INTRODUCTION:

Salbutamol sulphate (SAL), chemically known as bis [(1RS)-2-[(1,1-dimethylethyl) amino]-1-[4-hydroxy-3(hydroxymethyl) phenyl] ethanol] sulphate, is beta-adenocepter agonist used as an anti-asthmatic drug and it is official in Indian pharmacopoeia^1-3, Ambroxol hydrochloride (AMB), Trans-4-(2- amino-3, 5-dibromobenzylamino) cychlohexanol hydrochloride, is an expectorant and enhanced mucolytic agent use in the treatment of various respiratory disorders and it is official in Indian pharmacopoeia⁴. The official methods involve determination of SAL⁵ and AMB⁶ using potentiometry. Some procedures have been described for the assay of either SAL or AMB in single dosage forms or in the binary mixture along with other drugs^7-11.

One spectroscopic method has been reported for the determination of Salbutamol sulphate along with Brombexine hydrochloride and Etofylline in combined dosage forms¹². According to literature, no analytical method is reported for the analysis of SAL and AMB combination in pharmaceutical dosage forms. Therefore simple, rapid, economical and reliable methods for resolution as well as determination of these drugs in pure as well as tablet formulations are seems to be necessary. In the present work, a successful attempt has been made to estimate both these drugs simultaneously by two simple UV-spectrophotometric methods (Simultaneous equation method,¹³Area under curve method¹⁴) by using distilled water, hydrochloric acid (0.1N) and phosphate buffer (pH 6.8)^15,16 as solvent for assay study as well as dissolution study of a tablet formulation. The proposed methods were optimized and validated as per ICH guidelines¹⁷.

MATERIAL AND METHODS:

Instrument: A double-beam shimadzu uv- visible spectrophotometer, shimadzu-1700, with spectral bandwidth of 2 nm, wavelength accuracy ± 0.5 nm and a pair of 1-cm matched quartz cells were used to measure absorbance of solution.

Material: Standard gift samples of salbutamol sulphate and ambroxol Hydrochloride were provided by Macleod’s pharmaceuticals Ltd and Inventia Life Sci. Pvt. Ltd respectively. Combined dose tablet formulation containing salbutamol sulphate and ambroxol hydrochloride (Sal Mucolite, 2 mg of salbutamol sulphate and 30 mg of Ambroxol hydrochloride, Manufactured by Ceminova Remedies Pvt. Ltd), were purchased from local market.

Solvent Used: Distilled water, hydrochloric acid (0.1N), and phosphate buffer (pH 6.8) was used as solvent.

Preparation of stock solution:

Standard stock solution containing salbutamol sulphate and ambroxol hydrochloride were prepared by dissolving quantity of salbutamol sulphate equivalent to Salbutamol base 10 mg and 10 mg of ambroxol separately in 20ml of solvent in separate 100 ml volumetric flask and final volume of both solution were made up to 100 ml with same solvent to get stock solution containing each of 100µg/ml of salbutamol and ambroxol in two different 100 ml volumetric flasks.

Determination of absorption maxima:

By appropriate dilution of two standard drug solutions with distilled water, hydrochloric acid (0.1N) and phosphate buffer (pH 6.8) solutions containing 10 µg/ml of salbutamol and 10 µg/ml of ambroxol were scanned separately in the range of 200- 400 nm to determine the wavelength of maximum absorption for both the drugs in respective solvents showed absorbance maxima at 224.5nm for salbutamol and 244.5nm for ambroxol (Fig. 1).

Fig.1: Overlain spectra of Salbutamol and Ambroxol

Method I: Simultaneous Equation Method:

Two wavelengths selected for this method are 224.5 nm and 244.5 nm that are absorption maxima of salbutamol and ambroxol for distilled water, hydrochloric acid (0.1N), and phosphate buffer (pH 6.8) respectively. The stock solutions of both the drugs were further diluted separately with distilled water, hydrochloric acid (0.1N), phosphate buffer (pH 6.8) to get a series of standard solutions of 4-24 µg/ml and 10 – 60 µg/ml for SAL and AMB respectively. The absorbances of these standard solutions were measured at selected wavelength and calibration curves were plotted. Two simultaneous equations (in two variables C₁ and C₂) were formed using following absorptivity coefficient values. (Table No.-1)

Table No – 1: Absorptivity value for simultaneous equation method

Absorptivity Value (x)	Water	Hydrochloric Acid (0.1N)	Phosphate buffer (pH 6.8)
ax₁	0.02824	0.02775	0.02832
ax₂	0.00173	0.00074	0.00101
ay₁	0.02277	0.02364	0.02386
ay₂	0.02364	0.02394	0.02461

A₁ = ay₂ C₁ + ay₁ C₂………………….…………… (1)

A₂ = ax₂C₁+ ax₁C₂ …………….…….....………… (2)

By applying the Cramer's rule to equation 1 and 2, the concentration C₁ and C₂ can be obtained as follows,

A₂ ay₁– A₁ ay

C₁ = ----------------------…………………………….. (3)

ax₂ay₁- ax₁ay₂

A₁ax₂– A₂ax₁

C₂ = -------------------------- ……….……………….. (4)

ax₂ay₁- ax₁ay₂

Where,

C₁ = Concentrations of SAL,

C_{2 =} Concentrations of AMB,

A₁= Absorbance of mixture at 224.5 nm,

A₂ = Absorbance of mixture at 244.5 nm,

ax₁= Absorptivity value of SAL at 224.5 nm,

ax₂= Absorptivity value of SAL at 244.5 nm,

ay₁= Absorptivity value of AMB at 224.5 nm,

ay₂= Absorptivity value of AMB at 244.5 nm

Method II: Area under curve method:

From the overlain spectra of both drugs in distilled water, hydrochloric acid (0.1N), phosphate buffer (pH 6.8), area under the curve in the range of 229.5-219.5nm (for salbutamol) and 249.5-239.5 nm (for ambroxol) were selected for the analysis (Fig.1). The calibration curves for salbutamol and ambroxol were plotted in the concentration range of 4-24 μg/ml and 10-60μg/ml, respectively. The ‘X’ values for both the drugs were determined at the selected AUC range in distilled water, hydrochloric acid (0.1N), and phosphate buffer (pH 6.8). The ‘x’ value is the ratio of area under the curve at selected wavelength ranges with the concentration of component in g/lit. These ‘x’ values were the mean of six determinations (Table No.-2). A set of two equations obtained by using mean ‘x’ values are given below.

A₁= ay₂C₁ + ay₁C₂ (at λ229.5-219.5 nm)……………… (5)

A₂= ax₂C₁+ ax₁C₂ (at λ249.5-239.5 nm)………………. (6)

Where A₁ and A₂ were area under curve of sample at the wavelength range 229.5-219.5 nm and 249.5- 239.5 nm respectively. The concentration of Salbutamol and Ambroxol in sample was determined by using the equations (5) and (6).

Table No – 2: Absorptivity value for area under curve method

Absorptivity Value (x)	Water	Hydrochloric Acid (0.1N)	Phosphate buffer (pH 6.8)
ax₁	0.2710	0.2656	0.2680
ax₂	0.0195	0.0100	0.0138
ay₁	0.2484	0.2509	0.2528
ay₂	0.2276	0.2301	0.2348

ANALYSIS OF MARKETED TABLET FORMULATION:

For the estimation of drugs in the formulations, twenty tablets were weighed and average weight was calculated. The tablets were crushed to obtain fine powder. Tablet powder equivalent to 15 mg of ambroxol was transferred to 100.0 ml volumetric flask; 20 ml solvent was added and sonicated for 20 min. The volume was then made up to the mark with respective solvents. The resulting solution was filtered through whatmann filter paper and filtrate was appropriately diluted with respective solvents to get approximate concentration of 2 μg/ml of salbutamol and 30 μg/ml of ambroxol. In method I, the concentration of both salbutamol and ambroxol were determined by measuring absorbance of sample solutions at 224.5 nm (λmax of salbutamol) and 244.5 nm (λmax of ambroxol) using equations (3) and (4). For method II, the concentration of both salbutamol and ambroxol were determined by measuring area under curve in the range of 229.5-219.5 nm (for salbutamol) and 249.5-239.5 nm (for ambroxol) and values were substituted in equations (5) and (6) to obtain concentration of both the drugs. Results of tablet analysis are shown in (Table No.-3)

VALIDATION:

The proposed methods were validated as per ICH guidelines for validation of analytical procedures in order to determine the linearity, accuracy, precision and specificity for the analyte ¹⁷.

Linearity:

The linearity of measurement was evaluated by analyzing different concentration of the standard solution of salbutamol and ambroxol. For all two methods, the beer- lambert’s concentration range was found to be 4-24 μg/ml and 10-60μg/ml for salbutamol and ambroxol respectively.

Accuracy:

To ascertain the accuracy of the proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% &120). Pure salbutamol and ambroxol was added to the preanalyzed tablet powder at three spiking levels viz 80, 100, 120 %. Three replicate analyses were carried out at each level. The mean percent recovery was found in the range of 99.13-100.13 % for all the methods

Precision:

The reproducibility of the proposed methods was determined by performing tablet assay at different time intervals on same day (Intra-day assay precision) and on three different days (Inter-day assay precision). % RSD for estimation of Salbutamol and Ambroxol under intraday and interday variations was found to be less than 2.

Specificity:

The specificity of the method was confirmed by comparing the λ max of standard with that of salbutamol and ambroxol in the marketed formulation. There is no interference from the excipients commonly present in the tablets. Hence the developed method is specific and selective.

RESULTS AND DISCUSSION:

The maximum absorption for salbutamol and ambroxol in distilled water, hydrochloric acid (0.1N) and phosphate buffer (pH 6.8) were observed at 224.5nm and 244.5nm respectively. The high values of correlation coefficient in distilled water, hydrochloric acid (0.1N) and phosphate buffer 6.8 indicate linearity for salbutamol and ambroxol in all three solvents. Beer’s law was obeyed for distilled water, hydrochloric acid (0.1N) and phosphate buffer (pH 6.8) in the range of 4-24ug/ml for salbutamol and 10-60ug/ml for ambroxol. The accuracy of methods was determined by calculating mean percentages recovery. It was determined at 80, 100 and 120% level of standard dilution. The mean percent recovery was found in the range of 99.13-100.13% for all the methods. Precision was calculated as inter and intraday variations for salbutamol and ambroxol in all three solvents was found to be less than 2 % RSD.

Table No – 3: Result of marketed formulation analysis

Method	Solvents	Tablet Content	Label Claim	Amount Found (Mg)	Amount Found (%)	± S.D
Simultaneous equation method	Distilled Water	SAL	2	1.99	99.95	1.389
	Distilled Water	AMB	30	29.94	99.80	0.741
	Hydrochloric Acid (0.1N)	SAL	2	2.00	100.00	1.728
	Hydrochloric Acid (0.1N)	AMB	30	29.97	99.90	0.870
	Phosphate buffer (pH 6.8)	SAL	2	1.98	99.00	0.981
	Phosphate buffer (pH 6.8)	AMB	30	29.80	99.33	1.034
Area Under Curve Method	Distilled Water	SAL	2	1.99	99.50	1.485
	Distilled Water	AMB	30	30.10	100.33	1.781
	Hydrochloric Acid (0.1N)	SAL	2	1.99	99.50	1.622
	Hydrochloric Acid (0.1N)	AMB	30	29.88	99.60	0.707
	Phosphate buffer (pH 6.8)	SAL	2	2.01	100.50	1.904
	Phosphate buffer (pH 6.8)	AMB	30	29.97	99.90	1.533

* Mean of six estimation. SAL = Salbutamol, AMB= Ambroxol

CONCLUSION:

The two proposed methods based on the spectrophotometric, were validated as per ICH guidelines. The standard deviation and % RSD calculated for the proposed methods are low, indicating high degree of precision of the methods. The results of the recovery studies performed show the high degree of accuracy of the proposed methods. Hence, it can be concluded that the developed spectrophotometric methods are accurate, precise and selective and can be employed successfully for the simultaneous estimation of salbutamol and ambroxol for routine analysis as well as for dissolution study of a tablet formulation.

ACKNOWLEDGEMENT:

The authors gratefully acknowledge Dr. P. D. Patil, Chairman, Dr. D. Y. Patil Vidya Pratishthan Society and Dr. A. D. Deshpande, Director of Pharmacy for providing excellent infrastructure facility to carry out this research work. Thanks also go to Macleods Pharmaceutical Pvt. Ltd. Mumbai and Inventia Life Sci. Pvt. Ltd. Mumbai for providing pure drug samples.

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Received on 21.01.2011 Modified on 22.02.2011

Asian J. Research Chem. 4(5): May, 2011; Page 746-749