Validated RP - HPLC Method for Simultaneous Estimation of Tadalafil and Dapoxetine in Combined Pharmaceutical Dosage Forms

Aim: To develop a new, simple, fast, rapid, accurate, efficient and reproducible RP-HPLC method for the simultaneous estimation of Tadalafil and Dapoxetine. Background: Estimation of Tadalafil and Dapoxetine is a combination drug of choice used to treat preamature ejaculation in men. Objective: The main objective of the Simultaneous estimation of combined drug is to establish identity, physical characteristics and potency of the drugs and to demonstrate the suitability of the assay method to provide useful data to ensure the technique gives satisfactory and consistent results. Materials and methods: A HPLC (Inertsil, Water2695) with UV/VIS Detector/PDA detector, UV (lab India, UV 3000 + series) and Inertsil C18 250mm × 4.6mm × 5µm column was used. A new method was established for simultaneous estimation of Dapoxetine and Tadalafil by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Dapoxetine and Tadalafil by using inertsil C18 5µm (4.6*250mm) column, flow rate was 1ml/min, mobile phase ratio was Phosphate buffer (0.05M) pH 3: MEOH (30:70%v/v) (pH was adjusted with orthophosphoric acid), detection wave length was 260nm. Results: The results were in good agreement with those obtained with official HPLC with absorption maximum of 260 nm by preparing mobile phase 70:30 methanol: phosphate buffer with flow rate 1 ml/min and it run for 30 minutes by selecting column Inertsil C18 4.6mm×250 mm. All the results obtained with good precise, accurate and robustness as per international conference on Harmonization (ICH) guidelines. Conclusion: It can be concluded that the proposed RP-HPLC method is accurate, precise, sensitive, robust and reproducible for the simultaneous analysis of Tadalafil and Dapoxetine with less tailing factor and is also economical.Inertsil C18 column (4.6×250mm)5µ, flow rate was 1ml/min. Both samples scan in the range of 200 to 400 nm and maximum wavelength was identified at 260 nm.


INTRODUCTION
Tadalafil is a phosphodiesterase type 5 (PDE 5) inhibitor which works by increasing blood flow to the penis during sexual stimulation and enables its erection following sexual stimulation.[3] Chemicals and Reagents: Tadalafil and Dapoxetine HCL drug was obtained from MSN Laboratories, Hydrabad.We used HPLC grade acetonitrile, water and GR grade KH2PO4 and ortho phosphoric acid . 4 A HPLC (Inertsil, Water2695) with UV/VIS Detector/PDA detector, UV (lab India, UV 3000 + series) and Inertsil C18 250mm × 4.6mm × 5µm column was used.The HPLC system was equipped with Empower software for data processing. 6

Chromatographic Condition:
The mobile phase includes mobile phase 70:30 methanol: phosphate buffer was found to resolve Tadalafil and Dapoxetine.Ortho phosphoric acid was pre-owned for pH adjustment of buffer to 6.8.The mobile phase was strained through 0.45 nylon filter and then ultrasonicated for 30 min.The flow rate was set to 1.0ml/min. Preparation of Mobile Phase: Mix a mixture of above methanol 700 ml (70%) and 300 ml of buffer (HPLC grade-70%) and degassed in ultrasonic water bath for 5 minutes.Filter through 0.45 µ filter under vacuum filtration. 9 Preparation of Sample solution: Accurately weigh and transfer 10 mg of Dapoxetine and 10mg Tadalafil Tablet powder into a 10mL clean dry volumetric flask add about 7mL of Diluent and sonicate to dissolve it completely and make volume up to the standard with the same solvent.[12]

RESULTS AND DISCUSSION Method Development
The identification wavelength was adopted by dissolving the drug in mobile phase to get a concentration of 10µg/ml for individual and mixed standards.The resulting solution was scanned in Ultraviolet (U.V) range from 200-400nm.The overlay spectrum of Tadalafil and Dapoxetine was obtained and the isobestic point of Tadalafil and Dapoxetine showed absorbance's maxima at 260 nm.Chromatographic method development was optimized by various parameters both in Active Pharmaceutical Ingredient (API) and pharmaceutical dosage form in Figure 1.
The Optimized Chromatographic conditions by preparing mobile phase 70:30 methanol: phosphate buffer with flow rate 1 ml/min and it run for 10 minutes by selecting column Inertsil C18 4.5×250 mm 5.0 µm of ambient temperature.The retention time of Tadalafil and Dapoxetine was found to be 2.5 mins and 3.9 mins respectively was shown in figure no 2.

VALIDATION REPORT Linearity
The linearity study was performed for the concentration of 100 ppm to 500 ppm for Tadalafil and 1ppm to 5ppm for Dapoxetine was shown in Table no 1 Accuracy The accuracy study was performed for 50%, 100% and 150 % for Tadalafil and Dapoxetine.The percentage % retrieval was found to be 99.84% and 100.51% was shown in Table no 2 and 3.

Precision (Repeatability)
The precision evaluation was performed for five injections of Tadalafil and Dapoxetine.Each standard injection was injected into chromatographic system.Theintermediate precision study was performed for five injections was shown in Table no 4.

Intermediate Precision (Ruggedness)
The intermediate precision evaluation was performed for five injections of Tadalafil and Dapoxetine.Each standard injection was injected into chromatographic system.Theintermediate precision study was performed for five injections was shown in Table no 5.

LOD and LOQ
The LOD was performed for Tadalafil and Dapoxetine was estimated to be 2.9 and 3 respectively.The LOQ was performed for Tadalafil and Dapoxetine was estimated to be 10.03 and 10.1 respectively was shown in Fig No 3.

Robustness
The robustness was performed for the flow rate variations from 0.6ml/min to 1.2ml/min and mobile phase ratio variation from more organic phase to less organic phase ratio for Tadalafil and Dapoxetine which can be resulted that the variation in flow rate affected    3. The LOD was performed for Tadalafil and Dapoxetine was estimated to be 2.9 and 3 respectively and LOQ was found to be 10.03 and 10.1 respectively.

CONCLUSION
The developed analytical method was validated as per ICH guidelines and it meets the acceptance criteria of each parameter.It is concluded that the developed method is simple, linear, precise, accurate, robust and sensitive to analyze the simultaneous analysis of Tadalafil and Dapoxetine with less tailing factor and is also economical.Inertsil C18 250mm × 4.6mm × 5µm, flow rate was 1ml/min.Both samples scan in the range of 200 to 400 nm and maximum wavelength was identified at 260 nm.The main advantage of developed UV method over HPLC method is that it is less time consuming and also economical.

Fig No 1 .Fig No 2 .
Fig No 1.Spectrum showing overlapping spectrum of Tadalafil and Dapoxetine

Table no
1. Linearity Results for Tadalafil and Dapoxetine S.

Table no 2
. Accuracy Results for Tadalafil

Table no 4 .Results of Precision for Tadalafil and Dapoxetine injection Area Tadalafil Area Dapoxetine
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