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International Bulletin of Drug Research., 4(6): 92-105, 2014
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DEVELOPMENT AND VALIDATION OF STABILITY
INDICATING RP-HPLC METHOD FOR SIMULTANEOUS
ESTIMATION OF SILDENAFIL CITRATE AND
FLUOXETINE IN BULK & TABLET DOSAGE FORM.
ASHVIN DUDHREJIYA1*
, KARISHMA GANDHI2, MEERA MARU
3, NAVIN SHETH
4
________________________________________________________________________
ABSTRACT
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 20L. 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 ID: [email protected]; Mob: 9428712210
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INTRODUCTION
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
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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
pvt.ltd.
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 20L 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 30g/ml and
18g/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.
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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
Linearity
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%.
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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 30g/ml and 18g/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
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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)
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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
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Fig. 5(c). SC+FT oxidative degradation sample.
Fig.5 (d): SC+FT hydrolysis degradation sample.
Fig. 5(e): SC+FT thermal degradation sample.
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Table 1: Summary of Stability study
Condition
Drug
%Degradation
Sildenafil citrate Fluoxetine
API Formulation API Formulation
Acidic 0.13 1.19 0.19 2.11
Alkaline 1.56 1.45 3.6 3.47
Oxidative 3.12 3.7 7.96 8.33
Hydrolysis 0.87 1.04 0.95 0.76
Thermal 0.87 1.19 0.79 1.45
Linearity
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
conc(mcg/ml) area
15 704.668
22.5 1075.776
30 1413.078
37.5 1768.962
45 2121.48
R2 0.999921098
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Table 3: FT linearity. Fig.7: FT 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)
Compound Intra-day (SD, %RSD) Inter-day (SD, %RSD)
Conc.(µg/ml) 15 30 45 9 18 27
Sildenafil
citrate 6.96,0.98 14.59,1.03 36.89,1.74 7.54,1.06 12.01,0.85 13.72,0.64
Fluoxetine 4.40,0.99 9.20,1.03 23.26,1.74 4.7,1.07 7.57,0.85 8.60,0.644
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.
Theoretic
al conc. Net area
Concentration
found (µg/ml) %Recovery Avg. SD %RSD
80%
1134.729 24.00 100
100.02 0.535 0.53 1141.073 24.138 100.57
1129.105 23.88 99.5
Fluoxetine linearity
conc(mcg/ml) area
9 443.945
13.5 678.1
18 890.786
22.5 1116.004
27 1338.284
R2 0.99992105
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100%
1421.27 30.11 100.36
100.29 0.637 0.63 1430.605 30.29 100.9
1411.907 29.89 99.63
120%
1699.43 36.01 100.02
100.10 0.654 0.65 1713.152 36.30 100.8
1685.736 35.69 99.5
Table 6: Recovery study of FT by HPLC.
Theoritic
al conc. Net area
Concentration
found (µg/ml) %Recovery Avg. SD %RSD
80%
716.41 14.42 100.13
100.15 0.520 0.51 720.407 14.50 100.69
712.864 14.35 99.65
100%
897.188 18.07 100.38
100.38 0.665 0.66 903.125 18.19 101.05
891.34 17.95 99.72
120%
1072.76 21.62 100.09
100.07 0.810 0.80 1081.379 21.79 100.87
1064.073 21.44 99.25
Robustness
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.
Drug Parameter Change Condition 1 Change Condition 2
Sildenafil
citrate
Flow Rate 0.8 ml/min (n=3) 1.2ml/min (n=3)
Avg. area 1661.187 1136.449
STDEV 18.30101 12.404
%RSD 1.101683 1.09147
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Fluoxetine
Average 1049.344 716.933
STDEV 11.5795 7.820517
%RSD 1.103499 1.09083
Sildenafil
citrate
pH(4) 3.8 4.2
Average 1386.167 1413.079
SD 15.256 15.55152
%RSD 1.100589 1.100541
Fluoxetine
Average 873.824 890.794
SD 9.630511 9.782001
%RSD 1.102111 1.098122
Sildenafil
citrate
Mobile phase(50:50) 48:48 52:52
Average 1396.84 1437.142
SD 15.37301 15.79951
%RSD 1.100556 1.09937
Fluoxetine
Average 880.5607 905.9537
SD 9.705011 9.98301
%RSD 1.10214 1.101934
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
Parameters
Tailing Factor 1.37 1.38
Theoretical Plates 7026 7280
RetentionTime (minutes) 3.68 5.31
Resolution - 7.705
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CONCLUSION
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.
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