Waters spherisorb ods2 column

DEX release was measured by dispersing the DEX-coated nanoparticles (10 mg) into 1 mL of a buffer media (acetate buffer pH=5, and phosphate buffer pH=7.3) and incubated at 37°C in Eppendorf.
Daily, the suspension was centrifuged at 4000 g for 10 mins at room temperature; the supernatant was withdrawn for quantification and the medium was replaced with an equal volume of fresh buffer and the nanoparticles resuspended.
The amount of DEX/DEX-P released from the coating layer was determined using reverse-phase High Performance Liquid using an Agilent Technologies^® HPLC (1100 series) equipped with a Waters-Spherisorb ODS2 column (Pore size – 80 Å, 5 µm, and packing dimension of 4.6 mm×150 mm). The injection volume was 10 µL. The mobile phase was a mixture of PBS-acetonitrile-glacial acetic acid 70:26:4 at a flow rate of 1 mL/min; a UV detector at 244 nm was employed.
Stock solutions of DEX-P and DEX with a concentration of 1 mg/mL were prepared separately and a series of standards ranging from 0.4 to 250 µg/mL were prepared for calibration.

The HPLC system (Agilent 1100 Series; Agilent Technologies, Waldbronn, Germany) was fitted with the appropriate column, depending on the drug under test (fluticasone propionate: Waters Symmetry C18 column [5 μm, 250 × 4.6 mm, 40°C, 1.5 mL/min; Waters Corporation, Milford, MA]; salbutamol sulfate: Genesis Phenyl column [4 μm, 150 × 4.6 mm, 22°C, 1 mL/min; Crawford Scientific, Lanarkshire, UK]; budesonide: Supelcosil LC-18 column [5 μm, 50 × 4.6 mm, 22°C, 2 mL/min; Sigma-Aldrich, St. Louis, MO]; formoterol fumarate: Waters Spherisorb ODS 2 column [3 μm, 125 × 4.6 mm, 30°C, 1.5 mL/min; Waters Corporation]; beclomethasone dipropionate: Phenomenex Luna C18(2) column [3 μm, 150 × 4.6 mm, 25°C, 1 mL/min; Phenomenex, Inc., Torrance, CA]).
Bracketing standards of each of the drugs were made and run, and the accuracy of the standards was checked. System suitability was then checked by using five replicate injections of the bracketing standards. The relative standard deviations had to be either at or below 2%, and the USP tailing factor of the drug peak had to be no greater than the predetermined values (fluticasone propionate, budesonide, and beclomethasone dipropionate: 1.5, salbutamol sulfate and formoterol fumarate: 2.0) for the results of the run to be considered valid.