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Udenafil (Zydena®), Dapoxetine HCl (Priligy®), Liquid Chromatography, Jordan Market, Validation Udenafil is a new drug for (ED) erectile dysfunction and chemically known 3- {1-CH3-7-oxo-3-propyl-1-H,4-H,7-H-pyrazolo- [4,3- d]- pyrimidin-5-yl}-N-[2-(1-methylpyrrolidin-2-yl)-ethyl]-4-propoxybenzene-1-sulfone-amide [1,2]. PDE-5 inhibitions are the first choice line in therapy of sexual ED [1-5]. Udenafil is a five new drug amongst the PDE-5 inhibitors group and fulfillment this needed for a many molecule having benefits of the existing agents with a good tolerably image [2]. Udenafil has been registered in Jordan market by JFDA under the trade name of Zydena tablet 100 and 200 mg also it has been registered in South Korea and some of European countries and India. Zydena has a molecular mass of 516.661 g/mol and solubility of 98e-02 g/l. Figure 1 represents structural formula of udenafil [1,2]. Dapoxetine HCl, marketed as Priligy as trade name and chemically known a (1-S)-N, N-di-CH3-3-naphthalen-1- yloxy-1-phenyl-propan-1- amine HCl as seen in (Figure 2) is can be used for treatment of premature ejaculation in masculine humans [7]. Dapoxetine is a short acting Selective Serotonin Reuptake Inhibitor (SSRI). SSRI's are a group of components typically used as CNS medication exactly anti-depressants and their treatment of depression, anxiety and personality disorders, they can also by simulation of CNS, effective it can use in treating premature ejaculation problems, some cases of impotency in men and some cases hypnotics [7-9]. In previous studies shows that some methods reported for assay of udenafil and dapoxetine alone such as, quantification and stability indicating by reversed phase high pressure liquid chromatography/Rp-HPLC method for the determination of udenafil as active ingredient in in pharmaceutical preparation, some sensitive methods are recorded and some simple HPLC methods for validation of udenafil in human plasma or serum, more of stress degradation investigations on Udenafil and Estimation of stability- indicating LC analysis for drug formulation and pharmaceutical as dispensing dosage form and udenafil pharmacokinetics or dynamics in healthy volunteers, validation of a RP-HPLC assay for the determination of dapoxetine HCl in drug formulation, quantification of dapoxetine, an investigation parameters for treatment of CNS disorders as depression [8-10].

• Safety during pregnancy or breastfeeding has not been established.
• Use caution if you have a history of heart problems.
• Report any signs of allergic reactions such as rash or swelling.
• The medication’s efficacy varies among individuals.

Many studies used term of Internal Standard (IS) which represent a chemical substance may be a drug which can be used for calibration curve by plotting the ratio of the target API signal to e internal standard signal as a function of the amount of the standards in the assay [8,11].

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.

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Reaction and effect of excipients of the drug formulation was planned for whether it can be interfering with the assay of method. 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).

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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 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). 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. 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).

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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.

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The samples were injected in liquid chromatography system and deliberated signal from the samples was correlated with those of blank samples. 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 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). 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 priligy 90 mg was focused for HCl.

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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).

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The transducer (detector) which used of UV/Vis (JW-asco UV 3070) this model operated and fixed at a wavelength of 246 nm. 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.

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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 priligy 90 mg was 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.

• Priligy should be used as part of a comprehensive treatment plan.
• Regular evaluation helps determine ongoing suitability.
• Do not alter the dose without professional guidance.
• Combined therapy may include counseling for best results.

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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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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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 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).

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Udenafil (Zydena®), Dapoxetine HCl (Priligy®), Liquid Chromatography, Jordan Market, Validation Udenafil is a new drug for (ED) erectile dysfunction and chemically known 3- {1-CH3-7-oxo-3-propyl-1-H,4-H,7-H-pyrazolo- [4,3- d]- pyrimidin-5-yl}-N-[2-(1-methylpyrrolidin-2-yl)-ethyl]-4-propoxybenzene-1-sulfone-amide [1,2]. PDE-5 inhibitions are the first choice line in therapy of sexual ED [1-5]. Udenafil is a five new drug amongst the PDE-5 inhibitors group and fulfillment this needed for a many molecule having benefits of the existing agents with a good tolerably image [2]. Udenafil has been registered in Jordan market by JFDA under the trade name of Zydena tablet 100 and 200 mg also it has been registered in South Korea and some of European countries and India. Zydena has a molecular mass of 516.661 g/mol and solubility of 98e-02 g/l.

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Figure 1 represents structural formula of udenafil [1,2]. Dapoxetine HCl, marketed as Priligy as trade name and chemically known a (1-S)-N, N-di-CH3-3-naphthalen-1- yloxy-1-phenyl-propan-1- amine HCl as seen in (Figure 2) is can be used for treatment of premature ejaculation in masculine humans [7]. Dapoxetine is a short acting Selective Serotonin Reuptake Inhibitor (SSRI). SSRI's are a group of components typically used as CNS medication exactly anti-depressants and their treatment of depression, anxiety and personality disorders, they can also by simulation of CNS, effective it can use in treating premature ejaculation problems, some cases of impotency in men and some cases hypnotics [7-9]. In previous studies shows that some methods reported for assay of udenafil and dapoxetine alone such as, quantification and stability indicating by reversed phase high pressure liquid chromatography/Rp-HPLC method for the determination of udenafil as active ingredient in in pharmaceutical preparation, some sensitive methods are recorded and some simple HPLC methods for validation of udenafil in human plasma or serum, more of stress degradation investigations on Udenafil and Estimation of stability- indicating LC analysis for drug formulation and pharmaceutical as dispensing dosage form and udenafil pharmacokinetics or dynamics in healthy volunteers, validation of a RP-HPLC assay for the determination of dapoxetine HCl in drug formulation, quantification of dapoxetine, an investigation parameters for treatment of CNS disorders as depression [8-10]. Reaction and effect of excipients of the drug formulation was planned for whether it can be interfering with the assay of method.

7. Product details

Many studies used term of Internal Standard (IS) which represent a chemical substance may be a drug which can be used for calibration curve by plotting the ratio of the target API signal to e internal standard signal as a function of the amount of the standards in the assay [8,11]. 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.

Precautions & Warnings

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. 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). 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 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.