## 3 Results and Discussion
### Development of MW-FLR Method
#### 3.2.2 Optimization of Method Variables  
The effect of solvent on the fluorescence of DUV was studied. These solvents were water, acetonitrile, dioxane, diethyl ether, chloroform, dichloromethane, dichloroethane, isopropanol, ethanol, and methanol. The highest and comparable fluorescence intensity was achieved with polar solvents (water, acetonitrile, isopropanol, ethanol, and methanol); however, lower fluorescence intensity was achieved with less/non-polar solvents (dioxane, diethyl ether, chloroform, dichloromethane, and dichloromethane); Figure 2. The varying effects of the solvents on the DUV's fluorescence intensity were related to the varied potencies of the interactions between the solvents and the DUV. Furthermore, DUV molecules often have non-symmetric dipoles and exhibit varying rates of relaxation in various solvents. Furthermore, the intermolecular arrangement of the fluorophores in the excited state, and possible formation of hydrogen bonding may cause temporal effects on the fluorescence spectra of DUV [30]. Among the better solvents, water was selected as a solvent in all subsequent experiments because it is inexpensive and more importantly it is environmentally and health safety, which imposes a green approach property to the method. In fluorimetric analysis, it has been documented that adding an organized surfactant or macromolecule to the drug solution may increase its fluorescence intensity and accordingly enhance the method sensitivity [28]. Therefore, different surfactants of different categories and macromolecules were tested in the present study. These surfactants were anionic (sodium dodecyl sulphate: SDS), cationic (dodecyl dimethyl ammonium bromide: DDAB), non-ionic (cremophor-RH 40, cremophor-EL and tween-20) and macromolecule (carboxymethyl cellulose: CMC). The results (Figure 3) revealed that none of these media had an enhancement effect on the aqueous medium of DUV. Accordingly, adding an organized medium was ruled out from all the subsequent experiments. Figure 2: Effect of solvent on the fluorescence intensity of DUV solution (200 ng/mL in methanol). Values are mean of 3 determinations. The effect of pH on the fluorescence intensity of DUV was studied by adding Britton-Robinson buffer solution of different pH values (pH 2-12) to the solution and subsequently measuring the fluorescence. The results (Figure 4) indicated that the highly acidic (pH 2-3) and highly alkaline (pH 10-12) solutions had a quenching effect on the fluorescence intensity of the DUV solution. These effects were attributed to either protonation of the nitrogen atoms of the fluorophore moiety in the DUV structure (Figure 1) at high acidic pH values or a possible degradative effect on the entire structure at high alkaline pH values. The fluorescence quenching effect due to the protonation of nitrogen atoms in acidic pH was due to the delocalization of electrons on the fluorophore and accordingly decrease their liability for excitation by the exciting light wavelength. Besides, the documented effect of pH on the inter- and intra-molecular interactions [31]. The highest fluorescence intensity was achieved at pH values in the range of 4-9. The achieved fluorescence intensities using a buffer solution (pH 4-9) were almost comparable to that achieved using a water DUV solution without buffer. Accordingly, all the subsequent experiments were carried out without adding a buffer solution to the DUV solution. This added advantages to the procedures such as less cost, eco-friendly, and health safe. Figure 4: Effect of pH on the fluorescence intensity of DUV solution (200 ng/mL in methanol). Values are mean of 3 determinations. Figure 3: Effect of type of surfactant on the fluorescence intensity of DUV solution (200 ng/mL in methanol). Values are mean of 3 determinations. Effect of Sample Matrix  
To assess the applicability of the proposed MW-FLR method for the accurate determination of DUV in both capsules and plasma samples, the effect of matrices on the accuracy of the method was studied.