INTRODUCTION:

Nitazoxanide is a synthetic anti parasitic agent for oral administration. Nitazoxanide is rapidly hydrolyzed to an active metabolite, tinoxanide (desactyl-nitazoxanide). Tizoxanide then undergoes conjugation, primarily by glucuronidation. Nitazoxanide is reduced by multiple enzyme system including pyruvate-oxidoreductase and is more easily reduced and acitve against a broader spectrum of organism. Nitazoxanide, also known by the brand names Alinia and Annita, is a synthetic nitrothiazolyl-salicylamide derivative and an antiprotozoal agent. Ofloxacin is a fluoroquinolone antibiotic considered to be a second-generation fluoroquinolone with broad antibacterial spectrum. Ofloxacin involves the inhibition of bacterial toposomerase IV and DNA gyrase enzymes required for DNA replication, transcription and recombination^1-8. Nitazoxanide and Ofloxacin are markedly of combined tablet dosage form in the ratio of 500mg and 200 mg respectively. Literature survey reveals that Nitazoxanide can be estimated by spectrophotometrically^9,10 and by HPLC^11-14 individually or with other drugs in bulk drugs, literature survey reveals that various analytical methods have been reported for the estimation of Ofloxacin in single and in combination dosage form such as, Spectrophotometric^15,16,HPLC^17-20 and LC/MS/MS^21,22

Nitazoxanide

Ofloxacin

EXPERIMENT:

Working standards of Nitazoxanide and Ofloxacin were obtained from Mitila Drugs Pvt. Ltd. Glenmark Pharmaceuticals Ltd. respectively. Tablet Nizonide-O (700 mg) was procured from the local market. HPLC grade Acetonitrile, Grade make SD-Fine Chem. Ltd. Water HPLC-grade from Milli-QRO system were procured from the market.

INSTRUMENTS:

The separation was carried out on HPLC system WATERS ALLIANCE 2695 with Cosmosil 5 C₁₈ 250 X 4.6mm column and waters 2996 Photodiode Array detector using filtered and degassed mixture of 0.005M tetra butyl ammonium hydrogen sulphate and acetonitrile (45:55) was pumped at flow rate of 1.0 ml/min; the detection was monitored at 240nm.

Standard preparation:

Weighed accurately 25mg of Nitazoxanide transferred carefully into a 25ml volumetric flask. Added 10ml of mobile phase and sonicated for 15 minutes, cooled to room temperature and diluted 25ml with mobile phase, mixed well. Weighed accurately 20mg of Ofloxacin and transferred carefully into a 50ml volumetric flask, added 35ml of mobile phase. Sonicated for 15 minutes, Cooled to room temperature and diluted 50ml with mobile phase, mixed well.

Assay preparation for commercial formulation:

Weighed and finely powdered not less than 20 tablets. Transferred an accurately weighed portion of the powder equivalent to about 5 mg to 100ml volumetric flask, added 70 ml of mobile phase. Sonicated for 15 minutes and cooled to room temperature. Diluted to 100ml with mobile phase. Mixed well and filtered through Whatman No.1 filter paper. Discarded first few ml of the filtrate.

Method validation:

The method was validated for statistical parameters i.e. precision, accuracy, specificity, linearity, and robustness criteria. Results of the method validation experiments are given in Table.1. The precision of the method was determined by knowing percentage RSD of means of three replicate solutions of all the three independent samples.

Table.1.Validation and system suitability parameters

Parameters	Nitazoxanide	Ofloxacin
Linearity range µg/ml	400 to 600	160 to 240
Correlation Coefficient (r²) ± S.D	0.9996	0.9997
Retention time (min) ± S.D	6.067	2.104
Resolution	25.64
Tailing factor	0.9312	1.3196
Theoretical Plate	17975.232	5492.72
Limit of detection (µg/ml)	8.4	6.27
Limit of Quantification (µg/ml)	25.53	19.0
Precision (RSD %) intraday (n=6)	0.2525	0.4202

Linearity:

Linearity was demonstrated by analyzing six different concentrations of active compound. Peak areas were recorded for all the peaks and calibration plot was constructed by plotting peak area vs concentrations of Nitazoxanide and Ofloxacin which were found to be linear in the range of 400 to 600 mcg/ml and 160 to 240 mcg/ml for Nitazoxanide and Ofloxacin.(Fig.no.1)

Accuracy:

The accuracy of the method is determined by adding known amount of standard to that of sample (above and below the normal level) at 3 different levels to cover both above and below (80% to 120%) the normal levels expected in the sample.

Fig.1.Linearity curve for Nitazoxanide and Ofloxacin

Specificity:

The specificity of the RP-HPLC method was determined by complete separation of Nitazoxanide and Ofloxacin as shown in Fig.no.2 with parameters like retention time (t_r), resolution (Rs) and tailing factor (T). Here tailing factor for peaks of Nitazoxanide and Ofloxacin was less than 2 % and resolution was satisfactory. The peaks obtained for Nitazoxanide and Ofloxacin were sharp and have clear base line separation.

Fig.2. HPLC Chromatogram of Nitazoxanide and Ofloxacin

Detection limit and quantification limit:

A Calibration curve was prepared by using concentration in the expected detection limit range of 400 to 600 mcg/ml and 160 to 240 mcg/ml for nitazoxanide and for ofloxacin respectively. Detection limit of Nitazoxanide and Ofloxacin was 8.4 mcg/ml and 6.27 mcg/ml respectively. quantification limit of Nitazoxanide and Ofloxacin was 25.53 mcg/ml and 19.0 mcg/ml respectively.

Method development and Optimization:

To investigate the appropriate wavelength for simultaneous determination of nitazoxanide and for ofloxacin, solutions of these compounds in the mobile phase were scanned by UV–visible spectrophotometry (shimadzu, uv-1700, pharmaspec. Cuvetts: 1 cm matched quartz cells) in the range 200–400 nm. From the overlaid UV spectra, suitable wavelength considered for monitoring the drugs was 240 nm (Fig.no.3)

Fig.3. Overlay spectra of Nitazoxanide and Ofloxacin

Results and discussions:

Optimization of the mobile phase was performed based on resolution, asymmetric factor and peak area obtained for both Nitazoxanide and Ofloxacin. The mobile phase was a combination of 0.005M tetra butyl ammonium hydrogen sulphate and acetonitrile in the ratio of 45:55 was found to be satisfactory and gave two symmetric and well resolved peaks for Nitazoxanide and Ofloxacin. The retention time for Nitazoxanide and Ofloxacin were 6.051and 2.106, respectively (Fig.no.2). The asymmetric factors for Nitazoxanide and Ofloxacin were 1.06 and 1.2, respectively. The calibration curve for Nitazoxanide was obtained by plotting the peak area of Nitazoxanide versus the concentrations of Nitazoxanide over the range of 400 to 600 mcg/ml, and it was found to be linear with r² = 0.9996. Similarly, the calibration curve for Ofloxacin was obtained over the range of 160 to 240 mcg/ml and was found to be linear with r² = 0.9997.The recoveries of Nitazoxanide and Ofloxacin were found to be in the range of 99.08-99.81% and 100.18%-101.43% .The system suitability parameters such as theoretical plates and tailing factor were found to be 17975.23, 0.931 and 5492.72, 1.31 for Nitazoxanide and Ofloxacin respectively. Proposed study describes a new RP-HPLC method for estimation of Nitazoxanide and Ofloxacin combination in mixture using simple mobile phase. The method gives good resolution between both the compounds with a short analysis time. The method was validated and found to be simple, sensitive, accurate and precise. Percentage recovery shows that the method is free from interference of the excipients used in the formulation. Therefore, the proposed method can be used for routine analysis of Nitazoxanide and Ofloxacin their combined dosage form.

REFERENCES:

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Received on 09.07.2010 Modified on 21.07.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 1062-1064

Simultaneous Estimation of Nitazoxanide and Ofloxacin by RP-HPLC Method

INTRODUCTION: