Bioanalytical method validation of rapamycin in ocular matrix by QTRAP LC–MS/MS: Application to rabbit anterior tissue distribution by topical administration of rapamycin nanomicellar formulation

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 200 μL 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, 50 mm × 4.6 mm, 5 μm. 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 r2 > 0.9998 over the concentration range from 2.3 ng/mL to 1000.0 ng/mL. Rapamycin was found to be stable in ocular tissue homogenates for 6 weeks 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.