A Novel Validated Method for the Determination of Cyclosporine in Rabbit Ocular Tissues by LC-MS/MS

Ocular tissues commonly selected for analysis during pre-clinical pharmacokinetic studies for ophthalmic drugs range from very small, soft perfused tissues (e.g., ICB or retina/choroid) to much larger, tougher collagenous tissues (e.g., sclera and eyelid). It is essential that the quality of the homogenate and subsequent extraction be designed properly to ensure optimal extraction of the drug from the tissues. Key to achieving this goal is the use of appropriate equipment such as a powerful bead homogenizer that efficiently and effectively prepares tissue homogenates in a high throughput manner. The methodology discussed here using the powerful Omni Bead Ruptor Elite bead mill homogenizer allowed for the preparation of suitable homogenates for the extraction of Cyclosporine from eight different ocular tissues with a simple, user-friendly stepwise procedure. Due to the different phospholipid profiles between the tissues there were varying amounts of matrix suppression encountered during method development. While the stable labeled internal standard compensated quite well in most cases a Waters OstroTM phospholipid removal plate was needed to reduce the effect on analyte response.

Figure 2: LC-MS-MS chromatograms of cyclosporine and its internal standard in rabbit cornea at the LLOQ.