TMT-labeled peptides were fractionated by high pH reverse-phase HPLC using an ZORBAX Extend-C18 column (Catalog No. 770450-902, Santa Clara, CA) (5 μm particles, 4.6 mm inner diameter, 250 mm length). Briefly, labeled peptides were separated with a gradient of 2%–60% acetonitrile in 10 mM ammonium bicarbonate into 80 fractions for 80 min. Fractionated peptides were combined into 18 fractions for total proteome analysis or 8 fractions for crotonylome analysis.
Extend c18 column
Manufactured by Agilent Technologies
Sourced in United States, Japan
The Extend-C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of chemical compounds. It features a C18 bonded phase that provides excellent retention and selectivity for a variety of analytes. The column is suitable for use in both reversed-phase and normal-phase HPLC applications.
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Lab products found in correlation
Agilent 6530 quadrupole time of flight, by Agilent Technologies (4 mentions)
P 1020, by Jasco (3 mentions)
Agilent 1200 series hplc system, by Agilent Technologies (3 mentions)
Drx 500, by Bruker (3 mentions)
Agilent 1260 infinity hplc system, by Agilent Technologies (3 mentions)
Masshunter qualitative analysis software, by Agilent Technologies (3 mentions)
Tensor 27 infrared spectrophotometer, by Bruker (2 mentions)
G6230 time of flight mass spectrometer, by Agilent Technologies (2 mentions)
Silica gel, by Qingdao Marine Chemical (2 mentions)
Avance 3 600, by Bruker (2 mentions)
Mci gel chp 20p, by Mitsubishi Chemical Corporation (2 mentions)
1260 series hplc system, by Agilent Technologies (2 mentions)
Sephadex lh 20, by Cytiva (2 mentions)
Agilent 1200 system, by Agilent Technologies (1 mentions)
Chirascan spectropolarimeter, by Applied Photophysics (1 mentions)
Lcq deca xp max, by Thermo Fisher Scientific (1 mentions)
Cholesterol esterase, by Worthington (1 mentions)
Accurate mass q tof, by Agilent Technologies (1 mentions)
Agilent 1260 infinity, by Agilent Technologies (1 mentions)
Accq tag derivatization kit, by Waters Corporation (1 mentions)
51 protocols using extend c18 column
1
High pH Reverse-Phase HPLC Fractionation
2
MSH Purification and Quantification
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MSH was purified from C. glutamicum RES167 using thiopropyl sepharose 6B followed by Sephadex LH‐20 chromatography as previously described (Yin et al., 2010 ). The MSH concentration was determined using the thiol‐specific fluorescent‐labeling high‐performance liquid chromatography (HPLC) method as previously described (Feng et al., 2006 ; Yin et al., 2010 ). The HPLC used in this study was equipped with an Extend‐C18 column (ZORBAX, 250 × 4.6 mm) and was operated with aqueous acetic acid‐methanol gradient elution (eluent flow rate of 0.9 ml/min). The bimane derivative of MSH was eluted at approximately 15 min in this system.
3
In Vitro Release Study of AZ from NLC
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The in vitro release study of AZ from its NLC formulations was carried out by using the dialysis bag technique. The specified amount (1 mL equivalent to 5 mg) of prepared AZ-NLC formulations was placed in the dialysis bag (14 kDa molecular weight cut-off (MWCO)), then immersed in the 500 mL distilled water as dissolution medium (USP Dissolution Tester, apparatus II; Erweka, Germany). The dissolution medium was maintained at 37 °C and the rotation speed of the paddle was set to 75 rpm. Aliquots of 5 mL were taken from the dissolution medium after 0.5, 1, 2, 4, 6, 8, 12, and 24 h. Three samples were tested to determine the concentrations of AZ by the high-performance liquid chromatography (HPLC) method [85 (link)]. AZ concentrations were determined by the HPLC method, validated, and developed in our labs in terms of accuracy, linearity, and precision. Analysis was conducted using Agilent 1200 system with a diode array detector, Zorbax Extend C18 column (4.6 × 150 mm, 5 μm), a quaternary pump, and an auto-sampler (Palo Alto, CA, USA). The system was controlled by ChemStation software (Rev. B.01.03 SR2 (204). Isocratic elution at a 0.6 mL/min flow rate was utilized. The mobile phase was composed of acetonitrile/water in 0.1% formic acid (9:1). The wavelength was set at 210 nm. The AZ level in the injected samples was measured concerning the calibration curves.
4
High-pH HPLC Fractionation of TMT-Labeled Peptides
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TMT-labelled peptides were fractionated by high pH reverse-phase HPLC using an ZORBAX Extend-C18 column (Catalog No. 770450-902, Santa Clara, CA) (5 μm particles, 4.6 mm inner diameter, 250 mm length). Briefly, labelled peptides were separated with a gradient of 2-60% acetonitrile in 10 mM ammonium bicarbonate into 80 fractions for 80 minutes. Fractionated peptides were combined into 18 fractions for total proteome analysis or 8 fractions for crotonylome analysis.
5
Spectroscopic Characterization of Compounds
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Optical rotations were measured on Jasco P-1020 automatic digital polarimeter. CD spectra were recorded on a Chirascan spectropolarimeter (Applied Photophysics, Leatherhead, Surrey, UK). UV data were obtained from HPLC online analysis. IR spectra were obtained on a Bruker Tensor-27 infrared spectrophotometer with KBr pellets. NMR spectra were carried out on a Bruker Avance III 600 or DRX-500 spectrometer with deuterated solvent signals used as internal standards. ESIMS and HRESIMS were measured using Agilent G6230 time-of-flight mass spectrometer. Preparative MPLC was performed on a Büchi apparatus equipped with Büchi fraction collector C-660, Büchi pump module C-605 and manager C-615. Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., China), MCI gel CHP-20P (75–150 μm, Mitsubishi Chemical Corporation, Japan), Chromatorex C-18 (40–75 μm, Fuji Silysia Chemical Ltd., Japan) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for column chromatography. Fractions were monitored and analyzed using TLC, in combination with an Agilent 1200 series HPLC system equipped by an Extend-C18 column (5 μm, 4.6 × 150 mm).
6
Ovary Cholesterol Quantification Protocol
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Ovaries were weighted and put into a tube with 1ml chloroform-methanol (2:1, V/V) solution. After thoroughly homogenized, the substances were removed by centrifugation and the fluid was dried using nitrogen gas (N2). To get total cholesterol, 1U cholesterol esterase (Worthington) were added and the reaction was proceeded at 37°C for 1 hour. 1ml hexane was used to extract the total lipid. The hexane phase was dried and re-dissolved in 50μl acrylonitrile- isopropanol (4:1). 20μl fluid was used to detect total cholesterol content. Standard curve were done by accurate weighting and gradient diluting the standard cholesterol (Sigma). All of the HPLC analyses used an Agilent 1200 system. The reactions were analyzed using an Agilent Extend-C18 column (5um, 4.6×250mm) thermostatted at 35°C, a solvent system of methanol: Acetonitrile (9:1, v/v) and a flow rate of 0.8ml/min. The mass detection was carried out by a Thermo Finnigan LCQ Deca XP MAX. The source type was Atmospheric Pressure Chemical Ionization (APCI) in positive polarity mode. The capillary temperature and APCI vaporizer temperature was 150°C and 450°C respectively.
7
In Vitro Lidocaine Release Assay
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In vitro drug release profile of LH and LNP was studied using a dialysis method. Briefly, 1 mL of each formulation was placed in a Spectra/Por Float-A-Lyzer G2 dialysis device (8–10 kDa MWCO, Spectrum Labs, United States) and dialyzed against 30 mL of PBS (pH7.4). At each time point between 0.25 and 48 h, 10 mL of PBS containing released lidocaine were taken out and another 10 mL of fresh PBS was added. The amount of released lidocaine was determined by HPLC method using an Agilent Extend C18 column (4.6 mm × 150 mm, 5 μm), solvent A (10 mM ammonium bicarbonate solution) and B (acetonitrile) with an isocratic gradient ratio of 50:50. The peak of lidocaine was detected at the wavelength of 214 nm. Drug release profiles were constructed by plotting the amount of drug released over time.
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In vitro drug release profile of LH and LNP was studied using a dialysis method. Briefly, 1 mL of each formulation was placed in a Spectra/Por Float-A-Lyzer G2 dialysis device (8–10 kDa MWCO, Spectrum Labs, United States) and dialyzed against 30 mL of PBS (pH7.4). At each time point between 0.25 and 48 h, 10 mL of PBS containing released lidocaine were taken out and another 10 mL of fresh PBS was added. The amount of released lidocaine was determined by HPLC method using an Agilent Extend C18 column (4.6 mm × 150 mm, 5 μm), solvent A (10 mM ammonium bicarbonate solution) and B (acetonitrile) with an isocratic gradient ratio of 50:50. The peak of lidocaine was detected at the wavelength of 214 nm. Drug release profiles were constructed by plotting the amount of drug released over time.
8
Triterpene Analysis by HPLC-MS
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The extracts were qualitatively analyzed triterpene by high-performance liquid chromatography mass spectrometry (HPLC–MS; 1,290 series–6,530 Accurate-Mass Q-TOF, Agilent Technologies, Waldbronn, Germany) with an Extend C18 column (2.1 mm × 50 mm, 1.8-μm pore size; Agilent Technologies), which is equipped with an electrospray ionization source (ESI). The analysis parameters were set using negative ion modes, with spectra acquired for mass-to-charge values of m/z = 50–3,000. HPLC analysis was applied by modifying the previous method4 (link). HPLC conditions are as follows: solvent A, 0.2% acetic acid in water; solvent B, acetronitrile; 0–15 min (35%–40% B), 15–45 min (40%–60% B), 45–55 min (60%–80% B), 55–56 min (80%–35% B), 56–60 min (35% B); flow rate, 1.0 mL/min, and detection was performed at 280 nm.
9
HPLC Analysis of Organic Compounds
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Analyses were achieved using an Agilent Technologies 1260 series HPLC system (Agilent Technologies, Santa Clara, CA, USA) incorporated into a DAD. Sample separation was carried out with an Agilent Extend-C18 column (250 × 4.6 mm i.d., 5 μm). The mobile phase consisted of 0.1% formic acid in water solution (A) and acetonitrile (B) with the following gradient elution program: 0–10 min, 30–50% A; 10–15 min, 50–60% A; 15–19 min, 60–50% A; 19–20 min, 50–30% A. The flow rate was set to 1 ml/min with 10 μl of injection volume. The detection wavelength was 254 nm, and the column temperature was 30°C.
10
Methanolic Extract Analysis of Myrrh by RP-HPLC-DAD and LC/MS
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The methanolic extract of myrrh was evaporated to dryness, then subjected to analysis by analytical reversed phase-high performance liquid chromatographic–diode array detection (RP-HPLC–DAD) followed by iquid chromatography–mass spectrometry (LC/MS). The extract analysis was performed on the Agilent 1260 Infinity HPLC system (Agilent, Böblingen, Germany) coupled with the Agilent 6530 Quadrupole Time of Flight (Agilent, Singapore). Separation was performed using Agilent Extend-C18 column (2.1 mm × 50 mm, 1.8 μm) with the following elution gradient; 0–1 min, 5% B; 1–11 min, 5–100% B; 11–13 min, 95%B; 13–15 min, 5%B; 15–16 min, 5%B using mobile phase A (0.1% HCOOH in water) and mobile phase B (0.1% HCOOH in methanol). The injection volume was 10 µL and the flow rate was 300 µL/min. The acquisition method MS1 was achieved in positive mode in the mass range from 100–600 m/z. The mass spectrometer parameters were set as follows: gas temperature = 300 °C; gas flow = 8 I/min; nebulizer = 35 psig; sheath gas temperature = 350, and sheath gas flow rate = 11. The MS/MS fragmentation of the identified nine compounds was conducted using a similar protocol applying soft fragmentation energy (20 eV).
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