1. INTRODUCTION:

Nitazoxanide 1-5 chemically -N-(5-nitro-2-thiazoyal) salicylamide acetate) It is used as an antiprotazoal, anthelmenthic, giardiasis2 and cryptosporidiosis2 in immune-compromised patient, including those with AIDS or HIV infection. It has been used in helmentic infection3-7. It is not official in any pharmacopoeia and extensive literature survey revealed its estimation by UV spectrophotometric method in bulk drugs. ⁽¹⁾

2 EXPERIMENTAL:

2.1. Apparatus

Spectrometric method was carried out using Systronic series a double beam UV/Visible spectrophotometer.

2.2. Marketed formulation

The marketed formulation studied was Nizonide tablets manufactured by Alembic Pharma. and Nitacure tablet manufactured by Glenmark company. Each tablet contains 500mg Nitazoxanide.

2.3. Standard solutions

Standard stock solutions of Nitazoxanide each of 1mg/ml concentration in solvent Dimethylformamide were prepared. From these stock solutions appropriate dilutions in the range of 5-25 μg/ml for Nitazoxanide were prepared and analyzed.

2.4. Solvent used

Phosphate buffer 7.4 PH is prepared by addition of potassium dihydrogen phosphate and sodium hydroxide in quantity of 25 ml and 19.55 ml respectively. Make the volume upto 100 ml with distilled water.

2.5. Preparation of standard stock solution: -

Standard stock solution of Nitazoxanide were prepared by dissolving 10 mg of drug in 10 ml of dimethylformamide to get concentration of 1.0 mg/ml. 1 ml of stock solution was further diluted to 10 ml with phosphate buffer 7.4 pH to get a working standard solution of concentration 100 mg/ml of drugs. This solution used as standard working mixture solution. From these stock solutions prepared appropriate dilutions in the range of 5-25 μg/ml of Nitazoxanide and analyzed.

2.6. Selection of analytical wavelength

From the sample and standard stock solutions appropriate dilutions of Nitazoxanide drug were made to obtain final concentration each containing 10 mg/ml of Nitazoxanide Solutions were scanned in the wavelength range of 200 – 400 nm. Using appropriate dilutions of standard stock solution, the dilutions were scanned. The wavelength of Nitazoxanide was 346 nm.

2.7. Selection of analytical concentration range and preparation of calibration curves

For each drug appropriate aliquots were pipetted out from standard working solutions into a series of 10 ml volumetric flasks. The volume was made up to the mark with phosphate buffer 7.4 ph to get a set of solutions. The Absorbanceof these solutions were measured at the selected wavelength i.e. 346 nm and plotted against concentration. The concentration range over which the drugs obeyed Beer’s law was chosen. The range was found to be 5 to 25 mg/ml for Nitazoxanide drug.

3. VALIDATION OF THE METHOD:

3.1. Linearity

Linearity of the proposed method was verified by analyzing five different concentrations in the range of 5-25μg/ml for Nitazoxanide. Each concentration was made in triplicate.

3.2. Precision

The intraday precision of the developed method was evaluated by analyzing samples of five different concentrations of nitazoxanide (20 μg/ml) on the same day. The inter day precision was evaluated from the same concentration on three consecutive days.

3.3. Accuracy

The accuracy of the method was performed by conducting the recovery studies (80, 100 and 120%) of pure drugs from two different marketed formulations Nitacure and Nizonide, by standard addition method. The actual and measured concentrations were then compared.

3.4. Limit of detection and limit of quantitation

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated as an exact value. The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy.

The detection limit (LOD) for the proposed methods were calculated using the following equation

LOD = 3.3s/k

Where, s is the standard deviation of replicate determination values under the same conditions as for sample analysis in the absence of the analyte and k is the sensitivity, namely the slope of the calibration graph.

In accordance with the formula, the detection limits obtained for the absorbance’s were calculated and listed in Table 1. The limits of quantitation, LOQ, defined as

LOQ = 10s/k

3.6. Stability

Stability was observed by scanning the drug solutions in selected solvent system in time scan mode of UV spectrophotometer for 12 hours.

4 RESULT AND DISCUSSION:

Selection of analytical concentration range and preparation of calibration curves

4.1 Linearity

Table 1. Linearity regression data for calibration curves

Parameter

Nitazoxanide

λ (nm)

Beer’s law limit (μg/ml)

Régression Equation

Slope

Corrélations coefficient

346 nm

5-25

Y=0.010x-0.03

0.01

0.990

Fig .no.1.Calibration curve of Nitazoxanide (5-25 µg/ml)

4.2. Precision

It was ascertained by replicate estimation of marketed formulation (five times) and results are shown as:

Table No.2. Intra-day variability

Nitazoxanide
Concentration mg/ml)	Mean (n=5)±SD	%RSD
20	0.3228±0.003	0.97

Intra-day variability of 20 mg/ml (n=5)

Table.No.3.Interday Variability

Nitazoxanide
Concentration (mg/ml)	Mean (n=5)±SD	%RSD
20	0.322±0.0010	0.33

Interday Variability of 20 mg/ml (n=5)

4.3. Limit of Detection (LOD)

The LOD was found to be 1.0132 µg/ml for Nitazoxanide.

4.4. Limit of Quantification (LOQ)

The LOQ was found to be 3.1 µg/ml for Nitazoxanide.

4.5. Recovery Studies

For the recovery study of two different brands, Nitacure and Nizonide, the results obtained for 80%, 100% and 120% levels the % RSD obtained were 0.98, 0.39, 0.38 for Nitacure and 0.09, 0.83 and 0.50 for Nizonide listed in table no.4.

5. CONCLUSION:

The proposed method gives accurate and precise results for determination of Nitazoxanide in marketed formulation (tablet) without prior separation and is easily applied for routine analysis. The most striking feature of this method is its simplicity and rapidity. Method validation has been demonstrated by variety of tests for linearity, accuracy, precision and stability.

6. ACKNOWLEDGEMENT:

The authors express their gratitude to Dr. S.R. Chaudhari, Principal, Amrutvahini College of Pharmacy, Sangamner. Also extends thanks to Mrs. Bhagyashri Mahesh Deshpande for her constant support and encouragement.

7. REFERENCES:

1. Martindale the complete drug reference, thirty fourth edition, edited by Sean c. sweetman. page no.612.1

2. Shailendra Bindaiya, Shravan Bankey and Deepti Jain Simultaneous determination of nitazoxanide and ofloxacin in tablet by ultraviolet spectrophotometry (Dual wavelength method) International Journal of ChemTech Research. Vol.2, No.1, pp 11-15, Jan-Mar 2010

3. Development and validation of spectrophotometric method, The Nanotechnology Journal, K.M. Patel, C.N Patel and H.N. Patel, page no.247

4. United States Pharmacopoeias, United States Pharmacopoeial convention. INC. Twin Brook Parkway. Rockville. 2004. Page no.1355.

5. British Pharmacopoeia; 2003, Licensing Division HMSO, Norwich, 2003. Page no.357,1668

6. The Merck Index an Encyclopaedia of Chemicals, Drugs and Biological, 13rd edition Merck and co. Inc., White House Station, New Jersey, 2000; 1177.

7. U.S.F.D.A Guideline for industry analytical procedure and method validation: Chemistry, Manufacturing and Control and Documentation, 2000.

Received on 09.07.2011 Modified on 20.08.2011

Research J. Pharm. and Tech. 4(11): Nov. 2011; Page 1714-1716