Brimonidine, HR97-biotin, and HR97-brimonidine at a range of concentrations (3.125, 6.25, 12.5, 25, 50, 100 µg/mL) were dissolved in pH 6.5 PBS solution. The solutions (400 µL) were then mixed thoroughly with 400 µL of 1 mg/mL mNPs in pH 6.5 PBS solution and transferred to the inner reservoir of the rapid equilibrium dialysis (RED) device inserts (8 K MWCO). The outer reservoir was filled with 800 µL of pH 6.5 PBS solution. The samples were incubated on an orbital shaker with temperature control at 37 °C and 300 rpm for 48 h (n = 3). The solutions from outer reservoir (free drug) were than collected and transferred to an autosampler vial for HPLC analysis (Prominence LC2030, Shimadzu, Columbia, MD) with photodiode-array detection (PDA) system. Separation was achieved with a Luna® 5 µm C18(2) 100 Å LC column 250 × 4.6 mm (Phenomenex, Torrance, CA) at 40 °C using isocratic flow. The amount of bound drug was used to calculate the binding capacity (mol drug/mg melanin) and the dissociation constant (Kd) as previously described20 (link),34 (link).
Luna 5 m c18 2 100 lc column
Manufactured by Phenomenex
The Luna® 5 µm C18(2) 100 Å LC column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a 5 µm particle size and 100 Å pore size, which provides efficient chromatographic performance. The column is intended for use in HPLC applications.
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Lab products found in correlation
3 protocols using luna 5 m c18 2 100 lc column
1
Assessing Binding Capacity of Melanin-Based Nanoparticles
2
Stability of Brimonidine in Ocular Fluids
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Two pairs of human donor eyes were obtained from the Lions Gift of Sight under protocol IRB00056984 approved by the Johns Hopkins University School of Medicine Institutional Review Board. Both donors were male with an mean age of 74.5. The post-mortem times ranged from 35–40 h. The eyes were kept at 4 °C during transport and arrived within 48 h post-mortem. The vitreous and aqueous were first isolated and subsequently combined and filtered through the 0.02 µm syringe filter to remove cell debris. HR97-brimonidine (1 mg/mL) was incubated with human aqueous or vitreous (700 µL) at 37 °C (n = 3). On days 0, 1, 7, 14, 21 and 28, 100 µL of the solutions were collected, diluted with 900 µL of acetonitrile, and characterized by HPLC (Prominence LC2030, Shimadzu) with Luna® 5 µm C18(2) 100 Å LC column 250 × 4.6 mm (Phenomenex). The elution flow rate was 1 mL/min and with gradient of 10/90/90/10% solvent B (TFA 0.1% in ACN) in 1/11/13/15 min at λmax = 250 nm for HR97-brimonidine (RT = 4.6 min). The area under the curve (AUC) on day 0 was used to normalize the AUC calculated on days 1, 7, 14, 21 and 28.
3
Quantification of Tomatine and Tomatidine in Tomato Extracts
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The choice of the best extracts for use as natural pesticides was made after the detection of tomatine and tomatidine with High Pressure Liquid Chromatography (HPLC) with a Photodiode Array Detector (PDA) (Shimadzu LC-2030C, Kyoto, Japan). For this study, freeze-dried extracts of the leaves, stem, and green and red tomatoes were dissolved in 0.2 N HCl, and the alkaloids were precipitated with 5% NH 4 OH until a pH = 10 was reached. Subsequently, the mixture was centrifuged at 3000 rpm for 20 min at 4 • C, and the supernatant was rejected. The washing process was repeated twice. Any residual supernatant was removed by evaporation at 45 • C using a rotary evaporator (Buchi R200 rotavapor), and the collected precipitate was redissolved in methanol. Prior to HPLC, the samples were filtered through a 0.45 µm PTFE syringe filter. The column used was Phenomenex Luna ® 5 µm C18 (2) 100 Å, LC Column 250 × 4.6 mm; the mobile phase was methanol (isocratic flow) with a flow rate of 1 mL/min, and the injection volume was 10 µL. The absorbance was detected at 200 nm. All samples were studied in triplicate. The contents of the alkaloids were calculated using calibration curves of standard tomatine and tomatidine samples. The calibration curve was quite linear over the concentration range from 0.02 to 1 mg•mL -1 (r = 0.9987), and the limit of detection was 0.01 mg•mL -1 .
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