DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC ... · PDF fileDEVELOPMENT AND VALIDATION OF STABILITY ... FLUOXETINE IN BULK & TABLET DOSAGE FORM. ... HPLC Method Validation

International Bulletin of Drug Research., 4(6): 92-105, 2014

92

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

mailto:[email protected]


93

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


94

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.


95

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%.


96

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


97

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)


98

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


99

Fig. 5(c). SC+FT oxidative degradation sample.

Fig.5 (d): SC+FT hydrolysis degradation sample.

Fig. 5(e): SC+FT thermal degradation sample.


100

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


101

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


102

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


103

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


104

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.

REFERENCES

1. Tripathi KD, Essential of Medical Pharmacology, 4th ed., Jaypee Brothers, new delhi,

522-553 (2001).

2. 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).

3. Crossfire B, 2D structure of sildenafil gave one hit: information about the synthesis,

The Bioorganic & Medicinal Chemistry Letters, 1819 (1996).

4. ICH Steering Committee, ICH Q2B Validation of Analytical Procedures,

methodology. European Agency for the Evaluation of Medicinal Products,

International Commission on Harmonization, Switzerland, (1996).

5. 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.

6. 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).

7. 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).

8. Abourashed, Abdel-Azim M, Habib, HPTLC determination of sildenafil in

pharmaceutical products, journal of planner chromatography, 18, 372-376(2007).

9. Hefnawy, Rapid determination of Sildenafil citrate in pharmaceutical preparation

using monolithic silica HPLC column, Journal of Liquid Chromatography Related

Technologies, 26,1082-6076(2003).

10. Khan, A validated HPLC method for the measurement of Sildenafil citrate in different

formulations, Pakistan Journal of Science, 62 192-194(2010).


105

11. Patel S, Patel NJ, Simultaneous RP-HPLC and HPTLC estimation of Fluoxetine HCl

and Olanzapine in tablet dosage, Inventi Rapid, Pharm Analysis & Quality

Assurance, 54 (2010).

12. Kalaichelvi, Radha GT, Bindhu T, Quantitative UV Spectrophotometric estimation of

Sildenafil citrate by hydrotropic technique, International Journal of Pharmacy and

Pharmaceutical Sciences, 4, 171-172 (2012).

13. Salem H, spectrochemical methods for the determination of Sildenafil citrate(viagra)

in bulk powder and in pharmaceutical dosage form, japanese journal of applied

science, 8, 2, 28-43 (2008).

14. Patel Sejal, Simultaneous RP-HPLC and HPTLC estimation of fluoxetine HCl and

Olanzapine in tablet dosage form, Indian Journal of Pharmaceutical Sciences, 71, 4,

477-480 (2008).

15. Sharma BK, Instrumental Method of Chemical Analysis, 21st edn., Goel Publishing

Housing, Krishna Prakashan Ltd., 3-10 (2007).

16. Baokar Shrikrishna, Patil, Jagatap Rashmi, Validation of simple and Rapid UV-

Spectrophotometric method with stress degradation study for Sildenafil citrate,

Research J. Pharm. and Tech., 214-218 (2012).

17. Shuklab Shubhanjali, Moorthya Hari Narayana, Sushant Kumar, RP-HPLC method

development and its validation for simultaneous estimation of Alprazolam and

Fluoxetine HCl in pharmaceutical dosage form, Eurasian J. Anal. Chem, 5, 3, 239-

245(2010).

18. Kumbhar Smita, Prafulla B, Choudhari, Simultaneous estimation of Fluoxetine and

Norfluoxetine in plasma by RP-HPLC employing pre-column derivatization for UV-

sensitivity enhancement, open access journal, 14 (2012).

19. Rubesh kumar, Duganath N, Kiran CH, Sridhar c, Simultaneous estimation of

Fluoxetine HCl and Olanzapine in bulk drug and pharmaceutical formulation by using

UV-Visible spectroscopy method, International Journal of Pharmaceutical Sciences

and Drug Research, 3, 1, 52-55 (2011).

Documents

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC ... · PDF fileDEVELOPMENT AND VALIDATION OF STABILITY ... FLUOXETINE IN BULK & TABLET DOSAGE FORM. ... HPLC Method Validation