Chemometric Access for RP-HPLC Simultaneous Estimation of Tadalafil and Dapoxetine Applying Response Surface Methodology

Aims: A RP-HPLC method was developed and validated for simultaneous estimation of Tadalafil and Dapoxetine applying statistical experimental design. Methodology: Multivariate optimization of the experimental conditions of RP-HPLC method was using Design of experiments. Independent three factors like phosphate buffer pH, mobile phase composition and flow rate were applied to design mathematical models. To study the response surface methodology by using Central composite design (CCD). In depth the effects of these independent factors was studied using CCD. Simultaneously optimize the retention time and resolution of the analytes was applying Desirability function. Results: The predicted and optimized data from contour picture containing phosphate buffer (pH 3.4) and acetonitrile in the ratio of 40:60%v/v respectively. Flow rate was found to be 0.8 ml/min. Baseline separation of both analytes with run time of less than 10.0 min and good resolution were achieved using these optimum conditions. Original Research Article Jayaseelan et al.; JPRI, 33(43B): 278-288, 2021; Article no.JPRI.73369 279 Conclusion: Method was validated according to ICH guidelines by using optimized assay conditions. Therefore, the reports distinctly indicated that Quality by design access could be satisfactorily used to optimize RP-HPLC method for simultaneous estimation of Tadalafil and Dapoxetine.


Chemicals and Reagents
Pure Active pharmaceutical ingredients of Dimenhydrinate and Cinnarizine were obtained as a gift samples from Nebulae Hi-Tech Laboratories, Chennai, Tamilnadu, India. Combination tablet of uphold was procured from the local market. HPLC grade methanol, Acetonitrile, water and ammonium acetate were purchased from Merck Chemicals India Pvt. Limited, Mumbai, India.

Instrumentation and Chromatographic Conditions
Analysis was performed with a Shimadzu LC2010 CHT separation module equipped with LC solution software, Pump LC2010 binary and UV detector set at 240 nm. Compounds were separated on an Intek chromasol column (250 × 4.6 mm i.d., 5μm particle size) under reversed phase partition conditions. The mobile phase was Acetonitrile and ammonium acetate buffer. The flow rate was 1.0 ml/min and the run time was set as 12 minutes. Samples were injected by using Rheodyne injector with 10 μL loop and detection was carried out at 290 nm. Prior to analysis mobile phase were degassed by the use of a sonicator (Ultrasonic Cleaner, Power Sonic 420) and filtered through a 0.45μ nylon filter. Chromatography was carrying out in column temperature maintained at 30 ± 5˚C.

Preparation of Working Standard Stock Solution
About 20.2 mg of Tadalafil and 67.3 mg of Dapoxetine were weighed accurately and transferred into a 50 ml volumetric flask. 10 ml of the mobile phase was added and sonicated for 15 min. and the volume was made up to 50 ml with the mobile phase. From this, pipette out 4.5 ml of the solution and transferred into 50 ml volumetric flask. Then it was made up to the volume with mobile phase to get a concentration of 36.36 µg/ml for Tadalafil and 121.14 µg/ml for Dapoxetine.

Preparation of Sample Solution
Ten tablets were weighed and powdered (Uphold tablets containing Tadalafil 10 mg and Dapoxetine 30 mg). The tablet powder equivalent to 67.3 mg was weighed accurately and transferred into 50 ml volumetric flask. 10 ml of the mobile phase was added and sonicated for 15 minutes and the volume was made up to 50ml with the mobile phase. 4.5 ml of the above solution was pipetted out and transferred into a 50 ml standard flask and made up to the volume with the same. Finally, it was filtered through a 0.45µ membrane filter. Hence, the final concentrations were attaining 36.36 µg/ml for Tadalafil & 121.14 µg/ml for Dapoxetine.

Method Validation
The developed RP-HPLC method was validated by using ICH guidelines [22][23] for validation of analytical procedure to determine the linearity, LOD, LOQ, Precision, accuracy, Robustness and Ruggedness.

System suitability
System suitability tests are an integral part of any chromatographic analysis method which is used to verify reproducibility of the chromatographic system. To ascertain its effectiveness, certain system suitability test parameters were checked by repeatedly injecting the drug solution at the concentrations of 36 µg /ml and 121 µg / ml respectively for Tadalafil and Dapoxetine to check the reproducibility of the system. 20 µl standard solutions were injected.

Linearity
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. The linearity study was conducted for standard stock solutions of Tadalafil and Dapoxetine. For the construction of calibration curves, five calibration standard solutions were prepared over the concentration range of 32-48 µg / ml for Tadalafil and 107-161 µg / ml for Dapoxetine.

Limit of detection
The limit of detection is the lowest level of analyte that can be detected, but not necessarily determined in a quantitative fashion, by using a specific method under the required experimental conditions. The lowest detection limit was calculated by using the following formula. LOQ = 3.3 x std. dev / slope. Preparation of calibration curve from the serial dilutions of standard was repeated for three times. Limit of quantification was calculated by using the value of the slope and the standard deviation of intercept.

Limit of quantification
The limit of quantification is the lowest concentration of analyte in a sample that may be determined with acceptable accuracy and precision when the required procedure is applied. The lowest detection limit was calculated by using the following formula. LOQ = 10 x std.dev / slope.

Content estimation (assay)
About 36 µg / ml of Tadalafil and 121 µg / ml of Dapoxetine standard and sample (Uphold tablets containing Tadalafil 10 mg and Dapoxetine 30 mg) solutions were prepared separately and 20 µl of each standard and sample solution were injected The percentage purity was calculated by using the peak area.

Precision
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samples of the same homogeneous sample under prescribed conditions. Precision study was conducted by injecting standard solutions of Tadalafil and Dapoxetine five times at a concentration of 36 µg / ml and 121 µg / ml respectively. The peak area was used to determine standard deviation.

Accuracy
The accuracy of an analytical method may be defined as the closeness of the test results obtained by the method to the true value. It is the measure of the exactness of the analytical method developed. Accuracy may often express as percent recovery by the assay of a known amount of analyte added. The accuracy of the method was checked by spiking the sample with reference compound. It was evaluated in triplicate at the concentration levels (80%, 100% & 120%) of the target test concentrations (36 µg / ml of Tadalafil and 121 µg / ml of Dapoxtine). 20 µl solutions of each concentration were injected and the chromatograms were recorded.

Robustness
The Robustness study indicated that the factors selected remained unaffected by small variation of flow rate and the organic composition of mobile phase. The condition studied were flow rate (± 0.2 ml/min) and composition of mobile phase (± 3%).

Ruggedness
Ruggedness is a measure of reproducibility of test results under normal, expected operational conditions from laboratory to laboratory and from analyst to analyst.

RESULTS AND DISCUSSION
HPLC method of analysis of Tadalafil and Dapoxetine was carried out by means of a central composite design. CCD was selecting ample to its pliability. Optimize an HPLC partition by acquiring improved interpretation of the main factors by using CCD and their interconnection results. Previous theory from the article used to chosen the key factors. Acetonitrile concentration (A) (50-60%), Phosphate buffer pH (B) (3.0-3.4) and flow rate (C) (0.8-.1.2 ml/min) were the factors chosen for optimization. The capacity factor for 1 st peak i.e. Tadalafil (k1), the resolution between two pairs Tadalafil and Dapoxetine (Rs1, 2) and the retention time of last peak i.e. Dapoxetine (tR2), were chosen for response variables Table 1. Data was showed the ranges of the factors and response [2]. The effects of uncontrolled variables that may introduce a bias on the measurements randomized order to minimize all experiments were conducted. The estimation of experimental error replicates (n=6) of the central points were performed.
Linear, quadratic and cross terms the model can be expressed as the following Y = β0 + β1 X1 + β2 X2 + β3 X3 + β12 X1 X2 + β13 X1 X3 + β23 X2 X3+ β11 X1 2 + β22 X2 2 + β33 X3 2 with three factors an experimental design where Y is the response to be modeled, β is the regression coefficients and X1, X2 and X3 represents factors A, B and C respectively. ANOVA provides statistical parameters for the reduced models datas were shown in table 2. The pointless terms (p > 0.05) were removed from the model through reverse removing process to procure an easy and realistic model. Considering R 2 always reduces when a regressor variable is removed from a regression model. In statistical modeling the adjusted R 2 which precedes the number of regressor variables into report, is usually chosen [24]. Adjusted R 2 values were found to be within the standard limits of R 2 ≥ 0.81 [25], which declared that the experimental data indicated a fine suited with the second order polynomial equations. p value was procured > 0.05 for all the reduced models, suggested that these models were significant. The signal (response) to noise (deviation) ratio is given adequate precision value. A ratio more than 4 is advisable [26]. In the present study, the adequate precision value was found to be in the range of 9.696 -13.494 which suggested an adequate signal. Hence the model was significant for the partition procedure. Measurement of reproducibility of the model is denoted as coefficient of variation (% C.V). As a common rule a model can be examined reasonably reproducible the % C.V value is less than 10%. Our study, the % C.V value was found to be for all models within the limit (1.01 -5.87). The interconnection with the biggest perfect coefficients among the suited model was AC (+0.922AC) of tR2 model shown in Table 2 [27]. The interconnection between A and C positive sign was statistically significant (< 0.0001) for tR2.
The present study revealed that changing the concentration of acetonitrile from low to high reported in a fast decrease in the retention time Dapoxetine and big levels of flow rate. In addition at small level of factor A, an raise in the flow rate resulted in a decline in retention time. Hence, when the acetonitrile concentration was set at the smallest level, the flow rate had to be at its biggest level to reduce the run time. So that obtain good interpretation of the reports, the predicted models were represented in the form of perturbation plots 2 (Fig. a, b and c) and 3D response surface plots 3 (Figs. a, b and c). Variables giving quadratic and interconnection terms with the biggest perfect coefficients in the suited models were selected for the axes of the response surface plots.
Perturbation plot produce silhouette scene of the response surface plots where it appears how the response replace as each factor moves from a selected reference point, with all factors held constant at the reference value. The steepest slope or inflection showed the sensitiveness of the response to a specific factor. Acetonitrile concentration (factor A) had the most important effect on retention time tR2 followed by factor C and then B was shown in Fig. 2c. The remaining factors (buffer concentration and flow rate) had important effect on Rs1, 2 and k1. In (Fig. 3a, 3b) k1 and Rs1,2 values incremented as the level of buffer concentration incremented and k1, Rs2,3 values declined as the level of flow rate incremented. Analysis of the perturbation plots and response plots of optimization models revealed that factor A and C had important effect on separation of anlytes, whereas the factor B i.e. the buffer pH was of little important.

Multi Criteria Decision Making
Global optimization of three responses and select different optimal conditions for the analysis of samples were using by Derringer's desirability function.

Optimal Condition for Assay
To substantiate the flexibility of the development strategy and to search for evaluating analytes, standard was accepted by varying the response objective and their major values (Table-4). For illustration, the high value of Rt2 had to be chosen for the partition of Dapoxetine from the components. The desirability function was reduced at overall desirability of about D= 0.921. The global desirability function response surface was shown in Fig. 4. Each individual response goal was seen in Table 4.
The correlative producing the high value were acetonitrile concentration 60%v/v, Phosphate buffer pH 3.4 and flow rate 0.8 ml/min. Hence, C18 column with Phosphate buffer (pH 3.4): acetonitrile 40: 60%v/v as mobile phase at a flow rate of 0.8 ml/min and UV detection at 288 nm using were the optimal assay condition. Between experimental and predicted responses agreement report was shown in Table 4. Fig. 5 was shown the corresponding chromatogram.

Validation Report of the Methods
The system suitability parameters results were within the limit. Linearity report showed good correlation between analytes peak area and concentration with r 2 > 0.9998 (n = 6). 0.0097 µg /ml and 0.0071 µg /ml for Tadalafil and Dapoxetine respectively for LOD value were found. 0.0294 µg /ml and 0.0215 µg /ml for Tadalafil and Dapoxetine respectively for LOQ values were found. Determination of purity of Tadalafil and Dapxetine in tablet formulation was performed by assay (content determination) From the calibration curve nominal concentration was choosen and quantification of Tadalafil and Dapoxetine were performed. The tablet formulation Uphold contains (10 mg of Tadalafil and 30mg of Dapoxetine) was selected for the analysis. The % of drugs present in the tablet dosage form were found in the range of 99.84 -100.72%. The assay percentage RSD values were found 0.9679 and 1.2999 for Tadalafil and Dapoxetine.

CONCLUSION
An easy and fast RP-HPLC method was developed and validated prosperosuly for the simultaneous estimation of tadalafil and dapoxetine. Design of Experiments and Central Composite Design were used successfully for the estimation. Statistical analysis reported that the model represents the circumstance completely great. The responses variations were correctly interact to the factors variations. This method can be validated for linearity, precision, accuracy and selectivity as per ICH guidelines. The method has verified to be suitable and successful for the quality control of tadalafil and dapoxetine in pharmaceutical dosage form.

DISCLAIMER
The company name used for this research is commonly and predominantly selected in our area of research and country. There is absolutely no conflict of interest between the authors and company because we do not intend to use this company as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.