title: Development and validation of UHPLC method for the determination of cyclosporine A in biological samples.
abstract: 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.
authors.affiliation: Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk, Poland.
authors.forename: Oliwia
authors.initials: O
authors.lastname: Szerkus
authors.forename: Eliza
authors.initials: E
authors.lastname: Wolska
authors.forename: Wiktoria
authors.initials: W
authors.lastname: Struck-Lewicka
authors.forename: Danuta
authors.initials: D
authors.lastname: Siluk
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Radwańska
authors.forename: Paweł
authors.initials: P
authors.lastname: Wiczling
authors.forename: Juliusz
authors.initials: J
authors.lastname: Chorążewicz
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Sznitowska
authors.forename: Michał J
authors.initials: MJ
authors.lastname: Markuszewski
authors.forename: Roman
authors.initials: R
authors.lastname: Kaliszan
mesh_terms: D000818:Animals; D001082:Aqueous Humor / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods; D016572:Cyclosporine / Q000032:analysis*; D005123:Eye / Q000737:chemistry; D007166:Immunosuppressive Agents / Q000032:analysis*; D007765:Lacrimal Apparatus / Q000737:chemistry; D008297:Male; D011817:Rabbits
chemical_list: D007166:Immunosuppressive Agents; D016572:Cyclosporine
keywords: cyclosporine A (CsA); cyclosporine D (CsD); ocular tissues; rabbits; ultra high-performance liquid chromatography (UHPLC)