Quantitative LC analysis of cyclosporine A in ocular samples

An isocratic reversed-phase HPLC method with ultraviolet detection at 205 nm has been developed for analysis of cyclosporine A (CyA) in rabbit ocular samples. Neither internal standard nor extraction was needed for sample preparation. Acetonitrile (ACN; 1 mL) was added to 250 μL aqueous and vitreous samples to precipitate proteins. The supernatant was dried and the residue was reconstituted in mobile phase and injected for HPLC analysis. Chromatography was performed on an octadecyl silane-A (ODSA) C₁₈ (4.6 × 250 mm, 5 μm) column. The column temperature was fixed at 70 °C and the mobile phase was ACN 65%, methanol 20% and water 15% at a flow rate of 1.5 mL min^-1. The calibration curve for CyA in rabbit ocular samples was linear over the concentration range 0.2 and 10 μg mL^-1 with a correlation coefficient of 0.9992. Intra-day and inter-day precision were 4.61-7.83% and 5.27-10.70%, respectively. Intra-day and inter-day accuracy were 89.2-108% and 83.4-111%, respectively. The limits of detection (LOD) and quantification (LOQ) were 5.7 and 38 ng mL^-1, respectively. The method was successfully used for analysis of CyA in real aqueous and vitreous humor samples from New Zealand albino rabbits. The method is therefore suitable for analysis of CyA in ocular samples.