Research Journal of Recent Sciences ________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 281-288 (2012)
Res.J.Recent.Sci.

Stability-Indicating LC Method for the Determination of Epinastine in Bulk
Drug and in Pharmaceutical Dosage Form
Ahirrao V.K. and Pawar R.P.
Department of Chemistry, Deogiri Science College, Aurangabad 431004, MS, INDIA

Available online at: www.isca.in
(Received 8th Decembert 2011, revised 10th January 2012, accepted 6th Fabruary 2012)

Abstract
A novel stability-indicating LC assay method was developed and validated for quantitative determination of epinastine in
bulk drugs and in pharmaceutical dosage form in the presence of degradation products generated from forced degradation
studies. An isocratic, reversed phase LC method was developed to separate the drug from the degradation products, using an
YMC ODS A- C18 (250 mm x 4.6 mm, 5 µm) column, and 0.05% v/v trifluroacetic acid and acetonitrile (65:35 v/v) as a
mobile phase. The detection was carried out at the wavelength of 220 nm. The epinastine was subjected to stress conditions
of hydrolysis (acid, base), oxidation, photolysis and thermal degradation. Degradation was observed for epinastine in base,
thermal and in 30% H2O2 conditions. The drug was found to be stable in the other stress conditions attempted. The
degradation products were well resolved from main peak. The percentage recovery of epinastine was ranged from (99.57%
to 100.25%). The developed method was validated with respect to the linearity, accuracy (recovery), precision, specificity
and robustness. The forced degradation studies prove the stability indicating power of the method.
Keywords: Column liquid chromatography, forced degradation, epinastine, stability indicating, method validation.

Introduction
Epinastine is chemically (RS)-3-amino-9,13b-dihydro-1Hdibenz(c,f)imidazo(1,5-a) azepine1. Epinastine is a selective
H1-receptor antagonist and also has an antiallergic effect by
inhibiting the release of allergy-inducing substances such as
histamine. Usually epinastine is used to treat bronchial
asthma, allergic rhinitis, urticaria, eczema, dermatitis,
pruritus cutaneous, prurigo and psoriasis vulgaris with
pruritus 2-6.
A literature survey reveals that several methods were
reported for the estimation of epinastine in plasma, serum
and in tablet by high-performance liquid chromatography
and by capillary electrophoresis7-8. Liquid Chromatographic
and Ultraviolet Derivative spectrophotometric methods for
determination of epinastine hydrochloride in coated tablets
were reported9-10. Potentiometric determination of epinastine
was also reported11. Literature survey reveals that, there is no
stability-indicating HPLC assay method for determination of
epinastine in bulk drug and pharmaceutical dosage form in
presence of its degradants formed in stress conditions of
hydrolysis (acid and alkali), oxidative, photolysis and
thermal for degradation of epinastine. In the present research
article, we report the development and validation of a
stability indicating HPLC method for the determination of
epinastine as bulk drug and pharmaceutical dosage form. It
separates the drugs from the degradation products form under
ICH suggested stress conditions (hydrolysis, oxidations,
photolysis and thermal stress)12-13. We developed a rapid,

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robust and economic method which separates the degradation
products from main peak. The main advantages of developed
method are that method is useful for routine analysis in
quality control labs due to short run time. The developed
method is stability indicating and can be used for assessing
the stability of epinastine in bulk drugs and pharmaceutical
dosage form The developed method was validated with
respective linearity, accuracy, precision, LOD, LOQ and
robustness.

Material and Methods
Epinastine bulk drug (purity 99.8) was obtained from
Transchem Ltd (Mumbai, India) and Alesion tablet (20 mg)
were obtained from market. Trifluroacetic acid and
Hydrochloric acid were obtained from Merck fine chemicals,
India Limited. Acetonitrile, hydrogen peroxide, sodium
hydroxide were obtained from Rankem laboratories, India.
All chemicals and reagent were used have analytical or
HPLC grade. UV cabinet was used of Kumar made, (India).
Milli-Q-Water was used throughout the experiment.
Chromatographic Conditions: HPLC system used was an
Agilent Technology (1100 series, Germany), system
equipped with auto sampler, quaternary pump, degasser and
a UV Detector. The out-put signal was monitored and
processed using Agilent Chemstation software. The
chromatographic column YMC ODS A (250 x 4.6 mm, 5
um, YMC Co. Ltd) was used. The instrumental setting of

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Vol. 1(ISC-2011), 281-288 (2012)
Res.J.Recent.Sci
flow was 1 ml/min. The injection volume was 10 µL. The
detection wavelength was 220 nm.
Mobile Phase: The Mobile Phase consists of buffer and
acetonitrile in the ratio of (65:35 v/v). The buffer used in the
mobile phase contained 0.05%v/v Trifluroacetic acid in
double -distilled water. The mobile phase was premixed and
filtered through a 0.45 µm nylon filter and degassed.
Preparation of Standard stock solutions: All solutions
were prepared on a weight basis and solution concentrations
were also measured on weight basis to avoid the use of an
internal standard. Solution of epinastine was prepared by
dissolving the drug in the diluents and diluting them to the
desired concentration. Diluent was composed of water and
acetonitrile in the ratio of (70:30 v/v). A 75 mg of epinastine
was accurately weighed, transferred in a 50 ml volumetric
flask, dissolved and diluted to 50 ml with the diluent, from
this stock solution 5 ml of solution transferred in to 100 ml
volumetric flask and diluted to volume with diluent. This
final solution contained 75 µg/ml of epinastine.
Sample solution (Tablets): Twenty tablets of Epinastine
(Alesion 20) were finely ground using agate mortar and
pestle. The ground material, which was equivalent to 75 mg
of the active pharmaceutical ingredient, was extracted into
diluent in a 50 ml volumetric flask by vortex mixing
followed by ultra sonication and make up the volume by
diluent. The solution was filtered through 0.45-micron filter
and an appropriate concentration of sample (75 µg/ml
concentration) was prepared in diluent at the time of analysis.
Specificity/ Selectivity: Specificity is the ability of the
method to assess unequivocally the analyte in the presence of
components, which may be expected to be present.
Typically, these might include degradation products, matrix
etc14. The specificity of the developed LC method for
Epinastine was carried out in the presence of its degradation
products. Stress studies were performed for epinastine bulk
drug to provide an indication of the stability indicating
property and specificity of the proposed method. Intentional
degradation was attempted to stress condition exposing it
with acid (1N hydrochloric acid), alkali (0.1 N NaOH),
hydrogen peroxide (30%), heat (800C) and UV light (254 nm
and 366 nm wavelength) to evaluate the ability of the
proposed method to separate epinastine from its degradation
products. For light study, study period was 48 h where as for
acid, oxidation and base 3 h, for heat 24 h. Peak purity of test
was carried out for epinastine by using PDA detector in
stress samples. Assay studies were carried out for stress
samples against epinaststine reference standard and the mass
balance (% assay + % sum of all impurities + % sum of all
degradants) was calculated. \The excipient mixture present in
Alesion 20 tablets was injected in the optimized conditions to
show the specificity of the method in formulation of
epinastine.

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Procedure for forced degradation study of Epinastine:
Acidic Degradation: About 75 mg of epinastine was
accurately weighed and dissolved in 10 ml diluent, then 10
ml of 1.0 N. HCl added and kept at 60 oC about 3 h in water
bath, the solution was allowed to attend ambient temperature
then solution was neutralized by 1.0 N NaOH to pH 7 and
volume made up to 50 ml with diluent, from this solution 5
ml of solution transferred in to 100 ml volumetric flask and
diluted to volume with diluent.
Alkali Degradation: About 75 mg of epinastine was
accurately weighed and dissolved in 10 ml diluent, then 10
ml of 0.1 N. NaOH added and kept at 60 oC 3 h in water bath,
the solution was allowed to attended ambient temperature.
Then solution was neutralized by 0.1N HCl to pH 7 and
volume made up to 50 ml with diluent, from this solution 5
ml of solution transferred in to 100 ml volumetric flask and
diluted to volume with diluent .
Oxidative Degradation: About 75 mg of epinastine was
accurately weighed and dissolved in 10 ml diluent , then 10
ml of 30% H2O2 solution added and kept 60oC about 3 h in
water bath, the solution was allowed to attended ambient
temperature. Then volume made up to 50 ml with diluent,
from this stock solution 5 ml of solution transferred in to 100
ml volumetric flask and diluted to volume with diluent.
Thermal Degradation: About 75 mg of drug substance kept
at 80oC for 48 h then the solution was prepared to achieve
final concentration 150 µg/ ml.
UV Degradation: About 75 mg of drug substance were
exposed to UV short (254 nm) and UV long (366 nm) light
for 48 h. then the solution was prepared to achieve final
concentration 150 µg/ ml..

Results and Discussion
Optimization of chromatographic conditions: The primary
target in developing this stability-indicating HPLC method is
to achieve the resolution between epinastine and its
degradation products. To develop stability indicating method
different stationary phases like C18, CN, different mobile
phases containing buffers like phosphate, ammonium acetate
and trifluoroacetic acid with different pH (3-5) and organic
modifier (acetonitrile) were used. The data is shown in table
1 and 2.
Our objective of chromatographic method development was
to achieve peak tailing factor less than 2 and retention time in
between 3 to 10 minutes.
To achieve the separation of degradation products used
stationary phases of C-18 and combination of mobile phase
0.05%v/v trifluroacetic acid with acetonitrile. The separation
of degradation product and epinastine was achieved on YMC

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Vol. 1(ISC-2011), 281-288 (2012)
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ODS A column and 0.05% v/v trifluroacetic acid: acetonitrile
(65:35 v/v) as a mobile phase and the column temperature
30oC. The tailing factor obtained was less than 2 and
retention time was also about 5 min for main peak and less
than 20 min for degradation products, which would reduce
the total run time and ultimately increase productivity and
reduce the cost of analysis as per sample. Forced degradation
study showed the method is highly specific and the entire
degradation products were well resolved from the main peak.
The developed method was found to be specific and method
was validated as per ICH guidelines 14.
Result of forced degradation experiments: From the
development studies, it was determined that aqueous
solution of trifluoroacetic acid 0.05% v/v and acetonitrile in
the ration of 65:35 (v/v), the flow rate of mobile phase was
1.0 ml/min. and column temperature at 30oC. The analyte
peak shape with less tailing, resolution from degradants and
the chromatographic analysis time was less than 20 min. In
optimized conditions analyte and its degradants were well
separated. Typical retention time of epinastine about 5.4 min.
Though conditions used for forced degradation were
attenuated to achieve degradation in the range of 10-30%,
this could not be achieved in case of acidic and photolytic
degradation even after exposure for prolonged duration15.
During the initial forced degradation experiments, it was
observed that alkali hydrolysis was a fast reaction for
epinastine almost complete degradation occurred when 1.0N
NaOH solution is used. The drug showed extensive
degradation in alkali hydrolysis, oxidative condition and
thermal. Table 3 indicates the extent of degradation, peak
purity and assay of epinastine under various stress
conditions. Chromatographic peak purity data was obtained
from the spectral analysis report and a peak purity value
greater than 990 indicates a homogeneous peak. The peak
purity values for analyte peak in the range of 999-1000
indicating homogeneous peaks and thus establishing the
specificity of assay method. Figure: 2 to 8 shows the
chromatograms of diluent, epinastine tablet solution, alkali
hydrolysis blank, alkali hydrolysis degraded drug product,
oxidative blank, oxidative degraded drug product and
thermally degraded drug product respectively.
Method Validation: Precision: Assay of method precision
(intra-day precision) was evaluated by carrying out six
independent assays of tablets. The intermediate precision
(inter-day precision) of the method was also evaluated using
two different analysts, different HPLC systems and different
days in the same laboratory. The percentage of R.S.D and
mean of six assay values obtained by two analysts were 0.15,
99.69 and 0.19, 99.52 respectively. Data is shown in table 4.
Accuracy (Recovery test): Accuracy of the method was
studied by recovery experiments. The recovery experiments
were performed by adding known amounts of the drugs in
the placebo.

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The recovery was performed at three levels, 80%, 100%,
and 120% of the label claim of the tablet (20 mg of
epinastine). The recovery samples were prepared as
aforementioned procedure, and then 5 ml of epinastine
solutions were transferred into a 50 ml volumetric flask
and volume made up with diluent. Three samples were
prepared for each recovery level. The solutions were
then analyzed, and the percentage recoveries were
calculated from the calibration curve. The recovery
values for epinastine ranged from 90.57% to 100.25%
and the RSD % for nine determinations is 0.9%. Data is
shown in table 3.
Linearity The linearity of the response of the drug was
verified at seven concentration level, ranging from LOQ200% of the targeted level (75 µg/ml), of the assay
concentration. Linearity solutions were injected in triplicate.
The calibration graphs were obtained by plotting peak area
verses the concentration, data was treated by least-squares
linear regression analysis. The equation of the calibration
curve for analyte obtained y = 5267.5x – 4505.5, the
calibration graphs were found to be linear in the
aforementioned concentrations. The coefficient of
determination is 0.999.
Limit of detection and Limit of quantification (LOD and
LOQ): For determining the limit of detection (LOD) and
limit of quantification (LOQ), a specific calibration curve
was constructed using samples containing the analyte in the
range of LOD and LOQ. The LOD and LOQ for the
epinastine in HPLC method was 0.05 and 0.18 µg/ml
respectively.
LOD and LOQ were calculated by using following
equations.
LOD = Cd x Syx /b
LOQ = Cq x Syx /b
Where, Cd/Cq is coefficient for LOD/LOQ; Syx is residual
variance due to regression; b is slope.
Precision at limit of quantification was checked by analyzing
six test solutions prepared at LOQ level and calculating the
percentage relative standard deviation of area which was less
than 1.8%.
Robustness: To determine the robustness of the
developed method experimental condition were
purposely altered and the resolution between epinastine
and base degradation product were evaluated. The flow
rate of the mobile phase was 1.0 ml/min. To study the
effect of flow rate on the resolution, it was changed by 0.1
units from 0.9 to 1.1 ml/min.

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Vol. 1(ISC-2011), 281-288 (2012)
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The effect of percent organic strength on resolution was
studied by varying acetonitrile from -5 to +5%. The effect
of column temperature on resolution was studied at 25 oC
and 35 oC instead of 30oC, while the other mobile phase
components
were
held
constant
stated
in
chromatographic condition. The resolution between
epinastine and base degradation product in robustness
study was not less than 3.0 at in all conditions. Also assay
was performed at each condition in triplicate. The data is
shown in table 6.
Stability of analytical solution: The stability of the
standard solutions and the sample solutions was tested at
intervals of 24, 48 and 72 h. The stability of solutions was
determined by comparing results of the assay of the
freshly prepared solutions. The RSD for the assay results
determined up to 72 h for epinastine was 0.47 %. The
assay values were within 0.7 % after 72 h. The results
indicate that the solutions were stable for 72 h at ambient
temperature.

dermatitis patients, European J. of dermatology, 17(1)
33-36, (2007)
4.

Kanai K., Asano K., Watanabe S., Kyo Y. and Suzaki
H., Epinastine hydrochloride antagonism against
interleukin-4-mediated T cell cytokine imbalance in
vitro, Int. Arch Allergy Immuno., 140, 43-52 (2006)

5.

Takahashi N., Aizawa H., Inoue H., Matsumoto K.,
Nakano H., Hirose T., Nishima S., Hara N., Effects of
epinastine hydrochloride on cholinergic neuro-effector
transmission in canine tracheal smooth muscle,
European Journal of Pharmacology, 358 (1) 55-61
(1998)

6.

Fraunfelder F.W., Epinastine hydrochloride for atopic
disease, Drugs Today, 40 (8) 677-683 (2004)

7.

Ohtani H., Kotaki H., Sawada Y., Iga T., Quantitative
determination of epinastine in plasma by highperformance liquid chromatography, J. of Chrom. B:
Biomedical Sciences and Applications, 683 (2) 281-284
(1996)

8.

Vera-Candioti L., Olivieri A.C., Goicoechea H.C.,
Simultaneous multiresponse optimization applied to
epinastine determination in human serum by using
capillary electrophoresis, Anal Chim Acta, 595 (1-2)
310-318, (2007)

9.

Ghisleni D.D., Steppe M., Elfrides E.S. Schapoval.
Development and validation of Liquid chromatographic
and ultraviolet derivative spectrophotometric methods
for determination of epinastine hydrochloride in coated
tablets, Journal of AOAC International , 90(5) 12661271 (2007)

10.

Saleh O.A., El-Azzouny A.A., Badawy A.M. and
Aboul-Enein H.Y., A validated HPLC method for
separation
and
determination
of
epinastine
hydrochloride enantiomers, Journal of Liquid
Chromatography and Related technologies, 33(3) 413422 (2010)

Conclusion
The developed method is stability indicating and can be used
for assessing the stability of epinastine in bulk drugs and
pharmaceutical dosage form. The developed method can be
conveniently used for the assay determination of epinastine
in bulk drugs and pharmaceutical dosage form. The
developed HPLC method was specific, selective robust,
rugged and precise method. The developed HPLC method
can be conveniently used for assessing stability, assay,
related substances and dissolution of tablets of the
pharmaceutical dosage form containing epinastine in quality
control.

Acknowledgment
The authors are grateful to the Transchem Ltd (Mumbai,
India) for making available the samples of epinastatine
hydrochloride and Department of chemistry, Deogiri College
of Scinece Aurangabad, India for providing laboratory
facility for this research work.

References
1.

Merck Index, Merck and Co, Inc, USA, 13 th edn.,
(2001)

11.

2.

Oshima K., Kazuhito, Asano K., Kanai K., Suzuki M.
and Suzaki H., Influence of epinastine hydrochloride,
an H1-receptor antagonist, on the function of mite
allergen-pulsed murine bone marrow-derived dendritic
cells in Vitro and in vivo, Mediators of inflammation,
(2009)

Basaveswara Rao M.V., Reddy B.C.K., Srinivas Rao
T., and Kalyani P., Determination of epinastine
hydrobromide assay by potentiometric method,
Rasayan Journal of Chemistry, 2(2) 361-363, (2009)

12.

International Conference on Harmonization of
Technical
Requirements
for
Registration
of
Pharmaceuticals for Human Use, Stability testing of
New Drug Substances and Products, QIA (R2), (2003)

3.

Toyoda M., Nakamura M., Nakagawa H., Distribution
to the skin of epinastine hydrochloride in atopic

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Vol. 1(ISC-2011), 281-288 (2012)
Res.J.Recent.Sci
13.

Drug Information Branch (HFD-210), Validation of
analytical procedure: Methodology, Step 4 In ICH
Harmonized Tripartite Guidelines Q2B. Center for
Drug Evaluation and Research, Rockville, MD, (1996)

14.

ICH-Guidelines: Text on validation of analytical
procedures. Q2 (R1)

15.

Bakshi M., and Singh S., Development of validated
stability-indicating assay methods critical review, J.
Pharm. Biomed Anal., 28, 1011-1040 (2002).

Figure-1
Chemical structure of epinastine

Figure-2
Chromatograms of diluents
DAD1 A, Sig=220,4 Ref=off (DEC08\V01JAN40.D)
mAU
350
300
250
200
150
100
50
0

2.5

5

7.5

10

12.5

15

17.5

20

22.5

min

20

22.5

min

Figure-3
Chromatogram of epinastine tablet Soln.
DAD1 A, Sig=220,4 Ref=off (DEC08\V01JAN26.D)

5.447 - Epinastatine

mAU
350
300
250
200
150
100
50
0

2.5

5

7.5

10

12.5

15

17.5

Figure- 4
Chromatogram of base hydrolysis blank

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Research Journal of Recent Sciences ____________________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 281-288 (2012)
Res.J.Recent.Sci
DAD1 A, Sig=220,4 Ref=off (DEC08\V01JAN20.D)
mAU
350
300
250
200
150
100
50
0

2.5

5

7.5

10

12.5

15

17.5

20

22.5

min

22.5

min

22.5

min

Figure- 5
Chromatogram of base hydrolysis of epinastine
DAD1 A, Sig=220,4 Ref=off (DEC08\V01JAN16.D)
5.478 - Epinastatine

mAU
350
300

3.124 - B-D

250

50

17.143 - B-D

100

6.522 - B-D

150

2.598 - B-D
2.948 - B-D

200

0

2.5

5

7.5

10

12.5

15

17.5

20

Figure-6
Chromatogram of oxidative degradation blank
DAD1 A, Sig=220,4 Ref=off (DEC08\V01JAN21.D)
mAU
350
300
250
200
150
100
50
0

2.5

5

7.5

10

12.5

15

17.5

20

Figure- 7
Chromatogram of oxidative degradation of epinastine

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Vol. 1(ISC-2011), 281-288 (2012)
Res.J.Recent.Sci
DAD1 A, Sig=220,4 Ref=off (DEC08\V01JAN17.D)
5.463 - Epinastatine

mAU
350
300
250

100
50

16.139 - O-D

150

4.706 - O-D

3.017 - O-D
3.310 - O-D
3.471 - O-D
- O-D
3.7663.885
- O-D
4.147
O-D
4.244-- O-D

200

0

2.5

5

7.5

10

12.5

15

17.5

20

22.5

min

Figure- 8
Chromatogram of thermal degradation of epinastine
DAD1 A, Sig=220,4 Ref=off (DEC08\V01JAN05.D)
5.544 - Epinastine

mAU

400

300

6.549 - H-D

2.281

100

2.950 - H-D
3.147 - H-D

2.595

200

0
0

2

4

6

8

10

min

Table-1
The chromatographic behavior of Epinastine in different mobile phases
Mobile Phase

Retention time (Min.)

Water : Acetonitrile (60:40)

3.2

0.05M Ammonium Acetate pH 4.5with

4.5

Acetic acid : Acetonitrile (60:40)
0.05M Ammonium Formate pH 3.5with

9.2

Formic acid : Acetonitrile (70:30)
Water : Methanol (40:60)

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Table-2
The chromatographic behavior of Epinastine on different columns
Mobile Phase
Retention
Time (Min)
YMC ODS A C18;
5cm × 4.6 mm i.d.,5 µ particle
Zorbax SB C18;
25cm × 4.6 mm i.d.,5 µ particle
Kromosil C18;
25cm × 4.6 mm i.d., 5µ particle
Waters Symmetry C18;
25cm × 4.6 mm i.d., 5 µ particle

5.4
4.7
3.6
3.6
Table-3
Results of forced degradation study
Epinastine (%)
Peak puritya
degradation

Stress condition
Acidic
Alkali
Oxidative
Thermal
UV-short
UV-long
a

No degradation
27.88
18.12
17.20
No degradation
No degradation

Assay (%)

999.936
999. 706
999.532
999.379
999.609
999.977

99.18
73.26
81.23
82.52
99.89
100.32

peak purity values in the range of 990-1000 indicate a homogeneous peak

Average assay

Assay
% (n=6)
99.69
99.52

Chemist 1
Chemist 2

Table-4
Precision data
% RSD Standard
peak area
0.12
0.15
0.22
0.19

% RSD (n=6)

Table-5
Results of recovery experiment
Found
Amount of standard Spiked (mg)
(mg)
16
20
24

Recovery%
(n=3)

15.88
20.05
23.92

99.57
100.25
99.87

Table-6
Results of robustness study
Sr. no.

Parameter

Variation

Assay %

Resolutiona

n=3
1

Flow rate
(± 10% of the set flow)

a) At 0.9 ml/min
b) At 1.1 ml/min

99.38
99.66

3.8
4.2

2

Mobile phase composition

a) At 38.5 ml

99.62

3.3

(± 5% of organic modifier)

b) At 31.5 ml

100.04

4.5

Temperature (± 5° C of set temperature)

a) At 25°C

99.89

4.0

b) At 35°C

99.53

4.4

3
a

Resolution between epinastine and base degradant peak at RRT 1.2

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