Agilent 1260 hplc

Titers for expressed antibodies were determined by ÄKTA chromatography system (Cytiva, Marlborough, MA, USA) or Agilent HPLC-1260 (Agilent Technologies, Santa Clara, CA) with HiTrap^TM MabSelectSuRe^TM 1 mL column (Cytiva, CAT#11003493). The wash buffer was phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 2.7 mM KH₂PO₄, pH 7.2) and elution buffer was 150 mM Glycine, 40 mM NaCl, pH 3.5. The eluted antibody solution was neutralized with 10% of 2M HEPES, pH 8.0, and subjected to a size exclusion chromatography analysis with Superdex200 10/300 GL (Cytiva, CAT# GE28-9909-44) with 40 min at 0.75 mL/min flow rate in Agilent HPLC-1200. The antibody eluants were also analyzed by SDS-PAGE and visualized by coomassie blue staining.

The dissolution of curcumin from the prepared nanostructures was performed using USP type II dissolution apparatus (Hanson Research, USA) which was rotated at 50 rpm. The bath temperature was kept at 37^°C. In order to determine the ability of the formulations to enhance curcumin dissolution, non-sink condition was selected to perform the study; at which water was used as a discriminative dissolution medium. Briefly, a sample equivalent to 3 mg from each formulation was introduced into 300 mL of the dissolution medium and aliquots of 0.5 mL were taken at time intervals of (5, 10, 15, 30, 45 and 60 min), filtered through 0.2 μm PTFE filter and quantified for curcumin content by validated HPLC method utilizing (Agilent-HPLC1260, USA), mobile phase composed of acetonitrile and 2% v/v acetic acid (50:50) pumped at 0.6 mL/min, C18 Column (5 μm, 3mm X 30mm) kept at 35^°C, 20 μL injection volume and the detection wave length was 425 nm.

A total of 1.5 mL of blood was taken from the rabbits’ ear veins at various intervals following the administration of various formulations to prevent clotting, and blood samples were centrifuged at 4000 rpm for 15 min to obtain plasma. The separated plasma tubes were stored at minus 20 °C. The chromatographic system was high-performance liquid chromatography (HPLC; Agilent 1260; Agilent, Santa Clara, CA, USA) with a UV detector. Yehia et al. performed an internal standard analysis of all samples at room temperature using HiQsil C18 (25 cm) and levofloxacin [41 (link)].
After removing 225 µL of rabbit plasma, an internal standard of 25 µL of levofloxacin solution was added. Following each stage, 4 µL of dichloromethane was added, and this solution was vortexed for 3 min and centrifuged for 5 min at 5 °C and 5000 rpm (VWR VV3 S540, Avantor, Carpenteria, CA, USA). The supernatant was evaporated in a vacuum oven (VACUCELL VUS-B2V-M/VU 22, MMM Group, Ettlingen, Germany). The residue was dried and injected into an Agilent HPLC 1260 after it had been reconstituted with 200 µL of the mobile phase. Using HPLC data, plasma concentration–time profile was made using the PK solver (free add-in Excel program) application and all the kinetic parameters were calculated.

50 μg protein from each sample was digested into peptides with trypsin (Promega, Madison, USA). Peptides were subsequently cleaned up with C18 desalting columns (The Nest Group Inc., USA) and then lyophilized to dryness. iTRAQ labeling was performed according to the manufacturer’s instructions for the iTRAQ reagents 8-plex kit (AB Sciex Inc., Foster City, California, USA). WT was labeled with iTRAQ tags 113 and 117; BZR1-1D#6 was labeled with iTRAQ tags 115 and 119; BZR1-1D#23 was labeled with iTRAQ tags 116 and 121 (Figure S1). For each sample of WT and transgenic lines, three independent biological replicates were performed.
After labeling, samples from the same set were combined together and lyophilized. The peptide mixtures were dissolved in strong cation exchange (SCX) solvent A (25% (v/v) acetonitrile, 10 mM ammonium formate and 0.1% (v/v) formic acid, pH 2.8). The samples were fractionated using Agilent HPLC 1260 with a polysulfoethyl A column (2.1 mm × 100 mm, 5 μL, 300 Å; PolyLC, Columbia, MD, USA) at a flow rate of 0.2 mL/min. After that, peptides were eluted with a linear gradient of 0–20% solvent B (25% (v/v) acetonitrile and 500 mM ammonium formate, pH 6.8) over 50 min, followed by ramping up to 100% solvent B in 5 min. 12 fractions were collected and lyophilized for LC−MS/MS analysis.

Twenty milligrams of SN-ppF3 was hydrolyzed with 2 mL of 4 M trifluoroacetic acid (TFA) (Sigma, St. Louis, MO, USA) in a glass test tube for 5 hours at 95°C water bath. The hydrolysate was then evaporate with a stream of nitrogen gas at 80°C, washed with absolute methanol and redissolved with 1 mL of 75% Acetonitrile (ACN).It was filtered through 0.45 µM syringe filter and separated using Agilent Carbohydrate Analysis Column on Agilent HPLC 1260 (Agilent Technologies, Santa Clara CA, USA), equipped with refractive index detector. The column and detector temperature were set at 30°C and sample was eluted isocratically with 75% ACN at a flow rate of 1.4 mL/min. The possible identity of monosaccharides in the samples was determined by comparing the retention time of the peaks with those of standards (rhamnose, fucose, xylose, arabinose, glucose, galactose and mannose).