Ocular tissues, ranging from small, soft perfused tissues to larger, tougher collagenous ones, are commonly selected for analysis during preclinical pharmacokinetic studies for ophthalmic drugs. The process of tissue homogenization and subsequent extraction needs to be optimized for proper downstream drug detection from these various matrices. In this study, bead mill homogenizers were selected for use for its efficiency and effectiveness in preparing complete homogenates. This methodology allows for the extraction of Cyclosporine from eight different ocular tissues – cornea, conjunctiva, iris-ciliary body (ICB), sclera, eyelid, lens, retina-choroid, and lacrimal gland. Due to the different phospholipid profiles between the tissues, however, varying amounts of matrix suppression were encountered during method development; to reduce the effect on analyte response, a phospholipid removal plate was used. The developed LC-MS/MS assay is robust, as well as demonstrates analytical reproducibility and efficiency for the quantification of Cyclosporine in the assayed ocular tissues. Acceptable selectivity, matrix factor, and dilution integrity were demonstrated in detail. Similarly, the methodology was flexible enough to allow for reanalysis of over-range samples as well as in posterior tissues with expected lower Cyclosporine levels.

For research use only. Not for use in diagnostic procedures.