Tadalafil （TAD）（Fig.1）is （6R，12aS）-6-（1，3-benzodioxol-5-yl）-2，3，6，7，12，12a-hexahydro-2-methylpyrazino［1′，2′∶1，6］pyrido［3，4-b］indole-1，4-dione［1］. It is a selective long-acting phosphodiesterase type 5 （PDE5）inhibitor ［2，3］，safe and effective in treatment of erectile dysfunction［4-7］. Dapoxetine HCl （DAP）（Fig.1）is a short-acting selective serotonin reuptake inhibitor developed for the treatment of premature ejaculation ［8，9］. DAP has a unique pharmacokinetic profile as it is rapidly absorbed and eliminated after oral administration［10-13］and it does not have any pharmacokinetic interaction with PDE5inhibitors. Thus it was recently co-formulated with several PDE5inhibitors ［14］. As being recently co-formulated （TAD and DAP），the literature in hand shows only three HPLC methods with UV detection for their determination in dosage form［15-17］.

Fig.1 Molecular structures of tadalafil （TAD）and dapoxetine HCl （DAP）

Both drugs are natively fluorescent［18，19］which encourages us to develop an HPLC method coupled with fluorescence detection to provide more sensitivity over the reported HPLC-UV methods. Furthermore，the higher sensitivity of the proposed method allows the determination of both drugs in human plasma. Therefore，the aim of the present work is to develop a new，simple and sensitive HPLC method with fluorescence detection for the separation and quantitation of TAD and DAP in dosage form as well as spiked human plasma using avanafil which is a new PDE5inhibitor as an internal standard （IS）. Besides，the method was validated according to the International Conference on Harmonization （ICH）guidelines［20］.

1 Experimental

1.1 Chemicals and materials

The standards of TAD，DAP and IS were kindly supplied by El Andalus Medical Company （6th of October City，Giza，Egypt）. Super Tadarise?tablets are produced by Sunrise Remedies Pvt. Ltd.（India），and labeled to contain 20 mg of TAD and 60 mg of DAP per tablet. Other chemicals included potassium dihydrogen phosphate （Adwic，Egypt），o-phosphoric acid（Adwic，Egypt），methanol （HPLC Grade，Sigma-Aldrich，Germany），acetonitrile （HPLC Grade，Germany），triethylamine （HPLC Grade，Sigma-Aldrich，Germany）.

1.2 Standard solutions

TAD，DAP and IS stock standard solutions（100 μg／mL）were prepared. They were separately and accurately prepared by weighing 10.0 mg of each drug into 100-mL volumetric flask，dissolved and filled to volume with methanol.

1.3 HPLC conditions

Samples were loaded into a manual Rheodyne injector （model G1328B，USA）equipped with a 20-μL injector loop using a 100-μL Agilent syringe. HPLC separation and quantitation were made on Eclipse C18column （150 mm×4.6 mm，5 μm）with a mobile phase consisting of acetonitrile-0.15% triethylamine （40 ∶60，v／v）. The pH was adjusted to 4 using o-phosphoric acid. The mobile phase was filtered using 0.45 μm membrane filters （Millipore，USA）and degassed by ultrasonic vibration for 15 min prior to use. An isocratic pump model G1310A was used to deliver the mobile phase at a flow rate of 1.0 mL／min.The fluorescence detector model G1321A was set at different excitation／emission wavelengths using time programming. At 0 min，the fluorescence detection was set at 236／370 for IS；at 4.3 min it was changed to 236／410 for DAP；and finally at 5.5 min，it was set at 236／330 for TAD. Data acquisition was performed using Agilent LC Chem-Station software. All determinations were performed at room temperature.

1.4 Procedure

1.4.1 Construction of calibration curves

Different aliquots （0.001-3 mL）of each TAD and DAP stock standard solutions （100 μg／mL）were accurately transferred into two series of 10-mL volumetric flasks. An aliquot of 0.3 mL of IS stock standard solution （100 μg／mL）was added to each volumetric flask. The volumes were adjusted with the mobile phase to produce concentrations of 0.01-30 μg／mL of each drug along with 3 μg／mL of IS. An aliquot of 20 μL of each solution was injected triplicate into the chromatographic system. The previously described conditions were adopted.

1.4.2 Application to pharmaceutical formulations

Ten Super Tadarise?tablets （labeled to contain 20 mg TAD and 60 mg DAP per tablet）were accurately weighed and finely powdered. An accurate weight equivalent to 10 mg of TAD with 30 mg of DAP was transferred into a 100-mL volumetric flask，extracted with 50 mL methanol in an ultrasonic bath for 10 min and diluted to volume with the same solvent. The solution was sonicated again for 10 min in the ultrasonic bath and filtered. An aliquot （0.4 mL）was accurately transferred into a 10-mL volumetric flask and an aliquot （0.3 mL）of IS stock standard solution was added，then the volume was completed with the mobile phase to produce a solution of mass concentrations of 4 μg／mL TAD and 12 μg／mL DAP.Then，the procedure described under construction of calibration curves was followed，and the concentrations of both drugs were calculated from the corresponding regression equations.

1.4.3 Application to spiked human plasma

Aliquots of 0.9 mL human plasma were transferred into a series of centrifuge tubes and spiked with 0.1 mL of mixtures of TAD （0.5-20 μg／mL），DAP （0.1 - 20 μg／mL）and IS （5 μg／mL）so that the final mass concentrations of the drugs were 0.05-2 μg／mL and 0.01-2 μg／mL for TAD and DAP，respectively，and 0.5 μg／mL for IS. In addition，1 mL of acetonitrile was added as the precipitating agent for proteins and then centrifuged at 6 000 r／min for 30 min. The supernatant was then taken and filtered through 0.45 μm Millipore filter into a series of Eppendorf tubes. An aliquot of 20 μL of each solution was injected into the chromatographic system in triplicate and processed according to the previously described conditions.

2 Results and discussion

Tadalafil and dapoxetine HCl co-formulation was recently introduced into the market. They are both highly fluorescent which enables their separation and quantitation by HPLC with fluorescence detection. Fluorescence detection always shows better sensitivity and higher selectivity compared to UV detection. Its high sensitivity allows the determination of the studied drugs in spiked human plasma. Furthermore，the higher selectivity helps eliminating the interfering signals from some endogenous amino acids.

2.1 Method optimization

Several factors affecting the HPLC separation were optimized to achieve the best resolution and quantitation of the studied compounds. First was the composition and pH of the mobile phase. Our first selection was ethanol-water mixture of different ratios in an approach to develop a green HPLC and eco-friendly method，but it resulted in unacceptable resolution values. Upon trying methanol，bad resolution and high elution time were obtained. Finally，acetonitrile was tried in different volume percentages from 40% to 70%. It was found that 40% acetonitrile provided optimum separation. For optimization of the pH，pKavalues of the studied drugs were considered. The pKavalues are 16.68［21］and 8.6［22］for TAD and DAP，respectively. Accordingly，pH lower than 6.5 would be suitable for separation. The mobile phases having different pH values were prepared and tried（adjusted by o-phosphoric acid），the optimum value was found to be 4. In order to reach the suitable symmetry and minimize the tailing，triethylamine was added in different volume percentages as 0.1%，0.15% and 0.2%. It was found that 0.15% was optimum for best symmetry and consequently better resolution.

For achieving optimum sensitivity，different excitation and emission wavelengths were tried for each drug along with the IS. It was found that 236／330 nm，236／410 nm and 236／370 nm gave good detector response for TAD，DAP and IS，respectively.

Finally，it was found that acetonitrile-0.15%triethylamine （40 ∶60，v／v；pH adjusted to 4 using o-phosphoric acid）with a flow rate of 1 mL／min and fluorescence detection at 236／330 nm（TAD），236／410 nm （DAP）and 236／370 nm（IS）was most suitable to get resolved and sharp peaks （Fig.2）. System suitability parameters are presented in Table 1.

Fig.2 Chromatogram of 10 μg／mL of avanafil（tR ＝3.875 min），5 μg／mL of dapoxetine HCl （tR ＝4.819 min）and 3 μg／mL of tadalafil （tR ＝6.642 min）

2.2 Validation

2.2.1 Linearity

The linear relationships were obtained for both TAD and DAP between the relative peak areas and the corresponding mass concentrations. The regression equations were computed. All the regression parameters are summarized and shown in Table 2.

Table 1 System suitability parameters of the proposed HPLC method

Table 2 Regression and validation parameters for the determination of pure tadalafil and dapoxetine HCl by the proposed HPLC method

2.2.2 Accuracy and precision

The accuracy of the results was checked by applying the proposed method for determination of the studied drugs within their linear ranges. The concentrations were calculated from the corresponding regression equations with mean recoveries of 100.09% and 100.46% （n ＝5）for tadalafil and dapoxetine HCl，respectively （Table 3）.

The repeatability and intermediate precision were assessed by the analysis of TAD and DAP with the mass concentrations of 10，15 and 25 μg／mL. They were analyzed in triplicate in thesame and within three successive days. The proposed method showed good results as indicated by the low RSD values （Table 2）.

Table 3 Accuracy of the proposed HPLC method for the determination of tadalafil and dapoxetine HCl standards

2.2. 3 Application to pharmaceutical formulations

The proposed method has been successfully applied to the determination of TAD and DAP in Super Tadarise?tablets without interference from excipients with mean percentage recoveries of 99.250 0% and 99.252 5%，in a respective order.Standard addition technique was further applied to check the accuracy of the dosage form，results are presented in Table 4.

Table 4 Results obtained by applying the proposed HPLC method to the determination of tadalafil and dapoxetine HCl by the standard addition technique

The results obtained from the analysis of the pure samples by the proposed HPLC method were statistically compared with those obtained by applying a reported HPLC method［15］. The calculated t and F values were less than the tabulated ones at 95% confidence level，which reveals that there is no significant difference between the two methods with respect to accuracy and precision（Table 5）. The comparison of LOD values of the proposed method and the reported one shows the higher sensitivity of the proposed method.

2.2.4 Application to spiked plasma

For determination of the studied drugs in spiked human plasma samples，the linear relationship and range should be first assessed in the same matrix （human plasma）. Therefore，different mass concentrations of the two drugs along with IS were spiked into plasma. The precipitation of plasma proteins was achieved through the addition of acetonitrile. Different volumes of acetonitrile were tried. It was found that 1 mL of acetonitrile was enough for complete precipitation of plasma protein. The supernatant was then separated by chromatography using the previously optimized conditions. Then，the peak area ratios of each drug to IS were plotted against the mass concentration，and the linear relationships were obtained. The regression parameters are presented in Table 6. The mean recoveries （n ＝5）were 98.17% and 98.83% for TAD and DAP respectively （Table 7）.

Table 5 Statistical comparison of the results obtained by the proposed HPLC method and the reported HPLC method［15］for the analysis of tadalafil and dapoxetine HCl in pure form

Table 6 Regression parameters for the determination of tadalafil and dapoxetine HCl in spiked human plasma by the proposed HPLC method

Table 7 Accuracies of the proposed HPLC method for the determination of tadalafil and dapoxetine HCl in spiked human plasma

The proposed method showed high sensitivity for determination of the studied compounds in spiked human plasma with LOD values in Table 6.The peak plasma mass concentrations are reported to be 0.378 and 0.338 μg／mL for TAD ［23］and DAP ［24］respectively，above the minimum linear range of the proposed method. By being a selective technique， any possible interference arising from endogenous amino acids has been overcome. The typical chromatograms of TAD，DAP and IS in a spiked human plasma sample are shown in Fig.3.

Fig.3 HPLC chromatograms of （a）blank plasma and （b）avanafil （IS）（tR ＝3.189 min），dapoxetine HCl（tR ＝4.450 min）and tadalafil （tR ＝6.250 min）extracted from spiked plasma （0.5 μg／mL）

3 Conclusions

The proposed method is simple，selective and sensitive. The method was successfully applied to the analysis of tadalafil and dapoxetine HCl in tablets without any interference from excipients and additives which favor their applications in quality control labs. It exhibits higher sensitivity than reported HPLC methods as it could quantitatively determine as minimum as 10 ng／mL of both drugs with high accuracy，which allows the successful application of the proposed method to the determination of the studied drugs in spiked human plasma. The fluorescence detection also presents a selective tool that enables drug determination without any interference from plasma matrix.

［1］ Maryadele J，Neil O. The Merck Index：An Encyclopedia of Chemicals，Drugs and Biologicals. Whitehouse Station：Division of Merck and Co.，2006

［2］ Sweetman S C. Martindale：The Complete Drug Reference.London：Pharmaceutical Press，2007

［3］ Wright P J. Int J Clin Pract，2006，60（8）：967

［4］ Brock G B，McMahon C G，Chen K K，et al. J Urol，2002，168：1332

［5］ Carson C C，Rajfer J，Eardley I，et al. BJU Int，2004，93（9）：1276

［6］ Lewis R W，Sadovsky R，Eardley I，et al. J Sex Med，2005，2（4）：517

［7］ Gupta M，Kovar A，Meibohm B. J Clin Pharmacol，2005，45（9）：987

［8］ Gengo P J，Giuliano F，McKenna K. J Urol，2005，173（4）：239

［9］ Kendirci M，Salem E，Hellstrom W J. Ther Clin Risk Manag，2007，3（2）：277

［10］ Hiemke C，Harter S. Pharmacol Ther，2000，85：11

［11］ Modi N B，Dresser M J，Simon M，et al. J Clin Pharmacol，2006，46：301

［12］ Pryor J L，Althof S E，Steidle C，et al. Lancet，2006，368（9539）：929

［13］ Buvat J，Tesfaye F，Rothman M，et al. Eur Urol，2009，55（4）：957

［14］ Dresser M J，Desai D，GidwanI S，et al. Int J Impot Res，2006，18（1）：104

［15］ Giri A D，Bhusari V K，Dhaneshwar S R. Int J Pharm Pharm Sci，2012，4（2）：654

［16］ Rajeshwari M，Chenthilnathan A，Rama K. Int J Pharm Biol Sci，2014，4（2）：72

［17］ Rajpara C S，Akhtar J，Khandhar A. PharmaTutor，PHARMATUTOR-ART-1387

［18］ Kavitha A，Vijaya D D，Hima B S，et al. Asian J Pharm Clin Res，2013，6（3）：326

［19］ Hamilton C L，Cornpropst J D. J Chromatogr B，1993，612（2）：253

［20］ I. C. H. Q2 （R1）Guideline，Validation of Analytical Procedures：Text and Methodology，International Conference on Harmonization. Geneva，Switzerland，2005

［21］ Singh S，Prasad B，Savaliya A A，et al. TrAC-Trend Anal Chem，2009，28（1）：13

［22］ Pimple S，Shah M，Joshi A，et al. Int J Pharm Sci Rev Res，2014，26（2）：328

［23］ Forgue S T，Patterson B E，Bedding A W，et al. Br J Clin Pharmacol，2005，61（3）：280

［24］ Dresser M J，Kang D，Staehr P，et al. J Clin Pharmacol，2006，46（9）：1023