74

IJPAR |Volume 2 | Issue 2 | April – June -2013 ISSN: 2320-2831

Available Online at: www.ijpar.com

[Research article]
Analytical Method Development And Validation of Dutasteride and
Tamsulosin Hcl in Combination And Its Stress Degradation Studies
*
Raja Sundararajan, Christopher Vasanth Kumar, Jayaveera.
GITAM Institute of Pharmacy, GITAM University, Visakhapatnam – 530045, Andrapradesh,
India.

ABSTRACT
Asimple,specific, sensitive,precise and reproducible Reverse Phase High Performance liquid Chromatography
method has been developed for simultaneous estimation of Dutasteride and Tamsulosin Hcl. Dutasteride and
Tamsulosin is Anti-hyperplasia and Anti-hypertensive drug.The determinationwas carried out byusingsymmetryC-
18columnwith Methanol:0.1M Monobasic potassiumdihydrogenphosphate buffer(75:25) Adjusted the pH to 2.5
with Ortho phosporic acid as the mobile phase and with the detection wavelength of274 nmrespectively.The flow
rate is 0.7 ml/min.TheRetentiontime of Dutasteride,Tamsulosin Hcl was 2.218 minand 6.599 min
respectively.Linearityforthe Dutasteride and Tamsulosin Hcl were found inthe rangeof 25-75µgmand 20-60µgm
respectively.The limitof quantificationforbothdrugs wasfound to be30,24µg respectively.The recoveries of
Tamsulosin and Dutasteride were found to be inthe range of 99.81-99.90 %and98.00-102.00%, respectively. The
proposed methodwas validated suitablyand canbeused forroutine analysis. The degradation studies indicated
Dutasteride andTamsulosin Hcl to besusceptible to neutralhydrolysis, while Dutasteride and Tamsulosin Hcl
showed degradation inacid, H2O2,photolytic and inpresenceof UV radiation.The degradation productsof
Dutasteride andTamsulosin Hcl inacidic and photolytic conditions were well resolved from the pure
drugwithsignificant differences in the irretention time values. This method can be successfully employed
forsimultaneous quantitative analysis of Dutasteride and Tamsulosin Hcl in formulations.
Keywords: HPLC, simultaneous estimation, Dutasteride and Tamsulosin Hcl, stress degradation studies.

INTRODUCTION (R)-5-(2-{[2-(2-ethoxyphenoxy) ethyl] amino}

Dutasteride (DUT) and Tamsulosin (TAM) propyl)-2-methoxybenzene-1-sulfonamide, mono
combination tablets are newly marketed and both hydrochloride. Tamsulosin is an Alpha-blockers help
areused as Alpha-blockers help relieve BPH relieve BPH symptoms agents two compounds are
symptoms agents. DUT is chemically (5α, 17β)-N- active in the metabolism of body.In recent times,
{2, 5bis (trifluoromethyl) phenyl}-3-oxo-4- there is an increased tendency towards the
azaandrost-1-ene-17-carboxamide.Dutasteride is 5- development of stability indicating assays, using the
alphareductase enzyme inhibitors. Tamsulosin is a approach of stress testing as enshrined in the

* Corresponding author: Rajasundara Rajan.

E-mail address: [email protected]
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International Conference on Harmonization of DUT and 40 mg of TAM were prepared by

1,2. dissolving in mobile phase, in a 10 ml volumetric
ICH)guideline Even this approach is being
extended to drug combinations inderivative flask and made up to the volume. Final dilutions
3
were made to 50 µg/ml of DUT, 40 µg/ml of TAM
spectroscopy . There is no stability-indicating assay
method reported yet for this combination developed were prepared by dissolving in mobile phase, in a 10
4-6. ml volumetric flask and made up to the volume were
using the ICH approach of stress testing There are
stored under refrigeration. From the above solutions
several LC-/MSMS procedures known for the
7-9
. the dilutions of working standards were made from
analysis individualy .There is several RP-HPLC, 25-75 µg/ml DUT and 20-60 µg/ml for TAM
10-
TLC procedures known for the analysis individualy respectively.
13
.It is needed to develop a method without any
drawbacks and only very few methods have been CALIBRATION CURVE
reported for estimation of DUT and for TAM by The calibration curves were constructed for the
simultaneous estimation individually and by HPLC determination of the linearity and the curves were
method. plotted with the concentration range verses area
must obey Beer’s law. The linearity was evaluated
MATERIALS AND METHODS by analysis of the serially diluted sample in the
Pharmaceutical grade DUT and TAM were supplied range of 25-75 µg/ml and 20-60 µg/ml for DUT and
by Orchid chemicals, Chennai, India, with the purity TAM respectively. An aliquot was injected using
of 99.70% and 99.50% respectively on dried basis. mixture of Methanol and Buffer solution in the ratio
Methanol HPLC grade of merck, NaOH AR grade, of 75:25 v/v. The retention times were 2.218 min
Potassium di hydrogen ortho phosphate AR grade, and 6.599 min for DUT and TAM respectively with a
ortho phosphoric acid AR grade, Hydrochloric acid good resolution of 12.76.
AR grade, Purified Water milli-Q water, were used
for the analytical purpose. Waters HPLC System ANALYSIS OF FORMULATIONS
2965, with Symmetry C18 column (150x4.6 mm, 0.5 Analysis of the tablets was conducted in only one
µm) and Dual λ absorbance Detector 2996 worked in brand. Twenty tablets of the brand were weighed
room temperature. and powdered separately. A quantity equivalent to
480 mg of DUT and TAM were transferred to 10 ml
volumetric flask and dissolved 10 ml of Mobile
CHROMATOGRAPHIC CONDITIONS
phase.
The separation the drugs were performed by using
symmetry C 18 (150 × 4.6 mm, 5μm particle size) STRESS DEGRADATION STUDIES
column. Mobile Phase consisted of a mixture of Forced degradation studies of both the drugs were
Methanol: water (75: 25 v/v) with a Flow rate of 0.7 carried out under conditions of Acid hydrolysis,
ml/minute. Volume of injection is 20:l and the Alkali hydrolysis, Peroxide oxidation, UV light and
detection wavelength was 274 nm. photolysis. DUT and TAM were weighed
(50 mg and 40mg) and transferred into 10 ml
Preparation of Phosphate buffer
volumetric flasks and diluted up to the mark with
Weighed 7.0 grams of KH2PO4 into a 1000ml beaker,
mobile phase.
dissolved and diluted to 1000ml with HPLC water.
Adjusted the pH to 2.5 with Ortho phosporic acid. Acid hydrolysis
Mobile Phase was prepared by mixing 750 ml of To 10 ml of the drug solution, 10 ml of 1 m
Methanol HPLC Grade with 250 ml of Potassium di hydrochloric acid was added and the mixture was
hydrogen ortho Phosphate buffer in a 1000 ml refluxed on a water bath for 60 minutes at 90º. The
standard flask to get the proportion of 75:25 v/v. forced degradation in acidic media was performed in
The mobile phase was filtered through 0.45 micron the dark in order to exclude the possible degrative
membrane filter and degassed by Ultrasonication effect of light. The resulting solution was neutralized
for 15 min. The standard stock solutions of 50 mg by the base, to avoid any interference of acid or base.
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20 µl of the resulting solution was injected into suitability tests were carried out by five replicate
HPLC and the chromatograms were recorded. injections, with a constant concentration 50 μg/ml, 40
μg/ml DUT and TAM. The % Relative standard
Alkali hydrolysis deviation of peak area and the retention time were
To 10 ml of the drug solution, 10 ml of 0.1 m sodium within the limit of ±2%. This indicates that the
hydroxide was added and the mixture was refluxed method was system suitable. The linearity of DUT
on a water bath for 60 minutes at 90º. The forced and TAM were determined by calibration curves and
degradation in basic media was performed in the dark the linearity based on the area observed in the range
in order to exclude the possible degrative effect of of 25-75 μg/ml and 20-60 μg/ml respectively. The
light. The resulting solution was neutralized by the 2
regression co-efficient value (r ) for DUT and TAM
acid, to avoid any interference of acid or base. 20 µl
is 0.9999 and 0.9997 respectively The limit of
of the resulting solution was injected into HPLC and
quantification was determined by injecting minimum
the chromatograms were recorded.
concentration of the drugs .The limit of quantification
Peroxide oxidation was found to be 30, 24 µg/ml for DUT and TAM
To 10 ml of the drug solution, 10 ml of 3% v/v respectively the results are tabulated in Table 1.
hydrogen peroxide was added and the solution was Precision was measured for both inter and intra-day,
kept aside for an hour. After an hour, 20 µl of the and checked with repeatability and the %RSD for the
resulting solution was injected and the chromatogram repeatability was found to be 0.73% and 0.80% for
was recorded. DUT and TAM respectively. The RSD was found to
within the limit and tabulated in Table 2.Since both
UV treatment the drugs were stable for 24 hr only even under
10 ml of the drug solution was taken in a beaker and refrigeration condition; only intra-day precision
the solution was kept in an up chamber at shorter studies were conducted the results are tabulated in
wavelength region for an hour. After an hour, 20 µl Table 2. Analysis of the tablets was performed in
of the resulting solution was injected and the one brand containing 500 mg and 400 mg of the
chromatogram was recorded. drugs as label claim. An average quantity of DUT
and TAM were 500.943±0.004 and 400.037±0.002
Sun light treatment respectively and has conformation with the label
10 ml of the drug solution was taken in a beaker and claim. The results are tabulated in Table 3. The
the solution was kept in sunlight for an hour. After an accuracy was studied by the recovery studies. The
hour, 20 µl of the resulting solution was injected and recovery studies are usually made by spiking the
the chromatogram was recorded. known amount of pure drug with the formulation.
It is usually done by adding 50%,80%,100 %, 120
% and 150 % of the pure drug with the formulation
RESULT AND DISCUSSION
taken for analysis. The average % recovery for DUT
In method development phase, initially both the drugs
and TAM was found to be 99.88 % and 99.99 % for
showing asymmetry factor more than 2 in Methanol:
Brand drug.The results are tabulated below in Table
water, with a run time of more than 8.0 min. Then the
4. Forced degradation studies of both the drugs DUT
mobile phase was shifted to Methanol: Phosphate
and TAM were carried out under conditions of Acid
buffer, showed a good result. At the reported Mobile
hydrolysis RT 2.218 min, 6.605min, Alkali
phase proportion of 75:25, DUT and TAM showed a
hydrolysis RT 2.222 min, 6.573 min, Peroxide
retention time of 2.219 min and 6.607 min
oxidation RT 2.221 min, 6.608 min, UV light RT
respectively at the flow rate of 0.7 ml/min. The
2.220 min, 6.573 min and photolysis RT 2.209 min,
wavelength for the determination was selected at 274
6.531 min. All the results of accelerated degradation
nm for both the drugs. The tailing factor, resolution
study for Dutasteride and were Tamsulosin given in
and peak shape were found to be good in the finally
Table.5. The peaks are shown in Fig 2-6.
reported condition for both the drugs. The peaks are
shown in Fig 1. As per ICH guidelines, system
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TABLE 1: SYSTEM SUITABILITY PARAMETERS

PARAMETERS DUT TAM

Calibration Range (mcg/ml) 25-75 20-60

Correlation Coefficient(r2) 0.9999 0.9997

Retention time(Min) 2.219±0.2 6.607±0.2

Regression equation(y=mx+c)

Slope (m) 12591 9665

Intercept(c) -6215 -9883

Theoretical Plates 2659 3496

Resolution factor 12.76 --

Tailing Factor 1.7 1.03

Selectivity 1.78 --

Repeatability %RSD (n=5) 0.73% 0.80%

Limit of quantification 25 20

System suitability parameters are the data’s performed to check the system testing and
the data’s are based on the ICH Guidelines

TABLE: 2 PRECISION STUDY

DUT TAM
Conc μg/ml % RSD Conc μg/ml %RSD

50 0.73 40 0.80

Precision studies are done to confirm the repeatability and the stability in a day.
RSD stands for Relative standard deviation taken for three readings.
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TABLE 3: ANALYSIS OF MARKETED FORMULATION

DUT TAM
% assay
% assay
Formulation Label claim mg/tab Label claim mg/tab
± RSD
± RSD

Brand 500 99.88±0.08 400 99.99±0.02

Formulation analysis was done in brand. * stands for the average reading taken in three readings.

TABLE 4: RECOVERY STUDIES OF TAM AND DUT COMBINED DOSAGE FORM

DUT TAM

Formulation % added % recovery % added % recovery

± RSD ± RSD
50 99.81±0.085 50 100.15±0.014
100 99.93±0.090 100 100.09±0.012
Brand 150 99.90±0.105 150 100.22±0.045

Recovery experiment data for Tamsulosin and Dutasteride showing the amount of drug
recovered from sample solution at each level (n=3), percentage recovery and the average
percentage recovery.

TABLE 5: ACCELERATED DEGRADATION STUDIES

5.1. DUTASTERIDE:

Alkali Peroxide Heat Photolytic

Test No Unstressed Acid stress
Stress Stress stress Stress

Average
weight 480mg

Weight
480.2mg 481.2mg 480.8mg 482.0mg 481.5mg 479.8mg
Taken(in mg)

Area 546148 505212 489264 492754 482926 502814

Assay
(in mg) 50 46.19 44.73 45.05 44.15 45.97
Assay
101.5 92.38 89.46 90.10 88.30 91.94
(in %)
%Degradation NA 3.81 5.27 4.95 5.85 4.03
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5.2 TAMSULOSIN HCL

Alkali Peroxide Heat Photo

hotolytic
Test No UnstreessedAcid stress
Stress stress stress Stress
ss

Average
480mg
Weight

Weight
Taken(in mg) 480.2 481.2 480.8 482mg 481.5mg 479.8mg

Area
363218 303182 331830 298214 320818 299830

Assay
(in mg) 40 33.488 36.6526 33.071 33.456 30.098

Assay
(in %) 100 83.7206 91.6315 82.6791 88.59 82.882

%Degradation NA 16.2794 8.3685 17.3209 11.41 17.118

Fig 1: A Typical
ical Chr
Chromatogram for Dutasteride and Tamsulosin
n
(Concenttration of 50 mcg DUT and 40 mcg TAM)
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Fig 2: A Typical Chrom

matogram for Dutasteride and Tamsulosin - Acid
id stress

Fig 3: A Typical Chrom

matogram for Dutasteride and Tamsulosin - Alkali
ali stress
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Fig 4: A Typical Chromaatogram for Dutasteride and Tamsulosin - Peroxi

oxide stress

Fig 5: A Typical Chroomatogram for Dutasteride and Tamsulosin - UV stress

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Fig 6: A Typical Chrom

matogram for Dutasteride and Tamsulosin - Photolytic stress

CONCLUSIONS with no interference from excipieents. In almost all the

The isocratic RP-HPLC C method ddeveloped for cases, chromatographic pattern n was
w similar .The
quantitative determination of Tamsu msulosin and method could be applied with h success
su even to the
Dutasteride simple, specific, sensi nsitive, precise, analysis of marketed products,
ts, as no interference
in was
reproducible and validated stability-indic
indicating HPLC observed due to excipientsnts or other components
method for simultaneous estimation
mation of Dutasteride present.
and Tamsulosin in the presence oof degradation
products. The method was completelyy validated and ACKNOWLEDGEMENTS
ENTS
satisfactory results were obtained for aall the method The authors are grateful ful to Dr.Reddys Lab
validation data tested. A clear separation
on of the drugs Hyderabad, India. For providingding Tamsulosin and
and degradation products was achieveed in the tablet Dutasteride a gift sample.

REFERENCES
[1] Vijaya P. Godse, Ashok V. Bhosale, Yogesh S. Bafana, Dhanaraj D. Borkar, ICH guidance in practice:
Validated stability-indicating
indicating HPLC method for simultaneous determination of Olmesartan medoximil
andHydrochlorothiazide in combination drug products.Eurasian J. Anal. Chem. 5(2): 137-144,
137 2010
[2] ICH, Q1 (B) (1999) Stability testing: Photo stability testing of new drug substances.
substances And products, in:
International Conference on Harmonization, IFPMA, Geneva, 199.
[3] M. Kazemipour, M. Ansari, H. Ramezani1 and M. Moradalizadeh
Moradalizadeh, Simultaneous determination of
Dutasteride and Tamsulosin in tablet by first and third derivative spectrophotometry and H-point
H standard
addition methods: Research in Pharmaceutical Sciences, May 2012; 7(2): 95 95-102.
[4] A. Pathak and S.J. Rajput Development of a Stability-Indicating
Indicating HPLC Method for Simultaneous
Determination of Olanzapine and Fluoxetine in Combined Dosage Forms. Journ Journal
al of Chromatographic
Science, Vol. 47, August 2009
2009.
[5] Bhatia M. Sudesh and Kokil S. Uttamrao
Uttamrao, Determination and validation of valsartan and its degradation
products by isocratic HPLC. J. Chem. Metrl
Metrl. 3:1 (2009) 1-12.
[6] Faiyaz Shakeel, Sanjula Baboota, Alka Ah
Ahuja,
uja, Javed Ali and Sheikh Shafiq, Accelerated stability testing of

www.ijpar.com
83
Raja Sundararajan et al / Int. J. of Pharmacy and Analytical Research Vol-2(2) 2013 [74-83]

celecoxib nano emulsion containing Cremophor-EL. African Journal of Pharmacy and Pharmacology Vol.2
pp. 179-183, October, 2008.
[7] Ramakrishna NVS, Vishwottam KN ,Puran S, Koteshwar M, Manoj S,Santosh M. Selective and Rapid
Chromatography –tandem mass spectrometry assay of Dutasteride in Human plasma. Journal of
Chromatography B 2004 Sep; 809(1):117-24.
[8] Macek J, Klíma J and Ptácek P. Rapid determination of tamsulosin in human plasma by high-performance
liquid chromatography using extraction with butyl acetate. Journal of Chromatography B 2004 Oct;
809(2):307- 11.
[9] Nageswara rao R, kumar Talluri MVN, Narasu Raju A, Ramanjen eyulu GS, Shinde and Dhananjay D.
Development of a validated RP-LC/ESI-MS-MS method for separation, identification and determination of
related substances of Tamsulosin in bulk drugs and formulations. Journal of Pharmaceutical and
Biomedical Analysis 2008 Jan; 46(1):94-103.
[10] Chandorkar JG, Kotwal VB, Dhande NS, Gurav SG, Pande VV and Yadav PVA sensitive HPLC method
for simultaneous estimation of Tamsulosin hydrochloride and its impurity. Journal of pharmaceutical
analysis 2008; 21(3):307-10.
[11] Sanjay Dinkar Sawant, Minal Rushikesh Ghante, Asmita Shripad Deshpande and Neha Manish Munot.
Development and validation of RP-HPLC method for the estimation of Tamsulosin hydrochloride in bulk
drug & pharmaceutical dosage form. Journal of pharmacy research 2010 Oct; 3(11):2776-78.
[12] Patel DB, Patel NJ, Prajapati and Patel . RP-HPLC method for the estimation of Dutasteride in 5 tablet
dosage form. Indian journal of pharmaceutical science 2010 Jan; 72(1):113-116.
[13] Patel DB and Patel NJ. Validated RP-HPLC and TLC methods for simultaneous estimation of
Tamsulosin hydrochloride and finesteride in combined dosage form. Indian journal of pharmaceutical
science 2010 Feb; 60:197-05.
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