The in vivo model on rabbit eyes and the in vitro cytotoxicity on fibroblasts were used to compare irritation effect of aqueous and oily (Miglyol 812) solutions of surfactants. Tween 20, Tween 80 and Cremophor EL were tested in different concentrations (0.1, 1 or 5%) and the in vitro test demonstrated that surfactants in oil are less cytotoxic than in aqueous solutions. In the in vivo study, the aqueous solutions of surfactants were characterized as non-irritant while small changes in conjunctiva were observed after application the oily solutions of surfactants and the preparations were classified as slightly irritant, however this effect was similar when Miglyol was applied alone. In conclusion, it is reported that the MTT assay does not correlate well with the Draize scores.
The aim of the study was to develop and validate a simple and rapid method for the determination of cyclosporine A (CsA) in ocular rabbit tissues using reversed-phase ultra-high-performance liquid chromatography (UHPLC) with UV detection. Previous publications on chromatographic methods of CsA determination in ocular tissues involved only reversed-phase HPLC separation, usually in combination with such detection techniques as radio-immunoassay and mass spectrometry. The application of the UHPLC technique allowed us to significantly decrease the analysis time. Cyclosporine D (CsD) was applied as the internal standard. Satisfactory separation was achieved on an XB-C18 Kinetex column at 60°C with the use of gradient elution mode. The retention times of CsA and CsD were found to be 4.5 and 5.1 min, respectively. The developed assay is specific, sensitive (limit of detection = 6 ng/mL and limit of quantitation = 18 ng/mL) and linear within the analyte concentration range of 0.018-5 µg/mL, with a correlation coefficient of 0.999. High sensitivity, low injection volume (10 μL), short time of analysis (6.5 min) and simplicity make this method useful for the fast analysis of CsA in rabbit ocular tissues and fluids: lacrimal fluid, aqueous humor, cornea, conjunctiva and eye globe.
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