3.1.1. Method I
Different factors that influence the fluorescence of DPP were studied, e.g., type and concentration of organized media, pH, time, temperature, and type of diluting solvent.
a. Effect of organized media
The effect of various organized media including micellar solutions such as: CTAB (cationic surfactant), SDS (anionic surfactant) and tween-80 (nonionic surfactant) and different macromolecules, e.g. HP β-CD, a-cyclodextrin (α-CD), carboxy methyl cellulose (CMC) on the relative fluorescence intensity (RFI) of DPP were tested. SDS gave a marked increase in the RFI286 nm of DPP (Fig. 3-a). The reason of this behavior may be due to the electrostatic attraction between the negatively charged anionic surfactant, SDS, and the positively charged DPP molecules leading to decrease the repulsion between the surfactant molecule head groups and completion of the micellization process as shown in (Fig. 4). The fluorescence enhancement caused by HP β-CD was also large (but still smaller than SDS). This finding suggested that addition of HP β-CD to the micellar mobile phase (method II) may lead to increase the sensitivity of the method for DPP determination.
The effect of SDS concentration was tested through using different volumes of 1% SDS in the range of 0.25 to 2.5 mL. It was observed that increasing volume of SDS resulted in an increase in RFI286 up to 1 mL, after which RFI286 was nearly constant. Therefore, 1 mL of 1% SDS solution was chosen as the optimum volume for determination of DPP (Fig. 3-b).
b. Effect of pH, type, and concentration of buffer
The effect of pH on the enhanced fluorescence of DPP was studied using Teorell and Stenhagen buffer covering the pH range from 2 to 10 (Fig. 3-c). It was found that fluorescence enhancement usually achieved at acidic pH values (pKa of DPP = 8.87) where the drug presents in the ionized cationic form. Buffer with pH 5 gave the maximum RFI286 nm. The electrostatic attraction between the negatively charged SDS and the positively charged DPP molecules will produce an additional limitation of the DPP molecules movement and increase its rigidity leading to a great enhancement in the fluorescence intensity.
Different buffers including acetate buffer, citrate buffer, and Teorell and Stenhagen buffer at pH 5 were tried to evaluate the effect of buffer type. It was found that maximum and constant RFI was achieved by using Teorell and Stenhagen buffer (pH 5) (Fig. 3-d). The influence of Teorell and Stenhagen buffer volume on the RFI286 nm of DPP was also studied (Fig. 3-e). It was observed that increasing volumes of buffer solution resulted in an increase in RFI286 nm up to 1 mL, after that a decrease in RFI286 nm was observed. Therefore, 1 mL of Teorell and Stenhagen buffer pH 5 was chosen as the optimum for determination of DPP.
c. Effects of time and temperature
The effect of time on the fluorescence of DPP was determined by monitoring RFI286 nm for 60 minutes. It was observed that the enhanced fluorescence is developed immediately and is stable throughout the time of the study.
The influence of temperature was also assessed by varying reaction temperature in the range 10 to 60°C. It was observed that increasing the temperature had led to a marked decrease in RFI286 nm. This may be attributed to increasing the possibility of the internal/external conversion to occur, so allowing non-radiative deactivation of the excited singlet state to occur easily and an increase in the loss of energy through collision with other unexcited drug or matrix molecules, and so, fluorescence quenching [61]. Lower temperatures (< 25oC) increased the RFI286 nm but its application is difficult. Therefore, all experiments were carried out at room temperature (25°C).
d. Diluting solvent effect
The influence of different diluting solvents (ethanol, methanol, acetonitrile, and water) on the RFI286 nm of DPP was also investigated. The results revealed that the best solvent for dilution was water, as it provided the highest RFI286 nm and the lowest blank reading. Obvious decrease in RFI286 nm was observed when dilution was made by other organic solvents. The primary reason for this effect could be attributed to the denaturizing effect of organic solvents on the formed micelle as they dissolve in water and change their properties leading to decreasing micelles formation. In addition, organic solvents may also result in a decrease in micellar size [68].
e. Order of Addition
Different addition orders were tried and RFI286 nm were measured. The following order (drug→ buffer → SDS) achieved the maximum fluorescence intensity as the drug must be positively charged by the buffer to facilitate binding to micelles' surface and then reaching the core (Fig. 3-f).
3.1.2. Method II
Different factors were optimized regarding the system suitability parameters. The optimum condition gave shorter retention times (tR) with best resolution and tailing factor while number of theoretical plates, capacity factor and selectivity factor were usually acceptable.
a. Effect of type and concentration of surfactant
Different type of surfactants were tried. Surfactants are either used alone in the mobile phase or in combinaion (SDS, tween 20, Brij*35 and mixture of SDS and Brij*35). SDS can separate CFF and PAR while DPP was retained on the column and cannot be eluted by SDS. Tween 20 can separate the 3 drugs but runtime was very long. Only Brij*35 can separate the 3 drugs within a suitable runtime (Fig. 5). Different concentrations of Brij*35 in the range of 20 to 40 mM were tried. 30 mM Brij*35 was chosen as the optimum concentration with respect to retention time and tailing factor as shown (Fig. 6).
b. Effect of pH and type of buffer
Micellar mobile phases containing TEA/phosphoric acid with different pH values were tried. Only acidic mobile phases in the range of pH 2.5-5 showed short retention times and more symmetric peaks. Mobile phases with pH values < 4 were excluded due to bad resolution (Rs < 1.5). Mobile phase with pH 4 was chosen as optimum for separation of the studied drugs regarding tR and T (Fig. 7). Different buffers including TEA/phosphoric, citrate, acetate, and phosphate at pH 4 were tried. Phosphate buffer was chosen as optimum buffer with respect to the retention time and tailing factor (Fig. 8).
c. Effect of concentration of hydroxypropyl β-cyclodextrin
Different concentrations of HP β-CD (0.1 to 5.0 mM) in the mobile phase were tried and peak area of DPP using fluorescence detector were recorded. 0.5 mM HP β-CD was chosen as the optimum concentration that achieved the maximum degree of enhancement of DPP fluorescence, so enabling its determination in the dosage form simultaneously with PAR and CFF (Fig. 9).
d. Effect of type and concentration of organic modifier in the mobile phase
Different types of organic modifier were tried (methanol, ethanol, propanol, butanol, and acetonitrile). MeOH was chosen as the best organic solvent with respect to the retention time and tailing factor (Fig. 10). Moreover different ratios of methanol (MeOH) in the mobile phase (5–25%) were tried. Retention time, tailing factor and resolution values were compared. Although increasing the percent of MeOH showed improvements in retention time and tailing factor especially for CFF and PAR, concentrations higher than 10% had bad impact on resolution. MeOH concentration of 5% and 10% shows nearly similar results regarding tR, T and Rs, however 5% MeOH was chosen as the best to decrease the percent of organic modifier consumed by the method and so increasing the greenness of the method (Fig. 11).
e. Effect of temperature
Different column temperatures were tried in the range of 25 to 40°C. Column temperature of 40°C was chosen as optimum as it decreased the back pressure of the mobile phase and prevent the precipitation of the mobile phase into the system.
f. Effect of flow rate
Flow rate was increased gradually to decrease run time, enhance peak shape with maintaining acceptable resolution reaching 0.8 mL. min− 1after which the back pressure was very high.
The following experimental parameters were set as the optimum: [HP β-CD] = 0.5mM, [Brij*35] = 30 mM, 5%MeOH and 95% phosphate buffer pH 4. The optimum conditions were tried on a ternary mixture containing 16 µg. mL− 1 CFF and 300 µg. mL− 1 PAR and 2 µg. mL− 1 DPP (Fig. 12), which is the same ratio of drugs in their tablet, and results for system suitability tests were calculated as shown in Table 1.
Table 1: System suitability parameters for the simultaneous determination of CFF, PAR and DPP (method II)
Parameter | CFF | PAR | DPP | Reference value [71] |
Retention time, tR (min) | 2.323 ± 0.05 | 4.21 ± 0.02 | 5.34 ± 0.06 | - |
Tailing factor, T | 1.6 | 1.62 | 1.7 | ≤ 2 |
Capacity factor, k′ | 3.65 | 7.42 | 9.67 | ˃ 2 |
Selectivity factor, α | 1.81 | 1.81 | 1.24 | ˃ 1 |
Theoretical plates, N | 2370 | 2264 | 2120 | ≥ 2000 |
HETP, mm | 0.633 | 0.663 | 0.708 | - |
Resolution, Rs | 6.53 | 6.53 | 2.13 | ˃ 2 |