RESUMO
A novel, fast and sensitive 3200 QTRAP LC-MS/MS method was validated for rapamycin analysis in the rabbit eye following 0.2% administration of nanomicellar eye drop formulation. The LC-MS/MS technique was developed with electrospray ionization (ESI) in positive mode. Rapamycin was extracted from individual eye tissues and fluids by a simple protein precipitation method. Samples were reconstituted in 200µL of 80% of acetonitrile in water containing 0.05% formic acid. Twenty microliter of the sample was injected on LC-MS/MS. Chromatographic separations was achieved on reversed phase C 8 Xterra column, 50mm×4.6mm, 5µm. Multiple reactions monitoring (MRM) transition m/z 936.6/409.3 for rapamycin and 734.4/576.5 for erythromycin were employed as internal standard. The calibration curves were linear r(2)>0.9998 over the concentration range from 2.3ng/mL to 1000.0ng/mL. Rapamycin was found to be stable in ocular tissue homogenates for 6weeks at a refrigerated -80°C and -20°C temperatures. Rapamycin concentration was found to be 2260.7±507.1 (mean±S.D.)ng/g tissue and 585.5±80.1 (mean±S.D.)ng/g tissue in the cornea and iris ciliary muscle, respectively. This method has two advantages. First, a volatile base was used in the extraction procedure, which is easy to evaporate and generate consistent results. Second, the sodium adduct is employed that was stable in non-ammoniated mobile phase. The method demonstrates that absorption of rapamycin by a topical application of 0.2% rapamycin nanomicellar formulation generates therapeutically effective concentrations in the anterior segment of the eye.
