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RESEARCH ARTICLE

 

Simultaneous Spectrophotometric Estimation of Ambroxal Hydrochloride and Salbutamol Sulphate by Second Order Derivative Method in Combined Dosage Form

 

Rajan V. Rele*

Central Research Laboratory, D.G. Ruparel College, Matunga, Mumbai 400016.

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

 

ABSTRACT:

The objective of the study was to develop a simple, accurate, precise and rapid a UV spectrophotometric i.e. second order derivative method for the determination of ambroxal hydrochloride and salbutamol sulphate in combined dosage form i.e. tablets by using distilled water as a solvent. The method was further validated by ICH guidelines. The proposed second order derivative method involves the measurement of absorbance of one drug at zero crossing point of other; hence wavelengths 221.1 nm and 236.4 nm were selected for the estimation of ambroxal hydrochloride and salbutamol sulphate respectively. The linearity of the proposed method was found in the concentration range of 1 to 10 µg /ml (r2= 0.9989) for ambroxal hydrochloride and 1 to 14 µg /ml (r2= 0.9981) for salbutamol sulphate respectively. The percentage mean recovery was found to be 100.813 % for ambroxal hydrochloride and 100.065 % for salbutamol sulphate respectively. The method was also statistically validated for its linearity, accuracy and precision. Both intra and inter day variations showed less percentage (%) RSD values indicating high grade of precision of this method.

 

KEYWORDS: UV spectrophotometric estimation, second order derivative method, ambroxal hydrochloride, Salbutamol sulphate

 

 

INTRODUCTION:

Ambroxal Hydrochloride is trans-4-[(2Amino-3,5-dibromobenzyl) amino] cyclohexanol. It shows molecular formula as C13H18Br2N2O.HCl with molecular weight 414.57. It is official in BP1 and IP2. Ambroxal is a metabolite of bromhexine. It is an expectoration improver and mucolytic agent used in the treatment of acute and chronic disorders characterized by the production of excess or thick mucus.

 

Salbutamol sulphate is chemically known as bis [(1RS)‐2‐[(1, 1‐ dimethylethyl) amino]‐1‐[4‐hydroxy‐3‐ (hydroxymethyl) phenyl] ethanol] sulphate, is beta‐adrenoceptor agonist used for the relief of broncho‐spasm in conditions such as asthma and chronic obstructive pulmonary disease.

 

 

 

Received on 26.02.2015       Modified on 16.03.2015

Accepted on 19.03.2015      © RJPT All right reserved

Research J. Pharm. and Tech. 8(4): April, 2015; Page 432-436

DOI: 10.5958/0974-360X.2015.00072.4

 

 

It is official in IP 2, is used to increase the volume and reduce the viscosity of tenacious sputum and is used as expectorant for productive cough. In literature survey reveals UV spectrophotometric3 and HPLC4-6 for simultaneous determination of ambroxal hydrochloride and salbutamol sulphate in combined dosage form. Combination of ambroxal hydrochloride and salbutamol sulphate is used for the treatment of asthma and bronchitis

 

MATERIALS AND METHOD:

Instrument and reagents:

Spectral scan was made on a Shimadzu UV-spectrophotometer, model 1800 (Shimadzu, Japan) with spectral band width of 0.5 nm with automatic wavelength corrections by using a pair of 10 mm quartz cells. All spectral measurements were done by using UV-Probe 2.42 software. Reference standard of ambroxal hydrochloride and salbutamol sulphate were obtained from reputed firm with certificate of analysis.

 

Preparation of Standard Drug Solutions:

25 mg standard ambroxal hydrochloride was weighed accurately and transferred to a 25 ml volumetric flask and sonicated with 30 ml distilled water for 15 minutes. The volume was made up to the mark with distilled water to give a stock solution of ambroxal hydrochloride of concentration 1000 μg /ml. From this solution, 10 ml of solution was pipetted out and transferred into 100 ml volumetric flask. The volume was made up to mark with distilled water to give a working standard solution of concentration 100 μg/ml.

 

Similarly 25 mg standard salbutamol sulphate was weighed accurately and transferred to a 25 ml volumetric flask and sonicated with 30 ml of distilled water for 15 minutes. The volume was made up to the mark with distilled water to give a stock solution of distilled water of concentration 1000 μg /ml. From this solution, 10 ml of solution was pipetted out and transferred into 100 ml volumetric flask. The volume was made up to mark with distilled water to give a working standard solution of concentration 100 μg/ml.

 

Estimation from Tablets:

Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 30 mg of ambroxal hydrochloride and 2 mg of salbutamol sulphate was weighed and transferred in 100 ml of volumetric flask. A 30 ml of distilled water was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with distilled water to give concentration as 300 μg /ml of ambroxal hydrochloride and 20 μg/ml of salbutamol sulphate respectively. For working sample solution 1 ml of such solution was diluted to 100 ml and such solution was used for analysis.

 

EXPERIMENTAL:

Method: Second order derivative method:

(a) For ambroxal hydrochloride:

For the selection of analytical wavelength, 100 μg/ml solution of ambroxal hydrochloride was scanned in the spectrum mode from 350 nm to 190 nm by using distilled water as blank. The second order derivative spectrum was obtained by using derivative mode by UV probe 2.42 software. From the spectrum, the amplitude of the second derivative spectrum was measured at 221.1 nm.

 

(b) For salbutamol sulphate:

For the selection of analytical wavelength, 100 μg/ml solution of salbutamol sulphate was scanned in the spectrum mode from 350 nm to 190 nm by using distilled water as blank. The second order derivative spectrum was obtained by using derivative mode by UV probe 2.42 software. From the spectrum, the amplitude of the second derivative spectrum was measured at 236.4 nm.

 

Preparation of calibration curves:

 Series of solutions containing 1 – 10 µg/ ml of ambroxal hydrochloride and 1 -14 µg/ ml of salbutamol sulphate were used to determine linearity of the proposed method respectively.  Solutions were scanned in the spectrum mode and absorbance spectra were converted to second order derivative spectra. The overlain spectra of ambroxal hydrochloride and salbutamol sulphate were given in Fig. 1(a), 1(b) respectively. 


 

 

 

Fig. 1(a): Overlay spectra of second order derivative of ambroxal hydrochloride in the concentration range of 2 and 10 µg/ ml.

 

Fig. 1(b): Overlay spectra of second order derivative of salbutamol sulphate in the concentration range of 2 and 14  µg/ ml.

 

 

 

After observing the overlain second order derivative spectra of ambroxal hydrochloride and salbutamol sulphate, the zero crossing points of both drugs were selected for analysis of other drug. The second wave length selected was 221.1 nm, the zero crossing point of salbutamol sulphate where ambroxal hydrochloride showed considerable absorbance. The second wavelength was 236.4 nm, the zero crossing point of ambroxal hydrochloride, where salbutamol sulphate showed considerable absorbance. The calibration curves were plotted of amplitude against concentrations [Fig. 2 (a), 2(b)].

 

 

 

Fig.2 (a): Calibration curve of ambroxal hydrochloride in the concentration range of 2-12 µg/ml.

 

Fig.2 (b): Calibration curve of salbutamol sulphate in the concentration range of 2-14 µg/ml.

 

Results of the analysis are given in table 1.

Table 1: Values of results of optical and regression of drugs

Parameter

Ambroxal

hydrochloride

Salbutamol sulphate

Detection Wavelength (nm)

221.1

236.4

Beer Law Limits (µg/ml)

1-10

1-14

Correlation coefficient(r2)

0.9989

0.9981

Regression equation  (y=b+ac)

 

 

Slope (a)

0.0007

0.0003

Intercept (b)

-0.00003

-0.00001

 

Estimation from capsules:

Powdered from twenty capsules were collected and weighed accurately and average weight of powder from each capsule was determined. Powder equivalent to 30 mg of ambroxal hydrochloride and 2 mg of salbutamol sulphate was weighed and transferred in 100 ml of volumetric flask. A 30 ml of distilled water was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with distilled water to give concentration as 300 μg /ml of ambroxal hydrochloride and 20 μg /ml of salbutamol sulphate respectively. A 10 ml of such solutions was diluted to 100 ml. It was scanned in the range of 200-350 nm against distilled water as blank. The absorbance spectra were converted to second order derivative spectra. Calculations were done as per the equations. The concentrations of ambroxal hydrochloride and salbutamol sulphate present in capsules were calculated by substituting the values of absorbance in linearity equations.

(a) For ambroxal hydrochloride Y = 0.0007x – 0.00003

(b) For salbutamol sulphate Y = 0.0003x - 0.00001

 

Method Validation:

These methods were validated according to ICH guidelines.

 

Accuracy:

To ascertain the accuracy of proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%). Percentage recovery for ambroxal hydrochloride and salbutamol sulphate was found in the range of 99.651 % to 100.403% and 100.332% to 100.750 % respectively. (Table2).

 

Linearity:

The linearity of measurement was evaluated by analyzing different concentration of the standard solutions of ambroxal hydrochloride and salbutamol sulphate. For both the drugs concentration range was found to be 1-10 µg/ml for ambroxal hydrochloride and 1-14 µg/ml for salbutamol sulphate.

 

Precision:

The method precision was established by carrying out the analysis of powder blend from capsules containing   30 mg of ambroxal hydrochloride and 2 mg of salbutamol sulphate. The assay was carried out for the drugs by using proposed analytical method in six replicates. The values of relative standard deviation were 0.0031 % for ambroxal hydrochloride and0.1467% for salbutamol sulphate in respectively indicating the sample repeatability of the method. The results obtained are tabulated in table 3.

 

Intra-day precision was estimated by assaying tablets powder blend containing 30 mg of ambroxal hydrochloride and 2 mg of salbutamol sulphate. The assay was carried out for the drugs by using proposed analytical method in six replicates. The results were average for statistical evaluation.

 

Inter-day precision was estimated by assaying tablets powder blend containing 30 mg of ambroxal hydrochloride and 2 mg of salbutamol sulphate for three consecutive days (i.e. 1st, 3rd and 5th days). The statistical validation data for intra and inter day precision is summarized in table 4.

 

Both intra- day and inter-day precision variation found to be less in % RSD values. It indicates high degree of precision of the method.

 

Table 2:Statistical evaluation of the data subjected to accuracy

Level of %

recovery

Amount present in µg/ml

Amount added in µg/ml

Amount found in µg/ml

% Recovery

Mean %

recovery

AMB

SAL

AMB

SAL

AMB

SAL

AMB

SAL

AMB

SAL

80%

10

2

8

1.6

18.116

3.609

100.644

100.250

 

100.403

 

 

100.453

10

2

8

1.6

18.092

3.615

100.511

100.416

10

2

8

1.6

18.141

3.623

100.783

100.694

100%

10

2

10

2

20.108

4.079

100.054

101.975

 

99.651

 

 

100.750

10

2

10

2

19.987

4.020

99.350

100.500

10

2

10

2

19.991

3.991

99.550

99.775

120%

10

2

12

2.4

22.111

4.408

100.504

100.181

 

100.340

 

 

100.332

10

2

12

2.4

22.123

4.419

100.559

100.431

10

2

12

2.4

21.991

4.417

99.959

100.386

AMB  = Ambroxal hydrochloride,     SAL=Salbutamol sulphate

 

Table 3: Statistical evaluation of the data subjected to method of precision

Sr.No.

Sample No.

% Assay

 

 

Ambroxal

hydrochloride

Salbutamol sulphate

1

1

100.18

100.214

2

2

100.125

100.009

3

3

99.92

99.890

4

4

100.22

100.148

5

5

100.025

99.920

6

6

100.018

100.22

Mean % assay

100.0813

100.0658

%R.S.D.

0.0031

0.1467

 

Table 4: Summary of validation parameter for intra-day and inter-day

Sr. No.

Parameters

Ambroxal

hydrochloride

Salbutamol sulphate

1

Intra-day precision 

(N=3)amount found ±

% R.S.D.

99.75%

 

0.1912

99.75%

 

0.1514

2

Inter-day precision 

(N=3)amount found ±

 % R.S.D.

98.774

 

0.1142

98.925%

 

0.1142

 

 

 

RESULT AND DISCUSSION:

The developed second order derivative spectrophotometric method for simultaneous determination of ambroxal hydrochloride and salbutamol sulphate in tablet formulation was found to be simple and convenient for the routine analysis of two drugs. The method is used to eliminate the spectral interference from one of the two drugs while estimating the other drug by selecting the zero crossing point on the derivative spectra of each drug as the selected wavelength. The proposed method is accurate, precise and reproducible. It is confirmed from validation data as given in tables 1 to 4. The % RSD was found to be less than 1, which indicates validity of method. Linearity was observed by linear regression equation method for ambroxal hydrochloride and salbutamol sulphate in different concentration range. The correlation coefficient of these drugs was found to be close to 1.00, indicating good linearity figure 2 (a) and 2 (b).

 

The assay results obtained by proposed method is shown in table 2 are in good agreement. Hence proposed method can be used for routine analysis of these two drugs in combined dosage form. Method is simple, accurate, precise, reliable, rapid, sensitive, reproducible and economical. It is validate as per ICH guidelines.

 

CONCLUSION:

The proposed method is simple, precise, accurate and rapid for the determination of ambroxal hydrochloride and salbutamol sulphate in combined dosage form. This method can be adopted as an alternative to the existing methods. It can be easily and conveniently adopted for routine quality control analysis.

 

ACKNOWLEDGEMENT:

Authors express sincere thanks to the principal of D.G. Ruparel College, Dr. Tushar Desai, for encouragement and providing laboratory facilities.

 

REFERENCES:

1.       British pharmacopoeia. Licensing division HMSO ,Norwich. 2003.

2.       Indian Pharmacopeia, Controller of Publication, Delhi, 2007, Vol- 1, II, III.

3.       P.A. Patel, M.N. Dole, P.S. Shedpure, S.D. Sawant. Spectrophotometric simultaneous estimation of salbutamol and ambroxol in bulk and formulation. Asian Journal of  Pharmaceutical and  Clinical Research, 4( 3); 2011: 42-45.

4.       Avinash V. Deosarkar, Shruti D. Deshpande, Sanjay G.Walode, Deepali S. Tuljapure, Sainath G. Tekale, Vijay M. Waghmode, Simultaneous quantification of salbutamol sulphate and ambroxol hydrochloride by RP-HPLC and HPTLC in bulk drug and dosage form. International  Journal of Pharmacy and Pharmaceutical Sciences, 4 (l4); 2012:307-311.

5.       V. Sri Kalyani, D. Meena Bharathi, M. Anusha, B. Chandra Priyanka and Buchi N. Nalluri, Development and validation of RP HPLC PDA method for the simultaneous estimation of salbutamol sulphate and ambroxol hydrochloride in pharmaceutical dosage forms.  Journal of Chemical and Pharmaceutical Research, , 5(11); 2013:443-449.

6.       Parag G Bhortake, Rama S Lokhande. Simultaneous Determination of Salbutamol Sulphate and Ambroxol Hydrochloride in Solid Dosage Form by RP-HPLC. International  Journal of Pharma Research and Health Sciences, , 2 (6); 2014: 408-412.