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Manasa.

et al

Volume 1(3), 2014, Page 100-105

International Journal of Medicine and


Nanotechnology
Access online at www.medtechnano.com

Original Article

Analytical Method Development and Validation of


Hplc & Its Stability Studies

Exemestane by Rp-

T.Mani Manasa* , Pritosh Pattnaik, Arabinda patnaik, K.V. Subrahmanyam


Samskruti College Of Pharmacy, Ghatkesar, Hyderabad , Telengana, India.
Article Received on: 09-11-2014

Accepted on: 29-12-2014

ABSTRACT
A simple, sensitive, precise and Reverse phase high performance liquid chromatographic method has been developed for the quantitative analysis of
Exemestane drug present in drug substance. A suitable HPLC having a isocratic system equipped with manual injector , UV detector is used for this
work. The HPLC separation was achieved on HITACHI L2130 with D Elite 2000 Software with Isocratic with UV-Visible Detector (L-2400).
Develosil ODS HG -5 RP 15cm 4.6 mm 5m particle size and column temperature 25c used as stationary phase. The mobile phase used in this
analysis consists of a mixture of phosphate Buffer (0.01M potassium dihydrogen phosphate & pH adjusted to 2.95 with ortho phosphoric acid ) and
Acetronitrile in a ratio of 30:70. Stock sample is prepared by using acetonitrile and buffer and working sample used is about 100ppm. Flow rate
maintained is about 1.0ml/minute and wave length is about 246nm. Sample colour is Ambient. Injection volume injected about 20L with run time
10 minutes . The proposed method provided linear responses within the concentration range 140ppm for Exemestane . LOD and LOQ values for the
active substance were 0.5 and 1.0 g ml, respectively. Regression equations for the drug substance is about 0.994 in all cases. The precision of the
method was demonstrated using intra-day and inter day assay RSD% values which were in acceptance limit (2%) in all instances. The proposed
method was found to be accurate, precise, reproducible and specific and it can also be used for routine quality control analysis
Key words:
RP-HPLC, Exemestane, method development and validation , acetonitrile , buffer .

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Corresponding Author
T.Mani Manasa *
Samskruti College Of Pharmacy,
Ghatkesar, Hyderabad ,
Telengana, India Hyderabad
Phone: +91- 919618034189
E-mail:tmanumanasa@gmail.com

INTRODUCTION
Exemestane1 is an oral steroidal aromatase inhibitor2
used in the adjuvant treatment of hormonallyresponsive (also called hormone-receptor-positive,
estrogen-responsive) breast cancer in postmenopausal

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Volume 1(3), 2014, Page 100-105

women. It acts as a false substrate for the aromatase


enzyme3, and is processed to an intermediate that
binds irreversibly to the active site of the enzyme
causing its inactivation. According to literature
survey4 it was found that method development and
validation of Exemestame by RP-HPLC5 was not
accurate showing deviation in system suitability
parameters. It was found that all the methods were
developed using isocratic elution technique in RPHPLC. Hence the objective of the present work is to
develop a new method, its validation and forced
degradation studies of Exemestame by RP-HPLC and
its application to marketed formulation.

Acetonitrile and buffer were mixed in the ratio of


70:30 and filtered through 0.45m nylon membrane
filter and degassed in a sonicator for 10 minutes. The
final pH of the mobile phase was adjusted to 2.95.
PREPARATION OF BUFFER SOLUTION7:

2.625 gms of Potassium dihydrogen phosphate &


0.2625 gms dipotassium hydrogen phosphate was
weighed accurately and placed in a beaker filled with
500 ml of HPLC grade water. Shake well, this forms
5mM phosphate buffer, to this 500l of ortho
phosphoric acid was added (0.1 % ortho phosphoric
acid) and sonicated for 15 min. This solution was
filtered through 0.45m filter.
PREPARATION OF STANDARD SOLUTION8:

5mg of Exemestane (API) was weighed accurately


and taken into 5ml of volumetric flask. To this 3ml of
diluent was added and sonicated for few minutes and
the volume was made up to 5ml using diluent and it
was filtered through 0.45m filter paper.

Preparation of Working Stock Solution:(100g/ml)


Transfer 5 ml of stock solution previously prepared
into 50ml of volumetric flask. To this 25ml of diluent
[ACN: buffer (70:30)] was added, sonicated for 15
min and volume is made up to 50 ml using diluent.
60

PREPARATION OF MOBILE PHASE :

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50
40
30
20
10

8.67

Optimised Chromatographic conditions:


The mobile phase Buffer:ACN(30:70), were used in
this study. The conditions optimized were: flow rate
(1 ml/minute), Injection volume ( 20l ), wavelength
(246 nm, monitored by UV detector) and run time was
10 min, column temperature was maintained at 250C.
Retention time was found to be 3.33 min. Develosil
ODS HG-5 RP C18, 5m, 15cmx4.6mm i.d. column
was used as stationary phase in the study.

Preparation of Stock Solution:(1000g/ml)

3.33

Instruments and Reagents: The chromatographic


separation was performed on HPLC (Hitachi L2130)
with D2000 Elite Software with Isocratic--Gradient
with UV-Visible Detector Develosil ODS HG-5 RP
C18, 5m, 15cmx4.6mm i.d. column was used as a
stationary phase. PH Analyzer (Buchi Melting Point
540/545), Electronic Balance, Ultra Sonicator (
Bandelin Ultra Sonic Cleaner) has been used in the
work. Exemestane Active Pharmaceutical Ingredient
(API) was provided by Dr. Reddys Laboratories,
Hyderabad, A.P. Chemicals were obtained from Merck
Laboratories. Acetonitrile, Methanol, & water of HPLC
grade were from Standard reagents, Hyderabad.
Ortho-phosphoric acid (S.D Fine chemicals, Mumbai).

2.31

MATERIALS AND METHOD

Intensity (mV)

Fig 1: EXEMESTANE

5mg of Exemestane (API) was weighed accurately


and taken into 5ml of volumetric flask. To this 3ml of
diluent was added and shaken well. It was sonicated
for few minutes and the volume was made up to 5ml
using diluent. It gives 1000g/ml concentration.Then
the Solution was filtered through 0.45 m Millipore
HVLP filter and finally 5.0 ml of this solution was
diluted to 50 ml with mobile phase to make final
concentration 100g/ml Exemestane.

0
0

10

Retention Time (min)

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Volume 1(3), 2014, Page 100-105

Fig 2 : Chromatogram of Exemestane (100 ppm) in optimized


conditions (RT 3.33 min)
9

Method Validation: As per the ICH guidelines, the


method validation parameters checked were linearity,
accuracy, precision, limit of detection, limit of
quantitation.
Preparation of Calibration Curves
Calibration curve was prepared by taking
appropriate aliquots of standard Exemestane stock
solution in different 10 ml volumetric flask and
diluted up to the mark with diluents to obtain the final
concentrations of 40, 60, 80, 100, 120 ,140 g/ml of
Exemestane. Standard solutions (n=6) were injected,
the sample volume was 20 l with a flow rate of 1.0
ml/min., the results shown in fig

Mother Sample: Before carry out the degradation


studies the mother sample was prepared to find out the
% degradation. It is prepared as the same method
followed in sample or standard preparation.
PREPARATION OF STOCK SOLUTION:

10 mg of Exemestane was weighed accurately and


transferred in to 10 ml of volumetric flask. To this 5
ml of acetonitrile was added and remaining volume
was made up to 10 ml using buffer solution. (1000
g/ml) from this, 1 ml of solution was transferred into
another 10 ml volumetric flask and the volume was
made up to 10 ml using diluent solution (100 g/ml)
PREPARATION OF DILUENT:

20 ml of diluent was prepared by taking 10 ml of


acetonitrile and 10 ml of buffer solution and mixed
sonicated for few minutes.
ACID DEGRADATION:
PREPARATION OF 0.1 N HCl FROM 1 N HCl:

500 ml of 1 N HCl. was transferred into 5 ml of


volumetric flask and volume made up to 5 ml using
water.
PROCEDURE:

INJ 1: Acid control solution

INJ 2: Acid control solution

0.5 ml diluent (ACN: buffer) was taken in aINJ. To


this 0.5ml of 0.1 N HCl was added. Then mixed it
thoroughly. The two INJs are kept aside for 6 hours
without disturbing and then injected for analysis. The
results are shown in the graph.
3.58

70
60
50
40
30
20
10

2.59

The protocol was strictly adhered to for forced


degradation of Exemestane10 Active Pharmaceutical
Ingredient (API). The API (Exemestane) was
subjected to stress conditions in various ways to
observe the rate and extent of degradation that is
likely to occur in the course of storage and/or after
administration to body. This is one type of accelerated
stability studies that helps us determining the fate of
the drug that is likely to happen after long time
storage, within a very short time as compare to the
real time or long term stability testing. The various
degradation pathways studied are acid hydrolysis,
basic hydrolysis , oxidative ,Thermal and Photolytic
(UV) degradation.

2.03
2.23

Forced degradation studies:

0.5 ml stock (100g/ml) solution was taken in aINJ.


To this 0.5 ml of 0.1 N HCl was added. Then mixed it
thoroughly.

Intensity (mV)

Fig 3: Calibration curve of Exemestane

0
0

10

Retention Time (min)

Fig 4:Chromatogram showing degradation in 0.1 M HCl

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BASIC DEGRADATION:
PREPARATION OF 0.1N NaOH: 200mg of NaOH

40

2.59

2.24

2.01

0
0

10

Retention Time (min)

Fig 6 :Chromatogram showing degradation in 30 % H2O2

FOR THERMAL DEGRADATION:

INJ 3: Alkali control solution

0.5 ml stock (100g/ml) solution was taken in aINJ.


To this 0.5 ml of 0.1 N NaOH was added. Then mixed
it thoroughly.
INJ 4: Alkali control solution

3.58

0.5 ml diluent (ACN: buffer) was taken in aINJ. To


this 0.5ml of 0.1 N NaOH was added. Then mixed it
thoroughly. The two INJs are kept aside for 6 hours
without disturbing and then injected for analysis. The
results are shown in the graph.
80
Intensity (mV)

60

20

was weighed accurately and transferred in to a 50 ml


volumetric flask and dissolved in nanopure water and
volume made up to 50 ml.
PROCEDURE:

3.57

Intensity (mV)

80

60

40

INJ 8: 0.5 ml of stock solution + 0.5 ml of diluent


heated for 1 hr an automatic heater at
60C
maintaining constant temperature and then injected for
analysis. The results are shown in the graph.
INJ 9: 0.5 ml of stock solution + 0.5 ml of diluent
heated for 1 hr an automatic heater at 80C
maintaining constant temperature and it were then
injected for analysis. The results are shown in the
graph.
INJ 10: 0.5 ml of stock solution + 0.5 ml of diluent
heated for 30 min at an automatic heater at 100C
maintaining constant temperature and it was then
injected for analysis. The results are shown in the
graph.

10

Retention Time (min)

25
20
15
10

Fig 5: Chromatogram showing degradation in 0.1 M NaOH

2.59

30

Intensity (mV)

0
0

3.55

35

2.60

2.03
2.25

20

0
0

FOR OXIDATIVE DEGRADATION:

10

Retention Time (min)

30% H2O2 was taken from the lab itself

Fig 7: Chromatogram showing thermal degradation

PROCEDURE

FOR PHOTOLYTIC DEGRADATION:

INJ 5: Peroxide control solution

INJ 7: 0.5 ml of stock solution + 0.5 ml of diluents


Placed under UV light for 1 hr and then injected. The
results are shown in the graph.
70

3.27

0.5 ml stock (100g/ml) solution was taken in aINJ.


To this 0.5 ml of 30% H202 was added. Then mixed it
thoroughly.

50
40
30

10

2.37

20
2.05

0.5 ml diluent (ACN: buffer) was taken in aINJ. To


this 0.5ml of 30% H202 was added. Then mixed it
thoroughly. The two INJs are kept aside for 6 hours
without disturbing and then injected for analysis.

Intensity (mV)

60

INJ 6: Peroxide control solution

0
0

10

Retention Time (min)

Fig 8 :Chromatogram showing uv degradation

The results are shown in the graph.

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RESULTS:

For the results shown in forced degradation


studies it was found that Exemestane was stable in
acidic conditions, alkaline conditions, peroxide
(H2O2) ,photolytic degradation conditions and also at
thermal conditions 60 C, 80 C & 100 C.
Results of force degradation studies of Exemestane
Method Validation
Linearity and Range :The calibration curve showed
good linearity in the range of 40-140 g/ml, for
Exemestane (API) with correlation coefficient R =
0.996 (Fig. ). A typical calibration curve has the
regression equation of y = 12079x + 54574 for
Exemestane. From the linearity table it was that the
drug obeys Beers law and from the linearity studies
the Specified Range for Exemestane was found to be
40 g/ml to 140 g/ml .The linearity graph determines
the accurate results with R2 value 0.994.
ACCURACY:
Recovery Study: To determine the accuracy of the
proposed method, recovery studies were carried out
by adding different amounts (80%, 100%, and 120%)
of pure drug of Exemestane were taken and added to
the pre-analyzed formulation of concentration
30g/ml. From that percentage recovery values were
calculated.From the results shown in accuracy table, it
was found that recovery value of pure drug from the
reanalyzed solution of formulation were between 90
% & 110 % which indicates that the method is
accurate also reveals that commonly used excipients
and additives present in the pharmaceutical
formulation were not in the proposed method.

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Precision RSD was found to be 0.077, 0.167, and


0.319 for 24g/ml, 30g/ml and 40g/ml sample
solution of Exemestane.
INTRA & INTER DAY PRECISION :

The intra & inter day variation of the method was


carried out & the high values of mean assay & low
values of standard deviation & % RSD (% RSD < 2%)
within a day & day to day variations for Exemestane
revealed that the proposed method is precise.
Table 1: Results of force degradation studies
Stress Time Assay of Assay of Mass
No. Of
condition
active
degraded Balance impurity
substance products
(%)
Peaks

Acid Hydrolysis
(0.1 MHCl)

6Hrs.

99.82

_______

99.82

Basic Hydrolysis
(0.I MNaOH)

6Hrs.

99.32

-----------

98.32

Thermal
600C 1Hr.
Degradation
800C 1Hr.

99.29

-----------

98.29

99.02

______

99.02

1000C 1Hr.

98.93

_____

98.93

Peroxide

6Hrs.

99.69

----------

99.69

UV

1Hr.

99.77

----------

99.77

PRECISION: Repeatability

LOD & LOQ:

The precision of each method was ascertained


separately from the peak areas & retention times
obtained by actual determination of six replicates of a
fixed amount of drug Exemestane(API). The percent
relative standard deviation were calculated for
Exemestane are presented. The precision was found to
be within the limits the limit were not more than
RSD<2%.

The limit of detection and limit of quantification


were determined based on their respective single to
noise ratio at 0.5 g/ml = 3.1 and 1.0 g/ml = 15.5
respectively.
METHOD ROBUSTNESS:
Influence of small changes in chromatographic
conditions such as change in flow rate ( 0.1ml/min),

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Volume 1(3), 2014, Page 100-105

Temperature (20C), Wavelength of detection (2nm)


& Acetonitrile content in mobile phase (2%) studied
to determine the robustness of the method are also in
favour of (% RSD < 2%) the developed RP-HPLC
method for the analysis of Exemestane( API).

2. Robinson A: A review of the use of exemestane in


early breast cancer. Ther Clin Risk Manag. 2009
Feb;5(1):91-8. Epub 2009 Mar 26.
3. Untch M, Jackisch C: Exemestane in early breast
cancer: a review. Ther Clin Risk Manag. 2008
Dec;4(6):1295-304.

SYSTEM SUITABILITY:

4. Instrumental Methods of Chemical Analysis


by B.K. Sharma, pp.75-78, 113-115.

The system stability parameter also reveals


that the values are within the specified limit for the
proposed method. Tailing factor limit upto 2 result
0.089,capacity factor limit >2 result 2.764,column
efficiency limit >2000 Plates result 86544

5. Instrumental Methods of Chemical Analysis,


Vth Ed., by Galen W. Ewing, 1.
6. Pharmaceutical Analysis, Ist edition, by
Takeru
Higuchi,
EinarBrochmann,
HanffenHanssen, 1-10.
7. Practical Pharmaceutical Chemistry, IV
edition, Volume II, by A.H. Beckett, J.B.
Stenlake, 275-298.

Table 2 : Validation Parameters by Rp-Hplc Method


Validation parameters

Exemestane
% interference <0.5 %

Specificity
Range (g/ml)

Linear range

40-140 g/ml

Working range

0.03-100 g/ml

Target range

44,55,60.5 g/ml

Target concentration

55 g/ml

Accuracy (% Recovery) 80, 100, 120


Precision
RSD)

(%Repeatability
Intraday(80,100,120
g/ml)
Inter
g/ml)

100.4,101.0,99.8

8. ICH, Q6A: Specifications: Test Procedures and


Acceptance Criteria for New Drug Substances and
New Drug Products: Chemical Substances
(Geneva, Oct. 1999).

9. ICH, Q3A(R2) Impurities in New Drug


Substances (Geneva, Oct. 2006).
10. Quality assurence, worth the effort, Inforum,
october2003 volume 7;number.4.

0.229
0.22, 0.14, 0.07

day(80,100,120 0.31, 0.50, 0.11

LOD (g/ml)

0.5

LOQ (g/ml)

1.0

CONCLUSION
A sensitive& selective RP-HPLC method has been
developed & validated for the analysis of Exemestane
API.Further the proposed RP-HPLC method has
proved for the excellent sensitivity, precision and
reproducibility. The result shows the developed
method is yet another suitable method for assay,
purity & stability which can help in the analysis of
Exemestane in different formulations.
REFERENCES:
1. http://www.drugbank.ca/drugs/DB00990

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