INTRODUCTION:

Ambroxol is chemically trans-4-(2-Amino-3,5-dibrombenzylamino)-cyclohexanolis expectorant class of drug used in asthama[1-4].OlopatadineHClchemically{(11Z)-11-[3-(dimethylamino)propylidene]-6,11-dihydrodibenzo[b,e] oxepin-2-yl}aceticacid. Antihistamine, antiallergy class of drug use in allergy[5-7]. Literature review reveals that several spectroscopic and Chromatographic method have been reported for estimation of olopatadine hydrochloride alone. Literature review also reveals that several spectroscopic and Chromatographic methods have been reported for estimation of ambroxol with other drugs [8-12]. But not a single method is related for this combination. Hence it was thought worthwhile to develop analytical methods for simultaneous determination of Ambroxol and olopatadine hydrochloride in their synthetic mixture[13-15].Figure 1 indicates chemical structure of analytes.

Ambroxol

Olopatadine

Fig. 1: Chemical structure of Analytes

Apparatus:

Two different HPLC systems were used for the study. The corresponding specifications were provided below

HPLC system 1:

The HPLC 1 apparatus was a Shimadzu chromatographic system equipped with an injection valve (Rheodyne 033381); Shimadzu LC 20AT UV dual _ absorbance detector (SPD 20A) was used. A reversed-phase C18 column (Phenomenex-Gemini 150mm x 4.6mm, 5 µm). Peak area integration was performed using LC solution software.

HPLC system 2:

The HPLC 2 apparatus was a Shimadzu chromatographic system equipped with an injection valve (Rheodyne 033381); Shimadzu LC 10AT UV dual-absorbance detector (SPD 10A) was used. A reversed-phase C18 column (Phenomenex-Gemini 150mm x 4.6mm, 5 µm). Peak area integration was performed using Sprinchrome software.

MATERIAL AND METHODS:

Ambroxol was provided as gift samples by Nirlife Ltd., ahmedabad , India. Olopatadine.HCl was provided as gift sample by Ajanta. Methanol, ammonium acetate and triethylamine were used as solvents to prepare the mobile phase. All chemicals used were of HPLC grade (S. D. Fine Chem. Ltd., Mumbai, India) used without further purification.

Preparation of standard stock solution:

For the preparation of standard Ambroxol stock solution, 50 mg Ambroxol was accurately weighed and dissolved in methanol in a 50 ml volumetric flask and volume was made up with methanol then pipette out 30 ml in 50 ml volumetric flask and make up to 50 ml with distilled water to make 600 µg/ml and for Olopatadine hydrochloride 50 mg weighed accurately dissolved in 50 ml volumetric flask and make up to 50 ml with methanol then take out 5 ml in 50 ml volumetric flask to make 100 µg/ml

Preparation of working standard solution:

From the stock solution of 600 µg/ ml, prepare calibration range of 78-150 µg/ ml for Olopatadine hydrochloride and from the stock solution of 100 µg/ ml, prepare calibration range of 3-25 µg/ml for Ambroxol.

Absorbance correction method (method I):

Absorbance correction method uses the absorbances at two selected wavelengths, one at λmax of one drug where other drug also shows considerable absorbance and other being the wavelength at which the first drug has practically nil absorbance. From the stock solutions, working standard solutions of OLO (19 μg/ml) and AMB (114μg/ml) were prepared by appropriate dilution and were scanned in the entire UV range to determine the suitable wavelengths. AMB and OLO have λmax at 330 nm and 245 nm respectively. Both the drugs were found to have considerable absorbance at 245 nm while at 330 nm only AMB has absorbance. The wavelengths selected for analysis were 330 nm and 245 nm for AMB and OLO respectively as shown in figure 2. A series of standard solutions ranging from 78-150 μg/ml of AMB and 13-25 µg/ml OLO were prepared separately and the absorbances of solutions were measured at 330 nm and 245 nm. A calibration curve was prepared by plotting absorbance versus corresponding concentration of drug. The concentration of two drugs in sample solution was calculated by using following equations:

C_AMB= A₁/ a_x1……………………... (3)

C_{OLO =}A₁-a_x2 C_AMB………………..... (4)

a_y2

Where, A₁and A₂ are the absorbances of mixture at 330 nm and 245 nm respectively, a_x1and a_x2 are absorptivities of AMB at 330 nm and 245 nm respectively,a_y2isabsorptivity of OLO at 245 nm, C_AMBis concentration of AMB, ^COLOis concentration of OLO.

Fig.2 Overlay UV spectra of AMB and OLO

RP-HPLC method (method II):

Calibration procedure:

The calibration curve was plotted with six concentrations of the series of standard solutions ranging from 78-150 μg/ml of AMB and 13-25 µg/ml OLO were prepared separately and chromatography was repeated thrice for each dilution. The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method. Before injecting solutions, the column was equilibrated for at least 30 min. with the mobile phase flowing through the system. Peak area were recorded for all the solutions. The correlation graph was constructed by plotting the peak area obtained at the optimum wavelength of detection versus the injected amounts.

Chromatographic condition:

The mobile phase was a mixture of Methanol: Ammonium acetate (85:15% v/v/v), pH of mobile phase was adjusted 5.5 using Glacial acetic acid and flow rate was 1.0mLmin−1. The UV detector wavelength was set at 244nm and the temperature was set at 26 ± 2°C.Figure 3 indicates chromatograph for Ambroxol and Olopatadine Hcl

Fig. 3: HPLC Chromatograph of Ambroxol (114 µg/ml) and Olopatadine Hydrochloride (19µg/ml)

RESULTS AND DISCUSSION:

The proposed methods were validated as per ICH guidline¹⁶.The plot of absorbances versus respective concentrations of AMB and OLO were found to be linear in the concentration range of 78-150 μg/ml and 13-25 μg/ml respectively drugs with correlation coefficient 0.9982 at 330 nm and 0.9984 at 245 nm for Absorbance correction method (method I) and for RP-HPLC method correlation coefficient 0.9992 for AMB and 0.995 for OLO linearity range was same as for method I. Precision was calculated as interday and intraday variations and % RSD was found to be less than 2 for method I and method II for both the drugs shown in Table 2 and Table 3. The accuracy of method was determined at 80, 100 and 120 % level. The % recovery ranges from 98.23 -101.4 for absorbance Correction Method as shown in Table 3 and for RP-HPLC method recovery study were found to be 98.38-100.2%. The % RSD of ruggedness for OLO ranges from 0.14-0.90%, while for AMB it was found to be 0.39 to 1.37% for method I and for method II 0.26 to 1.81% and 0.31 to 0.89% for OLO and AMB respectively. Results for all validation parameters are presented in Table 2, 3 and 4. These two _methods can be successfully _used for simultaneous estimation of OLO and AMB_{in their synthetic combined tablet dosage form.}Synthetic mixture were analyzed and results obtained were in the range of 98-102 % listed in Table 4. Accuracy data for RP-HPLC was shown in Table 6.

TABLE 2: Validation Parameter for Absorption Correction Method

PARAMETERS	AMB		OLO
	330nm	245nm	245nm
Linearity range	78-150 μg/ml		13-25μg/ml
Correlation Coefficient	0.9982	0.9986	0.9984
Precision	% RSD
Repeatability	0.96		0.85
Intraday	0.13-0.49		0.27-0.57
Interday	0.17-0.58		0.21-0.50
% Recovery	98.23-100.19%		98.89-101.4%
Ruggedness	0.39 to 1.37%		0.14-0.90%

TABLE 3: Summary of Validation Parameters for RP-HPLC Method

Parameters	AMB	OLO
Linearity range	78-150 μg/ml	13-25 μg/ml
Correlation coefficient	0.9992	0.995
Precision (% C.V.)
Repeatability	1.758	1.403
Intraday (n=3)	0.49-0.84%	0.31-0.67%
Interday (n=3)	0.57-0.94%	0.35-0.74%
Mean % recovery	98.38-100.2	98.5-100.1
Limitof Detection	0.98 µg/ml	0.78µg/ml
Limitof Quantification	3.22 µg/ml	0.42µg/ml

_{TABLE 4: Recovery Studies for
Absorption Correction Method}

Ambroxol	%Recovery	Olopatadine HCl	%Recovery
78	99.05	13	98.89
96	98.23	16	99.56
114	100.19	19	99.96
132	99.46	22	101.4
150	99.87	25	99.09

_{TABLE 5: Result For Simultaneous
Estimation of OLO and AMB in Synthetic Mixture by Method- I and Method-}II

Method	µg/mixture		% Of label claim ± S.D.
	AMB	OLO	AMB	OLO
Method I (AC)	114	19	99.75±0.04	99.89±0.02
MethodII (RP-HPLC)	114	19	100.9±0.09	98.88±0.08

AC- Absorption correcton method

TABLE 6: System Suitability Parameters of OLO and AMB by RP-HPLC Method

Sr. No	System suitability parameters		Observed Values	IP’2007 specification
1	Resolution (R_s)		3.785	>1.5
2	Number of theoretical plates(N)	For AMB	2525	Not less than 2000
		For OLO	2158
3	Tailing factor (T_f)	For AMB	1.286	Not greater than 2.0
		For OLO	1.182

ACKNOWLEDGEMENT:

The authors are thankful to Nirlife Ltd., Ahmadabad, India and by Ajanta Pharma for providing pure gift samples of AMB and OLO respectively.

REFERENCES:

1. Indian Pharmacopoeia. Volume 2. The Indian Pharmacopoeia Commission, Ghaziabad, Govt. of India Ministry of Health and Family Welfare; 2010. Pp.792-793.

2. British Pharmacopoeia. Department of Health; Published by the stationery Office on behalf of the medicines and Healthcare products Regulatory Agency (MHRA); Volume I , London, HMSO Publication ;2009. P.118-119.

3. European pharmacopoeia 7.0, European Directorate for the quality of medicine and healthcare, p. 1365-1366.

4. The United States Pharmacopoeia, 2010 USP 33-NF 28, Rockville MD USA, United States Pharmacopeia Convention Inc, 2007, p. 391-392.

5. Budavari S. The Merck Index;13^th ed., Merck and Co. Inc., New Jersey, monograph no.1745, p. 382.

6. http://www.chemicalbook.com/ProductMSDSDetailCB0689142_EN.htm

7. http://www.drugbank.ca/drugs/DB00768

8. http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=5281071

9. http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=2132

10. Avhad M, Bonde CG. Development and validation of simultaneous UV Spectroscopic method for the determination of Levofloxacin and Ambroxol in tablets. International Journal of ChemTech Research. 2009; 1(4):873-888.

11. Goswami J, Kakadiya J, Shah N. RP-HPLC Method Development and Validation for Simultaneous Estimation of Ambroxol Hydrochloride and Cefpodoxime Proxetile in Pharmaceutical Dosage form. American Journal PharmTech Research 2012;2(3):1043-1052.

12. Senthil R, Shan SH, Perumala P and Moorthy MTS. RP-HPLC method development and validation for the simultaneous estimation of azithromycin and ambroxol hydrochloride in tablets. International Journal of PharmTech Research. 2010;2(1):36-39.

13. Drops M, Mathrusri A , Bindu GH, Divya I. New Analytical Methods for the Determination of Olopatadine(An Anti-allergic Drug) in Eye. Drug Invention Today. 2012;4(8):441-443.

14. Dey S, Reddy YV, Swetha B, Sandeep KD, Murthy PN, Sudhir k, Dhiraj k , Patro S and Mohapatra S. Method Development and validation for the estimation of Olopatadine in bulk and pharmaceutical dosage form and its stress degradation studies using UV -Vis spectrophotometric method. International Journal of Pharmacy and Pharmaceutical Sciences. 2010;2 (4):212-218.

15. Varghese SJ, Susheel JK, Manikanta A. Stability-Indicating High-Performance Column Liquid Chromatography and High-Performance Thin-Layer Chromatography Methods for the Determination of Olopatadine Hydrochloride in Tablet Dosage Form. Journal of AOAC International. 2011;94(6):1815-1820.

16. ICH, Q2 (R1). Validation of Analytical Procedures: Text and Methodology, Geneva, 2005.

Received on 22.03.2013 Modified on 31.03.2013

Asian J. Research Chem. 6(4): April 2013; Page 389-392

Olopatadine HCl			Ambroxol
Conc.	Area	%Recovery	Conc.	Area	%Recovery
13	634.585	99.09	78	3260.245	98.5
16	672.333	98.38	96	3355.263	100.1
19	705.488	100.2	114	3436.333	99.9
22	729.534	98.5	132	3529.352	98.3
25	763.221	98.6	150	3625.856	99.8
	Y=10.482x+501.87			Y=5.0295+ 2.868
	R² = 0.995			R² = 0.9992