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Bangladesh Pharmaceutical Journal 15(1): 47-51, 2012
Development and Validation of a Simple and Rapid UV
Spectrophotometric Method for Assay of Nitazoxanide in
Pharmaceutical Dosage Forms
Fahima Aktar1, Md. Ruhul Kuddus1, Akter Hossen2, Md. Khalid Hossain1 and Mohammad A. Rashid1
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh 2Department of Pharmacy, State University of Bangladesh, 77, Satmosjid Road, Dhanmondi, Dhaka-1207, Bangladesh Abstract
This paper reports the development and validation of a simple UV spectrophotometric method for the assay of
nitazoxanide in tablets and powder for oral suspension. The method was linear in the range of 5 and 50 µg/ml
presenting a good correlation coefficient (r = 0.9996, n = 7). Precision and accuracy analyses showed low relative
standard deviation (< 2.50%) and good recoveries (94.01-113.9%). The procedure was found to be accurate, precise,
linear, robust, simple and cost effective. It did not require any polluting reagents and can be applied to assay of
nitazoxanide in pharmaceutical dosage forms notably tablets and powder for oral suspensions.
Key words: Nitazoxanide, tablets, suspensions, UV spectrophotometry.
Introduction
Nitazoxanide (NTZ) is a new antiparasitic and antiprotozoal agent having broad-spectrum of activity. It is a nitrothiazole derivative and its chemical name is 2-acetyloxyl-N-(5-nitro-2-thiazolyl) benzamide (Fig. 1) (Rossignol et al., 1976). NTZ was first described in 1975 by Jean Francois Rossignol and was initially developed as a veterinary anthelminthic with activity against intestinal Although no official method for the determination of nematodes, cestodes and trematodes. NTZ was approved this drug in oral formulation has been described yet, NTZ by the US Food and Drug Administration (FDA) in 2002 in pharmaceutical formulations (tablets and powder for for use in human beings (Fox et al., 2005). It is used for oral suspension) can be estimated by liquid treating both intestinal protozoal infections and chromatography (LC) (Gopu et al., 2007; Jadhav et al., helminthiasis (Raether et al., 2003). It is also used for 2007; Rane et al., 2008; Malesuik et al., 2008), LC-mass treating diarrhea caused by Giardia lamblia as well as for spectrometry (LC-MS) (Stockis et al., 1996; Zhao et al., cryptosporidiosis in immune-compromised patients, 2008), high performance-thin layer chromatography (HP- including those with AIDS or HIV infection (Cravier et TLC), RP-HPLC and UPLC (Kalta et al., 2008). Although al., 1978; O'Neil et al., 2001; Murphy et al., 1985; Stockis various analytical methods have been reported for et al., 1996; Rossignol et al., 1984). determination of NTZ in bulk as well as in pharmaceutical NTZ is a light yellow/pink crystalline powder that is formulations, the reported chromatographic methods poorly soluble in ethanol, practically insoluble in water necessitate sample pretreatment and time-consuming with a molecular mass of 307.283 gm/mole and molecular extraction steps prior to analysis of the drug (Sakamoto et al., 2008). Several of these methods require the use of 12H9N3O5S. After ingestion, it is converted to the active metabolites tizoxanide and tizoxanide hazardous and expensive chemicals, which make the glucuronide. In plasma, more than 99% of NTZ is bound process not only a challenge for the environment but also to proteins. It is available in the market as tablets and oral complex. Moreover, these methods require expensive equipments and considerably skilled personnel. Correspondence to: Mohammad A. Rashid; Tel.: 880-2-9661900-73, Extn.- 8130, 8131, 8137; Fax: 880-2-8615583; E-mail: [email protected] Aktar et al. / Bangladesh Pharmaceutical Journal 15(1): 47-51, 2012
In this paper, we describe a simple UV- specto- dispersion of the drug. Then the flasks were shaken photometric method for the determination of NTZ in tablet ultrasonically for 20.0 minutes and the solution was and powder for suspension formulations. The method has filtered through Whatman no.1 filter paper (Whatmann been optimized and validated as per the ICH guidelines International Limited, Kent, UK). The residue was washed well with ethanol for complete recovery of the drug. After filtration an aliquot of 1.0 ml of this solution was Materials and Methods
transferred into a 50.0 ml volumetric flask and the volume was adjusted upto the mark with ethanol. In case of Apparatus: A Shimadzu UV-Visible spectrophoto- powder for oral suspension, at first suspension was made meter (UVmini-1700, Shimadzu Corporation, Kyoto, by adding purified water (according to direction in the Japan) with matching quartz cells was used for all label). From this suspension, 5.0 ml aliquot was taken in 100.0 ml volumetric flask and about 70.0 ml of ethanol reagents: All chemicals and reagents was added, sonicated for 20 minutes and diluted up to the were of analytical or pharmaceutical grade. NTZ was mark (100.0 ml) with ethanol to get a concentration of 1.0 kindly supplied by Incepta Pharmaceuticals Ltd. mg/ml. The solution was filtered through Whatman no.1 (Bangladesh) and was used as the reference standard. A filter paper and 1.0 ml of 1.0 mg/ml NTZ was further standard solution of NTZ was prepared by dissolving 50 diluted to 50 ml in a volumetric flask with ethanol to mg of NTZ in 50 ml of ethanol, transferred to a 100 ml produce a final concentration of 20 µg/ml. The assay was volumetric flask and the volume was adjusted to the mark completed following the same proposed method used for with ethanol to obtain a stock solution of 500 µg/ml. Five different tablets and five powder samples for suspension Method validation
of NTZ coded as NTZ-ta to NTZ-te and NTZ-pa to NTZ- The developed analytical method has been validated for specificity, linearity, limit of detection, precision, λmax: An ultra violet spectrophoto- accuracy and robustness as mentioned below. metric scanning (190-380 nm) was carried out with the Specificity: The reference standard and quality control reference solution (5.0µg/ml) to select the λmax for samples of NTZ were subjected to stress conditions (i.e. detection of nitazoxanide. light, heat etc). Each stressed sample was measured to procedure: Aliquots of 500 µg/ml determine the content of NTZ and the results were from the standard stock solution of NTZ were pipetted compared with those for an unstressed time zero reference into a six series of 50.0 ml volumetric flasks. Then the solution. The reference assay value for each unstressed mixture was serially diluted with ethanol to get NTZ product was evaluated and the contents of degradation in solutions of 5.0, 10.0, 15.0, 20.0, 25.0 and 50.0 µg/ml. the stressed and control samples were estimated relative to The contents of each flask were mixed well and the assay value. The system response was examined for immediately transferred to the spectrophotometric cell. the interference or overlaps if any, with NTZ responses at Procedure for the determination of NTZ in Linearity: The linearity was evaluated with six pharmaceutical formulations: The test sample solutions standard solutions: 5.0, 10.0, 15.0, 20.0, 25.0 and 50.0 containing NTZ at a concentration of 20.0 µg/ml were µg/ml. The determination was repeated five times at each prepared. For this 20 tablets from each sample were concentration level. The linearity was evaluated by linear weighed and the average weight was calculated. The regression analysis, which was calculated by the least contents of 20 tablets were obtained by gently peeling of the hard shells and then crushed to a fine powder and an Limits of detection (LOD) and limit of quantification amount equivalent to 100 mg of NTZ was transferred to (LOQ): Limits of detection (LOD) and quantitation (LOQ) each of the 100.0 ml volumetric flask and 70.0 ml of for the assay were calculated using the following ethanol was added and left for 10.0 minutes for complete equations (Mustafa et al., 2000). Aktar et al. / Bangladesh Pharmaceutical Journal 15(1): 47-51, 2012
LOD = 3.3 ×So /b and LOQ = 10 × So/b, where So and The intra-day and inter-day precision and accuracy b are the standard deviation and the slope of the were determined and listed in Tables 3 and 4 respectively. calibration line. Since all the values of accuracy and % CV are well within the acceptable range of 15%, the results indicated that the accuracy of the proposed method were determined by method is reliable, reproducible and accurate. replicate analysis (n= 4) of calibration standards at three Calibration curve of NTZ
concentration levels (5.0, 20.0 and 50.0 µg/ml). Inter-day precision and accuracy were determined by assaying the calibration standard at the same concentration levels for four consecutive days. Precision and accuracy were based on the calculated relative standard deviation (RSD, %) and coefficient of variance (CV,%) of the observed concentration as compared to the theoretical one, respectively. For the method to be precise the %CV determined at each concentration level, should not exceed Conce ntration (microgram/ml)
Robustness: Robustness of the proposed method was determined by changing the pH of the medium by ± 0.2 Table 1. Absorbance of NTZ solutions of varying concentrations at
units and by maintaining the solutions at room temperature (25 ± 2 ºC) for 3 h to test the stability of NTZ in the working diluent (ethanol at pH 4.5). test: The potency of the tested tablet and powder for suspension formulations were determined by
Table 2. Regression data for calibration of NTZ
Results and Discussion
A simple and rapid UV spectrophotometric assay method has been developed and validated for analysis of nitazoxanide (NTZ) in tablets and powder for suspension. In order to verify the absence of interference of
Table 3. Intra-day precision and accuracy study of standard NTZ
excipients on the analysis of NTZ tablets and powder for Intra-day precision and accuracy (n = 4 replicates) suspension, a sample was prepared with all the excipients present in the tablets but without the drug (placebo). Absorption spectra did not show any potential interference A good linear relationship was evident between the absorbance and concentration in the range of 5.0 to 50.0 Table 4. Inter-day precision and accuracy study of NTZ
µg/ml (Fig. 2). The correlation coefficient was 0.9996 Intra-day precision and accuracy (n = 4 consecutive days) (Table 2) indicating good linearity. The representative Calculated concentration (µg/ml) Mean ± SD linear equation was Y=0.0469x + 0.0235, calculated by the least squares method. The limit of quantification (LOQ) was found as 0.907 µg/ml while the limit of Aktar et al. / Bangladesh Pharmaceutical Journal 15(1): 47-51, 2012
Table 5. Extinction coefficient of NTZ
Conclusion
The results obtained and the statistical parameters for determination of NTZ in pharmaceutical dosage forms demonstrated that the proposed UV spectrophotometry method is simple, accurate, fast and precise. The method showed high sensitivity, acceptable linearity and accuracy. Therefore it could be easily used for the analysis of pure drug. Moreover, the method uses simple reagents with In addition, the reliability of the proposed method was minimum steps and time for sample preparation, which also evaluated by means of the determination of the allow it to be useful for routine analyses and quality- extinction coefficient of NTZ using Beer-Lambert’s Law control assays of NTZ in tablets and powder for After the validation of the newly developed UV spectrophotometric method, the potency of marketed References
formulations was determined by the proposed validated
method and the results are shown in Table 6. Among the bioanalytical method validation for small molecules. AAPS different marketed brands used, the potency of all the Journal. 9, 109-114.
brands was found to be within the limit of 94.012 – Cravier, R. 1978. Development and validation of spectro- photometric methods for the estimation of nitazoxanide in 113.9837%. According to USP 2011, the potency range of tablet dosage forms. Eur. J. Med. Chem. 13, 539.
Fox, L.M. and Saravolatz, L.D. 2005. Nitazoxanide: a new
Table 6. Potency determination of the NTZ formulations
thiazolide antiparasitic agent. Oxford J. Clin. Infect. 40,
Gopu, C.L., Thomas, S., Paradkar, A.R., and Mahadik, K.R. 2007. A validated stability indicating HPTLC method for determination of nitazoxanide. J. Sci. Ind. Res. 66, 141-145.
Jadhav, A.S., Pathare, D.B. and Shingare, M.S. 2007. A validated stability indicating RP-LC method for nitazoxanide, a new antiparasitic compound. 66, 595-600.
Jadhav, V.Y., Gandhi, S.V., Dhavale, N.D. and Sabnis, S.S.2005. RP-HPLC Determination of nitazoxanide in bulk and different tablet formulations. Eurasian J. Anal. Chem. 3,
For the recovery analysis, the concentration present and the percentage recovery were calculated by average of Kalta, R.R., Sharma, R. and Chaturvedi, S.C. 2008. Simultaneous RP-HPLC determination of nitazoxanide and ofloxacin in four replicate analyses of each formulation. The studies combined tablet dosage form. Indian. J. Pharm. Sci. 70,
were carried out at different level of concentrations by spiking different amount of standard drug to the pre- Malesuik, M.D., Cardoso, S.G. and Steppe, M. 2008. analyzed sample and the contents were re-analyzed by Development of a validated stability-indicating LC method the proposed method and the results are summarized in for nitazoxanide in pharmaceutical formulations. Chromatographia 67, 131-136.
Moustafa, A.A.M. 2000. Spectrophotometric methods for the Table 7. Recovery studies of NTZ

determination of lansoprazole and pantoprazole sodium sesquihydrate. J. Pharm. Biomed. Anal. 22, 45-58.
Murphy, J.R. and Friedmann, J.C. 1985. Pre-clinical toxicology of nitazoxanide, a new antiparasitic compound. J. Appl. Toxicol. 5, 49-52.
Aktar et al. / Bangladesh Pharmaceutical Journal 15(1): 47-51, 2012
O'Neil, M.J. 2001. An Encyclopedia of Chemicals, Drugs and Sakamoto, T. and Hiyama, Y. 2008. Rapid determination of Biologicals. The Merck Index. 13th edition, pp.1177-1178. nitazoxanide in tablets using reversed-phase ultra-performance liquid chromatography (UPLC) and high- Raether, W. and Hanel, H. 2003. Nitroheterocyclic drugs with performance liquid chromatography. Die Pharmazie. 63,
broad spectrum activity. Parasitol. 90, 19-39.
Rane, V.P., Sangshetti, J.N., Patil, K.R., Yeole, R.D., and Stockis, A., Deroubaix, X., Lins, R., Jeanbaptiste, B., Calderon, Shinde, D.B. 2008. Stability-indicating LC determination of P. and Rossignol, J.F. 1996. Pharmacokinetics of nitazoxanide in bulk drug and in pharmaceutical dosage nitazoxanide after single oral dose administration in 6 form. Chromatographia 67, 455-459.
healthy volunteers. Int. J. Clin. Pharmacol. Ther. 34, 349-
Rossignol, J.F and Cavier, R. 1976. New derivative of 2- benzamido-5-nitrothiazoles. Chem Abstr. 83, 28216.
Zhao, Z., Zhang, L., Xue, F., Wang, X., Zheng, W., Zhang, T., Rossignol, J.F., Maisonneuve, H. and Amer, J. 1984. Fei, C., Zhang, K., Qiu, M., Xin, R., and Yang, F.J. 2008. Nitazoxanide in the treatment of Taenia saginata and Liquid chromatography-tandem mass spectrometric analysis Hymenolepis nana infections. Trop. Med. Hyg. 33, 511-
of nitazoxanide and its major metabolites in goat. J. Chromatography B. 875, 427-436.

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