The purity verification, quantification, and stability study of the compounds under analysis were carried out by reversed-phase liquid chromatography (RP-LC). The chromatography method for the simultaneous analysis of AFAT-Se and PTX was optimized on a Shimadzu LC system (Shimadzu, Kyoto, Japan) containing an SPD-M20A photodiode array (PDA) detector, using a Gemini NX C18 Phenomenex column (150 mm × 4.6 mm; 5 μm). The system was operated in isocratic mode with flow gradient (0.8–1.2 mL/min), room temperature, mobile phase containing ultrapure water, acetonitrile, and methanol (37:38:25, v/v), and the UV detection was set at 227 nm.
Gemini nx c18 column
Manufactured by Phenomenex
Sourced in United States, Japan, Germany
The Gemini-NX C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a silica-based stationary phase with a C18 alkyl bonded functional group, which provides excellent separation and resolution capabilities for a variety of samples.
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Lab products found in correlation
Ultimate 3000, by Thermo Fisher Scientific (2 mentions)
3200 qtrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000, by Phenomenex (2 mentions)
Lc 20 prominence hplc system, by Shimadzu (2 mentions)
Prism 8, by GraphPad (2 mentions)
Openlab cds software, by Agilent Technologies (2 mentions)
1260 infinity quaternary lc, by Phenomenex (2 mentions)
Lc 20at hplc system, by Shimadzu (2 mentions)
3200 q trap, by Thermo Fisher Scientific (2 mentions)
Yl 9100 series hplc instrument, by Younglin (1 mentions)
Ultremex scx column, by Phenomenex (1 mentions)
6520 accurate mass q tof mass spectrometer, by Agilent Technologies (1 mentions)
Agilent 1260 hplc, by Agilent Technologies (1 mentions)
1290 uhplc, by Agilent Technologies (1 mentions)
Agilent 1260 infinity, by Agilent Technologies (1 mentions)
Xbridge shield rp18 column, by Waters Corporation (1 mentions)
E2695 system, by Waters Corporation (1 mentions)
Xcalibur software, by Thermo Fisher Scientific (1 mentions)
Ibuprofen d3, by Merck Group (1 mentions)
1100 series preparative hplc, by Agilent Technologies (1 mentions)
69 protocols using gemini nx c18 column
1
Optimized RP-LC Analysis of AFAT-Se and PTX
2
Quantification of ACAT-Se in NPs
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A reversed-phase liquid chromatography (RP-LC) method was developed for the quantification of ACAT-Se in the NPs. The method was performed, at room temperature, on a Shimadzu LC system (Shimadzu, Kyoto, Japan) equipped with an SPD-M20A photodiode array (PDA) detector, using a Gemini NX C18 Phenomenex column (150 mm × 4.6 mm; 5 μm). The UV detection was set at 263 nm and the LC system was operated isocratically, using a mobile phase consisted of potassium phosphate buffer (pH 3.0, 15 mM), acetonitrile and methanol (70:20:10, v/v/v), run at a flow rate of 0.8 mL/min. The method was validated according to international guidelines for specificity, linearity, precision, accuracy, and robustness.
3
HPLC Analysis of Bioactive Compounds
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Chromatographic analysis was performed using an YL-9100 series HPLC instrument equipped with a sample injector and a photodiode array ultraviolet/visible (PDA-UV/Vis) detector (Young Lin, Korea). The analysis was conducted using Phenomenex Gemini NX-C18 column (4.6 mm × 250 mm; 5 μm, Phenomenex Inc., CA, USA) as the stationary phase, with at injection volume of 20 μL at room temperature. The mobile phase was composed of water (containing 0.1% formic acid) (A) and acetonitrile (B), with the following gradient system: 0–5 min linear change from 10% B to 10% B, 5–10 min linear change from 10% B to 20% B, 10–60 min linear change from 20% B to 40% B, 60–80 min linear change from 40% B to 70% B, 80–85 min linear change from 70% B to 100% B, and 85–90 min held at 100% B. The flow rate for HPLC analysis was set to 0.7 mL/min while the detection wavelength was set to 210 and 254 nm. Each sample was analyzed three times. Standard samples for HPLC were dissolved in 50% methanol, and NJ20 was prepared at a concentration of 10 mg/mL. For the quantitative analysis of compounds 1–9, standard compounds were prepared at concentrations between 0.0125 and 0.5 mg/mL, and calibration curves were constructed by plotting peak areas against the known concentrations of the standard compounds. The slope, intercept, and correlation coefficient (R) were calculated by linear regression analysis.
4
LC-MS Analysis of Compounds
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LS-MS analysis for all the compounds was performed
on a Thermo Finnigan
LCQ-Fleet ESI-ion trap (Thermofischer, Breda, The Netherlands) equipped
with a Phenomenex Gemini-NX C18 column, 50 × 2.0 mm, particle
size 3 μM (Phenomenex, Utrecht, The Netherlands). An acetonitrile/water
gradient containing 0.1% formic acid was used for elution (5–100%,
1–20 min, flow 0.2 mL min–1).
on a Thermo Finnigan
LCQ-Fleet ESI-ion trap (Thermofischer, Breda, The Netherlands) equipped
with a Phenomenex Gemini-NX C18 column, 50 × 2.0 mm, particle
size 3 μM (Phenomenex, Utrecht, The Netherlands). An acetonitrile/water
gradient containing 0.1% formic acid was used for elution (5–100%,
1–20 min, flow 0.2 mL min–1).
5
iTRAQ-Based Quantitative Proteomics Analysis
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iTRAQ Reagent 4-plex Multiplex Kits were used to label each independent biological replicate (IBR) in the groups. For each group, 15 subjects were recruited, plasma samples of which were randomly mixed to three pools (five individuals for each IBR). Each iTRAQ reagent was diluted in 150 μl of isopropanol before 100 μg of peptide was added. Peptides were labeled as follows: Three IBRs of the nCMS-HPu group were labeled with iTRAQ tags 114; three IBRs of the CMS-HPu group were labeled with iTRAQ tags 115; three IBRs of the nCMS-TPu group were labeled with iTRAQ tags 116, and three IBRs of the nCMS-HPn group were labeled with iTRAQ tags 117. Next, 100 μl ultra-pure HPLC-grade water was added to terminate the labeling after the samples were labeled for 1 h. Samples were combined into one tube for each group, then resolved into 50 fractions using a 5-μm particle Ultremex SCX column (Phenomenex, Torrance, CA, U.S.A.), and finally desalted using a Gemini-NX C18 column (4.6 mm × 250 mm, Phenomenex). After all the fractions were freeze-dried under a vacuum, they were resuspended with 30 μl of mobile phase A (2% acetonitrile [ACN], 0.1% formic acid [FA]), divided into 15 groups based on their peak intensities and then centrifuged at 12,000 rpm for 10 min prior to LC-MS/MS analysis.
6
Analytical Characterization of Phenolic Compounds in Liqueurs
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The individual phenolic compounds in the liqueurs were determined by reversed-phase high performance liquid chromatography (RP-HPLC) on a Waters Systems instrument (model Alliance e2695) coupled to diode array detection (DAD) and fluorescence detection (FD). For the separation of compounds, a Gemini NX C-18 column (150 mm × 4.6 mm × 3 μm) and a Gemini NX C-18 guard column (4.0 mm × 3 mm × 3 μm) were used, both manufactured by Phenomenex (Torrance, CA, USA). The gradient used was 0 min: 100% A; 18 min: 87.5% A, 2.5% B, 10.0% C; 30 min: 83.5% A, 3.2% B, 13.3% C; 36 min: 75.0% A, 5.0% B, 20.0% C; 48.5 minutes: 65.0% A, 8.3% B, 26.7% C; 50 min: 65.0% A, 8.3% B, 26.7% C; and 65 min: 100% A. Solvent A consisted of a solution of 25 mmol L−1 of potassium dihydrogen phosphate with the pH adjusted to 2.05 with phosphoric acid, solvent B was methanol, and solvent C was acetonitrile. The oven temperature was maintained at 40 °C and the solvent flow at 0.6 mL min−1, with a total run time of 65 min. The detection and quantification of the compounds was carried out using external standards.
The analysis was performed according to the methodology described by Natividade et al. [16 (link)], using the software program Empower™ 2 (Milford, MA, USA) for data treatment.
The analysis was performed according to the methodology described by Natividade et al. [16 (link)], using the software program Empower™ 2 (Milford, MA, USA) for data treatment.
7
Bimodular PKS Assay Protocol
8
HPLC Analysis of Pharmaceutical Compounds
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Example 1
Samples (20.0 mg) are dissolved in acetonitrile (10.0 mL) to make 2 mg/mL solution. For the system: solvent A was Water+0.05% trifluoroacetic acid (TFA); solvent B was Acetonitrile+0.05% TFA; the flow rate was 1.0 mL/min; and the detection method was UV @242 nm and UV Spectra from 190 to 400 nm. The samples were loaded onto an Agilent 1100 series HPLC with either (i) a Phenomenex Gemini-NX C18 Column (5 μm; 110 Å; 250×4.6 mm; 00G-4454-E0) or (ii) Phenomenex SecurityGuard Analytical Guard Column (KJO-4282) with Gemini C18 4×3.0 mm Guard Cartridge (AJO-7597). The solvent gradient profile is shown in Table 4:
A compound of the disclosure was formed into a pellet in the glassy state by heat molding. Crystalline powder of the conjugate compound was melted between 85° C. to 110° C. and pressed into a cylindrical mold of ˜1 mm height×1 mm diameter.
Compound 5 was formed into rods by melt extrusion and were cut to 1, 1.5 or 2 mm length. The resulting implants were loaded in the lumen of needles, terminally sterilized, and injected into the anterior chamber of rabbits. Implants settled into the inferior iridocorneal angle and were visualized by anterior chamber optical coherence tomography.
9
High-pH Reverse-Phase Fractionation for Proteomics
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High-pH reverse-phase fractionation (hpRP) chromatography was performed using a Dionex UltiMate 3000 high-performance liquid chromatography (HPLC) system with built-in micro collection options for autosampling and ultraviolet (UV) detection. iTRAQ-labelled tryptic peptide was reconstituted in buffer A (20 mM NH4HCO2, pH of 10) and loaded onto a Gemini-NX C18 column (3 μm, 2 × 150 mm, 110 A, Phenomenex) with 20 mM NH4HCO2 as buffer A and 80% acetonitrile (ACN) + 20% 20 mM NH4HCO2 as buffer B. The peptide was eluted at a flow rate of 200 μL/min with a gradient of 0–5% buffer B for 10 minutes and then 5–15% buffer B for 5 minutes. Elution with 15–50% buffer B lasted for 45 minutes, and that with 50–90% buffer B lasted for 10 minutes. Twenty-four fractions were collected at 1-minute intervals based on UV absorbance at 214 nm/280 nm and a multi-fraction cascade strategy. Fractions were dried and acidified in 50% trifluoroacetic acid (TFA) for nano-liquid chromatography with tandem mass spectrometry (nano-LC-MS/MS) analysis.
10
HPLC-MS/MS Mycotoxin Analysis Protocol
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For mycotoxin analysis, the samples were injected into an HPLC coupled to a 3200QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA). The column used to separate mycotoxins was a Gemini NX C18 column (150 × 2.0 mm I.D, 3.0 mm, Phenomenex, Palo Alto, CA, USA). The mobile phases consisted of water (A) and ACN (B), both with 0.1% formic acid and 5 mM ammonium formate at 0.25 mL/min with a linear gradient. The ions transitions used for the AFB1 and OTA identification and quantification were m/z 313.1/241.3 and 284.9 (AFB1) and m/z 404.3/102.1 and 358.1 (OTA) [33 (link)].
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