Development and validation of stability indicating rp-hplc method for simultaneous estimation of sildenafil citrate and fluoxetine in bulk & tablet dosage form

International Bulletin of Drug Research., 4(6): 92-105, 2014
A simple, precise, sensitive and reproducible, rapid stability indicating RP-HPLC method for the simultaneous estimation of Sildenafil citrate and Fluoxetine by forced degradation studies has been developed. The developed method separates degradation products. Sildenafil citrate and Fluoxetine and their combination drug product were exposed to acid, base, oxidation, high temperature, and hydrolytic stress conditions. The proposed HPLC method utilizes the Shimadzu HPLC system on a Hiber R C-18 columns (250mm × 4.6 mm i.d. with particle size of 5 µm) using mobile phase consisting of Acetonitrile : Potassium Dihydrogen Phosphate buffer with 25 mM Triethylamine pH adjusted to 4 with o-phosphoric acid (50:50 v/v) in an gradoamt e;itopm ,pde at a f;pw rate pf 1 ml/min, at 23°C with a load of 20L. The detection was carried out at 223 nm. The retention time of Sildenafil citrate and Fluoxetine was found to be around 3.68 min and 5.31 min, respectively. The number of theoretical plates and tailing factor for Sildenafil citrate and Fluoxetine were NLT 3000 and should NMT 2 respectively. The proposed method was validated according to ICH guidelines and the results were found to be within the acceptable range. Hence, the proposed method can be used for the routine quality control of the drugs and can also be applied to pharmacokinetic studies. KEYWORDS
Sildenafil citrate, Fluoxetine, Reverse phase HPLC, Validation. AFFILIATION
1,2,3&4. Department of pharmaceutical sciences, Saurashtra university, Rajkot. *Author for Correspondence: Email I: 9428712210
International Bulletin of Drug Research., 4(6): 92-105, 2014
Sildenafil citrate (SC) chemically is 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)Phenylsulfonyl]-4-methylpiperazine.[1], structure of SC is shown in Figure 1(a). It acts as a Vasodilator Agents and Phosphodiesterase Inhibitors. Fluoxetine (FT) chemically is (RS)-N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy] propan-1-amine[1] as shown in Figure 1(b). It is anti depressant. Fig. 1(a): Sildenafil citrate
Fig. 1(b): Fluoxetine
Sildenafil citrate and Fluoxetine is a combination of well-known Sildenafil citrate and Fluoxetine, a selective serotonin reuptake inhibitor (SSRI). Antidepressants of the SSRI class including Fluoxetine have long been used off-label to treat premature ejaculation. Delayed orgasm is a well known effect of such drugs. Fluoxetine in combination with Sildenafil citrate has been shown to be more effective than Fluoxetine alone in delaying ejaculation and providing sexual satisfaction[1]. The stability of a drug dosage form refers to the ability of a particular form to maintain its physical, chemical, therapeutic, and toxicological specification presented in the monograph on identity, strength, quality, and purity. The stability of a drug product should ordinarily be demonstrated by its manufacturer by methods appropriate for the purpose. Obviously, a stability testing problem is never simple. Stability testing is an important part of the process of drug product development. The purpose of the stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors, such as temperature, humidity, and sun light, and enables recommendation of storage conditions, retest period and shelf life to be established. [4] The literature survey reveals that many analytical methods are reported for the determination of SC and FT individually and in combination. The literature survey revealed spectroscopic [5–12] and chromatographic [13–18] methods. It was found that number of studies involving method development for estimation of Sildenafil citrate and Fluoxetine alone are available including RP-HPLC, HPTLC, UPLC, UV Spectrophotometric and LC-MS. But review of literature reveals that there is no any method reported till now for simultaneous estimation of Sildenafil citrate and Fluoxetine by stability indicating RP-HPLC in the fixed pharmaceutical dosage forms. The present paper describes a simple simultaneous method for the determination of Sildenafil citrate and Fluoxetine by stability indicating reverse phase HPLC in gradient mode in pharmaceutical dosage form, which could be applied especially to the determination of drugs in clinical data monitoring, and in pharmacokinetic investigations. The method would help the assay of drugs in a single run which reduces the time of analysis and does not require International Bulletin of Drug Research., 4(6): 92-105, 2014
separate methods for each drug. The method was also validated for the parameters as per ICH guidelines [19]. The stability indicating power of the method was established by comparing the chromatograms obtained under optimized conditions before forced degradation with those after forced degradation via acidic, basic, oxidative, thermal, and photolytic stress conditions. MATERIALS AND METHODS
Chemicals and Reagents
HPLC grade were used. All the other reagents used were of analytical grade. Milli Q water (Millipore Q Grade) was used throughout the analysis. Materials and Reagents
Methanol, water, Acetonitrile, and o-phosphoric acid used were of HPLC grade (S.d. fine chem. Ltd.) Sildenafil citrate (SC) and Fluoxetine (FT) API were obtained from Cadila pharmaceutical and Formulation (Malegra-FXT) was obtained from Fedelty Healthcare Tablet dosage form containing SC 100mg and FT 60mg was used as the sample during the method development process. Chromatographic Equipments and Conditions
A Shimadzu, a high pressure liquid chromatographic Kyoto, Japan Instrument was used for the analysis. The instrument was provided with a Hiber R C-18 columns (250mm × 4.6 mm i.d. with particle size of 5 µm), an LC 20 AD pump and an SPD-M20A-PDA detector were employed. A 20L Hamilton syringe was used for sample injection. Data acquisition was done by using Winchrom software. Mobile Phase Preparation
Mixture of ACN: Potassium Dihydrogen Phosphate buffer with 25 mM triethylamine pH adjusted to 4 with o-phosphoric acid (50:50 v/v) with 1.0 ml/min flow rate was selected as mobile phase. The mobile phase was filtered through 0.45  membrane. The mobile phase was ultra sonicated for 5 min. Instrumental Parameters
The separation was done on Hiber R C-18 columns (250mm × 4.6 mm i.d. with particle size of 5 µm). The flow rate of mobile phase was maintained at 1 ml/min, and the detection was carried out at 223 nm. All determinations were performed at a constant column temperature of 23ºC with a load of 20 L. The retention time of SC and FT was observed at 3.68 and 5.31 min respectively. Stress Study
All the stress decomposition studies were performed at a concentration of 30g/ml and 18g/ml for SC and FT respectively in mobile phase. Acid hydrolysis was performed in 0.1M hydrochloric acid. The study in alkaline condition was carried out in 0.1M sodium hydroxide. International Bulletin of Drug Research., 4(6): 92-105, 2014
Oxidative studies were carried out in 10% hydrogen peroxide. Hydrolytic degradation studies in 10 ml HPLC grade water, additionally the drug powder was exposed to dry heat at 105ºC. Samples were withdrawn at appropriate time, cooled and neutralized by adding base or acid, and subjected to RP- HPLC analysis after suitable dilution. Preparation of Standard Solutions and Calibration Graphs
30 mg of Sildenafil citrate and 18mg of Fluoxetine was weighed and transferred to 100ml volumetric flask. Acetonitrile: Buffer (50:50, V/V) (HPLC Grade) was added to dissolve the drug and final volume was made with the same solvent to obtain a concentration 300μg/ml and 180μg/ml respectively. 1ml of stock solution was withdraw & transfer in 10ml volumetric flask and volume was make up with above same solvent to achieve 30μg/ml and 18μg/ml concentration respectively and these solution was used for analysis. Calibration curve of Sildenafil citrate and Fluoxetine was plotted over a range of 15-45μg/ml and 9-27μg/ml respectively. Solution was made by pipette out 1.5, 2.25, 3.0, 3.75 and 4.5 ml of standard stock solution and dilute up to 10 ml. These solutions were injected in to column to get the chromatogram. Preparation of Test Solution
Five tablets were weighed, crushed and mixed in a mortar and pestle for 10min. A portion of powder equivalent to 30mg of Sildenafil citrate and 18mg of Fluoxetine weighed and transferred to 10ml volumetric flask. Acetonitrile (HPLC grade) was added to dissolve the drugs and final volume was made with the Buffer solution to obtain a concentration 300μg/ml of SC and 180 μg/ml of FT. Then the sample solution was filtered through 0.45μm cellulose acetate filter paper (0.45μm) before diluting further. Then final volume was made up to obtain a concentration 30μg/ml of SC and 18μg/ml of FT. HPLC Method Validation
The linearity was tested for the concentration range of 1.5, 2, 2.5, 3, 3.5, 4, 4.5μg/ml for SC and 0.9, 1.35, 1.7, 2.25, 2.7μg/ml for FT and the calibration curve was constructed and evaluated by its correlation coefficient. The correlation coefficient (r2) for all the calibration curves was consistently greater than 0.999 ±0.0004. Precision
Three injections of same concentration (30μg/ml for SC and 18μg/ml for FT) were given on the same day, and these studies were also repeated on different days to determine interday precision. Accuracy
Accuracy may often be expressed as % Recovery by the assay of known, added amount of analyte. It's measure of the exactness of the analytical method. The recovery experiments were carried out in triplicate by sparking previously analyzed samples of the SC (30µg/ml) & FT (18 µg/ml) solution with three different concentrations of standards at 80%, 100% and 120%. International Bulletin of Drug Research., 4(6): 92-105, 2014

Limit of Detection (LOD) and Limit of Quantification (LOQ)
Limit of detection was calculated by using the formula: LOD = 3.3 SD/S SD = standard deviation of the response S = slope of calibration curve of the analyte. Limit of quantification was calculated by using the formula: LOQ = 10 SD/S SD = standard deviation of the response S= slope of calibration curve of the analyte. Robustness
To determine robustness of the method the experimental conditions were deliberately changed. The flow rate of the mobile phase, pH of the mobile phase and mobile phase composition was varied parameters. The study was performed on same day. The area obtained from each variation was compared with that obtained under optimized conditions. Forced Degradation Studies of API and Formulation
Stress studies were carried by using 30g/ml and 18g/ml for SC and FT respectively in different conditions. Acidic and alkaline hydrolysis was carried out in 0.1M hydrochloric acid (HCl) and 0.1M sodium hydroxide (NaOH). The oxidative study was carried out in 10% hydrogen peroxide (H2O2) for 24hr. Hydrolytic studies on the drug were carried out in water. For thermal stress testing, both the drugs were spread in petridish and placed in the oven at 105ºC. Samples were withdrawn periodically and subjected to analysis after suitable dilution. RESULTS AND DISCUSSION
The main objective of the RP-HPLC method was to determine a validated stability indicating method for the estimation of SC and FT simultaneously in liquid pharmaceutical formulation and to obtain well-resolved peaks of SC, FT, and impurities/ degradants. The method should be accurate, reproducible, robust, stabile indicating, filter compatible, linear and straight forward enough for routine use in quality control laboratory. Method Development and Optimization
Different chromatographic conditions were experimented to achieve better efficiency of the chromatographic system. Parameters such as mobile phase composition, wavelength of detection, column, column temperature and pH of mobile phase were optimized. Several proportions of buffer and solvents were evaluated in order to obtain suitable composition of mobile phase. Different composition of mobile phases containing a mixture (v/v) of 1% o-phosphoric acid in Acetonitrile, and methanol was tried, but the mixture of Acetonitrile : Potassium Dihydrogen Phosphate buffer (50:50 v/v) was selected as optimal for obtaining well defined and well-resolved peaks of SC and FT at a flow rate of 1 ml/min on a Hiber C18 column. 223nm was selected as the optimum wavelength for detection and quantitation, at which best detector response for both SC and FT was obtained. The mean retention time International Bulletin of Drug Research., 4(6): 92-105, 2014
for SC and FT was found to be 3.68 min and 5.38 min, respectively. The chromatograms obtained are presented in figure 2, 3, 4. Fig. 2: Sildenafil citrate 30 ppm.
Fig. 3: Fluoxetine 18 ppm.
Fig. 4: Sildenafil citrate(30 ppm) + Fluoxetine(18 ppm)

International Bulletin of Drug Research., 4(6): 92-105, 2014
Stability Studies
All forced degradation studies were analysed at 30 g/ml and 18 g/ml for SC and FT respectively concentration level. The drug stability in pharmaceutical formulations is a function of storage conditions and chemical properties of the drug and its impurities. Conditions used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis. Stability data is required to show that the concentration and purity of analyte in sample at the analysis corresponds to the concentration and purity of the analyte at the time of sampling. SC and FT were subjected to acid, alkaline, oxidative, hydrolytic, and dry heat stress conditions. The chromatograms obtained after subjecting sample solution to degradation are presented in Figures 5(a), 5(b), 5(c), 5(d), 5(e). Summary of Stability study is presented in table 1. Fig. 5(a): SC+FT acid degradation sample.
Fig.5(b) SC+FT base degradation sample
International Bulletin of Drug Research., 4(6): 92-105, 2014
Fig. 5(c). SC+FT oxidative degradation sample.
Fig.5 (d): SC+FT hydrolysis degradation sample.
Fig. 5(e): SC+FT thermal degradation sample.
International Bulletin of Drug Research., 4(6): 92-105, 2014
Table 1: Summary of Stability study
Sildenafil citrate
Calibration curves were obtained for SC and FT from which the linear regression equation was computed and found to be Y=47.02x+6.068, r2=0.999 for SC and Y=49.48x+2.791, r2=0.999 for FT. see table 1 & 2, Fig. 6 & 7 Table 2: SC linearity. Fig. 6: SC linearity.
Sildenafil citrate linearity
International Bulletin of Drug Research., 4(6): 92-105, 2014
Table 3: FT linearity.
Fig.7: FT linearity.
Fluoxetine linearity
Accuracy and Precision
The percentage standard error which is an indicator of accuracy is ≤1.0 and is indicative of high accuracy. The calculated percentage relative standard deviation (%RSD) can be considered to be satisfactory. The percentage RSD values were <2%. The results obtained for the evaluation of accuracy and precision of the method are compiled in Tables 4, 5, 6. Table 4: Precision Data (n = 3)
Intra-day (SD, %RSD)
Inter-day (SD, %RSD)
6.96,0.98 14.59,1.03 36.89,1.74 7.54,1.06 12.01,0.85 Fluoxetine
SD and %RSD of concentrations 15, 30, 45 μg /ml prepared from the synthetic mixture of formulations of SC & FT. Table 5: Recovery study of SC by HPLC.
found (µg/ml)
International Bulletin of Drug Research., 4(6): 92-105, 2014
Table 6: Recovery study of FT by HPLC.
found (µg/ml)
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters, and provides an indication of its reliability during normal usage. The results are summarized in Table 7. Table 7: Robustness parameter of SC & FT by HPLC Method.
Change Condition 1
Change Condition 2
0.8 ml/min (n=3) Sildenafil
International Bulletin of Drug Research., 4(6): 92-105, 2014
Mobile phase(50:50) Sildenafil
System Suitability
The system suitability was carried out after the method development and validation have been completed. For this, parameters like theoretical plates, resolution, retention time, and peak symmetry of samples were measured as in Table 8. Table 8: System Suitability Parameters.
System Suitability
Theoretical Plates RetentionTime (minutes) International Bulletin of Drug Research., 4(6): 92-105, 2014
The reported RP-HPLC method was proved to be simple, rapid, and reproducible. The validation data indicate good precision, accuracy, and reliability of the method. The developed method offers several advantages in terms of simplicity in mobile phase, isocratic mode of elution, easy sample preparation steps, and comparative short run time which makes the method specific and reliable for its intended use in simultaneous determination of Sildenafil citrate and Fluoxetine in liquid oral dosage form. Quick stability indicating RP-HPLC method was developed for the simultaneous estimation of Sildenafil citrate and Fluoxetine in the presence of its degradation products, generated from forced degradation studies. There were no reported stability indicating methods for this combination of drugs in liquid dosage form; hence, this method has an advantage of being unique and novel. REFERENCES
Tripathi KD, Essential of Medical Pharmacology, 4th ed., Jaypee Brothers, new delhi, 522-553 (2001). Cheitlin MD, Brindis RG, Ganz P, use of sildenafil (viagra) in patients with cardiovascular disease, American College of Cardiology/American Heart Association, 1, 273-282(1999). Crossfire B, 2D structure of sildenafil gave one hit: information about the synthesis, The Bioorganic & Medicinal Chemistry Letters, 1819 (1996). ICH Steering Committee, ICH Q2B Validation of Analytical Procedures, methodology. European Agency for the Evaluation of Medicinal Products, International Commission on Harmonization, Switzerland, (1996). Baranowska I , simultaneous determination of sildenafil, its N-desmethyl metabolite and other drugs in human urine by RP-HPLC method, Chemia Analityczna, 52 (2007) 645. Croucha V, Development and validation of a rapid HPLC assay for the simultaneous determination of three psychoanaleptic drugs in pharmaceutical formulations, Inventi Rapid: Pharm Analysis & Quality Assurance, 3, 722-732(2008). Dhaduk DM, Rathod BG, Estimation of sildenafil citrate and Dapoxetin HCl in their combined dosage form by validated HPLC method, Inventi Rapid: Pharm Analysis & Quality Assurance, 510-512 (2012). Abourashed, Abdel-Azim M, Habib, HPTLC determination of sildenafil in pharmaceutical products, journal of planner chromatography, 18, 372-376(2007). Hefnawy, Rapid determination of Sildenafil citrate in pharmaceutical preparation using monolithic silica HPLC column, Journal of Liquid Chromatography Related Technologies, 26,1082-6076(2003). Khan, A validated HPLC method for the measurement of Sildenafil citrate in different formulations, Pakistan Journal of Science, 62 192-194(2010). International Bulletin of Drug Research., 4(6): 92-105, 2014
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