2.3.1. Drug Content

Drug content was analyzed using a high performance liquid chromatography (HPLC) assay. For the HPLC assay, aliquots (100 μL) were diluted with an equivalent volume of methanol, the solution was filtered (0.22 μm) and the filtrate injected onto a C18 reverse phase Onyx monolithic column (100 × 4.6 mm). A gradient mobile phase was used as follows: 0 min—acetonitrile, water = 60:40 v/v; 4 min—acetonitrile, water = 5:95 v/v; 7.5 min—acetonitrile, water = 60:40 v/v. The run time was 10.5 min, the retention time for CsA was 4.9 min, the flow rate 1.5 mL min⁻¹, the injection volume was 20 μL and the column temperature was 40 °C. Analytes were monitored at a UV absorption wavelength of 210 nm (Figure S1 in Supplementary Information). A standard curve was prepared using cyclosporine A concentrations ranging from 10 to 1250 μg mL⁻¹ in methanol (y = 26527x + 370.25 r² = 0.9975, limit of quantification = 0.01 mg mL⁻¹). The HPLC system was an Agilent 1200 Series (Agilent Technologies Ltd., Stockport, UK) and the data were analyzed by Agilent ChemStation software, version 07/09 (Agilent Technologies Ltd., Stockport, UK).