich and used directly. Thin layer chromatography (TLC) was performed on Merck aluminum sheets with silica gel 60 F254 and was visualized by ultraviolet light and staining with KMnO4 and ninhydrin. For the purification of compounds, column chromatography was performed on Merck silica gel 60 (70–230 mesh or 230–400 mesh). NMR spectra including, 1H and 13C NMR spectra were recorded on a Bruker DRX-400 and DRX-500 spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) measured and coupling constants (J) are given in hertz (Hz). MALDI-TOF mass were recorded using Shimadzu mass spectrometer. Reverse-phase HPLC analysis (RP-HPLC) was carried out at 230 nm on an Agilent HPLC system equipped with a C18 analytical column (250 × 22 mm, 10 microns). Two different linear gradients of 0.05% aq. trifluoroacetic acid (TFA) (eluent A) and 0.05% TFA in CH3CN (eluent B) were used with a flow rate of 5.0 mL/min at 25 °C.
C18 analytical column
Manufactured by Agilent Technologies
Sourced in United States
The C18 analytical column is a type of high-performance liquid chromatography (HPLC) column used for the separation and analysis of a wide range of organic compounds. The column is packed with a stationary phase material consisting of silica particles chemically bonded with octadecyl (C18) functional groups. This column is a common choice for reverse-phase chromatography applications.
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Lab products found in correlation
Silica gel 60, by Merck Group (1 mentions)
Hplc system, by Agilent Technologies (1 mentions)
Drx 500 spectrometer, by Bruker (1 mentions)
Silica gel 60 f254, by Merck Group (1 mentions)
Anhydrous organic solvents, by Merck Group (1 mentions)
Drx 400, by Bruker (1 mentions)
1200 series hplc, by Agilent Technologies (1 mentions)
3200 qtrap, by AB Sciex (1 mentions)
Agilent 6530 accurate mass q tof lc ms system, by Agilent Technologies (1 mentions)
1100 capillary hplc system, by Agilent Technologies (1 mentions)
Methanol, by Merck Group (1 mentions)
Lc 10ad, by Shimadzu (1 mentions)
High performance liquid chromatography (hplc), by Agilent Technologies (1 mentions)
12 protocols using c18 analytical column
1
Organic Synthesis Protocols and Characterization
2
Measurement of Preservatives in Doogh via RV-HPLC-UV
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The RV-HPLC-UV techniques were applied to measure preservatives in Doogh samples in accordance with the procedure described by Esfandiari et al. (2013) [11 (link)]. A 10 mL of homogenized Doogh was accurately transferred into a 25 mL volumetric flask and diluted with mixture of buffer acetate 0.1 M (pH = 4.5) : methanol (2 : 1, v/v). The solution was treated by ultrasonication, centrifuged in 5000 rpm, both for about 10 min. The mixtures were then filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membranous filters, and 20 μL was injected into the HPLC by auto sampler. Chromatographic analysis was performed on 1200 series HPLC from Agilent technology equipped with automated sample injector, C18 analytical column (250 × 4.6 mm, 5 μm), ultraviolet detector, high-pressure pump, and degasser. The separation was carried out with a flow rate of 0.8 mL/min, and the UV detector was set at 225, 250, and 303 nm for SB, PS, and natamycin, respectively. The peaks were diagnosed based on the retention time. The data was processed in the Millennium 32 software. Correlation coefficient value was 0.996 for all the preservatives.
3
Purification of Antimicrobial Compounds by HPLC
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Purified antimicrobial substances were separated by high performance liquid chromatography (HPLC) using a reversed phase C18 analytical column (ODS: 4.6 × 250 mm, Agilent, Santa Clara, CA) with mobile phases of 0.05% trifluoroacetic acid in acetonitrile (solvent A) and 0.05% trifluoroacetic in Milli-Q water (solvent B) at a flow rate of 200 μL min−1 using a gradient elution with UV detection at 210 nm. The elution conditions were 50–100% (A), 0–50 min, 50–0% (B), 0–50 min; 100% (A), 50–70 min, 0% (B) 50–70 min. Electrospray ionization/collision induced dissociation (ESI/CID) mass spectrometry was performed using a Surveyor-LCQ DECA XP Plus (Thermo Finnigan, San Jose, CA, USA). The electrospray source was operated at a capillary voltage of 15 V, a spray voltage of 5 kV, and a capillary temperature of 275°C. Helium was used as the collision gas for the CID experiment and the collision energy was set at 35% [34 (link)].
4
HPLC Analysis of Free Amino Acids and Theanine
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Free amino acid and theanine content was determined using an Agilent HPLC instrument (Agilent Technologies, USA). HPLC separation was carried out using a C18 analytical column (250 mm×4.6 mm, 5 μm, Agilent, USA) maintained at 30°C. Gradient elution was used to obtain adequate separation. The mobile phase consisted of solvents A (0.1 M NaAc:ACN 97:3, v/v, pH 6.5) and B (ACN:water 4:1, v/v). The flow rate was 2 mL/min. Absorbance at 254 nm was measured using a UV detector. The total run time was 35 min. The sample injection volume was 2.0 μL. The analytical data were processed using Agilent software. Each sample was done in triplicate, and the values were expressed as the mean (n = 3)[30 (link)].
5
Targeted Metabolomics by LC-MS/MS
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LC-MS analysis was conducted using Agilent 6530 Accurate-Mass Q-TOF LC-MS system equipped with a C18 analytical column of 50 mm × 2.1 mm, 1.8 μm particle size (Agilent 6530). Column oven temperature of 35°C and flow rate of 250 μL/min were maintained throughout the experiment. Water and acetonitrile, each containing 5 mM ammonium formate and 0.1% formic acid, were used as mobile phase A and B, respectively. The injection volume was 20 μL with a run time of 15 min. The linear gradient program was set as follows: 0 min, 100% A; 0–45 min, 0–100% B; 46–50 min, 100% B; 51–55 min, 100% A. The UHPLC was hyphenated to a triple quadrupole mass spectrometer 3200 QTrap (ABSciex) equipped with an electrospray ionization interface set at negative mode. The interface heater held at the temperature of 500°C and an ion-spray (IS) voltage of -4500 eV. The nebulizing gas (GS1), heating gas (GS2) and curtain gas pressures set at 40, 40, and 10 psi, respectively during the whole analysis. Nitrogen was used as collision and spray gas. Full scan data acquisition was performed, scanning from m/z 5 to 1500 in enhanced MS IDA EPI mode (Karim et al., 2014 (link)).
6
HPLC Analysis of Modified Placental DNA
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An Agilent 1100 capillary HPLC System equipped with a Synergi C18 analytical column was used for HPLC analysis of native and modified analog of human placental DNA. General chromatographic conditions were as follows: C18 column (2 mm × 150 mm with 4 μm particle size); eluant A, 5 mM aqueous ammonium acetate buffer, pH 7; eluant B, Acetonitrile (CH3N) gradient solution the CH3N concentration was raised from 0 to 4.0% in the first 5 min; from 4.0 to 6.5% over 30 min; held at 6.5% for 5 min, and then raised to 90% to wash residual material off the column at a constant flow rate of 500 μL/min. DNA bases were detected by diode array detector (DAD) at 254 nm, their absorption maximum. LC-MS analyzes of CEdG standard were carried out using a Micromass Quattro Ultima Triple Quadrupole Mass Spectrometer interfaced to an Agilent 1100 capillary HPLC System.
7
Metabolomic Profiling of Soybean Treated with B. japonicum
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A UHPLC-ESI-QQQ-MS/MS analysis of total metabolite of control and soybean plants treated with B. japonicum-406 was conducted. Leaves obtained from the soybean plants were ground to fine powder in the pestle and mortar using liquid nitrogen. The extraction was performed using a mixture of methanol and water (80/20, v/v) containing 1 ng/μL of the internal standard. The obtained extract was centrifuged to settle the debris and passed by a microfilter assembly. Chromatographic separation was performed on an Agilent 1200 ultra-performance liquid chromatography system (Agilent, Santa Clara, CA, USA) with a C18 analytical column (Agilent) and coupled with a Triple-Quad tandem mass spectrometer (6470) system. The mobile phase “A” consisted of 0.1% formic acid (v/v) in deionized water, whereas the mobile phase B consisted of 0.1% formic acid (v/v) in methanol. The chromatographic conditions were 95% A and 5% B for the first 5 min, solvent A decreased to 45% and B increased to 55% up to 22 min, solvent A 5% and B 95% over the course of 3 min and remained unchanged for one minute, solvent A 95% and B 5% for 3 min until the end of the run. The MS scan range was 50 to 1500 m/z. The acquired data were analyzed by MzMine 2.53 software. Compounds were identified using the NIST MS/MS library operated by the MS search program coupled with the MzMine 3 software.
8
Measuring Drug Concentrations and Nanoparticle Properties
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The concentrations of Res and Tem were measured by using high-performance liquid chromatography (HPLC) on a Shimadzu LC-10AD (Shimadzu, Japan) with an ultraviolet (UV) detector and an Agilent C-18 analytical column.7 (link),24 The mobile phase for Tem was methanol/0.5% acetic acid (20:80, v/v). The pump rate was 1.1 mL/min, and the retention time was 3.682 min. The wavelength of 330 nm was used to determine the concentration of Tem. The mobile phase for Res consisted of methanol (spectral grade, Merck, Germany)/double-distilled water/glacial acetic acid (48/52/0.05, v/v/v) and was pumped at a flow rate of 1.0 mL/min with the retention time of 5.855 min at a wavelength of 303 nm. The concentration of each drug was calculated based on the peak area with reference to a calibration curve.
The drug loading content and encapsulation efficiency of the nanoparticles were calculated as follows:
The drug loading content and encapsulation efficiency of the nanoparticles were calculated as follows:
9
Purity Analysis of TB-II Compound
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The purity of the TB-II sample was authenticated using HPLC-MS-Q-TOF, 1H-NMR, and 13C-NMR (Table S1 , Figure S2 and S3 ). The quantitation of TB-II was analyzed through HPLC using a C18 analytical column (Agilent Technologies, Santa Clara, CA, USA) equipped with an evaporative light-scattering (ELSD) detector at a flow rate of 1.0 mL/min. The mobile phase comprised acetonitrile and distilled water (v/v, 25:75). Approximately 10 µL of TB-II (0.5 mg/mL) was injected into an HPLC column to measure the purity of the sample (purity 97%, HPLC, Figure S2 ).
10
Quantification of Rivaroxaban in Plasma
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Analysis of RIV concentrations in plasma used an Agilent HPLC with electrospray ionization and tandem mass spectrometry detection (Pump G1312C, Autosampler 1367E, Degasser G1322A, G6460A triple quad mass spectrometer, Agilent Technologies, Santa Clara, CA, USA). Diphenhydramine was used as the internal standard (IS). The analysis conditions of HPLC-MS/MS included a reversed phase C18 analytical column (3.5 μm, 2.1 mm - 100 mm, Agilent, Santa Clara, CA, USA) and gradient elution conditions at a flow rate of 0.3 ml/min. The mobile phase was acetonitrile- 0.1% formic acid solution (30/70, v/v). The triple-quadrupole mass spectrometer was operated in negative ionization mode, and detection and quantification used multiple-reaction monitoring (MRM). Tandem mass spectrometry (MS/MS) detection was conducted by monitoring the fragmentation of 436.0 → 144.8 (m/z) for RIV and 256.2 → 167.2 (m/z) for IS. The productions were generated with a collision energy of 28 eV.
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