INTRODUCTION:

Ambroxol hydrochloride (AB) [Trans-4-[(2-amino-3, 5-dibromobenzyl) amino] (Fig.1) cyclohexanol hydrochloride] is a semi-synthetic derivative of vasicine obtained from Indian shrub Adhatoda vasica. It is a metabolic product of bromhexine and possesses mucokinetic (improvement in mucus transport) and secretolytic (liquifies secretions) properties. It promotes the removal of tenacious secretions in the respiratory tract and reduces mucus stasis (arresting the secretion of mucus)¹. Guaifenesin (GF), (RS)-3-(2-methoxyphenoxy) propane-1, 2-diol (Fig.2) reportedly increases the volume and reduces the viscosity of tenacious sputum². Levosalbutamol sulfate (LBS) 4-[(1r)-2-(tert-butylamino)-1-hydroxyethyl]-2-(hydroxymethyl) phenol(Fig.3) is a synthetic 2-adrenoceptor which predominantly stimulates β2-receptors and is used as a bronchodilator in the treatment of bronchial asthma³.

The above combination is indicated for clinical relief of cough associated with bronchitis, bronchial asthma, emphysema and other Broncho pulmonary disorders where bronchospasm, mucous plugging and problems of expectoration co-exist. AB, GF and LBS are the subjects of monographs in Indian Pharmacopoeia (IP)⁴. Several methods such as HPLC ^5-19, spectrophotometric ^20-34, HPTLC ^35-36 have been reported for the analysis of AB, GF, LBS as an individual drug or in combination, either in pure or in pharmaceutical forms as well as in biological fluids and tissues. Literature survey reveals that there is no single UV method reported for the determination of AB, GF and LBS in liquid formulation like syrup. Hence the objective of the present study was to develop spectrophotometric analysis for estimation of AB, GF and LBS in syrup by First derivative and Colorimetric method.

Figure 1: Chemical structure of Ambroxol hydrochloride (AB)

Figure 2: Chemical structure of Guaifenesin (GF)

Figure 3: Chemical structure of Levosalbutamol Sulphate (LBS)

MATERIALS AND METHODS:

Instrumentation

A double beam UV-visible Spectrophotometer (Shimadzu, UV-1700, Japan), attached to a computer software UV probe 2.0, with a spectral width of 2 nm, wavelength accuracy of 0.5 nm and pair of 1 cm matched quartz cells.

Chemicals and Reagents

AB, GF and LBS were kindly supplied by Acron Pharma, Ahmedabad, India. Ambro lite Levo syrup, Health kart Pharma (AB 30mg, GF 50mg, LBS 1.0 mg per 5 ml) was used as marketed formulation. Freshly prepared phosphate buffer solution (adjusted pH between 9.2). 8% w/v of Potassium Ferricyanide (PF) solution. 2% w/v of 4- Amino Antipyrine solution (AAP) And distilled water were used throughout the experiment. All reagents were used of an analytical grade.

Preparation of Standard Stock Solution

v For First Derivative Method

Accurately weighed 10 mg standard powder of AB, GF and LBS was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with distilled water.

v For Colorimetric Method

Accurately weighed 10 mg standard powder of AB, GF and LBS was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with phosphate buffer pH 9.2 to obtain standard solution having concentration 100µg/ml.

Preparation of Sample Solution

v For First Derivative Method

Quantity of the syrup formulation equivalent to 30 mg AB and 50 mg GF was taken and diluted up to 100 ml with dis. Water. The solution was filtered through Whatman filter paper No. 41 and the volume was adjusted up to the mark with distilled water. The above solution(1.0 ml) was transferred to 10 ml volumetric flask and diluted up to mark with distilled water to obtain 30 µg/ml AB and 50 µg/ml GF. Then absorbance was measure at 323, 276 nm.

v For Colorimetric Method

Quantity of the syrup formulation equivalent to 1 mg LBS was taken and diluted up to 50 ml with phosphate buffer pH 9.2. The solution was filtered through Whatman filter paper No. 41 and the volume was adjusted up to the mark with phosphate buffer. The above solution (5.0 ml) was transferred to 10 ml volumetric flask and diluted up to mark with phosphate buffer to obtain 10 µg/ml LBS. Then absorbance was measure at 503 nm. Then absorbance was measure at 503 nm.

Preparation of phosphate buffer Solution (pH 9.2)

Dissolve 5.04 g disodium hydrogen phosphate and 3.01 g of potassium dihydrogen phosphate in sufficient water to produce 1000 ml. Adjust the pH to 9.2 with glacial acetic acid.

Determination of Wavelength having Maximum Absorbance

v For First Derivative Method

The stock solutions of AB, GF and LBS were further diluted with distilled water to get concentration of 30 μg/ml. These solutions were scanned in the range 200-400nm, all the spectra were converted to first order derivative spectra (Δλ=1, scaling factor=10).

v For Colorimetric Method

The stock solutions of AB, GF and LBS was further diluted with phosphate buffer to get concentration of 30 μg/ml. After dilution AB and GF show no color but LBS show reddish brown color. These solutions were scanned in the range 400-800nm. LBS give the maximum absorbance at 503 nm at that wavelength AB and GF show zero absorbance, so measurement of LBS at 503 nm is possible without interference of AB and GF.

RESULTS AND DISCUSSION:

Method Development

v For First Derivative Method

The working standard solution of AB, GF and LBS were prepared separately in Dis. Water. They were scanned in the wavelength range of 200-400 nm. Maximum absorbance was obtained at 323, 276 for AB, GF respectively. LBS showed zero absorbance at these two wavelengths. So, these two wavelengths were employed for the determination of AB, GF. Overlain spectra of the drugs are shown in Figure 4 and 5.

v For Colorimetric Method

The working standard solution of LBS, AB and GF were prepared separately in phosphate buffer. It was scanned in the wavelength range of 400-800 nm. Maximum absorbance was obtained at 503 nm for LBS at that wavelength AB and GF were show zero absorbance. This wavelength was employed for the determination of LBS. Overlain spectra of the drugs are shown in Figure 6

Ø Effect of pH

The effect of pH on the absorption of the complex formed by the reaction of LBS with 4- AAP and Fe (III) was studied at different pH of HCl or NaOH in the range 2-11. It was found that the chelating complex was formed at pH 9.0.Therefore different buffers of pH 9.00 were prepared using carbonate, bicarbonate and phosphate buffers to investigate the sensitivity of the 4-AAP-LBS.Fe (III) complex. It was found that phosphate buffers solution increased the sensitivity of the complex. However; the optimum amount of phosphate buffers solution of pH 9.2 has been studied (Fig. 7).

Figure 7: Effect of pH on the absorption intensity of LBS (20 µg/ ml)-4-AAP-Fe (III) complex.

Ø Effect of 4-AAP Reagent Concentration

The effect of changing the 4-AAP reagent concentration on the absorbance of solution keeping a fixed amount of LBS, Fe (III) and pH 9.2 was studied. It was found that absorbance increases with increasing 4-AAP concentration and reached its maximum value on using 0.1 ml of 2 % 4-AAP (Fig. 8). This condition is used in the subsequent experiment.

Figure 8: Effect of 2% 4-AAP reagent amount on the absorption intensity of 20 µg/ml LBS in the presence of Fe (III).

Ø Effect of Potassium Ferricyanide (PF) Reagent Concentration

The effect of changing the PF reagent concentration on the absorbance of solution keeping a fixed amount of LBS, 4-AAP and pH 9.2 was studied. It was found that absorbance increases with increasing PF concentration and reached its maximum value on using 0.1 ml of 8 % PF (Fig. 9). This condition is used in the subsequent experiment.

Figure 9: Effect of 8 % PF reagent amount on the absorption intensity of 20 µg/ml LBS in the presence of 4-AAP.

Ø Effect of Reaction Time

The reaction time was determined by following the color development at room temperature. The absorbance was measured at different time (min) intervals against reagent blank treated similarly. It is evident from (Fig. 10) that the formation of stable colored complex for LBS was achieved after 10 min. Hence, this time was selected for further investigations.

Figure 10: Effect of developing time on the absorbance of 20 µg/ml LBS.

Ø Effect of order of addition

From the experiments in which the reagent was added in all possible sequences, it was concluded that the maximum absorbance is attained only with the following order: LBS – Fe (III) - 4AAP.pH 9.2.

Ø Composition of complex

Colorimetric methods determination for LBS are based on the reaction between LBS and 4-AAP to form antipyrine dyes where 4-AAP is found to be the most sensitive, fast, and precise colorimetric reagent. 4-AAP reacts with phenolic-type compounds. It was found that LBS reacted with 4-AAP in 1:1 ratio forming a new ligand having low sensitivity at 503 nm. This sensitivity has been increased in its complexation with Fe (III) to give intense reddish brown colored chelate as shown in (Fig. 11)

METHOD VALIDATION

Specificity of colorimetric method

This was carried out by analyzing the standard LBS solutions after spiking with appropriate amount of AB and GF. The concentration of LBS solution was determined by performing the assay as per the procedure mentioned in assay of marketed formulation (Table 2).

Linearity

The linearity was observed in the concentration range of 5-80 µg/ml, 5-80 µg/ml and 5-30 µg/ml respectively for AB, GF and LBS. The calibration curves at different wavelengths are shown in (Fig. 12, 13 and 14)

Figure 11 Probable mechanisam reaction for complex of LBS with 4-AAP and PF

Table 1: Regression Analysis Data and Summary of Validation Parameters of AB, GF and LBS

PARAMETERS	AB	GF	LBS
Wavelength (nm)	323	276	503
Beer’s Law limit (µg /ml)	5--80	5--80	5--30
Regression equation (Y= mx+c)	y = 0.000329x + 0.000331	y = 0.000426x + 0.000133	y = 0.0176x + 0.1804
Slop	0.000329	0.000426	0.0176
Intercept	0.000331	0.000133	0.1804
Correlation coefficient (r²)	0.9988	0.9955	0.9942
LOD (µg/ml)	0.011423	0.006463	0.01624
LOQ (µg/ml)	0.034614	0.0195847	0.0492
Repeatability (RSD, n=6)	0.414	0.779	0.891
Precision (RSD)%
Interday (n=3)	0.300-0.820	0.600-0.884	0.161-1.084
Intraday (n=3)	0.537-1.234	0.468-1.602	0.208-1.173

Table 2: Results of Specificity

Sr. No	Conc of AB (µg /ml)	Conc of GF (µg /ml)	Conc of LBS (µg /ml)	Absorbance at 550nm (n=3) ± RSD
1	0	0	10	0.358
2	0	50	0	0
3	30	0	0	0
4	30	50	10	0.354

Figure 12: Calibration Curve of AB at 323 nm

Figure 13: Calibration Curve of GF at 276 nm

Figure 14: Calibration Curve of LBS at 503 nm

Method Precision (% Repeatability)

The RSD values of AB was found to be 0.414 % at 323.0 nm. The RSD value of GF was found to be 0.779 % at 276.0 nm, The RSD value of LBS was found to be 0.891 % at 503.0 nm (Table 3). Low value of RSD indicates that proposed method is repeatable.

Table 3: Repeatability Data for AB, GF and LBS

Conc (20 µg /ml)	AB	GF	LBS
1	0.00702	0.00999	0.5441
2	0.00702	0.00996	0.552
3	0.00704	0.00979	0.5412
4	0.00707	0.00994	0.5511
5	0.00709	0.00991	0.5509
6	0.00708	0.00996	0.5521
Mean	0.00705	0.009918	0.54786
SD	0.0000308	0.000077	0.0048820
% RSD	0.414	0.779	0.891

Intermediate Precision (Reproducibility)

The RSD values of AB for interday (0.300-0.820 %) and intraday (0.537-1.234 %) at 323.0, The RSD values of GF for interday (0.600-0.884 %) and intraday (0.468-1.602 %) at 276.0 and the RSD values of LBS for interday (0.161-1.084%) and intraday (0.208-1.173 %) at 503.0 which reveals that the Method is precise.

LOD and LOQ

LOD and LOQ values for AB were found to be 0.0114and 0.0346µg/ml at 323.0 nm, LOD and LOQ values for GF were found to be 0.00646and 0.0195µg/ml at 276.0 nm and LOD and LOQ values for LBS were found to be 0.016and 0.049µg/ml at 503 nm. Low value of LOD & LOQ indicates that the method is sensitive. (Table 1)

Table 4: Recovery studies data of AB and GF

Drug	Amount Present In Formulation (µg/ml)	Amount Added %	% Recovery ± SD
AB	30	50%	101.95 ± 0.816
	30	100%	100.47 ± 1.247
	30	150%	101.543 ± 0.203
GF	30	50%	101.69 ± 1.684
	30	100%	99.56 ± 1.826
	30	150%	100.14 ± 2.100
LBS	10	50%	98.91 ± 1.037
	10	100%	99.56 ± 1.826
	10	150%	99.51 ± 0.861

Table 5 Analysis of AB, GF and LBS in syrup formulation (n=6)

SAMPLE NO	LABEL CLAIM			AMOUNT FOUND			% LABEL CLAIM
	AB	GF	LBS	AB	GF	LBS	AB	GF	LBS
1	30	50	10	30.87	50.90	9.93	102.90	101.8	99.3
2	30	50	10	30.14	50.46	10.03	100.47	100.92	100.3
3	30	50	10	30.75	50.08	10.02	102.50	100.16	100.2
4	30	50	10	30.78	50.06	10.03	102.60	100.12	100.3
5	30	50	10	30.75	50.11	10.01	102.50	100.22	100.1
6	30	50	10	30.47	50.11	10.05	101.57	100.22	100.5
MEAN				30.63	50.29	10.01	102.09	100.57	100.12
SD				0.273	0.335	0.042	0.912	0.670	0.421
RSD				0.891	0.666	0.420	0.893	0.665	0.422

Accuracy

v The recovery experiments were performed by the standard addition method. The mean recoveries were found to be 101.32 ± 0.762 and 100.46 ± 1.1033 for AB and GF respectively. The recoveries results indicate that the proposed method is accurate. Results of recovery studies are shown in (Table 4).

Assay

v For First Derivative Method

5 ml of marketed syrup was measured and transferred in 50ml volumetric flask and made up to the mark with distilled water. This solution was filtered through whatman filter paper. This stock solution further diluted to get final concentration 30 μg/ml of AB and 50μg/ml of GF. This solution was scanned in UV-Visible region of 200-350nm for estimation of AB and GF (Table 5).

v For Colorimetric Method

For determination of LBS concentration in syrup 5 ml of stock solution was transferred in 100ml volumetric flask, 85 ml of a buffer solution and 1.0 ml of 4-aminoantipyrine solution were added and mixed. Further 1.0 ml of potassium ferricyanide solution was transferred with vigorous swirling and then sufficient buffer solution was added to produce 100.0 ml. LBS sample solution (10 µg/ml) was prepared. The absorbance of the resulting solution was measured at the maximum of 503 nm using blank (Table 5).

CONCLUSION:

Based on the results, obtained from the analysis using proposed method, it can be concluded that the method has linear response in the range of 5-80 μg/ml, 5-80 μg/ml and 5-30 μg/ml for AB, GF and LBS, respectively. The result of the analysis of syrup formulation by the proposed method is highly reproducible and reliable and is in good agreement with label claim of the drugs. The additive present in the synthetic mixture did not interfere in the analysis. So the method can be used for the routine analysis of drugs in combined dosage form.

ACKNOWLEDGMENTS:

The authors are grateful to Acron Pharmaceuticals Ltd. Ahmadabad, Gujarat, India for providing gift samples of Ambroxol Hydrochloride, Guaifenesin and Levosalbutamol Sulphate and also to Department of Quality Assurance, Shree S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Mehsana, Gujarat, India for providing the facilities to carry the research work.

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Received on 01.04.2013 Modified on 12.04.2013

Asian J. Research Chem. 6(4): April 2013; Page 407-414