Simultaneous determination of Nitazoxanide and Ofloxacin in pharmaceutical preparations using UV-spectrophotometric and high performance thin layer chromatography methods

2.1 Instrumentation and chromatographic conditions

Chromatography was performed on 10 cm × 10 cm precoated silica gel 60 F₂₅₄ HPTLC plates. The chromatographic plates were prewashed with methanol and dried in an oven at 120 °C for 2 h before use. The samples were applied onto the plates as a band with 6 mm width using Camag 100 μl sample syringe (Hamilton, Switzerland) with a Linomat 5 applicator (Camag, Switzerland). Linear ascending development was carried out in a twin trough glass chamber (for 10 × 10 cm). Densitometric scanning was performed using Camag TLC scanner 3 in the range of 400–1500 ng/spot and operated by winCATS software (V 1.4.2, Camag). The chromatography estimation was performed using the following conditions: stationary phase was precoated with silica gel 60 F₂₅₄ aluminum sheets and the mobile phase used was chloroform:carbon tetra chloride:toluene:glacial acetic acid (10:5:3:0.5 v/v). The source of radiation was a deuterium lamp. Slit dimensions were 6 mm × 0.45 mm and the scanning speed 20 mm/s. Plates were evaluated densitometrically at 289 nm with a CAMAG Scanner III, in conjunction with the winCATS software for quantitation.

2.2 Preparation of standard and sample solution of Nitazoxanide and Ofloxacin

Accurately weighed 50 mg of Nitazoxanide was taken and transferred to a 10 ml volumetric flask, dissolved in methanol and diluted up to mark to obtain stock solution of 5000 μg/ml. From this solution, 0.5 ml was diluted to 20 ml to get 2500 μg/ml standard Nitazoxanide solutions. Accurately about 10 mg of Ofloxacin was weighed and transferred to a 10 ml amber colored volumetric flask and dissolved in methanol and volume was made up to mark to get stock solution of 1000 μg/ml. From this solution, 0.75 ml was further diluted to 10 ml to get 75 μg/ml standard Ofloxacin solutions. Accurately weighed 5 mg of rosiglitazone maleate was taken in a 25.0 ml volumetric flask. This was dissolved in minimum quantity of acetonitrile and diluted up to the mark with acetonitrile to get 500 μg/ml of rosiglitazone maleate. Rosiglitazone maleate was used as an internal standard.

Twenty Nitazete-O tablets were weighed and finely powdered. An amount of the tablet powder equivalent to 500 mg Nitazoxanide and 200 mg of Ofloxacin was weighed and transferred into a 100.0 ml standard volumetric flask. Sixty milliliters of methanol was added to the flask and was sonicated for 30 min. The solution was then cooled to room temperature and diluted up to the mark with methanol. The resultant solution was filtered through Whatman Grade I filter paper. The filtrate was used as sample solution.

2.3 UV–vis spectrophotometric

UV–vis spectrophotometer 1601 (Shimadzu, Japan) with spectral bandwidth of 2 nm and 10 mm matched quartz cells was used. By appropriate dilution of two standard drug solutions with methanol, solutions containing 50 μg/ml of Nitazoxanide and 20 μg/ml of Ofloxacin were scanned separately in the range of 200–400 nm to determine the wavelength of maximum absorption for both drugs. They were scanned in the wavelength range of 400–200 nm and the overlain spectrum was obtained. Two wavelengths 221.8 nm (λ_max of Nitazoxanide) and 244.3 nm (λ_max of Ofloxacin) were selected for the formation of simultaneous equation. The calibration curves were found to be linear in the concentration range of 5–25 μg/ml, for each drug. The absorptivity coefficients of each drug at both wavelengths were determined. The concentration of two drugs in the mixture was calculated using equations (Beckett and Stanlake, 1997).

In First order derivative spectroscopy method Nitazoxanide (50 μg/ml) and Ofloxacin (20 μg/ml), were prepared separately by appropriate dilution of standard stock solution and scanned in the spectrum mode from 400 nm to 200 nm. The absorption spectra thus obtained were derivatized from first to fourth order. First order derivative spectra were selected for the analysis of both drugs. From the overlain spectra of both drugs wavelengths selected for quantitation were 263.6 nm for Nitazoxanide (zero cross for Ofloxacin) and 269.2 nm for Ofloxacin (zero cross for Nitazoxanide). The calibration curves for Nitazoxanide and Ofloxacin were plotted in the concentration range of 5–50 μg/ml at wavelength 263.6 nm and 269.2 nm, respectively.

The methods were validated with respect to linearity, accuracy, precision and selectivity (ICH,1996). To ascertain the accuracy of the proposed methods, recovery studies were carried out by standard addition method at three different levels 80%, 100% and 120%. The mean percent recovery for Nitazoxanide and Ofloxacin by all the three methods was found in the range of 97.66–101.11%. The linearity of measurement was evaluated by analyzing different concentrations of the standard solution of Nitazoxanide and Ofloxacin. For simultaneous equation and area under the curve method, the Beer–Lambert's concentration range was found to be 5–25 μg/ml and for derivative spectrophotometer 5–50 μg/ml for both Nitazoxanide and Ofloxacin. The reproducibility of the proposed method was determined by performing tablet assay at different time intervals (morning, afternoon and evening) on same day (Intraday assay precision) and on three different days (Interday precision). Result of intraday and interday precision is expressed in % RSD (Table 1). Percent RSD for Intraday assay precision was found to be 0.0783 (for Nitazoxanide) and 0.1863 (for Ofloxacin) in simultaneous equation method; 0.1392 (for Nitazoxanide) and 0.1174 (for Ofloxacin) in first derivative spectrophotometric method. Interday assay precision was found to be 0.263 (for Nitazoxanide) and 0.131 (for Ofloxacin) in simultaneous equation method; 0.029 (for Nitazoxanide) and 0.048 (for Ofloxacin) in first derivative spectrophotometric method (Table 2).