Current Pharmaceutical Analysis, 2011, 7, 189-194 189 1573-4129/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd. Aqueous Humor Pharmacokinetics of Dorzolamide Loaded PLGA- Chitosan Nanoparticles by Ultra Performance Liquid Chromatography Musarrat H. Warsi a* , Akash Chaurasiya a , Gaurav K. Jain a , Shadab A. Pathan a , Prakash Chander b , Ajeet K. Singh c , Sushama Talegaonkar a , Farhan J. Ahmad a and Roop K. Khar a a Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi 110062, India b Waters India Pvt. Ltd., CS-08/09, 7th Floor, Lobe 2, Tower A, The Corenthum, Plot No. A-41, Sector 62, Noida 201301, UP, India c Matrix Laboratories Limited, Medha 502325, Hyderabad, India Abstract: Ocular pharmacokinetic analysis of drugs is useful for evaluation of efficacy of novel ophthalmic formulations. A simple and sensitive ultra performance liquid chromatographic (UPLC) method with ultraviolet (UV) detection was de- veloped and validated for the quantification of dorzolamide (DZA) in rabbit aqueous humor. The aqueous humor samples were deproteinized with acetonitrile prior to UPLC analysis. A chromatographic separation was achieved on a Waters Ac- quity BEH C18 (100 mm2.1 mm, 1.7 μm) column with a mobile phase consisting of 0.1% triethylamine (TEA) in water and acetonitrile (50:50, v/v) at a flow rate of 0.25 ml min -1 . Detection of DZA was carried out at 254 nm. The method de- veloped was linear (r 2 = 0.9996) over the concentration range of 5 - 800 ng mL -1 . Accuracy ranged from 98.23 to 99.53% and precision was between 0.12% and 0.36%. Limit of detection and quantitation of DZA in aqueous humor were 5 and 10 ng mL -1 , respectively. The method was applied to determine pharmacokinetics of DZA in aqueous humor, following topical instillation of DZA-solution (DZA-SOL) and DZA nanoparticles (DZA-NPs) to rabbit eyes. The pharmacokinetic results showed that DZA-NPs enhances the ocular bioavailability of DZA by 6-fold compared to DZA-SOL. Keywords: Aqueous humor, Dorzolamide, Nanoparticles, Ocular pharmacokinetics, Ultra performance liquid chromatogra- phy. 1. INTRODUCTION Oral carbonic anhydrase inhibitors (CAIs) are commonly used to reduce intraocular pressure (IOP) in glaucoma pa- tients [1]. Systemic CAI administration can lead to various side effects, such as numbness, fatigue, depression, loss of appetite and gastrointestinal irritation, and consequent poor patient compliance [2, 3]. Dorzolamide (S,S-5,6-dihydro-4H- 4-ethylamino-6-methylthieno [2,3-b] thiopyran-2-sulfona- mide-7,7-dioxide, DZA), available as 2% w/v aqueous eye drop solution, was the first commercially available topical CAI [4, 5]. DZA (Fig. 1) is a potent and selective inhibitor of carbonic anhydrase II (CA II), an enzyme that is present in the ciliary epithelium where it plays an important role in the formation of aqueous humor (AH) [1-3]. The inhibition of CA II by DZA that presumably takes place by slowing the formation of bicarbonate ions with subsequent reduction in sodium and fluid transport, decreases AH production and lowers IOP in patients with glaucoma or ocular hypertension. Various drug delivery systems, such as nanoparticles [6, 7], nanoemulsions [8] and in situ gelling system [9], have been investigated by many researchers in an effort to overcome poor bioavailability of DZA after topical application. *Address correspondence to this author at the Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi 110062, India; Tel: +919911362540; Fax: +91-11-26059663; E-mail: mhwarsi@gmail.com S S O O H 3 C H NH H 3 C H S O O NH 2 Fig. (1). Structure of dorzolamide. Potential benefits of these novel formulations include fewer drops per day and prolonged pre-corneal retention. Conse- quently, to evaluate the efficacy of novel formulations of DZA, it is highly desirable to have a fast and reliable bio- analytical assay for the determination of DZA in the ocular matrix. Quantification of DZA in biological fluid by chroma- tographic procedures like mass spectrometry [10] and HPLC [11] have been reported previously. As for DZA concentra- tion in aqueous humor the assay described in the literature was based on HPLC [11]. In the last years, ultra performance liquid chromatography (UPLC) has become an alternative and powerful technique for quantitative biological analyses [12-15]. UPLC is a new separation technique based upon well recognized principles of liquid chromatography, which utilizes <2μm particles for stationary phase. These particles operate at elevated mobile phase linear velocities to affect a dramatic increase in resolution, sensitivity and speed of analysis [12]. Because of its speed and sensitivity, this tech- nique is gaining a considerable attention in recent years for