Vol - 4, Issue - 3, Supl - 1 Apr-Jul 2013 ISSN: Tiwari et al PHARMA SCIENCE MONITOR
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1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES METHOD DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR ESTIMATION OF SILDENAFIL CITRATE AND FLUOXETINE HYDROCHLORIDE BY RP-HPLC IN BULK AND MARKETED FIXED DOSE COMBINATION Shashank K Tiwari*, Bapna M, Arshad Hala, C. Dave, Kunal D. Brahmbhatt Department of Quality Assurance, Shivam Pharmaceutical Studies and Research Centre, Valasan, Anand, Gujarat. ABSTRACT A simple, accurate and precise RP-HPLC method was developed for simultaneous estimation of Fluoxetine hydrochloride and Sildenafil Citrate. C 18 ODS column ( mm) was used as stationary phase. The mobile phase used was buffer + 0.1% triethyl amine: acetonitrile (45:55 v/v) at ph 3.5 adjusted by 5% o-phosphoric acid. The mobile phase was delivered at flow rate 1.0 ml/min. UV detection was set at 230 nm. The retention time of Sildenafil and Fluoxetine was found to be min and min respectively. Linearity was observed over the concentration range of µg/ml and µg/ml for sildenafil and fluoxetine respectively. Moreover, the % RSD for repeatability, inter and intraday precision was found to be less than 2%, which reveals that the method is precise. The % recovery was found to be 99.84%-99.94% for sildenafil and % for fluoxetine. However, the change in ph, flow rate and organic phase ratio also did not show any significant variance. Assay of the combined dosage form finalized the applicability of this method for simultaneous estimation of Sildenafil citrate and fluoxetine hydrochloride in combined dosage forms. Keywords: Fluoxetine hydrochloride, sildenafil citrate, method validation, HPLC, quantitative analysis. INTRODUCTION Serotonin (5-HT) has been implicated in etiology of many diseased states and may be particularly important in mental illness such as schizophrenia, eating disorder, obsessive compulsive disorder (OCD), Migraine and Panic disorder. Indeed many currently used treatment of this disorder are thought to act by modulating serotininergic tone. So this lead to realization of many treatment acting via serotinergic system such as selective serotonic reuptake inhibitor (SSRI) e.g. fluoxetine, sertaline, paroxetine. Selective serotonic reuptake inhibitor(ssri) have been reported to reduce libido in men and women Antidepressant-induced sexual dysfunction becomes an important issue in the IC Value
2 context of treatment effectiveness, as antidepressant medications are helpful only insofar as patients take them. Given the important clinical implications of premature discontinuation -- for example, higher rates of relapse and recurrence -- increasing attention is currently being devoted to the management of antidepressant-induced sexual dysfunction and other unwanted side effects of pharmacotherapy for depression [1-5]. The simultaneous determination of Sildenafil and Fluoxetine in their combined doses form has been reported in HPLC method. The present work describes the development of a validated RP-HPLC method which can quantify these components simultaneously from a combined dosage form which is fast, simple, precise and reliable method for routine analytical needs as compared to the early reported HPLC methods. The present RP- HPLC method was validated following the ICH guidelines [6-8]. Sildenafil Citrate Fluoxetine Hydrochloride Fig.1. Structure of Sildenafil Citrate and Fluoxetine hydrochloride MATERIAL AND METHODS Materials Fluoxetine Hydrochloride, Sildenafil Citrate API standards,malegra fxt plus tablets Materials Instruments & Apparatus IC Value
3 Shimadzu HPLC with C18 ODS column ( mm); Labindia Double beam UV Visible spectrophotometer; Soltec sonica Ultrasonicator; Systronics ph Meter. Borosil Measuring cylinder 10 ml, 50 ml, 100 ml. Borosil Pipette 1 ml, 2 ml, 5 ml, 10ml. Borosil Volumetric flask 10 ml, 100 ml. (all apparatus were previously calibrated and made up of glass.) [9, 10] Chromatographic conditions The mobile phase, a mixture of buffer + 0.1% triethyl amine: acetonitrile(45:55 v/v) at ph 3.5 pumped at a flow rate of 1.0 ml/min through the column (C18; 5ì, 4.6 X 250 mm, Shimadzu) at 300C. The mobile phase was degassed prior to use under vacuum by filtration through a 0.2ì nylon membrane. Concentrations were measured at 230 nm by UV detector at a sensitivity of Methodology [11-16] Preparation of standard stock solution Sildenafil Citrate Standard Stock Solution (1000 ppm): Stock solution was prepared by accurately weighing 100 mg standard powder of PCM and transferring to 100ml volumetric flask containing a few ml of Mobile Phase. Volume was made up to the mark with Mobile Phase to yield a solution containing 1000µg/ml of Sildenafil. Fluoxetine HCl Standard Stock Solution(600 ppm): 60 mg of standard powder of fluoxetine was accurately weighed and transferred to a 100ml volumetric flask containing few ml of Mobile Phase and volume made up to the mark with Mobile Phase resulting into the solution of 600µg/ml of Fluoxetine. Calibration curve for Sildenafil And Fluoxetine: Appropriate volume of aliquot from standard Sildenafil Citrate And Fluoxetine Hcl stock solution was transferred to same volumetric flask of 10 ml capacity. The volume was adjusted to the mark with mobile phase to give a solution containing 50,75,100,125 and 150μg/ml Sildenafill (50% to 150%) and 30,45,60,75 and 90μg/ml fluoxetine Hcl (50% to 150%). The mixed standard solution was chromatographed for 10 minutes using mobile phase at a flow rate of 1.0 ml/min.the calibration curve were plotted for peak area vs. concentration for both the drugs. IC Value
4 Optimized Chromatographic conditions Stationary phase : C18 ODS Column ( mm); Mobile phase buffer + 0.1% triethyl amine: acetonitrile(45:55 v/v) at ph 3.5 adjusted by 5% o-phosphoric acid; Detection wavelength: 230 nm; Injection volume : 20 μl ; Temperature of column : Room temperature ; Flow rate : 1.0 ml/min. Figure 2 Chromatogram of Sildenafil and Fluoxetine hydrochloride, buffer + 0.1% triethyl amine: acetonitrile(45:55 v/v) System Suitability test: System suitability parameters mainly include Resolution, Column efficiency in terms of theoretical plates and tailing factor. These parameters are described below. TABLE 1: DATA FOR SYSTEM SUITABILITY TEST FOR SILDENAFIL AND FLUOXETINE Sr System Suitability Parameters Observed Value Specifications No. 1. Resolution (Rs) > Number of theoretical Plates (N) 3. Tailing Factor (TF) Sildenafil 7180 Fluoxetine 7104 Sildenafil 1.37 Fluoxetine 1.35 Not Less than 2000 Not greater than 2 Validation of RP-HPLC Method IC Value
5 1) Linearity and Range The linearity range for Sildenafil was μg/ml and for Fluoxetine Hydrochloride it was found to be in the range of μg/ml. The Calibration Curve of standard Sildenafil and Fluoxetine is depicted in below figure 4. Figure 4 Calibration Curve of standard Sildenafil ( μg/ml) and Fluoxetine (30-90 μg/ml) IC Value
6 TABLE 2: LINEARITY FOR SILDENAFIL AND FLUOXETINE Conc. Area SD %RSD Conc. Area SD %RSD ) Precision I. Intraday precision The data for intraday precision of Sildenafil citrate and fluoxetine were presented by repetition of experiment on the same day at the three different timings for each drug. Conc Sildenafil Conc Fluoxetine Mean Area± SD %RSD Mean Area± SD %RSD ± ± ± ± ± ± II. Interday precision The data for interday precision of Sildenafil citrate and fluoxetine were obtained by repeatation of experiment on different days of a week IC Value
7 Conc Sildenafil Conc Fluoxetine Mean Area± SD %RSD Mean Area± SD %RSD ± ± ± ± ± ± ) Accuracy The data for accuracy of Sildenafil and Fluoxetine are presented in Table 3. The recovery range for Sildenafil and fluoxetine were found to be % and % from combined dosage form. Drug Accuracy level TABLE 3: ACCURACY TABLE Amt. taken Amt. added Recovered Conc. Mean % Recovery±SD Sildenafil Citrate 80% ± % ± % ±1.14 Fluoxetine 80% ± % ± % ± ) Limit Of Detection (L.O.D.) The limit of detection for Sildenafil and Fluoxetine was found to be 6.02 μg/ml and 3.88 μg/ml. 5) Limit Of Quantification (L.O.Q.) IC Value
8 The limit of quantification for Sildenafil and Fluoxetine was found to be μg/ml and μg/ml. 6) Robustness To evaluate robustness of the method few parameters were deliberately varied. The parameters included variation of flow rate, change in ph of the buffer, change in organic phase ratio. The average value of % RSD for determination of Sildenafil and Fluoxetine, less than 2 % revealed the robustness of the method. Parameter Variation Area %RSD Sildenafil Fluoxetine Silde Fluox Flow rate +0.2ml/min ml/min ph Mobile phase Assay of Combined Dosage Form The results are shown in Table 4. The data revealed % for Sildenafil and % for Fluoxetine. These data confirmed the applicability of this method for combined dosage forms. TABLE 5: ASSAY OF COMBINED DOSAGE FORM Formulation Drug Labeled Claim (mg/tab) Amount Found (mg/tab) %Assay Tablets Sildenafil Fluoxetine IC Value
9 TABLE 6: SUMMARY OF VALIDATION PARAMETERS Parameters Data obtained Sildenafil Fluoxetine HCl Theoretical plate Asymmetry factor Retention time (min) Linearity Linearity µg/ml 30-90µg/ml Range Correlation Coefficient Precision (%RSD) Intraday Interday Accuracy (% Recovery) LOD LOQ CONCLUSION A simple RP-HPLC method for simultaneous estimation of Sildenafil citrate And Fluoxetine Hydrochloride was developed and validated. The mobile phase used was phosphate buffer + 0.1% triethyl amine :Acetonitrile (45:55 v/v) at ph 3.5 adjusted by 5% o-phosphoric acid. UV detector was set at 230 nm. The retention time of Sildenafil and Fluoxetine was found to be 4.2 min and min respectively. The proposed method utilizes isocratic elution technique at room temperature.the method is simple, rapid, precise, and accurate for simultaneous estimation of Sildenafil Citrate and fluoxetine hydrochloride It can be used for the routine quality control of the formulation in the pharmaceutical industries. ACKNOWLEDGEMENT The authors are thankful to Molecule Laboratory, Ahmedabad. Also Thanks my Friends Brahmbhatt Kunal, Chaitanya Dave, Palmi Pendal for successful conduct & completion of this research work. REFERENCES 1. Tripathi K.D, Essential of Medical Pharmacology, 6th Edition, Jaypee brothers, 2005, pp Goyal R.K. et al, Elements of pharmacology, 16th Edition, B.S.Shah Prakashan, , pp IC Value
10 3. Drugs.com,Sildenafil Citrate.Updated on November Available from: Paracetamol. 4. Drug Bank, Fluoxetine Hydrochloride.Updated on February 2011.Available From: 5. Sildenafil monograph,merck Index 14 th edition, pg no Sharma B.K, Instrumental Methods of Chemical Analysis, GOEL Publication House, Meerut, pp Ahuja S, Sephen S, Modern Pharmaceutical Analysis Separation Science and Technology, 3rd Volume, 1st Indian Reprint, Academic Press, 2005, pp Skoog D.A. et al, Fundamentals of Analytical Chemistry, 8th Edition, Thomson Asia Pvt Ltd, Singapore, 2004, pp Connors KA, A Textbook of Pharmaceutical Analysis, 3rd Edition, John Wiley and sons, 1999, pp Beckett AH, Stenlake JB, Practical Pharmaceutical chemistry, 4th Edition, CBS publishers and distributors, 2001, pp Skoog DA, Holler FJ, Stanley R, Principles of instrumental analysis, 6th Edition, Thomson Asia Pvt Ltd, Singapore, 2007, pp Christian GD, Analytical Chemistry, 4th edition, John Wiley and Sons, Singapore, 2004, pp ICH guidelines, validation of analytical procedure Methodology Q2B, I.C.H. Harmonized Tripartite Guidelines, ICH, Q2A Text on validation of analytical procedures, Oct 1994; International Conference on Harmonization 15. ICH, Q3B validation of analytical procedures, methodology Nov 1996; International conference on Harmonization 16. Patel Nitin, Chaudhari Ankit, Development & Validation of first order spectrophotometric Method for simultaneous Estimation of Sildenafil Citrate and Fluoxetine Hydrochloride in Tablet dosage form. Inventi Rapid :Pharmaceutical Analysis & Quality Assurance Article ID- " Inventi:ppaqa/773/13 ", For Correspondence: Shashank Tiwari shashanktiwari1990@gmail.com IC Value
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