Absorbance Correction and Vierodt’s Spectrophotometric Methods for the Simultaneous Determination of Ofloxacin and Nitazoxanide in Combined Tablet Dosage Form

 

MD Game¹*, DM Sakarkar², KB Gabhane¹, KK Tapar¹, VN Shrikhande¹ and UA Deokate³

¹Vidyabharati College of Pharmacy, C.K.Naidu Road, Camp, Amravati – 444602 (M. S.) India             

² SN Naik College of Pharmacy, Pusad Dist. Yawatmol (M. S.) India           ³ Govt. College of Pharmacy, Amravati – 444602 (M. S.) India

*Corresponding Author E-mail: game_madhuri@yahoo.com

ABSTRACT:

Two simple, accurate, precise, rapid and economical methods have been developed for the simultaneous estimation of Nitazoxanide and Ofloxacin in combined tablet dosage form. First method is Vierodt’s method (Method A), where the wavelengths selected for quantitation were 347nm (λmax of Nitazoxanide) and 295nm (λmax of Ofloxacin). Second method is absorbance correction method (Method B), is based on correcting the absorbance of the component of interest from the total absorbance of the sample and the wavelengths used for estimation of Nitazoxanide and Ofloxacin were 380 nm. and 295 nm. Both the methods were validated statistically and recovery studies were carried out. Both drugs show linearity in the concentration range of 2 – 30 µg/ml. Linearity of Nitazoxanide and Ofloxacin were in the range of ± 20% of the test concentration. The proposed methods have been applied successfully to the analysis of the cited drugs in pharmaceutical formulations with good accuracy and precision. The methods herein described can be employed for quality control and routine analysis of drugs in pharmaceutical formulations.

 

 

INTRODUCTION

Nitazoxanide,^1-5 chemically (N- (5-nitro-2-thiazoyal) salicylamide acetate), is used as an antiprotozoal, antihelmentic, giardiasis² and cryptosporidiosis² in immune-compromised patient, including those with AIDS or HIV infection. It has been used in helmentic infection^3-7. It is not official in any pharmacopoeia and extensive literature survey revealed its estimation by UV spectrophotometric methods in bulk drugs. Ofloxacin^8-9 belongs to flouroquinolone group of antimicrobial agents. Chemically, it is (±)-9-fluoro-2, 3-dihydro-3-methyl-10- (4-methyl-1-piperazinyl)-7-oxo-7H-pyrido-[1,2,3-de]-1,4-benzoxazine -6-carboxylic acid. It is mainly used as antibacterial agent for the treatment of urinary tract infection and sexually transmitted diseases. Ofloxacin is official in BP⁸, USP9, and EP¹⁰. Literature survey revealed that a number of analytical methods have been reported for the estimation of Ofloxacin individually and in combination with various drugs in various dosage form such as Spectrophotometric ^11-14,Potentiometry and Conductometry¹⁰, HPLC ^15-20, Electrophoresis^{21, 22} and LC/MS/MS ^23,24.

 

The aim of the present work is to develop simple, rapid, precise and selective spectrophotometric methods for the estimation of Nitazoxanide and Ofloxacin in pharmaceutical dosage form.

 

MATERIAL AND METHODS:

Pure drugs of Nitazoxanide and Ofloxacin were obtained from Lupin Pharmaceuticals Aurangabad, Maharashtra; India. The commercial formulation of Nitazoxanide and Ofloxacin is available in the ratio of 2.5:1 named Nizonide-O in form of tablet dosage form. A Shimadzu UV-visible 1601 spectrophotometer with 1 cm matched quartz cells were used for all the spectral measurements. The solution of 0.1 N HCl was prepared in double distilled water as per I.P.1996 procedure. Stock standard solutions of Nitazoxanide and Ofloxacin (1mg/ml) were prepared separately in methanol. Working standard solutions (100μg/ml) were prepared by appropriate dilutions of the stock solutions in methanol. Further, accurate volumes of working standard solutions were transferred into the two sets of 10 ml calibrated flasks and their volumes were made by using 0.1 N HCl solution. The first series contained varying concentration of Nitazoxanide (2µg to 30µg) and the second series contained varying concentration of Ofloxacin (2µg to 30µg).From the two individual series of drug solutions, Nitazoxanide (15 µg/ml) and Ofloxacin (6 μg/ml) solutions were scanned over the range of 400-200 nm in 1 cm cells against solvent as blank. Figure-1 represents the overlain spectra of Nitazoxanide (15 µg/ml) and Ofloxacin (6 µg/ml).

Method A (Vierodt’s method) also called Simultaneous equation method. The wavelengths selected from the overlain spectrum for estimation of drugs were 347 nm as λmax for Nitazoxanide and 295 nm as λ max for Ofloxacin. Absorbances of two sets of varying concentration for Nitazoxanide and Ofloxacin individually and in combination were recorded at 347nm and 295 nm and the Beer- Lambert’s graphs were plotted. The calibration curves were found to be linear in the concentration range under study. The absorptivity values (A 1%, 1cm) of each drug at selected wavelengths were determined. In this method amount of each drug was estimated by substituting the absorbance and absorptivity values in the following simultaneous equation.

              A₂ay₁- A₁ay₂                                             A₁ax₂ – A₂ax₁

C_x  =    -------------------       ;  C_y   =     ----------------------

           ax₂ ay₁ – ax₁ ay₂                                    ay₁ ax₂– ay₂ ax₁

 

Where, Cx is concentration of Nitazoxanide in g/100 mL, Cy is concentration of Ofloxacin in g/100 ml, A₁ is absorbance of laboratory mixture at 347 nm, A₂ is absorbance of laboratory mixture at 295 nm, ax₁ is absorptivity value of Nitazoxanide at 347 nm, ax₂ is absorptivity value of Nitazoxanide at 295 nm, ay₁ is absorptivity value of Ofloxacin at 347 nm and ay₂ is absorptivity value of Ofloxacin at 295 nm.

 

Method B (absorption correction method) is based upon determination of identity, concentration and absorptivity of the absorbing interference and finally its contribution is calculated from the total absorbance of the mixture. Here firstly the concentration, identity, and absorptivity of the absorbing interference i.e Nitazoxanide was determined and then the concentration of the absorbing component of interest i.e. Ofloxacin was calculated from corrected absorbance. (Total absorbance minus absorbance of interfering substance).Wavelengths selected for estimation of drugs were 380 nm as detecting wavelength for Nitazoxanide and 295 nm as detecting wavelength for Ofloxacin. Absorbances of two sets of varying concentration for Nitazoxanide and Ofloxacin individually and in combination were recorded at 380 nm and 295 nm and the Beer-Lambert’s graphs were plotted. The calibrations curves were found to be linear in the concentration range under study. The absorptivity values (A 1%, 1cm) of each drug at selected wavelengths were determined. The concentration of each drug was calculated using equation C = A /A (1%, 1cm) where C, A, A (1%, 1cm) are the concentration, absorbance and % absorptivity at selected wavelengths for selected drugs.

 

In order to see the feasibility of proposed methods for simultaneous estimation of Nitazoxanide and Ofloxacin in marketed pharmaceutical formulations, the methods were first tried for estimation of both drugs in standard laboratory mixtures, obtained by mixing the aliquot portions of individual stock solutions to get final concentration of Nitazoxanide (15 µg/ml) and Ofloxacin (6 µg/ml) respectively. The results for both methods were found satisfactory and hence the methods were further applied to marketed preparations.

 

For analysis of tablet formulation, for both methods twenty tablets of Nizonide-O were accurately weighed and average weight of content per tablet was calculated. The contents of tablets were crushed to fine powder and mixed thoroughly, dissolved in solvent i. e. methanol with vigorous shaking and volume was made to 100 ml with the same solvent. The solution was filtered through Whatman filter paper No. 41.The aliquot portion of filtrate was further diluted with 0.1 N HCl to get final concentration of about 15 μg/ml of Nitazoxanide and 6 μg/ml of Ofloxacin and the absorbances of sample solution were measured at selected wavelengths and concentration of each drug was obtained by using mentioned formulae. The results of marketed formulation analysis and recovery studies are depicted in Table 1.Both the methods were validated statistically as per ICH/USP16 guidelines for parameters like accuracy, precision, ruggedness, specificity, linearity and range (Table 2). Accuracy was ascertained on the basis of recovery studies. Precision was studied by analyzing five replicates of sample solutions and concentrations were calculated. Ruggedness was established by carrying out experiment at different conditions like intra-day, inter-day and by different analyst. The recovery studies were carried out at different concentrations by spiking a known concentration of standard drug to the pre-analyzed sample and contents were reanalyzed by proposed methods.

 

Table 1:  Results of tablet formulation analysis

Sr. No.	Method	Drug	% Label Claim	±  S.D.
1	A	Nitazoxanide	100.47	0.732
		Ofloxacin	100.45	0.4711
2	B	Nitazoxanide	99.62	0.187
		Ofloxacin	101.22	0.209

Method A is Vierodt’s method and Method B is Absorbance correction method, Results are mean of five determinants (n = 5), SD is standard deviation

 

 

RESULTS AND DISCUSSION:

The proposed methods were successfully used to estimate Nitazoxanide and Ofloxacin in marketed tablet formulation. The results of marketed formulation analysis and recovery studies are depicted in Table-1. Both the methods were validated statistically as per ICH/USP16 guidelines and results for all the parameters like accuracy, precision, ruggedness, specificity, linearity and range were found satisfactory (Table-2). The assay values were in good agreement with the corresponding labeled claim. The recovery studies justified accuracy of the proposed methods.

 

 

Table 2: Optical characteristics and validation of the proposed methods

Sr. No.	Parameters	Method A		Method B
		Nitazoxanide	Ofloxacin	Nitazoxanide	Ofloxacin
1	Linearity Range	± 20% of test conc.	± 20% of test conc.	± 20% of test conc.	± 20% of test conc.
2	Beer’s law limit (mg/ml)	2 to 30 µg/ml	2 to 30 µg/ml	2 to 30 µg/ml	2 to 30 µg/ml
3	Accuracy (% Recovery)	99.51	100.2	99.85	100.42
Ruggedness, (% Label claim, n = 5)
4	Intraday	100.79	100.32	100.81	100.35
5	Interday	100.45	99.66	101.24	100.29
6	Different analyst	100.41	99.73	100.79	100.39

Method A is Vierodt’s method and Method B is Absorbance correction method.

 

 

Figure no: 1 Overlain spectra of Nitazoxanide (15 μg/ml) and Ofloxacin (6 µg/ml) in methanol for Method A (Vierodt’s method) and for Method B (Absorbance correction method)

 

 

 

CONCLUSION:

On observing the assay results and validation parameters, both these methods were found to be accurate, precise and specific. Hence, the methods can be employed for quality control and routine analysis of Ofloxacin and Nitazoxanide in pharmaceutical formulations.

 

ACKNOWLEDGEMENTS:

The authors are gratefully acknowledging Lupin Pharmaceuticals for providing the gift samples of Nitazoxanide and Ofloxacin. Authors are also thankful to Vidyabharati College of Pharmacy, Amaravati for providing necessary facilities for the research work.

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Accepted on 12.12.2009         © RJPT All right reserved