Development and Validation of UV Spectrophotometric Method of Ambroxol Hydrochloride in Bulk and Pharmaceutical Formulation

Chhotaram Seervi*, Kundan Pawar, PN Dhabale, ID Gonjari, Chandrakant Raut and Deepali Gharge

Govt. College of Pharmacy, Karad-415124 (Satara), Maharashtra, India.

*Corresponding Author E-mail: crseervi@rediffmail.com

ABSTRACT

Two simple, precise and economical UV methods have been developed for the estimation of Ambroxol Hydrochloride in bulk and pharmaceutical formulations. Ambroxol Hydrochloride has the absorbance maxima at 246.5nm (Method A), and in the first order derivative spectra showed sharp peak at 240 nm (Method B). Linearity for detector response was observed in the concentration range of 10-50 µg/ml for all two methods. The proposed methods were successfully applied for the estimation of Ambroxol Hydrochloride in commercial pharmaceutical preparation by using simple solvent Distilled water. The results of the tablet analysis were validated with respect to accuracy (recovery), linearity, Limit of Detection and Limit of Quantiﬁcation and specificity were found to be satisfactory.

KEYWORDS: Ambroxol Hydrochloride, UV spectrophotometry, Absorbance maxima, Derivative spectroscopy.

INTRODUCTION:

Chemically Ambroxol hydrochloride (AMB) is trans-4-((2-amino-3,5-dibromobenzyl) amino) cyclohexanol hydrochloride¹. Ambroxol is a metabolite of bromhexine with similar actions and uses². It is an expectoration improver and a mucolytic agent used in the treatment of acute and chronic disorders characterized by the production of excess or thick mucous. It has been successfully used for decades in the form of its hydrochloride as a secretion-releasing expectorant in a variety of respiratory disorders¹. Literature survey revealed that it is estimated individually by spectrophotometric methods in visible region by forming colour complex³only. Although simultaneous UV estimation of ambroxol hydrochloride and levocetirizine dihydrochloride has been reported by Prabu et. al. ⁴, No UV spectrophotometric methods have been reported for estimation of AMB in single component formulation. Hence, an attempt has been made to develop new UV methods for its estimation in pharmaceutical formulations with good accuracy, simplicity and precision.

MATERIALS AND METHODS:

Pure sample of Ambroxol hydrochloride was obtained from Dr. Reddy's Laboratories Ltd., Hyderabad as a gift sample. A Shimadzu UV-1700 UV/VIS Spectrophotometer was used with 1 cm matches quartz cell. Tablet of 30mg MUCOLITE tab was procured from local pharmacy.

Preparation of standard solution:

The pure drug accurately about 10 mg was weighed and dissolved 100 ml Distilled water to give the standard stock solution of concentration 100 µg/ml.

Method A: Absorption Maxima Method

The solutions were scanned in the range from 400-200 nm (method A), and the peaks were observed at 210nm, 307.5nm and 246.5 nm. The wavelength selected for the analysis of the drug was 246.5nm (Fig. 1). The calibration curves for AMB was plotted in the concentration range of 10-50 µg/ml at wavelength 246.5 nm. The drug followed the Beer’s- Lamberts law in the range of 10-50 µg/ml. By using the calibration curve, the concentration of the sample solution can be determined (Table no.1).

Method B: First Order Derivative Spectroscopy

In this method, 100 µg/ml stock solution of AMB was prepared by above procedure and scanned in the spectrum mode from 400 nm to 200 nm. The absorption spectra thus obtained were derivatized from first to fourth order. First order derivative spectra were selected for analysis of drug. First order derivative spectra of drug (Fig. 2), showed a sharp peak at 240 nm, which was selected for its quantitation. The calibration curves for AMB was plotted in the concentration range of 10-50 µg/ml at wavelength 240 nm. The concentration of the drug present in the sample solution was determined against the calibration curve in quantitation mode(Table no.1).

Analysis of the Tablet formulation:

For the estimation of Ambroxol hydrochloride in tablet formulation by two methods, 10 tablets of brand were weighed and triturate to fine powder. Tablet powder equivalent to 10mg of Ambroxol hydrochloride was weighed and the dissolved in 100 ml distilled water . It was kept for ultrasonication for 15 min; this was filtered through Whatman filter paper no. 41 to get the stock solution of 100 µg/ml. The filtrate was appropriately diluted with Distilled water to obtain 20 µg/ml of AMB . In Method-A, the concentration of AMB was determined by measuring the absorbance of the sample at 246.5 nm in zero order spectrum mode. By using the calibration curve, the concentration of the sample solution can be determined. Method-B, the concentration of AMB was determined by measuring the absorbance of the sample at 240 nm, in first order derivative mode. The results of the tablet analysis were calculated against the calibration curve in quantitation mode. Amount of drug estimated by this method is given in Table no.2.

Figure 1: Zero order spectra of Ambroxol hydrochloride

VALIDATION:

The methods were validated with respect to accuracy linearity, limit of detection (LOD) and limit of quantiﬁcation (LOQ) and Specificity.

Accuracy (recovery test):

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 AMB, by all the methods, was found in the range of 98.93-99.97(Table no.3)

Figure 2: First order derivative spectrum of Ambroxol Hydrochloride

with n=1

Linearity:

The linearity of measurement was evaluated by analyzing different concentration of the standard solution of AMB. Beer-Lambert’s concentration range was found to be 10-50 µg/ml for all two methods.

Limit of detection (LOD) and limit of quantification (LOQ):

The LOD and LOQ of Ambroxol hydrochloride were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines⁵.The LOD and LOQ was found to be as in table no.1

Specificity:

A 20 µg/ml solution of candidate drug in distilled water at UV detection by Method A at 246.5 nm will show an absorbance value of 0.4910 ± 0.0015. A 20 µg/ml solution of candidate drug in distilled water at UV detection Method B at 240 nm will show an absorbance value of 0.4499 ± 0.0035.

RESULTS AND DISCUSSION:

The methods discussed in the present work provide a convenient and accurate way for analysis of AMB in its pharmaceutical dosage form. Absorbance maxima of AMB at 246.5 nm (Method A) and in the first order derivative spectra, sharp peak at 240 nm (Method B) were selected for the analysis. Linearity for detector response was observed in the concentration range of 10-50 µg/ml for all two methods. Percent label claim for AMB in tablet analysis, by all the methods, was found close to 100 %. Standard deviation for six determinations of tablet sample,

Table No.1: Optical characteristics and Other Parameters

Parameters	Method A	Method B
λmax (nm) / wavelength range (nm)	246.5	240
Beer’s-Lambert’s range (µg/ml)	10-50	10-50
Coefficient of Correlation	0.9990	0.9997
Regression Equation Y = mx + c a. Slope(m)	0.0261	0.0217
b. Intercept(c)	-0.0076	0.0188
Sandell’s Sensitivity (mg/cm2/0.001 absorbance unit)⁶	0.03295	0.04608
LOD (µg/ml)	0.16	0.53
LOQ (µg/ml)	0.48	1.61

Where, A is zero order derivative spectrum method with n = 0. B is first order derivative method with n = 1.

Table No.2: Result of Analysis of Ambroxol hydrochloride in Tablet

Methods	Lable Claim (mg)	Amount Found* (mg)	% Estimated*	S.D.* (±)	R.S.D.*
A	30	29.9877	99.959	0.0015	0.00501
B	30	29.8150	99.383	0.0035	0.00012

Where, A is zero order derivative spectrum method with n = 0, B is first order derivative method with n = 1. * indicates mean of six determinations.

Table No.3: Recovery studies data.

Methods	Lable Claim (mg)	Level of Recovery (%)	Amount Added (mg)	Amount Found* (mg)	Recovery* (%)	S.D.* (±)	R.S.D.*
A	30	80	24	53.985	99.97	0.0031	0.0057
	30	100	30	58.996	99.32	0.0022	0.0036
	30	120	36	65.897	99.83	0.0046	0.0070
B	30	80	24	53.422	98.93	0.0051	0.0094
	30	100	30	59.720	99.53	0.0018	0.0030
	30	120	36	65.462	99.18	0.0112	0.0171

Where, A is zero order derivative spectrum method with n = 0, B is first order derivative method with n = 1. * indicates mean of six determinations.

by all the methods, was found to be less than ± 2.0 indicating the precision of the methods(Table no.2). Accuracy of proposed methods was ascertained by recovery studies and the results are expressed as % recovery. Percent recovery for AMB, by all the methods, was found in the range of close to 100% and values of standard deviation was satisfactorily low indicating the accuracy of all the methods(Table no.3).

CONCLUSIONS:

The developed method was found to be simple, sensitive, accurate, precise, reproducible, and can be used for routine quality control analysis of Ambroxol hydrochloride in bulk and pharmaceutical formulation.

ACKNOWLEDGEMENTS:

The authors are thankful to the Principal Dr. S. B. Bhise, Govt.College of Pharmacy, Karad, Dist. Satara, Maharashtra for providing necessary facilities and Dr. Reddy's Laboratories Ltd., Hyderabad for providing gift samples of Ambroxol hydrochloride.

REFERENCE:

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3. Kuchekar B.S. et al. Spectrophotometric estimation of ambroxol hydrochloride in tablets. Indian Journal of Pharmaceutical Sciences. 2003; 65(2): 193-195.

4. S Lakshmana Prabu et al. Simultaneous UV spectrophotometric estimation of ambroxol hydrochloride and levocetirizine dihydrochloride. Indian Journal of Pharmaceutical Sciences.2008;70(2):236-238.

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Received on 05.09.2009 Modified on 09.10.2009

Asian J. Research Chem. 2(4):Oct.-Dec. 2009 page 547-549