Gel tranches were de-stained, with protein reduced, alkylated, digested with trypsin, and peptides extracted and dried89 (link). Peptides were resuspended in 0.2% formic acid, 0.1% trifluoroacetic acid, and 0.002% zwittergent 3–16 (Calbiochem, San Diego, CA), and analyzed by nano-flow LC–MS/MS using a Q-Exactive Hybrid Quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to a Thermo UltiMate 3000 RSLCnano HPLC system. Peptides were loaded onto a 250 nL OPTI-PAK trap (Optimize Technologies, Oregon City, OR) packed with Michrom Magic C8, 5 µm solid phase (Michrom Bioresources, Auburn, CA). Chromatography was performed using 0.2% formic acid in solvents A (98% water, 2% acetonitrile) and B (80% acetonitrile, 10% isopropanol, 10% water), over a 2–45% B gradient for 60 min at 400 nL/min through a 100 µm × 35 cm PicoFrit column (New Objective, Woburn, MA) packed with Agilent Poroshell 120 EC-C18 (Agilent Scientific Instruments, Santa Clara, CA). MS1 survey scans 350–2000 m/z were acquired at 70,000 resolution targeting 3 × 106 ions and 60 ms maximum inject time, followed by data dependent high energy collisional dissociation MS2 on the top 15 ions at 17,500 resolution targeting 2 × 105 ions with 60 ms maximum inject time, using dynamic exclusion of measured ions for 60 s.
Agilent poroshell 120 ec c18
Manufactured by Agilent Technologies
Sourced in United States
The Agilent Poroshell 120 EC-C18 is a high-performance liquid chromatography (HPLC) column. It is designed for the separation and analysis of a wide range of compounds. The column features a porous shell particle technology that provides efficient and rapid separations.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Picofrit column, by New Objective (1 mentions)
Dionex ultimate 3000 uhplc system, by Thermo Fisher Scientific (1 mentions)
Tsq quantum ultra triple quadrupole mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Agilent 1260 hplc system, by Agilent Technologies (1 mentions)
Triart c18, by YMC (1 mentions)
7 protocols using agilent poroshell 120 ec c18
1
Peptide Analysis by Nano-flow LC-MS/MS
2
HPLC-MS Analysis of Acarbose
3
LC-MS/MS Analysis of Chlorogenic Acids
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LC-MS/MS analysis was performed as described previously by Nemzer et al. [13 (link)]. LC-MS/MS analysis was performed on a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) coupled to a Dionex UltiMate 3000 UHPLC system (Thermo Scientific). A C18 analytical column (Agilent Poroshell 120 EC-C18, 3 × 150 mm, 2.7 µm, Agilent Technologies, Santa Clara, CA, USA) was employed for the separation of the compounds as follows: 0–15 min, 7% B; 40 min, 50% B; 50 min, 70% B; 51 min, 0% B and kept for an additional 10 min at 0% B. Flow rate was 0.4 mL/min for 60 min. The MS instrument operated in both positive and negative ion modes with a capillary voltage of 3.2 kV. Precursor ions were scanned in the range of m/z 150–1200 at a resolution of 70,000 and an automatic gain control target value of 1.0 × 106. Precursor ions were fragmented in the higher-energy collisional activated dissociation cell, and the fragments were analyzed using an orbitrap analyzer. The major chlorogenic acids in each sample were identified manually based on their precursor mass-to-charge ratios and unique fragmentation spectra.
4
In Vitro Drug Release Evaluation of RDV-Loaded Thermogel and Cyclodextrin
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RDV-loaded cyclodextrin inclusion complex was prepared as a control before the drug release study. Literature methods were followed as references [21 (link),22 (link)]. The in vitro release profiles of RDV from the thermogels and cyclodextrin inclusion complexes were measured in Meilubio artificial tears (Dalian, China) at 35 °C. Briefly, 200 μL of RDV-loaded thermogel and cyclodextrin inclusion compound (both 1 mg/mL) were sealed into an MD25-3.5 dialysis membrane. Then, 2 mL of artificial tears was used as release medium. At predetermined time points, 1 mL of release medium was extracted, and 1 mL of artificial tears was replenished to maintain the volume. Cumulative release (%) profile of RDV was calculated using the Agilent 1200 HPLC system (Agilent Poroshell 120 EC-C18, 4.6 × 150 mm i.d., 4 µm) with a mobile phase of 0.05% TFA in H2O/acetonitrile (50:50 v/v) at 254 nm. The column temperature was 30 °C and the injection volume was set at 10 µL. The total run time was 31.0 min with a flow rate of 1.0 mL/min; the peak at around 18.5 min was assigned to RDV.
5
Quantification of CoQ10 in Bacterial Cultures
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The CoQ10estimation required an initial step of bacteria cell disruption, as described in Zhang et al. [56] (link), to extract the compound. A CoQ10 analytical standard (Sigma-Aldrich USA) was used to generate a standard curve. The amount of CoQ10 present in the cultures was estimated using the Agilent Poroshell 120 EC—C18 (100mmx 3.0 mm x 2.3um) (Agilent Technologies, USA) HPLC with a mobile phase of 65% methanol and 35% ethanol. The flow rate was set at 1.5 ml/min, and a wavelength of 275 nm was used for the detection of CoQ10. The dry cell weight of the compound was estimated using (Eq. (1) ) .
6
Quantitative HPLC-MS/MS Analysis
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Compound concentrations were determined using an Agilent 1260 HPLC system coupled to an Agilent 6460 triple-quadrupole mass spectrometer (Agilent Technologies, Singapore) and a Shimadzu UFLC XR instrument (Shimadzu, Tokyo, Japan) coupled to a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Chromatographic separations were performed on Agilent Poroshell 120 EC-C18 (3.0 × 50 mm, 2.7 µm; Agilent Technologies, Inc., Santa Clara, CA, USA) and YMC triart C18 (2.0 × 50 mm, 3 µm; YMC Co., Ltd., Kyoto, Japan) columns using 5 mM ammonium formate in water (pH 4) and methanol. The detailed analysis conditions are listed in Tables S2 and S3 .
7
Roscovitine Pharmacokinetics in Mice
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Plasma concentrations of roscovitine were determined by HPLC-DAD. Mice were treated with roscovitine and blood was collected after 10, 20, 30, 60, 120, and 240 min. For each time point, blood of three mice was pooled.
100 μl mice blood were mixed with 200 μl acetonitrile, vortexed (1 min), centrifuged (5 min, 10,000 g, RT) and the supernatant was analyzed by HPLC-DAD using an Agilent Series 1100 HPLC system (Waldbronn, Germany) consisting of a quaternary pump system (G1311 A QuatPump), an autosampler (G1329 A ALS), a column oven (G1316 A ColComp) and a UV-DAD detector (G1315 A DAD). Chromatographic separation was carried out with an Agilent poroshell 120 EC-C18 (100 × 3.0 mm, i.d. 2.7 μm) column (Waldbronn, Germany) and a mobile phase of acetonitrile and water (0.1% phosphoric acid, 1.0% tetrahydrofuran) 15:85 (v/v). The total run time was 7 min with an isocratic flow rate at 1.0 ml/min, and an injection volume of 10 μl. The column oven was set at 50°C. The UV detection wave-length was set at 292 nm. Data analysis and instrument control was carried out with Agilent ChemStation® software Rev. B04.02. The average retention time of roscovitine was 3.1 min. The concentration of roscovitine was determined according to an external standard calibration.
100 μl mice blood were mixed with 200 μl acetonitrile, vortexed (1 min), centrifuged (5 min, 10,000 g, RT) and the supernatant was analyzed by HPLC-DAD using an Agilent Series 1100 HPLC system (Waldbronn, Germany) consisting of a quaternary pump system (G1311 A QuatPump), an autosampler (G1329 A ALS), a column oven (G1316 A ColComp) and a UV-DAD detector (G1315 A DAD). Chromatographic separation was carried out with an Agilent poroshell 120 EC-C18 (100 × 3.0 mm, i.d. 2.7 μm) column (Waldbronn, Germany) and a mobile phase of acetonitrile and water (0.1% phosphoric acid, 1.0% tetrahydrofuran) 15:85 (v/v). The total run time was 7 min with an isocratic flow rate at 1.0 ml/min, and an injection volume of 10 μl. The column oven was set at 50°C. The UV detection wave-length was set at 292 nm. Data analysis and instrument control was carried out with Agilent ChemStation® software Rev. B04.02. The average retention time of roscovitine was 3.1 min. The concentration of roscovitine was determined according to an external standard calibration.
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