FLU (logP: 0.5, MW: 355.34 g/mol) was analysed using HPLC/UV, adapted by [16 (link)]. Briefly, FLU was extracted from cornea samples with 5 mL of an isotonic HEPES buffer solution (25 mM, pH 7.4) in an Ultra-Turrax (Model T25 Digital, IKA, Staufen, Germany) for 30 s and filtered (0.45 µm hydrophilic filters) directly into a vial. Then, the filtered samples were injected (20.0 µL) in an isocratic system using acetonitrile, methanol, and water (15:5:80) as a mobile phase with a flow rate of 0.8 mL/min. A C18 column (150 × 4.6 mm, 5 µm, Supelco Discovery BIO Wide-Pore) with the temperature settled at 40 °C was used, and the drug was detected at 210 nm. The standard curve was selective and linear (y = 30,676x + 2843.9 and R2 = 0.9992) for a concentration range of 0.5–10 µg/mL. The Limit of Detection and Limit of Quantification were determined to be 0.007 and 0.021 µg/mL, respectively.
Discovery bio wide pore
Manufactured by Merck Group
The Discovery BIO Wide-Pore is a laboratory equipment product offered by Merck Group. It is designed for use in various scientific applications. The core function of this product is to provide wide-pore separation capabilities for research and analysis purposes.
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3 protocols using discovery bio wide pore
1
HPLC-UV Analysis of Fluconazole in Cornea
2
NanoLC-MS/MS Peptide Fractionation and Analysis
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Desalted peptide mixtures were fractionated on an Ekspert 425 nanoLC system (Eksigent) equipped with a C18 column (Discovery BIO Wide Pore, Supelco). The mobile phases were solvent A (water, 0.1% FA) and B (acetonitrile, 0.1% FA). Injection was performed with 98% solvent A at a flow rate of 5 μl/min. Peptides were separated at 30◦C with the following gradient: 2% to 40% B for 105 min, 40–80% B for 5 min. The column was washed with 80% solvent B for 5 min and equilibrated with 98% solvent A. Peptide separation was monitored online with the coupled TripleTOF 5600 mass spectrometer (Sciex). The total ion chromatogram acquisition was made in information-dependent acquisition (IDA) mode using the Analyst TF v.1.7 software (Sciex). Positive ion profiling was performed from m/z 350–1,500, followed by a MS/MS product ion scan from m/z 100–1,500 with the abundance threshold set at more than 100 cps. The accumulation time for ions was set at 250 ms for MS scans, and 100 ms for MS/MS scans. Target ions were excluded from the scan for 10s after detection. The IDA advanced “rolling collision energy (CE)” option was employed to automatically ramp up the CE value in the collision cell as the m/z value was increased. A maximum of 25 spectra were collected from candidate ions per cycle.
3
Milk Protein Profiling by LC-MS
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Milk protein composition was determined by LC/ ESI-MS method developed at INRA as described in detail by Fang et al. (2016) . Briefly, milk proteins were separated by reversed-phase HPLC using an Ultimate LC 3000 system (Thermo Fisher Scientific, Waltham, MA) with a Discovery BIOWide Pore (Supelco, Bellefonte, PA) C5 column (150 × 2.10 mm, 300 Å). Genetic variants and isoforms of the 6 major milk proteins (α S1 -CN, α S2 -CN, κ-CN, β-CN, α-LA, and β-LG) were identified using an ESI-TOF mass spectrometer micrOTOF II focus (Bruker Daltonics, Wissembourg, France). Relative concentrations of individual milk proteins and of α S1 -CN phosphorylation isoforms were estimated based on dividing the peak area of an individual protein by the total integrated peak area in the chromatogram (absorbance at 214 nm) of an individual milk sample. Mass signal intensity obtained from mass spectrometry was used to estimate the proportion of each α S2 -CN phosphorylation isoform as a fraction of total α S2 -CN. The relative concentration of each α S2 -CN phosphorylation isoform was estimated using the following equation:
[1]
The phosphorylation degrees of α S1 -CN and α S2 -CN were defined as the proportion of isoforms with higher degrees of phosphorylation, which were calculated as α S1 -CN PD = (α S1 -CN-9P/total α S1 -CN) × 100 and
[1]
The phosphorylation degrees of α S1 -CN and α S2 -CN were defined as the proportion of isoforms with higher degrees of phosphorylation, which were calculated as α S1 -CN PD = (α S1 -CN-9P/total α S1 -CN) × 100 and
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