Development and Validation of Analytical Method for Bromhexine Hydrochloride, Chlorpheniramine Maleate and Guaifenesin by Simultaneous Equation Spectroscopic Method

 

Ladmeera1*, Hasumati Raj2, Vineet Jain2

1Research Scholar 2014, Gujarat Technological University, Gujarat

2QualityAssurance Department, Shree Dhanvantary Pharmacy College, Kim, Surat

*Corresponding Author E-mail: meeralad235@gmail.com

 

ABSTRACT:

A simple, accurate and precise spectroscopic method was developed for simultaneous estimation of Bromhexine hydrochloride, Chlorpheniramine maleate and Guaifenesin in syrup using simultaneous equation Method. In this spectroscopic method, 249.00 nm and 261.00 nm and 274.00 nm wavelengths respectively were selected for measurement of slope and intercept according to its calibration curves. Bromhexine, Chlorpheniramine maleate and Guaifenesin show linearity in a concentration range of 02-10μg/ml, 01-05μg/ml and 25-125μg/ml at their respective λ max. Accuracy, precision and recovery studies were done by QC samples covering lower, medium and high concentrations of the linearity range. The relative standard deviation for accuracy, precision studies were found to be within the acceptance range (<2%). The limit of determination was 0.102μg/ml, 0.169μg/ml and 0.312μg/ml for Bromhexine, chlorpheniramine maleate and guaifenesin respectively. The limit of quantification was 0.311μg/ml, 0.947μg/ml and 0.514μg/ml for Bromhexine, chlorpheniramine maleate and guaifenesin respectively. Recovery of Bromhexine, guaifenesin and chlorpheniramine maleate were found to be 100.02 %, 101.17 and 100.66% respectively confirming the accuracy of the proposed method. The proposed method is recommended for routine analysis since they are rapid, simple, accurate and also sensitive and specific by no heating and no organic solvent extraction.

 

KEYWORDS: Bromhexine hydrochloride, Guaifenesin, Chlorpheniramine maleate, simultaneous estimation, Simultaneous equation method, analysis method.

 


INTRODUCTION:

Bromhexine HCl (BRH) is a mucolytic agent used in the treatment of respiratory disorders associated with viscid or excessive mucus. Bromhexine depolymerises muco-polysaccharides directly as well as by liberating lysosomal enzyme network of fibres in tenacious sputum is broken.[1]

 

Figure1: Structure of Bromhexine(3)

 

Guaifenesin may act as an irritant to gastric vagal receptors, and recruit efferent parasympathetic reflexes that cause glandular exocytosis of a less viscous mucus mixture. Cough may be provoked. Guaifenesin increases fluid and mucus removal from the upper respiratory tract by increasing the volume of secretion and reducing their adhesiveness and surface tension.

 

Figure2: Structure of Guaifenesin(3)

Chlorphenamine or chlorpheniramine, commonly marketed in the form of chlorpheniramine maleate, is a first-generation alkylamine antihistamine used in the prevention of the symptoms of allergic conditions such as rhinitis and urticaria. Chlorpheniramine competes with histamine for H1 receptor sites, there by antagonizing many histamine effects to reduce allergy signs and symptoms. So, it ultimately reduce the cough.

 

Figure 3: Structure of Chlorpheniramine maleate(3)

 

The review of literature regarding quantitative analysis of Bromhexine, Guaifenesin and Chlorpheniramine maleate revealed that no attempt was made to develop analytical methods for these three drugs. Some spectrometric methods and chromatographic methods have been reported for the estimation of the individual drugs.[6-44]The focus of the present study was to develop and validate a rapid, stable, specific, and economic spectroscopic method for the estimation of Bromhexine, Guaifenesin and Chlorpheniramine maleate in Syrup.

 

MATERIALS AND METHODOLOGY:

·         Bromhexine HCl was gifted by Galpha Laboratories Ltd., Ankleshwar.

·         Chlorpheniramine Maleate was gifted by Maharshee  Laboratories Pvt. Ltd., Bharuch.

·         Guaifenesin was gifted by Pan Drugs Limited, Nandesari.

·         A double beam UV/Visible spectrophotometer (Shimadzu model 2450, Japan) with spectral width of 2 nm, 1 cm quartz cells was used to measure absorbance of all the solutions.

·         Spectra were automatically obtained by UV-Probe system software.

·         An analytical balance (Sartorius CD2250, Gottingen, Germany) was used for weighing the samples.

·         Sonicator (D120/2H, TRANS-O-SONIC)

·         Class ‘A’ volumetric glassware were used (Borosillicte)

 

Standard Solution:

Preparation of the Stock Solution of Bromhexine HCl:

An accurately weighed quantity of  Bromhexine HCl (10 mg) was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with Methanol to obtain standard solution having concentration of Bromhexine (100μg/ml). Sonicate for 10 mins if require.

 

Preparation of the Stock Solution of Chlorpheniramine Maleate:

An accurately weighed quantity of Chlorpheniramine Maleate (10 mg) was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with Methanol to obtain standard solution having concentration of Chlorpheniramine Maleate (100μg/ml). Sonicate for 10 mins if require.

 

Preparation of the Stock Solution of Guaifenesin:

An accurately weighed quantity of Guaifenesin (15 mg) was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with Methanol to obtain standard solution having concentration of Guaifenesin (150μg/ml). Sonicate for 10 mins if require.

 

Preparation of Test Solution:

From the stock solution of BROM take 0.4ml and from stock solution of CHL take 0.2ml and from the stock solution of GUA take 3.33ml transferred in to 10ml volumetric flask and diluted up to mark with methanol to give a solution having strength of BROM was 4μg/ml, CHL was 2μg/ml and GUA was 50μg/ml.

 

Calibration curves for Bromhexine HCl:

Pipette out 0.2, 0.4, 0.6, 0.8and 1.0 ml of the stock solution of Bromhexine (100μg/ml), , 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml stock solution of Chlorpheniramine maleate (100μg/ml) and 1.66 ml, 3.33 ml, 5.0 ml, 6.66 ml, 8.33 ml stock solution of Guaifenesin (150μg/ml)into a series of 10ml volumetric flasks and the volume was adjusted to mark with methanol and measured absorbance at 249.00nm, 261nm and 274 nm. Plot the graph of absorbance versus respective concentration of Irbesartan and atorvastatin. Linearityrange of BROM, CHL and GUA was found with correlation co-efficient.

 

Development and validation of spectroscopic simultaneous equation method:

Selection of wavelength and Method Development For determination of Bromhexine Hydrochloride, Chlorpheniramine Maleate and Guaifenesin:

The standard solution of BROM, CHL and GUA were scanned separately between 200-400nm, and BROM showed absorbance maxima at 249.00 nm and CHL at 261.00 nm and GUA at 274.00 nm (figure 4)


 

 

Figure 4: Overlain linear zero order spectra of BRO (Green), CHL (Violet), GUA (Brown) in 2:1:25 ratio, respectively

 


The concentrations of three drugs are calculated using the following equations:

Where:

Sx, Sy and Sz are the slops of drugs X, Y and Z in their calibration curves:

 

A1*,A2* and A3* are the modified Absorbance

A1* = A1 – (Ix1 + Iy1 + Iz1)

A2* = A2 – (Ix2 + Iy2 + Iz2)

A3* = A3 – (Ix3 + Iy3 + Iz3)

 

A1,A2 and A3 are the Absorbance of test at λ1, λ2 and λ3 respectively

 

Ix, Iy and Iz are the Intercepts of drugs X, Y and Z in their calibration curves


Table 1: Calibration data for BROM, CHL and GUA at249.00 nm and 261.00 nm and 274.00 nm respectively. *(n=6)

Sr no.

Brom

Chlor

Gua

Abs. ± SD (n=6)

Brom (248 nm)

Chlor (261nm)

Gua (274nm)

1

2

1

25

0.108 ±0.0083

0.016 ±0.0051

0.211 ±0.0009

2

4

2

50

0.196 ±0.0021

0.037 ±0.0020

0.477 ±0.0020

3

6

3

75

0.286 ±0.0021

0.0535 ±0.0005

0.717 ±0.0013

4

8

4

10

0.366 ±0.0016

0.071 ±0.0075

0.932 ±0.0012

5

10

5

125

0.457 ±0.0018

0.0945 ±0.0010

1.190 ±0.0013

 

Table 2: Intraday and Interday precision data for estimation of BROM, CHL and GUA*(n=3)

Drug

Concentration (ppm)

Precision Mean ± %RSD

Intraday

Interday

Bromhexine HCl

02

0.181 ± 0.317

0.183 ± 0.546

04

0.373 ± 0.309

0.376 ± 0.405

06

0.525 ± 0.190

0.525 ± 0.290

Chlorpheniramine Maleate

01

0.252 ± 0.604

0.183 ± 0.821

02

0.425 ± 0.489

0.376 ± 0.405

03

0.622 ± 0.092

0.426 ± 0.590

Guaifenesin

25

0.432 ± 0.266

0.434 ± 0.351

50

0.653 ± 0.306

0.653 ± 0.233

75

0.944 ± 0.266

0.945 ± 0.161

 


Validation parameters:(45)

1. Linearity and Range:

The Zero order (fig.4) showed linear absorbance at 249.00 nm for BROM (02-10 µg/ml), 261.00 nm for CHL (01-05µg/ml) and 274 nm for GUA (25-125µg/ml) with correlation Coefficient (r2) of 0.9997, 0.9959 and 0.9988for BROM, CHL and GUA, respectively. (figure 5, 6 and 7) This method obeyed beer’s law in the concentration range 02-10µg/ml, 01-05µg/ml and 25-125µg/ml for BROM, CHL and GUA respectively. (Table 1)

 

Figure 5: Calibration curve for BROM at 249.00 nm

 

Figure 6: Calibration curve for CHL at 261.00 nm

 

2. Precision:

I. Intraday Precision:

The precision of the developed method was assessed by analysing samples of the same batch in nine determinations with three Standard solutions containing concentrations 1, 2, 3μg/ml for Chlorpheniramine Maleate 2, 4, 6μg/ml for Bromhexine HCl and 25, 50, 75μg/ml for Guaifenesin.  Three replicate (n=3) each on same day.It presented in Table 2. These% RSD value was found to be less than ±2. 0 indicated that the method is precise.

 

Figure7: Calibration curve for GUA at 274.00 nm

 

II. Interday Precision:

The precision of the developed method was assessed by analysing samples of the same batch in nine determinations with three Standard solutions containing concentrations 1, 2, 3  μg/ml for Chlorpheniramine Maleate, 2, 4, 6μg/ml for Bromhexine HCl and 25, 50, 75 μg/ml for Guaifenesin. Triplicate (n=3) per day for consecutive 3 days for inter-day precision. Interday precision data presented in Table 2. These % RSD value was found to be less than ±2.0indicated that the method is precise.

 

3. Accuracy:

Accuracy of the method was determined by recovery study from syrup at three level (80%, 100%, 120%)of standard  addition. The% recovery values are tabulated in Table 3, 4and 5 Percentage recovery for BROM, CHL and GUA by this method was found in the range of 99.77-100.05%, 99.50-102.22% and99.42-102.27%, respectively, the value of % RSD within the limit indicated that the method is accurate and percentage recovery shows that there is no interference from the excipients.

 

4. Limit of Detection and Quantitation:

The LOD for BROM, CHL and GUA was conformed to be 0.1026 µg/ml, 0.1696 µg/ml and 0.3126 µg/ml, respectively. The LOQ for BROM, CHL and GUA was conformed to be 0.3110 µg/ml, 0.5141 µg/mland 0.9473 µg/ml, respectively. The obtained LOD and LOQ results are presented in Table 6

 

5. Robustness and Ruggedness

The obtained Ruggedness and Robustness results are presented in table 7 The % R.S.D was found to be 0.21- 0.83 % for BROM, 0.23- 0.46 % for CHL and 0.11 – 0.35 % for GUA.  These %RSD value was found to be less than ± 2.0 indicated that the method is precise. No significant  changes  in  the  spectrums  were  observed,  proving that the developed method is rugged and robust.


 

Table 3: Recovery data of BRO*(n=3)

Conc. Of BRO from formulation (µg/ml)

Amount of Std. BRO added (µg/ml)

Total amount of BRO (µg/ml)

Total amount of BRO found (µg/ml)* Mean± SD

% Recovery (n=3)

% RSD BRO

2

1.6

3.6

3.62±0.0005

100.55

0.18

2

2.0

4.0

3.99±0.0005

99.78

0.17

2

2.4

4.4

4.39±0.0005

99.77

0.16

 

Table 4: Recovery data of CHL*(n=3)

Conc. of CHL from formulation (µg/ml)

Amount of Std. CHL added (µg/ml)

Total amount of CHL(µg/ml)

Total amount of CHL found (µg/ml)* Mean± SD

% Recovery (n=3)

% RSD CHL

1

0.8

1.8

1.82±0.0005

102.22

0.13

1

1.0

2.0

1.98±0.0005

99.50

0.13

1

1.2

2.2

2.18±0.0005

101.81

0.12

 

Table 5: Recovery data of GUA*(n=3)

Conc. of GUA from formulation (µg/ml)

Amount of Std. GUA added (µg/ml)

Total amount of GUA (µg/ml)

Total amount of GUA found (µg/ml)* Mean± SD

% Recovery (n=3)

% RSD GUA

25

20

45

44.74±0.001

99.42

0.51

25

25

50

50.14±0.001

100.29

0.29

25

30

55

56.25±0.005

102.27

0.15


 

Table 6 LOD and LOQ data of BROM, CHL and GUA *(n=10)

Conc. (μg/ml)

BRO(249.0 nm) (n=10)

CHL(261.0 nm) (n=10)

GUA(274.0 nm) (n=10)

DEX

CHL

GUA

Avg. ± SD

%RSD

Avg. ± SD

%RSD

Avg. ± SD

%RSD

2

1

25

0.181 ±0.0013

0.744

0.252 ±0.0009

0.386

0.432 ±0.0009

0.212

LOD(μg/ml)

0.1026

0.1696

0.3126

LOQ(μg/ml)

0.3110

0.5141

0.9473


 

Table 7: Robustness and Ruggedness data of BROM, CHL and GUA *(n=3)

Drugs

Concentration

Mean  ±% RSD

Instrument I

Instrument II

Bromhexine HCl

02

0.181 ± 0.635

0.182 ± 0.836

04

0.371 ± 0.410

0.372 ± 0.410

06

0.525 ± 0.219

0.526 ± 0.290

Chlor-pheniramine Maleate

01

0.250 ± 0.460

0.251 ± 0.229

02

0.426 ± 0.234

0.427 ± 0.468

03

0.621 ± 0.245

0.623 ± 0.245

Guaifenesin

25

0.433 ± 0.133

0.434 ± 0.351

50

0.651 ± 0.177

0.652 ± 0.234

75

0.942± 0.122

0.944 ± 0.323

 

 

 

 

Drugs

Concentration

Change in Wavelength (249 ± 0.2 nm,  261 ± 0.2 nm, 274 ± 0.2 nm) Mean  ±% RSD

Wavelength 249.20 nm

Wavelength 248.80 nm

Bromhexine HCl

02

0.182 ± 0.837

0.183 ± 0.833

04

0.371 ± 0.411

0.372 ± 0.819

06

0.524 ± 0.397

0.524 ± 0.190

 

 

261.20 nm

260.80 nm

Chlorpheniramine Maleate

01

0.252 ± 0.604

0.254 ± 0.988

02

0.424 ± 0.271

0.425 ± 0.359

03

0.621 ± 0.161

0.623 ± 0.424

 

 

274.20 nm

273.80 nm

Guaifenesin

25

0.433 ± 0.230

0.434 ± 0.479

50

0.652 ± 0.234

0.653 ± 0.306

75

0.942 ± 0.162

0.944 ± 0.105

 


Application of the proposed method for analysis of BROM, CHL and GUAIN combined capsule dosage form:

·         Pipette out 5 ml of liquid sample, Add 10 ml of methanol, basify with 10 ml 1 M NaOH solution.

·         Extract with 10 ml chloroform 4 times. Combine chloroform and evaporate.

·         Take all residue in 20 ml methanol and make the volume upto 100 ml.

·         Finally, measure absorbance at 249.0 nm, 261.0 nm and 274.0 nm.

·         Determine the concentration of drugs using equation

 

 

Drugs

% Assay ± % RSD(n=3)

IP Limit

Bromhexine HCl

100.83 ± 0.0010

98.5-101.5%

Chlorpheniramine Maleate

100.50 ± 0.0005

98.0-101.0%

Guaifenesin

100.46 ± 0.0058

98.0-101.5%

 


 

 

Summary of validation parameter:

Table 8: Summary of validation parameters

Parameter

Bromhexine HCl

Chlorpheniramine Maleate

Guaifenesin

λmax

249.0 nm

261.0 nm

274.0 nm

Linearity

2 – 10 ppm

1 – 5 ppm

25 – 125 ppm

Regression equation

y = 0.043x + 0.0223

y = 0.0189x – 0.0022

y = 0.0097x - 0.0183

Correlation coefficient (r2)

0.9997

0.9959

0.9988

Accuracy

100.02 ± 0.628

101.17 ± 1.466

100.66 ± 1.460

LOD (n=10)

0.1026 ppm

0.1696 ppm

0.3126 ppm

LOQ(n=10)

0.3110 ppm

0.5141 ppm

0.9473 ppm

Precision

Intra-day(n=3)

Inter-day (n=3)

%RSD

0.190 – 0.317

0.290 – 0.546

%RSD

0.092 – 0.604

0.321 – 0.821

%RSD

0.266 – 0.306

0.161 – 0.351

Assay

100.833 ± 0.001%

100.50 ± 0.0005%

100.46 ± 0.005%

Robustness(%RSD)

0.219 – 0.836%

0.234 – 0.468 %

0.122– 0.351 %

 


CONCLUSION:

A new, Simultaneous Equation method has been developed for estimation of Bromhexine hydrochloride, Chlorpheniramine maleate and Guaifenesin in syrup. The method was validated by employment of ICH(18) guidelines. The validation data is indicative of good precision and accuracy, and prove the reliability of the method.

 

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Received on 04.03.2015          Accepted on 21.03.2015        

© Asian Pharma Press All Right Reserved

Asian J. Res. Pharm. Sci. 5(2): April-June 2015; Page 76-82

DOI: 10.5958/2231-5659.2015.00013.2