Raja et al., IJPSR, 2012; Vol.

3(8): 2658-2663
ISSN: 0975-8232
IJPSR (2012), Vol. 3, Issue 08
(Research Article)

Received on 10 April, 2012; received in revised form 04 May, 2012; accepted 22 July, 2012

SIMULTANEOUS & STABILITY INDICATING METHOD FOR DETERMINATION OF

CETRIZINE HYDROCHLORIDE AND AMBROXOL HYDROCHLORIDE IN SYRUP
1

M. Gnana Raja *, G. Geetha , A. Sankaranarayanan , Karna Maga Ganiga Raju

3
Saravanamuthu Kumar

and P.

PSG College of Pharmacy , Coimbatore, Tamil

2
Nadu, India KMS Health Center , Padi,
Chennai, Tamil Nadu, India Apex Laboratories
3
Pvt. Ltd., , Chennai, Tamil Nadu, India
ABSTRACT
Keywords:
Cetirizine
Hydrochloride,
Ambroxol
hydrochloride,
HPLC,
Acetonitrile,
Chromatogra
phy
Correspondence to Author:
M. Gnana Raja
Manager, Analytical research
and development, KMS Health
Center, Padi, Chennai, Tamil
Nadu, India
E mail:
[email protected]

A simple isocratic HPLC method has been developed and

validated as per ICH Guidelines for simultaneous and stability
indicating determination of Cetirizine Hydrochloride and
Ambroxol hydrochloride in pharmaceutical liquid oral dosage
form syrup. Separation was achieved in Hypersil BDS C18
column, 5 , and 250 mm x 4.60 mm id with flow rate of 1.5
ml/minute and the detection at 230nm. 0.2 molar Ammonium
phosphate with the pH of 4.0 with dilute orthophosphoric acid
of about 65 percentage in acetonitrile combination was used
as a mobile phase. Developed method validated as per ICH
Guidelines, prior all the parameter system suitability was
carried with five repeated injection of standard solution at the
concentration of 50 g/mL and 30 g/mL Cetirizine
Hydrochloride and Ambroxol Hydrochloride respectively.
Percent RSD of five replicate injections of Cetirizine
Hydrochloride and Ambroxol Hydrochloride and Tailing factor
was found below 2.0% and 2.0 overall analyses respectively. A
linear response was observed over the concentration range
of 25.06 to 75.18 and 15.13 to
45.38ppm for the assay of Cetirizine Hydrochloride and
Ambroxol Hydrochloride respectively. The results of analysis
were validated statically and by recovery studies. Hence, the
proposed method was found to be accurate, precise,
reproducible and specific and can be used for simultaneous
analysis of these drugs in liquid formulation.

INTRODUCTION: Cetirizine Hydrochloride

is chemically
2-[2-[4-[(4-chlorophenyl)-phenylmethyl]
piperazin- 1- yl] ethoxy] acetic acid and the

active metabolite of the piperazine H1receptor antagonist Hydroxyzine, it is not

metabolized. Cetirizine inhibits the release of
histamine and of cytotoxic mediators from

Available online on
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989
898

platelets, as well as eosinophil chemotaxis

during the secondary phase of allergic
response. Also it is used to treat chronic
idiopathic,
urticaria,
perennial
allergic
rhinitis, seasonal allergic rhinitis, allergic
asthma, physical urticaria and atopic
dermatitis.

Cetirizine hydrochloride (Figure 1 oficial in

1
EP and BP
2
) is a highly selective antagonist of the
peripheral
histamine H1-reseptor on effectors cell in
the GIT, and blood vessels.

FIGURE
1:
CHEMICAL
STRUCTURE
OF
CETIRIZINE HYDROCHLORIDE

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999
999

Raja et al., IJPSR, 2012; Vol. 3(8):

2658-2663

Ambroxol Hydrochloride (Figure 2 official

1
2
in EP
and BP ) is chemically Trans-4-(2Amino-3,
5dibrombenzylamino)cyclohexanol Ambroxol is a clinically proven
systemically
active
mucolytic
agent.
Administered orally onset of action occurs
after about
30
minutes.
The
breakdown of acid
mucopolysaccharide
fibers
makes
the
sputum thinner and less viscous and
therefore more easily removed by coughing.
Although sputum volume decreases, its
viscosity remains low for as long as
treatment is maintained. It is a metabolite of
Bromhexine.

FIGURE
2:
CHEMICAL
STRUCTURE
AMBROXOL HYDROCHLORIDE

OF

Objective of study: As far as literature

concerned, numbers of methods are
reported
for
RP-HPLC
and
3-4
spectrophotometric
estimation
of
ambroxol hydrochloride
and
Cetirizine
Hydrochloride in combined dosage form,
5-6
Spectrophotometric
estimation
of
ambroxol
hydrochloride
and
cetirizine
hydrochloride
in
tablet
formulation.
Simultaneous
analysis
ambroxol
hydrochloride and cetirizine hydrochloride in
7-9
tablet dosage form by RP-HPLC
method.
10

A rapid, stability indicating RP-HPLC

method for simultaneous determination of
ambroxol
hydrochloride,
cetirizine
hydrochloride
and
antimicrobial
preservatives in liquid pharmaceutical
formulation,
high
performance
liquid
chromatographic method for cetirizine and
11
ambroxol in human plasma and urine . But
this study was worked on the simultaneous
and
stability
indicating
method
for
determination of cetirizine Hydrochloride
and ambroxol hydrochloride and validation
12
performed as per ICH , Q2B- Validation of
Analytical Procedure.
MATERIAL AND METHODS: HPLC grade

ISSN: 09758232

acetonitrile,
reagent
grade
Ortho
-phosphoric acid, ammonium phosphate,
sodium hydroxide, Hydrochloric acid, 6%
hydrogen peroxide were purchased from
Merck (Darmstadt, Germany).

Raja et al., IJPSR, 2012; Vol. 3(8):

ISSN: 09758232125% and
2658-2663
Distilled water, purified using
a Millipore
equivalent to 50%, 75%, 100%,
Milli-Q plus water system was used to
150%
of
the
nominal
concentration
prepare the mobile phase and standard
respectively.
solutions.
At low level concentration and highest level
The development and validation of the
concentration accuracy proved with six
assay was performed on a HPLC Quaternary
replicate preparation and medium level
system with VWD Model Agilent 1200 series,
triplicate preparations was done.
consisting of a Quaternary pump-G1311A, a
Degasser- G1322A, a ALS- G1329A, a TCCG1316A and Waters-2996 Photodiode Array
Detector, a Waters HPLC with PDA
Detectors, Waters
2695- Separation Module with EMPOWER
software.
The analytical column used to achieve
chromatographic separation was Hypersil
BDS column
250 x 4.6 mm, 5m particle size. The peak
purity
was determined on
a 2996
Photodiode Array Detector (PDA).
Proced
ure
Mobile phase preparation: 0.2 m
Ammonium phosphate bufer pH adjusted to
4.0 with orthophosphoric acid and 65
percentage of the buffer in acetonitrile used
as a mobile phase. 1.5 mL/minutes flow rate
of the mobile phase with the column
temperature of about 25C was giving the
consistent of retention time of about 18.6
and 5.8 of Cetirizine Hydrochloride and
Ambroxol Hydrochloride respectively.
Standard
and
Test
solution
preparation: Standard and sample solution
was prepared at the concentration of about
50g/mL
and
30g/mL
of
Cetirizine
Hydrochloride and Ambroxol Hydrochloride
respectively with the mobile phase as a
diluent by sonication of about 15 minutes
and the final solution filtered through 0.45
micron filter paper.
Method
Validation:
Precision
was
performed with six repeated
sample
preparation
of
nominal concentration,
accuracy was performed 50% to 150% of
five
concentration
level
Cetirizine
Hydrochloride and Ambroxol Hydrochloride
by the preparation sample was weighed as

The linearity was performed for the

standard
solution
of
Cetirizine
Hydrochloride and Ambroxol Hydrochloride
from the level of 50% to 150% of about five
different
concentrations
from
nominal
concentration of standard and sample
solution. Range of the method has been
captured from precision, accuracy
and
linearity section. Deliberate change of the
chromatographic condition will affect the
method reproducibility so the method
accuracy was proved by robustness and
ruggedness experiments of 20% of flow rate,
10 % of the organic modification, 5% of
the column temperature and pH of the
mobile phase, and the system suitability was
proved.
Degradation Study: Proving the method
as stability indicating method, sample and
placebo were forcefully degraded, Acid
degradation by 5 m hydrochloric acid,

Base hydrolysis by 5m sodium hydroxide

and 6% hydrogen peroxide oxidation, UV
visible light as per ICH guidelines in the
chamber, 105C of temperature was
maintained for 24 hours for thermal
degradation, saturated KNO3 was used for
degrading the sample by moisture and
aqueous degradation was done by water.
RESULTS & DISCUSSION:
Developed
analytical method was validated and system
suitability was performed prior to each
parameter and the result was found
satisfactory for all the parameter. Percent
RSD of overall analysis was about 1.6% to
1.0% and overall tailing factor was found 1.2
and 1.3 Cetirizine Hydrochloride and
Ambroxol Hydrochloride respectively. Refer
chromatogram of blank, standard and
sample Figure 1, Figure 2 and Figure 3.

FIGURE 1: CHROMATOGRAM
OF BLANK

FIGURE 2: CHROMATOGRAM OF
STANDARD

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100
1001

FIGURE 3: CHROMATOGRAM
OF SAMPLE

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101
1011

Six replicate preparations was done for

precision and the average assay of six
replicate preparations of about
99.6%, 99.7% and the percent RSD of assay
was found
1.6%, 1.0% for Cetirizine Hydrochloride and
Ambroxol
Hydrochloride
respectively
(Table 1).
Linearity of Cetirizine Hydrochloride and
Ambroxol
Hydrochloride
of
the
concentration of 25.1 to 75.02 and 15.1 to
45.4g/mL respectively, the correlation of
coefficient was found to be 0.999 and 0.999
respectively (Figure 4 and Figure 5).

TABLE 1: PRECISION
S. No.
CTH
AXH
1
99.5
2
98.2
3
101.1
4
100.5
5
99.8
6
99.2
Average
99.7
% RSD
1.0

Precision

Nomin
99.3 al
97.2
98.5
100.2
101.2
101.1
99.6
1.6

FIGURE 4 LINEARITY OF CETIRIZINE HYDROCHLORIDE

FIGURE 5 LINEARITY OF AMBROXOL HYDROCHLORIDE

Accuracy of the both the drugs was proved

50.0% to
150.0% form the nominal concentration and
the percent RSD of each level and average
of overall was found 0.3% and 1.8% .
Average percent recovery of each level was
found 99.2% to 101.0% (Table 2).
Ruggedness or intermediate precision was

proved with the sample preparation of six

replicates with intra day, another
analyst,
different
column
and
different system,
the percent RSD was found 0.7 and 0.9
for Cetirizine Hydrochloride and Ambroxol
Hydrochloride respectively (Table 3).

Robustness
was
proved
with
the
chromatographic condition is deliberately
changed of about 20% of flow rate, 10 %
of the organic modification, 5% of the
column temperature and pH of the mobile
phase, in this condition system suitability
was proved (Table 4).
Degradation study was performed for
placebo
and drug product, and the
placebo chromatogram shows no peak at
the interested peak retention time and
peak purity of Cetirizine Hydrochloride and
Ambroxol hydrochloride passes and the
percentage degradation is 4.21, 1.21 and
3.23, 1.02% in acid, 6% hydrogen

peroxide degradation and acid, Humidity

degradation of maximum and minimum
percentage
degradation
of
Cetirizine
Hydrochloride and Ambroxol hydrochloride
respectively (Table 5).
TABLE
2:
ACCURACYS. No.

1
2
3
4
5
6
Average
% RSD

CTH
AXH
50
97.5 % 100.2
98.6
97.9
101.2 98.5
102.1 99.5
100.2 101.5
100.8 102.3
100.1 100.0
1.7
1.7

The finalized chromatographic condition and

validation summary refer Table 6 and
Table 7 respectively.

CTH
AXH
75
%
99.2
99.5

Accura
CTH cyAXH
100
%
100.2 100.1

CTH
AXH
125
%
98.5
99.3

99.8

98.6

100.3

99.5

99.6

99.5

100.2

102.1

99.8

99.9

99.5

102.2

99.7
0.5

100.1
1.8

100.1
0.3

99.8
0.3

99.2
0.6

100.3
1.6

TABLE 3: INTERMEDIATE PRECISION

S. No.
Analyst II
CTH
AXH 99.3
1
99.9
2
97.2
100.2
3
98.5
100.3
4
100.2
101.2
5
101.2
102.2
6
101.1
101.2
Average
99.6
100.8
% RSD
1.6
0.9
TABLE 4: ROBUSTNESS

Analyst I
AXH
99.5
98.2
101.1
100.5
99.8
99.2
99.7
1.0

CTH

AX
150 H
99.5 % 102.3

102.3
102.4
99.1
98.1
99.7
100.2
1.8

CTH
99.5
100.2
100.3
101.2
99.8
99.3
100.1
0.7

System
Suitability
S. No.
Type of
Cetirizine Hydrochloride
modification
Hydrochloride
% RSD
Tailing factor 1.1
1
Organic variation +10%
1.3
2
Organic variation -10%
1.3
1.2
3
pH +5%
1.1
1.3
4
pH -5%
1.2
1.2
5
Column temperature +5%
1.0
1.2
6
Column temperature -5%
1.1
1.4
7
Flow +20%
1.4
1.2
8
Flow -20%
1.3
1.1
TABLE 5: DEGRADATION STUDY
S. No.
Type of Degradation
Degradant
1
Acid hydrolysis
4.21
3.23
2
Base hydrolysis
2.84
2.10
3
Oxidation
1.21
1.25
4
Humidity
2.31
1.02

99.5
99.5
101.2
102.2
101.2
101.0
1.2

Ambroxol

% Degradation
Cetirizine
5 N Hydrochloric acid Ambroxol
5 N Sodium hydroxide
6% hydrogen Peroxide
95% moisture by using saturated KNO3

TABLE 6: CHROMATOGRAPHIC CONDITION

S. No.
Variable
Condition
1
Column
250*4.6, 5
2
Phase
3
Mobile phase & Diluent
bufer
in
Acetonitrile.
4
Flow rate
5
Temperature
6
Injection volume
7
Wave length

Hypersil BDS,
BDS
65% of 50mM of Ammonium phosphate
1.5 mL / Minutes
25C
20 L
230 nm

TABLE 7: VALIDATION SUMMARY

S. No.
Parameter
Ambroxol
Hydrochloride
1
Precision (% RSD)
1.0
2
0.9
3
100.2
4
0.999

Cetirizine Hydrochloride
1.6
0.7
99.9
0.999

Intermediate Precision (%RSD)

Accuracy (Overall recovery)
Linearity

ACKNOWLEDGEMENTS: We are thankful

to Analytical Research and Development
Micro labs Pvt. Ltd, Hosur, India, for
providing as necessary facility to carry out
the above stated research work.
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CE:
1.
European Pharmacopoeia 6.0, (2007), 1156-58,
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British Pharmacopeia 2010, Vol. 1, 118-19, 44344.
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Kokil. Validated RP-HPLC and spectrophotometric
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hydrochloride
and
Cetirizine Hydrochloride in combined dosage form,
IJPS Res 2008, 70(5), pp 603-608.
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Manish
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How to cite this

article:
Raja MG, Geetha G., Sankaranarayanan A., Raju KMG
and Kumar PS. Simultaneous & Stability indicating
method for determination of Cetrizine Hydrochloride
and Ambroxol Hydrochloride in Syrup. Int J Pharm Sci
Res 2012; Vol. 3(8): 2658-2663.