WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Chaudhary et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 5.210

Volume 4, Issue 05, 1679-1694. Research Article ISSN 2278 4357

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR

SIMULTANEOUS ESTIMATION OF BROMHEXINE HYDROCHL
ORIDE, GUAIPHENESIN AND CHLORPHENIRAMINE MALEATE IN
TABLET.

Ankit B. Chaudhary*, Shweta M. Bhadani, Chintal M.Shah

Saraswati Institute of Pharmaceutical Sciences, Dhanap, Gandhinagar, 382355, India.

ABSTRACT
Article Received on
20 March 2015, A Reverse Phase High Performance Liquid Chromatographic method
Revised on 11 April 2015, was developed for the simultaneous estimation of Bromhexine
Accepted on 02 May 2015
Hydrochloride, Guaiphenesin and Chlorpheniramine Maleate. The
separation was achieved by C18 column (2504.6 mm, 5 m Particle
*Correspondence for Size) with the mobile phase consisting of 0.01 M Potassium
Author dihydrogen Phosphate Buffer pH-3 adjusted with 1% of Ortho
Dr. Ankit B. Chaudhary
Phosphoric Acid: Acetonitrile (40:60% v/v). Detection was carried out
Saraswati Institute of
at 254 nm. RP-HPLC method gives retention time of Bromhexine
Pharmaceutical Sciences,
Dhanap, Gandhinagar, Hydrochloride (BH), Guaiphenesin (GP) and Chlorpheniramine
382355, India. Maleate (CPM) was found to be 5.54min, 2.03min and 6.61 min
respectively. The method has been validated for ICH Guideline.
Linearity for Bromhexine Hydrochloride, Guaiphenesin and Chlorpheniramine Maleate were
found in the range of 10-60 g/ml, 125-750g/ml and 5-30 g/ml. The percentage recoveries
obtained for BH, GP and CPM were found to be in range of 99.40-101.95%, 100.63-101.51%
and100.64-101.51% respectively in tablet. Limit of detection and Limit of quantification was
found to be 1.449g/ml and 4.830g/ml for BH, 15.02g/ml and 50.08g/ml for GP and
0.795g/ml and 2.651g/ml for CPM respectively.

KEYWORDS: Reverse Phase High Performance Liquid Chromatography Method (RP-

HPLC), Bromhexine Hydrochloride (BH), Guaiphenesin (GP) and Chlorpheniramine Maleate
(CPM)

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INTRODUCTION
Bromhexine Hydrochloride (BH) is a 3, 5Dibromo-N (alpha) cyclohexyl-N (alpha)-Methyl
toluene-alpha -2-diamine Hydrochloride, represent the Class of Mucolytic Agent or
Expectorant Category. Bromhexine acts on the mucus at the formative stages in the glands,
within the mucus-secreting cells. Bromhexine disrupts the structure of acid muco
polysaccharide fibers in mucoid sputum and produces less viscous mucus, which is easier to
expectorate.[1] Guaiphenesin is a (RS) -3-(2-Methoxy phenoxy) propane-1, 2-diol. , represent
the Class of Bronchial Secretion Enhancer. Guaiphenesin is thought to act as an expectorant
by increasing the volume and reducing the viscosity of secretions in the trachea and bronchi.
It has been said to aid in the flow of respirator.[2] Chlorpheniramine Maleate is a [3-(4-
Chlorophenyl)-3- (pyridine-2-yl)propyl] dim ethylamine, represent the Class of Histamine H1
Antagonist . Chlorpheniramine binds to the histamine H1 receptor. This block the action of
endogenous histamine, which subsequently leads to temporary relief of the negative
symptoms brought on by histamine.[3] Literature Review related that few analytical Methods
[4-9]
have been reported for BH with other combination like UV Spectrophotometry ,
[10-19] [20-21]
HPLC , and HPTLC. Literature review related that few analytical Methods have
[22-30]
been reported for GP with other combination like HPLC , UV Spectrophotometry.[31-34]
Literature Review related that few analytical methods have been reported for CPM with other
[35-42]
combination like UV Spectrophotometry , HPLC.[43-53] The Present work describes a
validated reverse phase HPLC Method for Simultaneous determination of these drugs in
tablet.

Fig 1 Structure of BH Fig 2 Structure of GP Fig 3 Structure of CPM

EXPERIMENTAL WORK
Instrument and Apparatus
RP-HPLC instrument (Younglin, Korea) equipped with a PDA detector (YL-9160)
Rheodyne injector (20 l Capacity), C18, 250mm 4 .6mm, 5m (Particle size), Bischoff
Chromatography with YL-Clarity Model 1900 as software.

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Electronic weighing Balance (Sartorius CP224S with 0.1 mg sensitivity).

Ultra Sonicator (PCI make, Model-1.5L, 5H)
Uv Spectrophotometer (Shimadzu 1800)
pH meter (Systronics, Model- pH system 361)
Pipettes and volumetric flask: Borosil (10, 100, 500, 1000 ml)

Chemical and Materials

Table 1 List of Chemical and Materials
Material Company Grade
Bromhexine HCl (BH) Mediwin pharmaceutical Pure API
Laboratory ( Ahmedabad )
Chlorpheniramine Maleate (CPM) Mediwin pharmaceutical Pure API
laboratory ( Ahmedabad )
Guaiphenesin (GP) Kamron Analytical laboratory Pure API
(Ahmedabad)
Water SD fine Chem ltd, Mumbai HPLC Grade
Acetonitrile SD fine Chem ltd, Mumbai HPLC Grade
Methanol SD fine Chem ltd,Mumbai HPLC Grade
Potassium dihydrogen phosphate SD fine Chem ltd,Mumbai AR Grade
Ortho Phosphoric Acid SD fine Chem ltd,Mumbai AR Grade

Chromatographic Condition
Stationary phase: Water Spheris orb CNRP C18 Column (250 4.6 mm, 5m).
Mobile phase: Acetonitrile : 0.01 M Potassium dihydrogen phosphate pH-3 Adjusted
with 1% of O- phosphoric acid.(60:40 %v/v)
Detector: 254 nm
Injection volume: 20 l
Flow rate: 1.5 ml/min
Temperature: RT
Run time: 12 minutes

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Fig 4 Chromatogram of BH (20g/ml), GP (250g/ml) and CPM (10g/ml) in ACN:

Phosphate buffer pH- 3 with OPA (60:40 %V/V), Flow rate -1 ml/min

Preparation of Solutions
Preparation of Buffer (pH 3)(0.01 M KH2PO4)
An accurately weighed 0.6804 gm of potassium dihydrogen phosphate (0.01M) was
transferred into 500ml Volumetric Flask, Followed by addition of 300 ml of HPLC Grade
Water. Resulting solution was shaken for 5 min. Sonicated for 15 minute. Then resulting
solution filtered and transferred to another 500 ml Volumetric Flask& Volume was make up
to mark with HPLC grade Water. Then pH 3 was adjusted with ortho phosphoric acid (1%),
above solution filtered with vacuum filter using filter membrane (filter disc) (0.45m)
40 ml of Buffer and 60 ml Acetonitrile was mixed and solution was sonicated for degassing.

Composition of Mobile Phase

0.01 M Potassium dihydrogen phosphate Buffer pH-3 adjusted with 1%of O-phosphoric acid:
Acetonitrile (40: 60%V/V)

Preparation of Stock Solution (1000g/ml)

Weighed accurately 50mg of Bromhexine hydrochloride, 50 mg of Guaiphenesin and 50mg
of Chlorpheniramine Maleate was transferred into 3 different 50 ml volumetric flask and
dissolved into methanol up to mark.

Preparation of Sample Solution

Twenty tablets were weighed and crushed for fine powder. An accurately weighed powder
equivalent to 4 mg of Bromhexine Hydrochloride, 2 mg of Chlorpheniramine Maleate and 50
mg of Guaiphenesin was transferred into 20 ml volumetric flask and sample dissolves in

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methanol. Solution was sonicated for 15 minutes. Filtered the Solution with 0.45 Membrane
filter. Volume was made up to mark with methanol to give a 200g/ml of Bromhexine
hydrochloride, 2500 g/ml of Guaiphenesin and 100 g/ml of Chlorpheniramine Maleate.
From this solution 1ml solution was pipette out and transferred in 10 ml volumetric flask and
volume was made up to mark with methanol to obtain 20 g/ml of Bromhexine
hydrochloride,250 g/ml of Guaiphenesin and 10 g/ml of Chlorpheniramine Maleate.
Above solution was filtered through the membrane filter and injected using syringe.

Preparation of Calibration Curve

Standard stock solution of Bromhexine hydrochloride (1000g/ml), Guaiphenesin
(1000g/ml) and Chlorpheniramine Maleate (1000 g/ml) were diluted with methanol which
Contain 0.1, 0.2, 0.3, 0.4,0.5,0.6 ml,1.25, 2.50, 3.75, 5.00, 6.25, 7.5 ml and 0.05, 0.10, 0.15,
0.20, 0.25, 0.30 ml respectively into 10 ml volumetric flask and volume was made with
methanol .To get the final concentration of Bromhexine Hydrochloride was 10, 20, 30, 40,
50, 60 g/ml, Guaiphenesin was 125, 250, 375, 500, 625, 750 g/ml and Chlorpheniramine
Maleate was 5, 10, 15, 20, 25, 30 g/ml. All resulting solution were Scanned in HPLC and
Plot the graph of Area V s Concentration.

Calibration Curve were Plotted over a Concentration Range of 10-60g/ml, 125-750

g/ml and 5-30 g/ml of BH, GP and CPM Respectively.

Determination of Wavelength for Maximum Absorbance

The sensitivity of HPLC method that uses UV detection depends on proper selection of
detection wavelength. An ideal wavelength is that gives good response for drugs those are to
be detected.

In the present study, standard solution of BH, GP and CPM were scanned over the range of
200-400nm wavelengths. The both drugs showed good absorbance at 254 nm. So the 254 nm
was selected for simultaneous estimation of BH, GP and CPM in solid dosage forms.

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Fig 5 Overlain Spectra of BH (20g/ml), GP (250g/ml) and CPM (10g/ml)

A) VALIDATION
RESULT AND DISCUSSION
Linearity
Standard stock solution of Bromhexine hydrochloride (1000g/ml), Guaiphenesin
(1000g/ml) and Chlorpheniramine Maleate (1000 g/ml) were diluted 10 ml of Volumetric
flask. To get the final concentration of Bromhexine Hydrochloride was 10, 20, 30, 40, 50, 60
g/ml, Guaiphenesin was 125, 250, 375, 500, 625, 750 g/ml and Chlorpheniramine Maleate
was 5, 10, 15, 20, 25, 30 g/ml. All resulting solution were Scanned in HPLC and Plot the
graph of Area Vs Concentration.

Calibration Curve were Plotted over a Concentration Range of 10-60g/ml, 125-750 g/ml
and 5-30 g/ml of BH, GP and CPM Respectively.

Fig 6 Calibration Curve of BH

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Fig 7 Calibration curve of GP

Fig 8 Calibration Curve of CPM

Fig 9 Overlain Chromatogram of BH, GP and CPM

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Precision
Repeatability
Repeatability expresses the precision under the same operating conditions over a short
Interval of time. Six repeated injections of standard solutions were made and the response
factor of drug peaks and percentage RSD were calculated. Repeatability data for BH, GP, and
CPM are summarized in (Table 2)

Table 2 Repeatability study

Drugs Concentration Peak areaSD % RSD

(g/ml) (n=6)
BH 20 485526.32622.05 0.54
GP 250 10488216545.59 0.62
CPM 10 433924.52568.58 0.59

Intra Day & Inter Day Precision

Intraday precision was performed by analyzing three different concentrations (g/ml) within
linearity range, three times in a day. Intraday Precision data of BH, GP, and CPM are
summarized in Table 3, Table 4 and Table 5 respectively. % RSD for Intraday Precision were
found to be 0.25-1.38 for BH, 0.98-1.37 for GP and 0.40-0.53 for CPM.

Interday Precision was performed by analyzing three different concentrations (g/ml) within
linearity range, on three consecutive days. Interday Precision data of BH, GP and CPM are
summarized in Table 3, Table 4 and Table 5 respectively. %RSD for Interday Precision were
found to be 0.32-0.50 for BH, 0.92-1.37 for GP and 0.35-0.63 for CPM.

Table 3 Precision study of BH

Intraday Precision Inter Day Precision
Drug Concentration
MeanSD MeanSD
(g/ml) %RSD %RSD
(n=3) (n=3)
20 4817216661.01 1.38 479072.71646.98 0.34
BH 30 647374.74553.03 0.73 614816.33106.27 0.50
40 863713.72192.23 0.25 867147.32793.91 0.32

Table 4 Precision study of GP

Intraday Precision Inter Day Precision
Drug Concentration
MeanSD MeanSD
(g/ml) %RSD %RSD
(n=3) (n=3)
250 105482214528.85 1.37 107386814752.63 1.37
GP 375 129369111092.63 0.98 128620611929.41 0.92
500 142352916035.61 1.12 14165598684.56 0.61

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Table 5 Precision study of CPM

Drug Concentration Intraday Precision Inter Day Precision

(g/ml) MeanSD (n=3) %RSD MeanSD (n=3) %RSD
CPM 10 423493.31965.43 0.46 417124.71465.16 0.35
15 6173142504.19 0.40 614106.73918.86 0.63
20 812566.74312.48 0.53 8146954134.45 0.50

Accuracy (% Recovery)
Accuracy study was performed by addition of known amounts of standard drugs to a pre
analyzed sample of commercial product (standard addition method). Recovery by standard
addition method is shown in Table 6. The effect of the excipient and other additive present in
the validity of the suggested procedures was assessed by applying the standard addition
technique, which showed no interference during determination. Standard drug was added at
three different concentrations (10, 20, 30 g/ml of BH, 125,250,375 of GP and 5, 10, 15
g/ml for CPM to sample) and mixture were analyzed by proposed method. The good
recoveries prove the good accuracy of the proposed Methods.

Table 6 Recovery Study of BH, GP and CPM in Tablet

Drug Level Amount Amount Total %
taken added Amount RecoveryStudySD
(g/ml) (g/ml) Found (n=3)
(g/ml)
BH 50 20 10 30 99.400.13
100 20 20 40 101.950.12
150 20 30 50 101.660.10
50 250 125 375 100.630.55
GP 100 250 250 500 101.510.26
150 250 375 625 101.400.91
50 10 5 15 101.550.31
CPM 100 10 10 20 101.360.27
150 10 15 25 100.640.62

Limit of Detection
Limit of detection for BH, GP and CPM was found to be 1.449g/ml,15.02g/ml and 0.795
g/ml respectively.

Limit of Quantification
Limit of Quantification for BH, GP and CPM was found to be 4.830g/ml, 50.08g/ml and
2.651g/ml respectively

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Robustness
The method was found to be robust. Robustness was carried by varying parameters from the
optimized chromatographic conditions.

Table 7 Robustness Study

Factor Area
Value
BH GP CPM
1.49 493003 1063728 429073.3
Flow 1.50 483739 1053511 426420
Rate 1.51 486353.3 1050805 423729.6
( 0.1 ) Mean Area 487698.4 1056015 426407.62671.87
S.D (n=3) 4776.23 6815.47
%RSD 0.97 0.64 0.62
58:42 484145 1073711 429342
Mobile 60:40 483739 1053511 426420
Phase 62:38 485997.3 1058142 429468.6
(2) Mean Area 484627.1 1061788 428410.2
S.D (n=3) 1203.86 10581.9 1724.72
%RSD 0.24 0.99 0.40
pH 2.9 486436 1063114 429510.6
( 0.1) 3 483739 1053511 426420
3.1 481833 1052523 423151.3
MeanSD 484002.7 1056383
426360.63180.06
(n=3) 2312.8 5850.47
%RSD 0.47 0.55 0.74

Optical Regression Characteristics and Validation Parameters

Table 8 Optical Regression Characteristic and Validation Parameter
PARAMETERS BH GP CPM
Detection wavelength 254 nm
Calibration range (g/ml) 10-60 125-750 5-30
Regression Equation (y = mx + c) Y=18733x+87844 Y=3810.96x+116519.35 Y=36954x+25307
Slop (m) 18733 3810.96 36954
Intercept ( c ) 87844 116519.35 25307
Correlation coefficient (r) 0.997 0.998 0.998
Precision- Intraday (% RSD) 0.25-1.38 0.98-1.37 0.40-0.53
Precision-Interday ( %RSD) 0.32-0.50 0.92-1.37 0.35-0.63
Accuracy-Tablet (n=3) %Recovery S.D %Recovery S.D %Recovery S.D
(50%) 99.400.13 100.630.55 101.510.31
(100%) 101.950.12 101.510.26 101.360.27
(150%) 101.660.10 101.400.91 100.640.62
Limit of detection (g/ml) 1.449 15.02 0.795
Limit of quantification(g/ml) 4.830 50.08 2.651

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B) Application to Pharmaceutical Dosage form:

Applicability of the Proposed Method was tested by analyzing the tablet formulation. The
results are shown in Table 9.

Table 9 Assay of Pharmaceutical Dosage form

Formulation Parameter BH GP CPM
Label Claim
4 50 2
(mg)
Cheston DT
Amount
Tablet
found 4.040.04 50.020.21 2.010.01
(n=3)SD
%Drug
content 101.520.003 100.050.004 100.740.60
(n=3) S.D

CONCLUSION
The developed method was validated for various parameters like accuracy, recovery, linearity
as per ICH guidelines. The result obtain were within the acceptance criteria for the respective
parameters. The proposed method was applied for the estimation of Bromhexine
Hydrochloride, Guaiphenesin and Chlorpheniramine Maleate in marketed formulations. The
assay results conformed to the label claim of the formulation.

ACKNOWLEDGEMENTS
The Authors are thankful to Mediwin Pharmaceutical Lab. and Kamron Analytical Lab. At
Ahmedabad, for Providing Samples of Pure Drug API. The authors are also thankful to
Saraswati Institute of Pharmaceutical Sciences for providing necessary Equipment, Facility
and Chemicals.

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