DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC METHODS FOR ESTIMATION OF DORZOLAMIDE HCl IN BULK AND PHARMACEUTICAL DOSAGE FORMS

DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC METHODS FOR ESTIMATION OF DORZOLAMIDE HCl IN BULK AND PHARMACEUTICAL DOSAGE FORMS

1. M. Sharath*, G babu Jose, K. P. Channabasavaraj, J. S. Modiya

Department of Pharmaceutical Analysis, Bharathi College of Pharmacy, Bharathi Nagara, Mandya (District), Karnataka, India

ABSTRACT

Three Simple, precise, accurate and economical UV methods have been developed and validated for the quantitative estimation of Dorzolamide HCl in bulk and pharmaceutical dosage form. Dorzolamide HCl has the absorbance maxima at 253 nm in zero order spectra (Method A). In the first order derivative spectra, showed absorbance maxima at 238 nm (Method B) and in the second order derivative spectra, showed peak maxima at 278 nm (Method C). Distilled water was used as solvent for all the methods. Beer’s law was  found to be obeyed in the concentration range of 3-24 μg/ml. The developed method was validated according to ICH guidelines and was found to be accurate, economic and precise. The proposed method can be successfully applied for the estimation of Dorzolamide HCl in bulk and pharmaceutical dosage forms.

Derivative Spectroscopy

INTRODUCTION: Dorzolamide HCl ^{1, 2} is an anti-glaucoma agent and topically applied in the form of eye drops (fig. 1). Chemically is an [(4S, 6S)-4-(Ethylamino)-6-methyl-5, 6-dihydro-4Hthieno [2, 3b] thiopyran-2-sulphonamide 7, 7-dioxide hydrochloride]. Dorzolamide HCl is a carbonic anhydrase inhibitor and used to lower increased intraocular pressure in open-angle glaucoma and ocular hypertension.

FIG.1: CHEMICAL STRUCTURE OF DORZOLAMIDE HCL

Analytical methods are required to characterize drug substances and drug products composition during all phases of pharmaceutical development. Extensive literature survey reveals that only few methods ^{3, 4} were reported for determination of Dorzolamide HCl in bulk and in its pharmaceutical dosage form. Hence there is a need to develop new methods for its estimation in bulk and pharmaceutical dosage forms.

MATERIALS AND METHODS:

Instruments and reagents: A Shimadzu-1800 UV/Vis double beam Spectrophotometer with 1 cm matched quartz cells was used for all spectral measurements Distilled water was used as solvent for dilution. Authentic drug sample of Dorzolamide HCl  was given as a gift sample by Lake Chemicals Pvt. limited, (Bangalore, India)  Eye drop formulation [DORTAS (Brand name), Intas Pharmaceutical Limited, Jaipur, India was procured from a local pharmacy with labelled amount 2% solution (20mg/ml).

Preparation of working standard drug solution: The standard Dorzolamide HCl (100 mg) was weighed accurately and transferred to 100 ml volumetric flask containing few ml of Distilled water and it was sonicated for 5 min to dissolved completely and diluted up to the mark with Distilled water to obtain final concentration of 1000 μg/ml and the resulting solution was used as working standard solution.

Analysis of marketed formulation: The commercially available eye drops contains 2% solution of sterile Dorzolamide HCl (20mg/ml). From this eye drop 1ml Solution was carefully transferred in to volumetric flask of 20 ml capacity containing 10 ml of the Distilled water and sonicated for 5 min. and then final solution was made with Distilled water to get the solution of 1000 µg/ml From this solution, various dilutions of the sample solution were prepared and analysed.

Calibration curve:

Method A:

Zero order spectroscopy: The solutions were scanned in the range from 400-200 nm and the peak was observed at 253 nm. The wavelength selected for the analysis of the drug was 253 nm (Fig. 2). The drug followed the Beer’s- law in the range of 3-24 μg/ml. The different concentrations of the sample solution were determined by using the calibration curve (Fig. 3).

FIG. 2: ZERO ORDER SPECTRA OF DORZOLAMIDE HCl AT 253nm

FIG. 3: CALIBRATION CURVE OF DORZOLAMIDE HCl

Method B:

First Order Derivative Spectroscopic method: The first order derivative spectra at n=1, showed a sharp peak at 238nm as shown in (Fig. 4). The absorbance difference at n=1 (dA/dλ) is calculated by the inbuilt software of the instrument which was directly proportional to the concentration of the standard solution. The standard drug solution was diluted, so as to get the final concentration in the range of 3-24 μg/ml and derivatized in to first order derivative spectra mode. The calibration curve of dA/dλ against concentration of the drug showed linearity. Similarly absorbances of samples solution were measured and the amount of Dorzolamide HCl in the sample was determined from standard calibration curve (Fig. 5).

FIG. 4: FIRST ORDER DERIVATIVE SPECTRA OF DORZOLAMIDE HCl AT 238 nm

FIG. 5: CALIBRATION CURVE OF DORZOLAMIDE HCl

Method C:

Second Order Derivative Spectroscopic method: The Second order derivative spectra at n=2, showed a sharp peak at 278nm (Fig. 6). The absorbance difference at n=2 (d²A/dλ²) is calculated by the inbuilt software of the instrument which was directly proportional to the concentration of the standard solution. The standard drug solution was diluted so as to get the final concentration in the range of 3-24 μg/ml and derivatized in to second order derivative spectra mode. The calibration curve of d²A/dλ² against concentration of the drug showed linearity. Similarly absorbances of samples solution were measured and amount of Dorzolamide HCl was determined from standard calibration curve (Fig. 7).

FIGURE 6: SECOND ORDER DERIVATIVE SPECTRA OF DORZOLAMIDE HCl AT 278nm

FIG. 7: CALIBRATION CURVE OF DORZOLAMIDE HCl

RESULT AND DISCUSSION:

Validation of the method: All these methods were validated according to ICH guidelines ^{5, 6} in terms of linearity, precision, accuracy and ruggedness parameters.

 Linearity: The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method (Table 1). The linearity was found to be 3-24 µg/ml for zero, first and second order derivative spectrophotometeric methods. Optimum conditions, Optical characteristics and Statistical data of the Regression equation in UV method are given in table 2.

TABLE 1: RESULTS OF CALIBRATION CURVE FOR DORZOLAMIDE HCl

TABLE 2: OPTIMUM CONDITIONS, OPTICAL CHARACTERISTICS AND STATISTICAL DATA OF THE REGRESSION EQUATION IN UV METHOD

Accuracy: The accuracy of the method was assessed by recovery studies at three different levels i.e. 50%, 100%, 150%. The values of standard deviation were satisfactory and the recovery studies were close to 100% (Table-3). Hence these methods can be useful in routine analysis of Dorzolamide HCl in bulk drug and formulations.

Ruggedness: Ruggedness is a measure of the reproducibility of a test result under normal, expected operating condition from instrument to instrument and from analyst to analyst. The results of ruggedness testing are reported in the Table 4.

Precision: The precision of an analytical  method is  the degree of agreement  among individual test  results  when   the   method  is  applied  repeatedly  to   multiple  samplings  of homogenous samples. It provides an indication of random error results and was expressed as coefficient of variation (CV). The results of inter-day and intra-day were reported in Table 5.

TABLE: 3 ACCURACY RESULTS FOR DORZOLAMIDE HCl

**Average of six determinations

TABLE: 4 RUGGEDNESS RESULTS FOR DORZOLAMIDE HCl

** Average of six determinations

TABLE: 5 PRECISION RESULTS FOR DORZOLAMIDE HCl

** Average of three determinations

CONCLUSION: It can be concluded that the proposed methods were validated and found to be simple, sensitive, accurate, precise, reproducible, rugged and relatively inexpensive. So, the developed methods can be easily applied for the routine Quality Control analysis of Dorzolamide HCl in pharmaceutical preparations.

ACKNOWLEDGEMENT: We would like thank to Lake Chemicals Pvt. Ltd., Bangalore for providing pure sample of Dorzolamide HCl and also to the Principle Dr T. Tamizh Mani, Bharathi College of Pharmacy, Bharathi Nagara for providing facilities to carry work.

REFERENCES:

1. wikipedia.com.
2. rxlist.com.
3. Nevin Erk. Simultaneous determination of Dorzolamide HCl and Timolol Maleate in eye drops by two different spectroscopic methods. J Pharm Biomed Anal 2002; 28(2):391-397.
4. Lories IB. Application of TLC-densitometry, first-derivative UV-Spectrophotometery and Ratio Derivative Spectrophotometery for the determination of Dorzolamide Hydrochloride and Timolol Maleate. J Pharm Biomed Anal 2002; 27(5):737-746.
5. ICH, Q2A Text on validation of analytical procedures, Oct, 1994.
6. ICH, Q3B Validation of analytical procedures: methodology, Nov, 1996.
   

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