INTRODUCTION:
Nitazoxanide (NT), N-(5-nitro-2-thiazolyl) salicylamide acetate1,2 is a nitrothiazole derivative. Its chemical structure related to metronidazole. It is a broad spectrum antiprotozoal. It is not official in any Pharmacopoeias. The combination of Nitazoxanide and Ofloxacin is used in Diarrhea. Literature survey reveals that, spectrophotometric3 and RP-HPLC4 method is available for estimation of NT in single dosage form. Ofloxacin (OF) is chemically 9-fluoro-2, 3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-Oxo-7H-pyrido (1,2,3-di)-1,4-benzoxazine carboxylic acid. It is a fluoroquinolone derivative. It is used mainly as an antibacterial. It is official in USP5 (United State Pharmacopoeia), BP6(British Pharmacopoeia) and EP7 (European Pharmacopoeia). Literature survey reveals that spectrophotometric8, HPLC9, RP-HPLC10, and HPTLC11 methods are available for determination of Ofloxacin from pharmaceutical preparations and biological formulation.
More ever, the literature survey revealed that so far, no method has been reported for estimation of NT and OF in combined dosage form, hence two spectrophotometric methods have been developed for simultaneous estimation of NT and OF in tablet dosage form.
MATERIALS AND METHODS:
UV/ Visible double beam spectrophotometer, Shimadzu model-1700, with 1cm quartz cells was used. Pure sample of Nitazoxanide and Ofloxacin was obtained as gift sample from Indswift laboratories, Pvt Ltd Samba Jammu .The tablet dosage form, Nitazete-O (claim: 500mg NT and 200mgOF) was procured from the local market. Acetonitrile and Phosphate buffer AR grade was obtained from Merck Limited, Mumbai-400018.India.
UV-Spectrophotometry:
Standard stock solution of Nitazoxanide having concentration100 µg/ml was prepared by dissolving 10 mg of Standard Nitazoxanide in 100 ml solvent mixture. From this stock solution working standard solutions having concentration 20 µg/ml was prepared by appropriate dilution. Standard stock solution of Ofloxacin having concentration 100 µg/ml was prepared from standard Ofloxacin in a similar manner. Working Standard solutions of 20 µg/ml of each of the drug were scanned in the range 200-400 nm.to obtained the absorbance graph for both the drug (Fig.1 and Fig.2) and the overlain spectra (Fig.3).
Absorption ratio method (Method-1):
Absorption ratio method12,13,14 uses the absorbencies at two selected wavelengths, one of which is an isoabestic point and the other being the wavelength of maximum absorption of one of the two components. From the overlain spectra wavelength 320.6 nm (iso-bestic point) and 345.6 nm (λmax of Nitazoxanide) were selected for absorption ratio method. The absorbnces at 320.6 nm and 345.6 nm for Nitazoxanide were obtained. Similarly for Ofloxacin absorbencies are measured at 320.6 nm and 294.4 nm.The absorptivities value of each drug at both wavelengths was also determined which the mean of five independent reading.
Fig. 1. Absorbance graph of Nitazoxanide
The absorbances and absorptivity at these wavelengths were substituted in the following equation to obtained the concentration of both the drugs.
CNT = (QM- Qy)/ (Qx-Qy)х A1/ax1
COF = A1/ax1-CNT.
Where, CNT and COF are concentration of Nitazoxanide and Ofloxacin respectively, A1 is the absorbance of sample at 320.6nm, ax1 is the absorptivity of NT at 320.6nm, Qx was obtained by using the equation-(absorptivity of NT at 345.6nm) / (absorptivity of NT at 320.6nm).Qy was obtained from the equation-(absorptivity of OF at 345.6 nm /absorptivity of OF at 320.6 nm) and QM from (absorbance of sample at 345.6 nm/absorbance of sample at 320.6 nm).
Simultaneous Equation method (Method-2):
For selection of analytical wavelength for Simultaneous Equation Method12,13,15 , the wavelength 294.4 nm (λmax of OF) and 345.6 nm (λmax of NT) was selected. The absorbances of OF and NT were measured at 294.4 nm and 345.6 nm. The absorptivities of both the drugs at both the wavelengths were also measured.
The absorbance and the absorptivity values at these particular wavelengths were calculated and substituted in the following equation, to obtain respective concentration.
CNT= (A1 ax2- A2 ax1)/(ax2 ay1- ax1 ay2 )
COF = (A2 ay1 – A1 ay2 )/ (ax2 ay1- ax1 ay2)
Where, A1 is the absorbance of sample at 294.4 nm, A2 is the absorbance of sample at 345.6 nm,ax1 and ax2 are the absorptivity of NT at 294.4nm and 345.6 nm respectively, ay1 and ay2 are the absorptivity of OF at 294.4 nm and 345.6 nm respectively.
Table 1. Analysis Data of Tablet Formulation 
Parameters. Method 1. Method 2.
NT OF
NT OF
Label claim (mg) 500 200 500 200
% of label Claim* 100.16 99.37 99.78 100.50
± S.D. 1.110 0.732 1.271 0.614
% R.S.D 1.108 0.736 1.273 0.610
* Value for drug content (%) is the mean of three estimation.
Methd 1 is absorption ratio method, method 2 is simultaneous equation method, S.D is Standard deviation and R.S.D is relative standard deviation.
Fig. 2: Absorbance graph of Ofloxacin
Fig. 3: Overlain spectra of Nitazoxanide and Ofloxacin
Analysis of pharmaceutical Formulation:
For the simultaneous estimation of commercial formulation twenty tablets of Nitazete-O (Labail claim: Nitazoxanide 500 mg and Ofloxacin 200 mg) was taken. The average weight of tablet was determined. Tablets are finely powdered by mortar and Pastel. For method 1 and 2 powder equivalent to 50 mg NT was accurately weighed, transferred to a 50 ml volumetric flask, dissolved in 40ml solvent mixture for 20 minutes with vigorous shaking. Finally the volume was made up to the mark with solvent mixture. The solution was filtered through Whatman filter paper-41, first few drops were rejected. The filtrate was then appropriately diluted to get final concentration of 2 μg/ml of and 5 μg/ml of NT. Absorbance of this solution was measured at appropriate wavelengths and values were substituted in the respective formula to obtain concentration. The result of analysis was mentioned in Table 1.
Table 2. Recovery Studies
|
Methods |
Excess drug |
% Recovery* |
%R.S.D |
|||
|
NT |
OF |
NT |
OF |
NT |
OF |
|
|
Method 1 |
5 |
2 |
100.4 |
101.0 |
0.292 |
0.614 |
|
10 |
4 |
99.90 |
99.50 |
0.405 |
0.541 |
|
|
15 |
6 |
100.80 |
101.50 |
0.805 |
0.336 |
|
|
Method 2 |
5 |
2 |
100.4 |
101.50 |
0.415 |
0.252 |
|
10 |
4 |
100.6 |
99.84 |
0.675 |
0.415 |
|
|
15 |
6 |
99.35 |
99.69 |
0.647 |
0.097 |
|
* Recovery is mean of five estimation. Method 1 is absorption ratio method while Method 2 is simultaneous equation method and R.S.D is the relative standard deviation.
Recovery Study:
To check the accuracy of the developed methods and to study the interference of formulation additives, analytical recovery experiment was carried out by standard addition method. From the total amount of drug found, the percentage recovery was calculated. The results are reported in Table 2.
RESULT AND DISCUSSION:
Both the UV Spectrophotometric methods were found to be simple, accurate, economic and rapid for routine simultaneous estimation of Nitzoxanide and Ofloxacin, in tablet dosage forms. For both the methods, linearity was obtained in concentration range of 1-40 µg/ml and 1-30 µg/ml for Nitazoxanide and Ofloxacin respectively. Both the drug show good regression values at there respective wavelengths. Recovery was in the range of 99-101%; the value of standard deviation and % R. S. D are found to be < 2%; shows the high precession of the method.
Acknowledgement:
The authors are thankful to Indswift laboratories, Pvt Ltd Samba Jammu for providing gift samples of Nitazoxanide and Ofloxacin and one of the authors Mr. Yogendra Prasad is thankful to All India Council for Technical education (AICTE) for providing junior research fellowship.
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