WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Hina et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Volume 3, Issue 5, 1458-1467.

Research Article

ISSN 2278 4357

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR

SIMULTANEOUS ESTIMATION OF AMBROXOL,
CHLORPHENIRAMINE MALEATE AND PHENYLEPHRINE HCL IN
BULK AND LIQUID DOSAGE FORM BY R

P-HPLC METHOD

*Hina Bagada, Hitesh Panchal, Priya Patel , Aashka Jani

Department of pharmaceutical science, Saurashtra University, Rajkot-360005, Gujarat.

Article Received on
18 March 2014,
Revised on 10 April 2014,
Accepted on 01 May 2014

ABSTRACT
A simple Reverse phase liquid chromatography method has been
developed and subsequently validated for the determination of
Ambroxol, Chlorpheniramine Maleate and Phenylephrine HCl in liquid
dosage form. The saparation was carried out using mobile phase

*Correspondence for Author

Hina Bagada

consisting methanol : acetonitrile (50%:50%) and phosphate buffer 5

Department of pharmaceutical

pH containing 0.75% TEA set by formic acid(40%:60%v/v) gave

science, Saurashtra University,

resolution of peaks and satisfied retention time in HPLC.The good

Rajkot-360005, Gujarat

saparation carried out using C-18 (250mm 4.6mm i.d with particle
size of 5 m )used with flow rate 0.95 ml/min using UV detection at

261nm.the described method was linear over the concentration range of 32-48 g/ml (R2 =
0.998 )for Ambroxol, 6.4-12.8 g/ml (R2 = 0.997) for Chlorpheniramine maleate and 11.222.4 g/ml (R2 = 0.990) for Phenylephrine HCl. The saparation was achieved with in 15 min.
The mean Recovery studies was found to be 99.9-99.95% for AMB and 100.0-100.77% for
CPM and 100.0-100.72% for PH. Whereas the LOD and LOQ value was found to be
11.58g/ml and 35.092 g/ml for AMB & 0.66 g/ml and 2.02 g/ml for CPM And 7.04
g/ml and 21.36 g/ml for PH. The result of the study showed that the proposed RP-HPLC
method is simple, rapid, precise, accurate and cost effective which is useful for the routine
determination of the Ambroxol, Chlorpheniramine Maleate and Phenylephrine HCl in Bulk
and Liquid Dosage Form.
Keyword: RP-HPLC, Ambroxol, Chlorpheniramine Maleate, and Phenylephrine HCl,
Validation.

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INTRODUCTION
Ambroxol (AMB) is mucolytic agent and Expectorant, chemically, it is trans-4-[(2-amino-3,
5-dibromobenzyl) amino] cyclohexanol.it is effective in the treatment of the cough and cold.
Literature survey revealed that Ambroxol can be estimated spectrophotometric method, RPHPLC and HPTLC method.
Chlorpheniramine maleate is a H1-receptor antagonist, chemically, it is 2, 2-dichloro-N[(1R, 2R)-2- hydroxy1 hydroxyl methyl2 (4 nitro phenyl) ethyl] HCl, it is effective to relieve
symptoms such as watery eyes and runny nose. Literature survey revealed that
Chlorpheniramine maleate can be estimated spectrophotometric method, RP-HPLC and
HPTLC method.
Phenylephrine HCl is a selective 1-adrenergic receptor agonist. Chemically, it is (R)-1-(3hydroxyphenyl) 2methylaminoethanol hydrochloride. It acts as an agent to constriction of
blood vessels in the nasal mucosa is intended to relieve nasal congestion. Literature survey
revealed that Phenylephrine HCl can be estimated spectrophotometric method, RP-HPLC and
HPTLC method. But no method was developed for simultaneous estimation of all three drugs
in combined liquid dosage form.
OBJECTIVES
Extensive Literature review reveals that only few analytical method like UV spectroscopic
method, HPLC, HPTLC and LC/MS have been reported for the estimation of the Ambroxol,
Chlorpheniramine maleate and Phenylephrine HCl in single and combined dosage form with
other drug but there is no UV Spectrophotometric, RP-HPLC method and HPTLC method
have been developed for simultaneous estimation of Ambroxol, Chlorpheniramine Maleate,
and Phenylephrine HCl in combined liquid dosage form. Hence main aim of work is for the
development of newer, simple, sensitive, rapid, accurate, and reproducible method for routine
estimation of Ambroxol, Chlorpheniramine Maleate, and Phenylephrine HCl in combined
liquid dosage form.
MATERIAL AND METHODS
Reagent and Materials
Acetonitrile, Methanol of HPLC grade, Dipotassium hydrogen phosphate and potassium
dihydrogen phosphate and Triethyle amine. Ambroxol, Chlorpheniramine maleate and
Phenylephrine HCl was obtained from Department of Pharmaceutical Sciences store,

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Saurashtra University, Rajkot.

Instrumentation of RP-HPLC
The chromatographic saparation was carried out on HPLC system (simadzuLC 20 AD) with
UV visible dual absorbance detector (PDA), C18 (250 4.6,5 m) column. The mobile phase
consisting of Methanol: Acetonitrile (50:50%v/v): phosphate buffer 5 pH with 1%
triethylamine pH set by formic acid (40:60%v/v) with 0.95 ml/min flow rate. The detection
was carried out at 261nm.The volume of injection loop was 20 l prior to the injection of the
drug solution. The column was equilibrated for at least 30 min with mobile phase flowing
through the system. The column and the HPLC system was carried at the ambient
temperature (25C).
Preparation of Stock solution
100 mg of Ambroxol HCl, 15 mg of Chlorpheniramine Maleate and 35 mg of Phenylephrine
HCl were weighed separately and transferred in two different 50 ml volumetric flasks. Both
the drug were dissolved in 25 ml of methanol by ultra sonication and then volume was made
up to the mark with methanol to obtain final concentration 2000g/ml of Ambroxol HCl,
300g/ml of Chlorpheniramine Maleate and 700g/ml of Phenylephrine HCl.1ml of stock
solution was withdraw from each & transfer in 10 ml volumetric flask and volume was make
up with methanol to achieve 200 g/ml of Ambroxol HCl, 30 g/ml of Chlorpheniramine
Maleate and 70 g/ml of Phenylephrine HCl.
Preparation of Sample Solution
Accurately take 15.0mL of sample liquid solution was basified with 25.0mL of 0.1N NaOH
and extracted with three successions 15 mL of chloroform each in a separating funnel. The
organic layer was evaporated to dryness and aqueous layer was discarded. The dried residue
was diluted with 50.0mL of methanol. Finally 1.0 mL of the solution was diluted with 10.0
mL of methanol.
Method
For HPLC method, chromatographic conditions were optimized to obtain an adequate
saparation of eluted compound. Various mobile phases comprising of Methanol, acetonitrile
and phosphate buffer were tried in different compositions at various flow rates & different pH
to achieve sharp peak of Ambroxol HCl, Chlorpheniramine Maleate and Phenylephrine HCl.
The optimum wavelength for detection was set at 261 nm at which better resolution &

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sharpness achieve. Methanol: Acetonitrile (50:50): phosphate buffer 5 pH with 1%

triethylamine pH set by formic acid (40:60%v/v) with 0.95 ml/min flow rate was selected as
mobile phase because all system suitability parameter are fulfill tailing is less than 2,
resolution is greater than 2, number of theoretical plates are more then 2000 and retention
time of analytes is 2.8, 11.1 and 13.58.

Fig.1: Chromatogram of standard drugs of Ambroxol (36 g/ml), Chlorpheniramine

maleate (8 g/ml) and Phenylephrine HCl (14 g/ml)
Table 1: Table of system suitability parameters
System Suitability
Parameters
Tailing Factor
Theoretical Plates
Retention
Time(minutes)

AMB

CPM

PH

1.68
3078.9

1.7
2717.0

1.69
2548

11.1

13.58

2.8

METHOD VALIDATION
Linearity
Appropriate aliquots were pipette out from the standard stock solution in to a series of in 10
ml volumetric flask and volume was make up with methanol to achieve 32-48 g/ml of
Ambroxol HCl, 6.4-12.8 g/ml of Chlorpheniramine Maleate and 11.2-22.4 g/ml of
Phenylephrine HCl. Evaluation of three drugs was performed and peak areas are recorded for
all drugs. The slope and intercept value for calibration curve was y= 484418x + 06, R2=0.998
for Ambroxol, and y = 760844x + 101581,R2= 0.998 for Chlorpheniramine maleate, and y=
339783x+ 479944, R2= 0.990 for Phenylephrine HCl. The result show good correlation

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between peak area and concentration range indicated below regression graph are shown in fig
2, 3 and 4 respectively.

Fig 2: Linearity chromatogram of Ambroxol, Chlorpheniramine maleate and

Phenylephrine HCl standard solution

Fig.3: Calibration curve of Ambroxol

Fig 4: Calibration curve of Chlorpheniramine maleate

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Fig 5: Calibration curve of Phenylephrine HCl

LOD and LOQ
LOD and LOQ value was found to be 11.58g/ml and 35.092 g/ml for AMB & 0.66 g/ml
and 2.02 g/ml for CPM and 7.04 g/ml and 21.36 g/ml for PH.
Recovery study
The study of accuracy and reproducibility of proposed method were followed by recovery
studies. A fixed amount of sample mixture was added at 80%, 100% and 120% each level
was injected 3 times.
Table 2: Recovery study of Ambroxol HCl

AMB
80%

100%

120%

Total
area
14203109
14245778
14179444
15727554
15759818
15773408
17331545
17359655
17331545

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Conc.
(g/ml)
38.36
38.48
38.3
47.87
47.97
48.01
57.53
57.93
57.53

Mean
Area

SD

%RSD

99.95

14209444

33617.643

0.23

99.90

15753593

23552.223

1.49

99.9

17340915

16229.316

0.51

%Recovery Average
99.91
100.21
99.74
99.74
99.95
100.03
99.89
100.08
99.89

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Table 3: Recovery study of Chlorpheniramine maleate

CPM
80%

100%

120%

Total
area
9246335
9373403
9386622
10326625
10326068
10266549
11329276
11316883
11329276

Conc.
(g/ml)
10.21
10.35
10.36
12.83
12.84
12.76
15.38
15.35
15.36

%Recovery
99.74
101.11
101.25
100.28
100.27
99.69
100.04
99.98
99.99

Average

Mean
Area

SD

%RSD

100.7

9335453.3 77461.239

1.7

100.08

10306414

7155.1019

0.89

100.01

11325145

34525.226

0.54

Table 4: Recovery study of Phenylephrine HCl

PH
80%

100%

120%

Total
area
7176843
7207994
7234695
7905078
8112831
8121053
8835966
8879952
8902546

Conc.
(g/ml)
17.83
18.11
18.18
22.17
22.68
22.70
26.77
26.90
26.97

%Recovery
99.65
101.08
101.45
99.0
101.26
101.36
99.61
100.11
100.36

Average

Mean
Area

SD

%RSD

100.72

7206511

28954.51

0.71

100.54

8872821

33857.92

0.84

100.02

8046321

122388.8

1.2

Ruggedness and Robustness

The ruggedness of the method was determined by carried out the experiment by different
analyst. Robustness is determined by making slight changes in pH of buffer, change in flow
rate and mobile phase ratio. It was observed that there were not marked changes in
chromatograms, which indicated that the developed RP-HPLC method is robust and rugged.
Table 5: Assay of liquid dosage form
Formulation
AMB
CPM
PH

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Label
claim(mg/30ml)
30
4
10

Amt found
(mg/30ml)
29.7
3.94
9.91

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%Drug
content
99.00
99.04
99.10

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RESULT AND DISCUSSION

Ambroxol, Chlorpheniramine Maleate, and Phenylephrine HCl has absorbance wavelength at
261nm.The polynomial regression data for the calibration plot show good linear relationship
in the concentration range 32-48 g/ml for AMB, 6.4-12.8g/ml for CPM, 11.2-22.4 g/ml
for PH.Recovery study is carried out at three different level 80%, 100% and 120% by adding
pure drug to the previously analysed test sample. percentage recovery for all three drug was
determined by all the method and they were found to be acceptance criteria which are 99.999.95% for AMB, 100.0-100.77% for CPM and 100.0-100.72 for PH according to ICH
guidelines .The result shown in table no. 2,3 and 4.The reproducibility ,precision and
accuracy was found to be good ,which was evidence by low standard deviation,

Drug name
Linearity

Acceptance
criteria
-

Linearity Equation

Correlation
coefficient (r2)

0.997

Method

Accuracy (n = 3)
Precision (RSD
%)(n=6)Repeatabil
ity

Intraday Precision

Interday precision
Assay
LOD (g/ml)
LOQ (g/ml)
Robustness

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HPLC

%recovery found
to be within range
of 99.95-100.77
%relative
standard
deviation within
2%
%relative
standard
deviation within
2%
%relative
standard
deviation within
2%
Change in pH
Change in
wavelength
Change in flow
rate

AMB
32-48 g/ml
y= 484418x +
06

CPM
6.4-12.8g/ml
y = 760844x +
101581

R2=0.998

R2= 0.997

R2= 0.990

99.9-99.95

100.0-100.77

100.0-100.72

1.56

1.66

2.0

0.27

0.67

0.82

1.76

0.26

0.34

99.0
11.58 g/ml
35.092 g/ml
0.746

99.3
0.66 g/ml
2.02 g/ml
0.57

99.2
7.04 g/ml
21.36 g/ml
0.693

1.867

0.59

0.7

0.86

0.3

0.756

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PH
11.2-22.4 g/ml
339783x+479944

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CONCLUSION
The proposed study describes a new and simple RP-HPLC method for simultaneous
estimation of Ambroxol, Chlorpheniramine maleate and Phenylephrine HCl .The method
validated according to ICH guidelines, it is found to be simple, accurate and precise and
reproducible, sample recoveries from the formulation were in good agreement with their
respective label claims, which suggested that non interference of excipients in the estimation.
The most striking features of this method are its simplicity and rapidicity,not requiring
tedious sample preparation such as heating, degassing which are may needed for HPLC
procedure. All the above results indicate that, the method employed here are very simple,
accurate, economic and rapid for routine analysis and hence can adopted for the routine
quality assurance analysis.
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