A simple method for the subnanomolar quantitation of seven ophthalmic drugs in the rabbit eye

This study describes the development and validation of a new liquid chromatography-tandem mass spectrometry (MS/MS) method capable of simultaneous quantitation of seven ophthalmic drugs-pilocarpine, lidocaine, atropine, proparacaine, timolol, prednisolone, and triamcinolone acetonide-within regions of the rabbit eye. The complete validation of the method was performed using an Agilent 1100 series high-performance liquid chromatography system coupled to a 4000 QTRAP MS/MS detector in positive TurboIonSpray mode with pooled drug solutions. The method sensitivity, evaluated by the lower limit of quantitation in two simulated matrices, yielded lower limits of quantitation of 0.25 nmol L(-1) for most of the drugs. The precision in the low, medium, and high ranges of the calibration curves, the freeze-thaw stability over 1 month, the intraday precision, and the interday precision were all within a 15% limit. The method was used to quantitate the different drugs in the cornea, aqueous humor, vitreous humor, and remaining eye tissues of the rabbit eye. It was validated to a concentration of up to 1.36 ng/g in humors and 5.43 ng/g in tissues. The unprecedented low detection limit of the present method and its ease of implementation allow easy, robust, and reliable quantitation of multiple drugs for rapid in vitro and in vivo evaluation of the local pharmacokinetics of these compounds.