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In the present study a simultaneous determination of udenafil and dapoxetine HCL by RP-HPLC in pharmaceutical preparation found in Jordan Market as tablet dosage form. The proposed method is validated as per International Harmonization guidelines (ICH) [12,13]. Pure drug sample of Udenafil, Hikam Pharmaceuticals Group, Jordan, sample of B. Saudi Pharmaceutical Industries & Medical Appliances Corporation (SPIMACO) supply as a gift the dapoxetine HCl as pure drug sample of Batch No. Both was used without further purification because the assay certification shows that 99.91% (w/w) for udenafil and 98.99% (w/w) for dapoxetine HCl on dry phase. All chemicals or reagents which used in this study, were in term of HPLC grade and were purchased from Jordan and made by Merck Chemicals, Germany. Pregabalin was used as internal standard (IS) which obtained from Jordan Pharmaceutical Industries (JPM) B. 3584 as pure API powder, Figure 3 represent the structure formula of pregabalin. The High Pressure LC, system included a Pump (Model JW-asco PIU 3080), Intelligent-Sensitive Liquid Chromatography pump with auto-sampler programmed at 10-20 μl capacity per injection was applied in the system. The transducer (detector) which used of UV/Vis (JW-asco UV 3070) this model operated and fixed at a wavelength of 246 nm.

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The collected date was used (JBV) Jasso Berwyn Version 1.8, LC-Net- III/ADLC system. The column used was Thermo Hypersil type-3-BDS-C18 (200 mm × 4.6 mm, 5.0 μ) made in Germany. Optimization of High Performance Liquid Chromatography (HPLC)-Method The HPLC method was optimized by development a simultaneous analysis method for determination of Udenafil and dapoxetine HCl, simultaneously. For RP-HPLC, optimization the organic solvent Methanol (CH3OH) and pure water (H2O) was tested in ratio of 85: 15 but it found the result on the peak shape was not identical and correct due to the peak shape in this mixture is not good for the both of udenafil and dapoxetine. Hence pure water (H2O) was changed with a buffer solution (phosphate buffer).

Organic solvent CH3OH: Phosphate (PO4) buffer pH 4.6 adjusted with ortho- phosphoric acid in the ratio of 85: 15 it was found the result of sharp peak was obtained for udenafil but, the result of dapoxetine HCl was hump shape peak obtained. After that the phosphate buffer was changed with tri-ethyl amine. Solvent Methanol 0.2% tri-ethyl amine in H2O pH adjusted to 4.5 with ortho-phosphoric acid in the ratio of 85: 15 was tested, the resulting sharp peak was observed for udenafil but slight tailing in peak was cheapest priligy focused for HCl. In the Final of trails, CH3OH (Organic solvent of methanol) was changed to Acetonitrile (ACN) and the optimal mobile phase obtained was acetonitrile: 0.2% triethyl amine in water (H2O) at adjusted pH to 4.4 with ortho-phosphoric acid in the ratio 75: 25 V/V, with a flow rate 1.0 ml/min which provide an acceptable (tR) retention time), with high number of plates(N) and a better resolution (RS), more than another mobile phase tested before, for udenafil and dapoxetine HCl (Figure 3).

The results of Linearity in the optimization method was designed by injection of a six values of concentrations of the drug adapted in the mobile phase in three times or triplicate into the liquid chromatography system by keeping the injection volume stable. The peak areas were hatch against in the corresponding of the mention concentrations to achieve a good calibration curve. Another term of precision is intermediate precision of the method which was analyse by repeating readings on three diverse days. Limits of Detection (LOD) and Quantification (LOQ) LOD and LOQ serve the concentration of the analyse API sample that would profit signal-to-noise(S/N) ratios of three for LOD and ten for LOQ, subsequently. To quantify the LOD and LOQ, consecutive dilutions of mixed prepared standard solution of udenafil and dapoxetine HCl was destined from the stock solution standard. The samples were injected in liquid chromatography system and deliberated signal from the samples was correlated with those of blank samples.

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In the present study a simultaneous determination of udenafil and dapoxetine HCL by RP-HPLC in pharmaceutical preparation found in Jordan Market as tablet dosage form. The proposed method is validated as per International Harmonization guidelines (ICH) [12,13]. Pure drug sample of Udenafil, Hikam Pharmaceuticals Group, Jordan, sample of B. Saudi Pharmaceutical Industries & Medical Appliances Corporation (SPIMACO) supply as a gift the dapoxetine HCl as pure drug sample of Batch No. Both was used without further purification because the assay certification shows that 99.91% (w/w) for udenafil and 98.99% (w/w) for dapoxetine HCl on dry phase.

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All chemicals or reagents which used in this study, were in term of HPLC grade and were purchased from Jordan and made by Merck Chemicals, Germany. Pregabalin was used as internal standard (IS) which obtained from Jordan Pharmaceutical Industries (JPM) B. 3584 as pure API powder, Figure 3 represent the structure formula of pregabalin. The High Pressure LC, system included a Pump (Model JW-asco PIU 3080), Intelligent-Sensitive Liquid Chromatography pump with auto-sampler programmed at 10-20 μl capacity per injection was applied in the system. The transducer (detector) which used of UV/Vis (JW-asco UV 3070) this model operated and fixed at a wavelength of 246 nm.

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The collected date was used (JBV) Jasso Berwyn Version 1.8, LC-Net- III/ADLC system. The column used was Thermo Hypersil type-3-BDS-C18 (200 mm × 4.6 mm, 5.0 μ) made in Germany. Optimization of High Performance Liquid Chromatography (HPLC)-Method The HPLC method was optimized by development a simultaneous analysis method for determination of Udenafil and dapoxetine HCl, simultaneously. For RP-HPLC, optimization the organic solvent Methanol (CH3OH) and pure water (H2O) was tested in ratio of 85: 15 but it found the result on the peak shape was not identical and correct due to the peak shape in this mixture is not good for the both of udenafil and dapoxetine. Hence pure water (H2O) was changed with a buffer solution (phosphate buffer). To checkout one of the important parameters in validation according to ICH – guidelines, robustness of a HPLC method, petty parameter was purposely various. The parameters constituted changes of flow rate, percentage of the organic solvent (ACN) acetonitrile in the mobile phase and the other solvents from diverse part were appropriated. The specificity of the assay against the drug formula was entrenched over the study of resolution factor (Rs) of the drug peak and peak width from the adjacent resolved peak.

The peak purity of udenafil and dapoxetine HCl, was steadfast by priligy tablets analysed the spectrum at three diverse regions of the blot i.e., Start peak (S), apex peak (A) and last peak (Peak end E) (L). Reaction and effect of excipients of the drug formulation was planned for whether it can be interfering with the assay of method.

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Accuracy of this method was achieved by employ the method to drug analysed sample (Udenafil and Dapoxetine HCL consolidation tablet) to which known amount of Udenafil and Dapoxetine HCL standard powder reciprocal to 80, 100 and 120% (According to ICH-guidelines) of label claim had been added (Standard Addition Method -SAM), mixed and the powder was obtained and verified by running read out chromatogram in developed mobile phase.

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Accuracy of this method was achieved by employ the method to drug analysed sample (Udenafil and Dapoxetine HCL consolidation tablet) to which known amount of Udenafil and Dapoxetine HCL standard powder reciprocal to 80, 100 and 120% (According to ICH-guidelines) of label claim had been added (Standard Addition Method -SAM), mixed and the powder was obtained and verified by running read out chromatogram in developed mobile phase. Analysis and determination of a commercials formulation To determine exactly the contended of udenafil and dapoxetine HCl in current and common tablet in Jordanian market (trade name: Zydena200 mg/tablet, requirement label: 200 mg udenafil and 60 mg/tablet dapoxetine HCl), 20 tablets were weighed for this test, their mean substances were determined and powdered. The weight of the tablet grind equivalent to 200 mg of udenafil and 60 mg dapoxetine HCl was removed into a 100 mL volumetric flask containing 45-50 ml CH3OH (Methanol, sonicated for 25 min and diluted up to 50 mL with CH3OH. The obtaining solution was centrifuged at (4000 rpm) time of 7 min and the API-content of the supernatant was decisive (250 and 1400 μg/ml for udenafil and dapoxetine HCl, correspondingly). Supernatant was appropriated and then a suitable dilution for the sample solution after filtered using 0.50- micron filter type (Millipore-Milford).

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The mention above solution stock was further diluted to get sample solution of 15 and 4 μg/ml for udenafil and dapoxetine HCl, correspondingly. 30 μl volume of solution sample was priligy 30 mg online introduced by auto injection into HPLC, 6 times, with the same conditions described before. The areas of the peaks were measured at 246 nm and concentrations obtained in the samples were calculated using multilevel calibration curve indicating on the same HPLC system and in same time under the equivalent parameters and conditions using equation of linear regression. The development and validation for this method on estimation of udenafil and dapoxetine HCl simultaneously in marketed solid dosage form/tablets in the present study the mobile phase involving acetonitrile: 0.2% triethyl amine in H2O pH was adjusted to 4.4 with ortho-phosphoric acid as buffer solution in ratio of (75: 25). The mean values of the slope and intercept were 143712 ± 0.847 and 147669 ± 1.211 for udenafil and 42734 ± 0.9641 and 432842 ± 0.5422 for dapoxetine HCl, respectively.

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The experiments of precision parameters as repeatability and intermediate precision the results are shown in Table 1. Table 1: The results for repeatability and intermediate precision parameters The validated method was found to be precise as the Relative Standard Deviation (RSD) values for the parameters of repeatability and intermediate precision studies were < 2%, correspondingly according to the ICH-guidelines. The term N/S ratios (signal-to-noise) of 6:2 and 20:2 were pick up for the measure of parameters LOD and LOQ respectively. Any parameters selected (except columns from diverse suppliers) was modified at three levels according to ICH-guidelines (−1, 0 and 1). One parameter at the time was changed to evaluate the development. Analysis and determination of a commercials formulation To determine exactly the contended of udenafil and dapoxetine HCl in current and common tablet in Jordanian market (trade name: Zydena200 mg/tablet, requirement label: 200 mg udenafil and 60 mg/tablet dapoxetine HCl), 20 tablets were weighed for this test, their mean substances were determined and powdered.

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The weight of the tablet grind equivalent to 200 mg of udenafil and 60 mg dapoxetine HCl was removed into a 100 mL volumetric flask containing 45-50 ml CH3OH (Methanol, sonicated for 25 min and diluted up to 50 mL with CH3OH. The obtaining solution was centrifuged at (4000 rpm) time of 7 min and the API-content of the supernatant was decisive (250 and 1400 μg/ml for udenafil and dapoxetine HCl, correspondingly). Supernatant was appropriated and then a suitable dilution for the sample solution after filtered using 0.50- micron filter type (Millipore-Milford). The mention above solution stock was further diluted to get sample solution of 15 and 4 μg/ml for udenafil and dapoxetine HCl, correspondingly. 30 μl volume of solution sample was priligy 30 mg online introduced by auto injection into HPLC, 6 times, with the same conditions described before. The areas of the peaks were measured at 246 nm and concentrations obtained in the samples were calculated using multilevel calibration curve indicating on the same HPLC system and in same time under the equivalent parameters and conditions using equation of linear regression. The development and validation for this method on estimation of udenafil and dapoxetine HCl simultaneously in marketed solid dosage form/tablets in the present study the mobile phase involving acetonitrile: 0.2% triethyl amine in H2O pH was adjusted to 4.4 with ortho-phosphoric acid as buffer solution in ratio of (75: 25). The mean values of the slope and intercept were 143712 ± 0.847 and 147669 ± 1.211 for udenafil and 42734 ± 0.9641 and 432842 ± 0.5422 for dapoxetine HCl, respectively. The experiments of precision parameters as repeatability and intermediate precision the results are shown in Table 1. Table 1: The results for repeatability and intermediate precision parameters The validated method was found to be precise as the Relative Standard Deviation (RSD) values for the parameters of repeatability and intermediate precision studies were < 2%, correspondingly according to the ICH-guidelines. The term N/S ratios (signal-to-noise) of 6:2 and 20:2 were pick up for the measure of parameters LOD and LOQ respectively.

Any parameters selected (except columns from diverse suppliers) was modified at three levels according to ICH-guidelines (−1, 0 and 1). One parameter at the time was changed to evaluate the development.

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Organic solvent CH3OH: Phosphate (PO4) buffer pH 4.6 adjusted with ortho- phosphoric acid in the ratio of 85: 15 it was found the result of sharp peak was obtained for udenafil but, the result of dapoxetine HCl was hump shape peak obtained. After that the phosphate buffer was changed with tri-ethyl amine. Solvent Methanol 0.2% tri-ethyl amine in H2O pH adjusted to 4.5 with ortho-phosphoric acid in the ratio of 85: 15 was tested, the resulting sharp peak was observed for udenafil but slight tailing in peak was cheapest priligy focused for HCl. In the Final of trails, CH3OH (Organic solvent of methanol) was changed to Acetonitrile (ACN) and the optimal mobile phase obtained was acetonitrile: 0.2% triethyl amine in water (H2O) at adjusted pH to 4.4 with ortho-phosphoric acid in the ratio 75: 25 V/V, with a flow rate 1.0 ml/min which provide an acceptable (tR) retention time), with high number of plates(N) and a better resolution (RS), more than another mobile phase tested before, for udenafil and dapoxetine HCl (Figure 3). The results of Linearity in the optimization method was designed by injection of a six values of concentrations of the drug adapted in the mobile phase in three times or triplicate into the liquid chromatography system by keeping the injection volume stable.

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The peak areas were hatch against in the corresponding of the mention concentrations to achieve a good calibration curve. Another term of precision is intermediate precision of the method which was analyse by repeating readings on three diverse days. Limits of Detection (LOD) and Quantification (LOQ) LOD and LOQ serve the concentration of the analyse API sample that would profit signal-to-noise(S/N) ratios of three for LOD and ten for LOQ, subsequently. To quantify the LOD and LOQ, consecutive dilutions of mixed prepared standard solution of udenafil and dapoxetine HCl was destined from the stock solution standard. The samples were injected in liquid chromatography system and deliberated signal from the samples was correlated with those of blank samples.

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To checkout one of the important parameters in validation according to ICH – guidelines, robustness of a HPLC method, petty parameter was purposely various. The parameters constituted changes of flow rate, percentage of the organic solvent (ACN) acetonitrile in the mobile phase and the other solvents from diverse part were appropriated. The specificity of the assay against the drug formula was entrenched over the study of resolution factor (Rs) of the drug peak and peak width from the adjacent resolved peak. The peak purity of udenafil and dapoxetine HCl, was steadfast by priligy tablets analysed the spectrum at three diverse regions of the blot i.e., Start peak (S), apex peak (A) and last peak (Peak end E) (L). Reaction and effect of excipients of the drug formulation was planned for whether it can be interfering with the assay of method. Hence, repeat injections at 6 trails (n=6) of mixed of standard solution at three known concentration ranges were performed under small deliberate modified of three chromatographic factors or parameters.

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These deliberated changes show an insignificant variation in the areas of the peak (PA) and fewer changeability in retention time (tr) were detected and observed, as mention above three factors were slightly changed at three levels too which (-1.0, 0.0,1.0) (Table 2a, 2b, 2c and 2d). Table 2a: Robustness parameters testing (n=3) for udenafil (Zydena®) tablet Table 2b: Robustness parameters testing (n=3) for dapoxetine HCl (Priligy®) tablet Table 2c: Robustness parameters testing (n=3) for udenafil (Zydena®) tablet Table 2d: Robustness parameters testing (n=3) for dapoxetine HCl (Priligy®) tablet According to ICH-guidelines requirements, peak purity requirements of udenafil and dapoxetine HCl was determined by correlating their corresponding spectra at the peak start(S), apex(A) and peak end(last)(E) positions i.e., r (S, A)=0.9986 and r (M, E)=0.9990.

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Good correlation (R=0.9988) was achieved between the standard sample and targeted sample spectra of udenafil and dapoxetine HCl, correspondingly. Also, the none active ingredient called excipients from the pharmaceutical formulation were not intrusive or interacting with the assay components.

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Hence, repeat injections at 6 trails (n=6) of mixed of standard solution at three known concentration ranges were performed under small deliberate modified of three chromatographic factors or parameters. These deliberated changes show an insignificant variation in the areas of the peak (PA) and fewer changeability in retention time (tr) were detected and observed, as mention above three factors were slightly changed at three levels too which (-1.0, 0.0,1.0) (Table 2a, 2b, 2c and 2d). Table 2a: Robustness parameters testing (n=3) for udenafil (Zydena®) tablet Table 2b: Robustness parameters testing (n=3) for dapoxetine HCl (Priligy®) tablet Table 2c: Robustness parameters testing (n=3) for udenafil (Zydena®) tablet Table 2d: Robustness parameters testing (n=3) for dapoxetine HCl (Priligy®) tablet According to ICH-guidelines requirements, peak purity requirements of udenafil and dapoxetine HCl was determined by correlating their corresponding spectra at the peak start(S), apex(A) and peak end(last)(E) positions i.e., r (S, A)=0.9986 and r (M, E)=0.9990. Good correlation (R=0.9988) was achieved between the standard sample and targeted sample spectra of udenafil and dapoxetine HCl, correspondingly. Also, the none active ingredient called excipients from the pharmaceutical formulation were not intrusive or interacting with the assay components.

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As seen in the data obtained in Table 3a and 3b, there are a good recoveries parameters obtained of the udenafil collected date and dapoxetine HCl date in the range from 98.92 to 102.42% were realized at different combined concentrations. Table 3a: Recovery parameters studies for udenafil at n=6 Table 3b: Recovery parameters studies for dapoxetine HCL at n=6 The results obtained in this experiment of the quantity of udenafil and dapoxetine HCl in marketed tablets, expressed as a percentage of label request or claims were in acceptable agreement with the label request or claims by that implying that there are no interactions from any of additives or excipients which are ordinary present. As seen in the data obtained in Table 3a and 3b, there are a good recoveries parameters obtained of the udenafil collected date and dapoxetine HCl date in the range from 98.92 to 102.42% were realized at different combined concentrations. Table 3a: Recovery parameters studies for udenafil at n=6 Table 3b: Recovery parameters studies for dapoxetine HCL at n=6 The results obtained in this experiment of the quantity of udenafil and dapoxetine HCl in marketed tablets, expressed as a percentage of label request or claims were in acceptable agreement with the label request or claims by that implying that there are no interactions from any of additives or excipients which are ordinary present.