*2.2. Drug Analysis*

The amount of moxifloxacin was determined by the high-performance liquid chromatography (HPLC) method described in the literature [10]. A HPLC system (1525, Waters, Milford, MA, USA) consisting of a Discovery C18 column (250 mm × 4.6 mm, i.d, 5 μm) was used. Chromatographic separation was carried out using a mixture of methanol: acetonitrile: water (85:5:10, *v*/*v*/*v*), pH 2.75 adjusted with phosphoric acid was delivered at a flow rate of 1 mL min−<sup>1</sup> with an injection volume of 25 μL. The isocratic elution was done at 25 ◦C and monitored using UV detector at 290 nm.

### *2.3. Development of Pseudoternary Phase Diagram*

The pseudoternary phase diagram of four component mixtures of oil (ethyl oleate), surfactant (Tween 80), cosurfactant (Soluphor P) and water was constructed by titration technique to obtain concentration ranges that can result in large existence with the nanoemulsion region at room temperature [11]. A titration technique was employed for the preparation of the pseudoternary phase diagrams [12]. Briefly, Tween 80 was blended with Soluphor P in a fixed weight ratio of 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4 and 1:5, respectively. Aliquots of each Tween 80 and cosurfactant mixture (*S*mix) were then mixed with oil phase at room temperature (25 ◦C). The ratio of oil to *S*mix was varied as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 7:3, 2:8 and 1:9. Then, water was added to the above mixture in 5% increments and checked for formation of emulsion or liquid crystal or gel. The phases were identified using visual inspection, microscopic inspection, and measurement of droplets size. The resulting nanoemulsion was tightly sealed and stored at ambient temperature. The points corresponding to mixture components that resulted in nanoemulsion were noted and marked in a phase diagram region. The resulted phase diagrams of different surfactant: Cosurfactant ratios were compared and the ratio that could result in the large existence area of nanoemulsion domain in phase diagram was selected for further optimization.

### *2.4. Preparation of Moxifloxacin Nanoemulsion*

Moxifloxacin nanoemulsions (o/w) were formulated by appropriate quantity of deionized water, ethyl oleate, Tween 80 and Soluphor P. Required amount of moxifloxacin (0.5%, *w*/*v*) was dissolved in ethyl oleate. To *S*mix, an adequate amount of ethyl oleate was added and mixed. Then the mixture was titrated by gradual addition and mixing of distilled water in order to achieve the equilibrium immediately. Further, it was thoroughly stirred and vortexed to obtain nanoemulsion. To maintain sterility of the prepared formulation, benzalkonium chloride (0.005%, *w*/*w*) was used as a preservative and stored in multi-dose containers [13].
