Hypurity c18 analytical column

The high performance liquid chromatography (HPLC) consisted of a Dionex (Dionex Softron GmbH, Germany) HPLC system with a P 680 pump, an ASI-100 automated sample injector and a UVD 1704 detector. A 250 μl injector with a 20 μl loop was used. Reversed-phase-liquid chromatography was carried out using a Hypurity™ C₁₈ analytical column, 5 μm × 4.6 mm (Thermo Electron Corporation, Runcorn, UK 22105-154630). A column guard (Thermo electron 60140-412) was used to protect the analytical column. The ultraviolet detector was set to monitor at 215 nm wavelength. The mobile phase for the analysis was composed of ammonium formate 20 mM (pH = 4.2), ACN and MeOH (57:38:5 v/v), and was prepared fresh for each assay. The separation was facilitated via isocratic elution at 1.5 ml/min flow rate and the run time was eight minutes for each separation. 20 μl of the samples was injected for each run by means of an automated injector. The peak area ratios for the calibration curves and quantification were obtained and analyzed using Chromelon software (version 6.5).

The HPLC consisted of a Dionex (Dionex Softron GmbH, Germany) HPLC system with a P 680 pump, an ASI-100 automated sample injector and a UVD 1704 detector. A 250μl injector with a 20μl loop was used. Reversed-phase-liquid chromatography was carried out using a Hypurity™ C₁₈ analytical column, 5μm x 4.6mm (Thermo Electron Corporation, Runcorn, UK 22105-154630). A column guard (Thermo electron 60140-412) was used to protect the analytical column. The ultraviolet detector was set to monitor the 215 nm wavelength. A Packard Tri-Carb Liquid Scintillation Analyzer model 1900 TR (Packard instrument Co.) was used for radioactivity counting. A Millicell Electrical Resistance System (Fisher Scientific, Leicestershire, UK) was used for measuring the transepithelial electric resistance (TEER). Transwells (six-well transwell polycarbonate tissue culture treated plates, 4.67 cm², 24 mm diameter; 0.4 μm pore size) were purchased from Corning life Sciences (Costar High Wycombe, Bucks; UK).

Blood samples for Sul-121 analysis were obtained two weeks after implantation of the first osmotic minipump. Sul-121 analysis was performed on an Agilent 6460 A (Santa Clara, Ca, USA) triple quadrupole LC-MS/MS system, with a combined Agilent 1200 series LC system. A HyPURITY® C18 analytical column from ThermoFisher Scientific (Waltham, MA, USA) was used for chromatographic separation by means of a gradient with a flow of 0.5 mL/min and a run time of 2.4 min. Sample preparation was performed by means of protein precipitation.