Simultaneous Spectrophotometric Estimation of Ambroxal Hydrochloride and Guaiphenesin by Area Under Curve Method in Combined Dosage Form

 

Rajan V. Rele.*

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

 

ABSTRACT:

The objective of the study was to develop a simple, accurate, precise and rapid a UV spectrophotometric i.e. area under curve method for the determination of ambroxal and guaiphenesin in combined dosage form i.e. tablets by using distilled water as a solvent. The method was further validated by ICH guidelines. The proposed area under curve method involves the measurement of area at selected analytical wavelength ranges and performing the analysis using “Cramer’s rule and Matrix method” .The two analytical wavelengths ranges were used i.e. 239-248 nm and 268-277 nm for estimation of ambroxal hydrochloride and guaiphenesin respectively. The linearity of the proposed method was found in the concentration range of 1- 10 µg /ml (r²= 0.9999) for ambroxal hydrochloride and 10-100 µg /ml (r²=0.9999) for guaiphenesin respectively. The percentage mean recovery was found to be 100.124 % for ambroxal hydrochloride and 99.98 % for guaiphenesin respectively. The method was statistically validated for its linearity, accuracy and precision as per ICH guidelines.  Both intra and inter day variation showed less percentage (%) RSD values indicating high grade of precision of this method.

 

 

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.

Guaiphenesin  is, 3-(2-Methoxyphenoxy)-1,2-propanediol. It shows molecular formula as C₁₀H₁₀O₄ with molecular weight as 198.2. It is official in BP¹ and IP² and USP³ 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 spectrophotometric method⁴ for simultaneous determination of ambroxal hydrochloride and guaiphenesin in combined dosage form.

 

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 and guaiphenesin were obtained from reputed firm with certificate of analysis.

 

Preparation of standard drug solutions:

100 mg standard ambroxal hydrochloride was weighed accurately and transferred to a 100 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 100 mg standard guaiphenesin was weighed accurately and transferred to a 100 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 100 mg of guaiphenesin   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 1000 μg/ml of guaiphenesin respectively. For working sample solution 1 ml of such solution was diluted to 100 ml and such solution was used for analysis.

 

EXPERIMENTAL:

Method: Area under curve method:

Area under curve method involves the calculation of integrated values of absorbance with respect to the wavelength between two selected wavelengths such as λ₁ and λ₂. The area under curve between λ₁ and λ₂ was calculated by UV probe 2.42 software.

 

(a) For ambroxal hydrochloride:

For the selection of analytical wavelength range, 10 μg/ml solution of ambroxal hydrochloride was scanned in the spectrum mode from 350 nm to 190 nm by using distilled water as blank. On examination of the spectra, 239- 248 nm was selected as working wavelength range for ambroxal hydrochloride.

 

(b) For guaiphenesin:

For the selection of analytical wavelength range, 10 μg/ml solution of guaiphenesin was scanned in the spectrum mode from 350 nm to 190 nm by using distilled water acid as blank. On examination of the spectra, 268-277 nm was selected as working wavelength range for guaiphenesin.

 

Preparation of calibration curves:

Series of solutions containing 1 – 10 µg/ ml of ambroxal hydrochloride and 10 -100 µg/ ml of guaiphenesin were used to determine linearity of the proposed method respectively. Area under curve of above solutions of ambroxal hydrochloride and guaiphenesin were measured at their respective selected analytical wavelength ranges. [Fig. 1(a), 1(b)]. This area under curve (AUC) was then divide by concentration in g/lit to get X_amb for ofloxacin and X_guaip for guaiphenesin.

 

Fig 1(a): Spectrum showing area under curve of ambroxal hydrochloride in the concentration of 2 µg/ ml at 239-248 nm.

 

Fig. 1(b):Spectrum showing area under curve of guaiphenesin in the concentration of 60 µg/ ml at 268-277 nm.

 

After measuring the area under curve of ambroxal hydrochloride at 229-248 nm and 268-277 nm for guaiphenesin by using UV-Probe software 2.42, the calibration curves were plotted of area under curve against concentrations [Fig. 2 (a), 2(b)].

      

Fig.2 (a): Calibration curve of ambroxal hydrochloride  in the concentration range of 2-10 µg/ml.

 

Fig.2 (b): Calibration curve of guaiphenesin in the concentration range of 2-10 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	Guaiphenesin
Detection Wavelength range (nm)	239-248	268-277
Beer Law Limits (µg/ml)	1-10	10-100
Correlation coefficient(r2)	0.9999	0.9999
Regression equation (y=b+ac)
Slope (a)	0.011	0.0079
Intercept (b)	0.0004	0.0160

 

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 100 mg of guaiphenesin 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 1000 μg/ ml of guaiphenesin respectively. Appropriate aliquot was pipetted out from the sample solution and was further diluted to obtain mixture containing 3 µg/ml of ambroxal hydrochloride and 10 µg/ml of guaiphenesin respectively. The spectrum of sample solution containing ambroxal and guaiphenesin was recorded and areas under curves were recorded in wavelength ranges of 239-248 nm and 268-277 nm. The areas under curves were analyzed by applying “Crammer’s rule and “Matrix method”. It is defines as “The total area under curve of mixture at particular wavelength range is equal to sum of area under curve of individual component at same wavelength range.” (Fig. 3).    

 

Fig.3:Spectrum showing area under curve of mixture at 239-248 nm and 268-277 nm.

 

X = AUC of component between selected wavelength ranges

 

Concentration of that component in mg/lit

 

 C_amb = (X^guaip2.AUC^M268-277) – (X^guaip1.AUC^M239-248)

                          (X^amb1.X^guaip2) – (X^amb2.X^guai1)

 

C_guaip = (X^amb1.AUC^M268-277) – (X^amb2.AUC^M239-248)

                          (X^amb1.X^guaip2) – (X^amb2.X^guai1)

 

Where,

C_amb = Concentration of ambroxal hydrochloride

C_guaip = Concentration of guaiphenesin

X^amb1 = Area under curve of ambroxal hydrochloride at wavelength 239-248 nm

X^amb2 = Area under curve of ambroxal hydrochloride at wavelength 268-277 nm

X^guaip1 = Area under curve of guaiphenesin at wavelength 239-248 nm

X^guaip2 = Area under curve of guaiphenesin at wavelength 268-277 nm

AUC^M = Area under curve of mixture

 

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%). Percent recovery for ambroxal hydrochloride was found in the range of 100.03% to 100.07% and guaiphenesin was found in the range of 100.07% to 100.11 %. (Table 2).

 

 

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	GUI	AMB	GUI	AMB	GUI	AMB	GUI	AMB	GUI
80%	3.0	10	2.4	8	5.407	18.043	100.14	100.24	100.03	100.07
	3.0	10	2.4	8	50392	18.032	99.87	100.18
	3.0	10	2.4	8	5.405	17.976	100.1	99.87
100%	3.0	10	3.0	10	6.0054	20.036	100.09	100.18	100.07	100.07
	3.0	10	3.0	10	6.020	20.028	100.34	100.14
	3.0	10	3.0	10	5.986	19.978	99.78	99.89
120%	3.0	10	3.6	12	6.511	22.026	100.17	100.12	100.06	100.11
	3.0	10	3.6	12	6.609	22.026	100.15	100.28
	3.0	10	3.6	12	6.590	21.986	99.86	99.94

AMB  = Ambroxal hydrochloride,    

GUI = Guaiphenesin

 

 

Linearity:

The linearity of measurement was evaluated by analyzing different concentration of the standard solutions of ambroxal hydrochloride and guaiphenesin respectively. For both the drugs concentration range was found to be 1-10 µg/ml for ambroxal hydrochloride and 10-100 µg/ml for guaiphenesin.

 

Precision:

The method precision was established by carrying out the analysis of tablets powder blend containing 30 mg of ambroxal and 100 mg of guaiphenesin. The assay was carried out for the drugs by using proposed analytical method in six replicates. The values of relative standard deviation were well within limits 99.45 % and 100.54 % for ambroxal hydrochloride and 99.563 % and 100.34 % for guaiphenesin respectively indicating the sample repeatability of the method. The results obtained are tabulated in table 3.

 

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

Sr. No.	Sample No.	% Assay
		AMB	GUI
1	1	100.18	100.08
2	2	100.54	100.34
3	3	99.45	99.563
4	4	100.09	99.91
5	5	100.36	100.34
6	6	100.18	99.65
Mean % assay		100.124	99.98
%R.S.D.		0.4140	0.3337

 

Intra-day precision was estimated by assaying tablets powder blend containing 30 mg of ambroxal and 100 mg of guaiphenesin restively. 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 100 mg of guaiphenesin for three consecutive days (i.e. 1^st, 3^rd and 5^th days). The statistical validation data for intra and inter day precision is summarized in table 4.

 

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

Sr. No.	Parameters	AMB	GUI
1	Intra-day precision  (N=3)amount found ± % R.S.D.	100.11% 0.1418	100.07%  0.2103
2	Inter-day precision  (N=3)amount found ± % R.S.D.	98.15% 0.1857	98.69% 0.1847

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

 

RESULT AND DISCUSSION:

The developed area under curve spectrophotometric method for simultaneous determination of ambroxal hydrochloride and guaiphenesin in tablet formulation was found to be simple and convenient for the routine analysis of two drugs. 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 and guaiphenesin 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 and guaiphenesin 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        United States Pharmacopoeia. United States Pharmacopoeial Convention, Inc. Rockville,  2004.

4        Prasanthi N. L, Mohan Ch. Krishna, Manikiran S.S., Rao N. Rama. Estimation of  ambroxal hydrochloride and guaiphenesin in  tablet dosage  form by simultaneous equation method, International Journal of Research in Ayurveda and Pharmacy. 2010: 1(1): 140-146.

 

 

 

Received on 23.06.2014                Modified on 10.07.2014

Accepted on 25.07.2014                © RJPT All right reserved