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Abstract: Three new spectrophotometric methods Calibration curve method, first derivative method and AUC method for determination of nitazoxanide in bulk ...
Spectrophotometric Methods for the Estimation of Nitazoxanide in Bulk and Tablet Dosage Form International Science Press ISSN: 0976-5573

 Published by International Science Press

SPECTROPHOTOMETRIC METHODS FOR THE ESTIMATION OF NITAZOXANIDE IN BULK AND TABLET DOSAGE FORM K. V. SHAH*, D. R. BHIMANI, B. L. THUMAR & T.R.DESAI

R. K. College of Pharmacy, Kasturbadham, Rajkot, Gujarat, India-360 003

Abstract: Three new spectrophotometric methods Calibration curve method, first derivative method and AUC method for determination of nitazoxanide in bulk and in tablet dosage form have been developed and validated for linearity, accuracy and precision. The first method employs formation of calibration curve at λmax of the drug, which is 416.0 nm. The second method is by using first derivative spectroscopy, which is adopted to eliminate spectral interference. Here calibration curve was plotted at 441.0 nm. The last method employs formation of calibration curve by plotting graph of AUC against concentration. The drug obeys the linearity with absorbances in the concentration ranges employed for these methods. The methods have been validated statistically and by recovery studies. These methods were found to be simple, sensitive, rapid, accurate, reproducible and economical. Keywords: Nitazoxanide, Calibration Curve Method, First Derivative Method, Area under the Curve Method

Introduction

Materials

Nitazoxanide (NTZ), chemically N-(5-Nitro-2-thiazolyl) salicylamide acetate (Sweetman 2005) is used as anthelmintic and antiprotozoal (Budavari 2001). It was found initially to have activity against tapeworms; subsequent in vitro studies suggested that it was effective against a number of intestinal helminthes and protozoans. Nitazoxanide inhibit the growth of the sporozoites and oocytes of C. parvum and inhibit the growth of the trophozoites of G. intestinalis, E. histolytica, and T. vaginalis in vitro.

A Shimadzu UV/Visible spectrophotometer model 1601 (Japan) was employed with spectral bandwidth of 2 nm and wavelength accuracy of ± 0.5 nm with automatic wavelength correction with a pair of 10mm quartz cells. A Shimadzu electronic analytical balance (AX-200) was used for weighing the sample. An ultrasonic cleaner (Art No.400014CL) was used for sonicating the tablet powder. Analytical grade methanol (make: Qualigens) was used for making solution.

Nitazoxanide may have some efficacy against Fasciola hepatica infections and has been used to treat infections with G. intestinalis that is resistant to metronidazole and albendazole (Abbound et al., 2001; Rossignol et al., 1984). Nitazoxanide is not official in I.P., B.P. and U.S.P. But reported method requires condensation reaction, so tedious and time consuming (Adagu et al., 2002; Favennec et al., 2003; Abbound et al., 2001; Rossignol et al., 2001; Rossignol et al., 1998; Bailey et al., 2004; Beckett et al., 2007). The paper presents three simple, accurate, reproducible, speedy and economical methods for the estimation of NTZ in bulk and in formulation. *

Corresponding Author E-mail: [email protected]

Standard Stock solutions (100 µg/ml) of NTZ were prepared by dissolving separately 10 mg of drug in 100 ml of methanol: water (50:50). The scanning of 20µg/ml solution of NTZ was carried out in the range of 600-200 nm against blank for obtaining the spectrum of the drug. (Fig.1). The maximum absorbance of NTZ was obtained at 416.0nm.

Method-A Suitable dilutions of 2, 5,10,20,30 µg/ml of NTZ were prepared from the stock solution. In the quantitation mode calibration curve was plotted and exhibited λmax and co-efficients of correlations were found to be 0.9978 in the quantitation mode of the instrument. The optical characteristics and regression values for the calibration curve are presented in Table 1.

International Journal of Pharmaceutical Formulation and Analysis, September 2010

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K. V. Shah, D. R. Bhimani, B. L. Thumar & T.R.Desai

Method-B Suitable dilutions of 2, 5,10,20,30 µg/ml of NTZ were prepared from the stock solution. The scanning of 20µg/ ml solution of NTZ was carried out in the range of 600200 nm against blank for obtaining the spectrum of the drug. This spectrum was derivatised and λmax was found to be 441.0nm (Fig.2). In the derivative option of the quantitation mode calibration curve was plotted at 441.0 nm and exhibited linearity with absorbances in the range of 2-30 µg/ml. Co-efficients of correlations were found to be 0.9975 in the quantitation mode of the instrument. The optical characteristics and regression values for the calibration curve are presented in Table 1.

Method-C Suitable dilutions of 2, 5, 10, 20, 30 µg/ml of NTZ were prepared from the stock solution. These solutions were scanned in the spectrum mode of the instrument and AUC were found for all the above dilutions in the range of 440.0-390.0 nm (Fig.3) and calibration curve was prepared, because in this range drug shows maximum area for measurement. It follows linearity in the range of in the range of 2-30 µg/ml and co-efficients of correlations were found to be 0.9986. The optical characteristics and regression values for the calibration curve are presented in Table 1.

Preparation and Analysis of Tablet Sample Solution Twenty tablets (brand name Nitacure-500 and manufactured by Alembic Ltd., Vadodara) were weighed and crushed to a fine powder. An accurately weighed powder sample equivalent to 10 mg was transferred to a 100 ml volumetric flask and dissolved in about 25 ml of methanol. After the immediate dissolution, the volume was made up to the mark with methanol : water (50:50). The solution was kept for sonication for about 20 minutes. The solution was filtered through Whatmann filter paper No.41 (20 µ). From this stock solution 20.0 µg/ml solution of NTZ was prepared. Concentration of this solution was obtained in the quantitation mode of the instrument against standard calibration curve for first two methods. For AUC method concentration was found by using working curve equation of y = 2.8214 x + 1.5006. The analysis procedure was repeated six times with the same batch of tablets. The results of analysis and statistical validation for the marketed tablet formulation are reported in Table 2. The results of recovery studies conducted by the addition of different amounts of pure drugs at 80%, 100% and 120% levels to a tablet solution were found to be 58

satisfactory and are given in the Table-3. The results of recovery studies indicated that the method is accurate and reproducible.

Results and Discussion The objectives of the proposed work is to develop some new and sensitive analytical methods for the determination and validation of Nitazoxanide in bulk and pharmaceutical dosage forms. The proposed method for estimation of NTZ by calibration curve method, derivative spectroscopy and Area under the curve method was found to be simple, accurate, economical, selective, sensitive and reproducible. The optical characteristics such as Beer’s law limits, slope, intercept and correlation coefficient are summarized in Table 1. In methanol : water (50:50) mixture Nitazoxanide shows colored solution and λ max was 416 nm. For calibration curve method 416 nm was used as detection wavelength. For first derivative method absorbance was determined at 441 nm for standard drug concentration and also for unknown solution. AUC was determined for the wavelength range 440 to 390 nm for determination of concentration using area under curve method. In all the methods correlation coefficient obtained was 0.99750.9986 with concentration range of 2-30 µg/ml. Once the standard calibration curve was plotted, analysis requires only the scanning of the sample solution against standard calibration curve and instrument gives results directly on the screen and it does not require any calculations for first two methods so these are less tedious compare to AUC method, because in AUC method concentration can be found by putting values of the AUC in the working curve equation. All three method shows linearity in the concentration range used at detection wavelength. Regression characteristics like mean, standard deviation and standard error of mean were calculated for all the methods and it is given in Table 2 and Table 3 for standard drug solution and tablet analysis respectively. By using these three methods mean percentage of drug found in standard drug solution was 100.34, 99.16 and 100.32 for calibration curve method, first derivative method and area under curve method respectively. In tablet analysis mean percentage of drug found was 100.05, 100.29 and 99.96 using calibration curve method, first derivative method and area under curve method respectively. Standard deviation obtained by these methods was also within limit for standard and unknown drug solution. The relative standard deviation values are below 2%, indicating the precision of the method. For calibration curve method, first derivative method and area under curve method, standard deviation values

International Journal of Pharmaceutical Formulation and Analysis, September 2010

Spectrophotometric Methods for the Estimation of Nitazoxanide in Bulk and Tablet Dosage Form

obtained was 0.9267, 0.8346 and 1.270 respectively with standard drug solution and it was 1.372, 1.255 and 1.468 respectively for tablet assay. From this data it is observed that AUC method requires calculation, but it is more precise than other two methods. The validation of the proposed method was further confirmed by recovery studies. The statistical parameters and recovery study data clearly indicate the reproducibility and accuracy of the methods. Analysis of the samples containing NTZ showed no interference from the common excipients. Hence, all the three methods can be employed for routine analysis of the drugs in quality control, R and D laboratories.

Table 1 Regression and Optical Characteristics of Nitazoxanide Parameters

Method-A

Method-B

λmax in Methanol: Water (50:50)

416.0 nm

441.0 nm 440.0-390.0 nm

Beer’s Law range 2-30 µg/ml

Method-C

2-30 µg/ml

2-30 µg/ml

Regression Values: i. Slope

0.0636

–0.0018

2.8214

ii. Intercept

0.0336

–0.0012

1.5006

iii. Regression

0.9978

0.9975

0.9986

coefficient (r ) 2

Table 2 Assay Results of Nitazoxanide Drugs Solution by Method-‘A’, ‘B’& ‘C’ Name of Component

Amount Present (g/ml)

Method

Mean* % Content

Standard Deviation

% Co-efficient of Variation

Standard Error

20

Method-A

100.34

0.9267

0.9236

0.3783

20

Method-B

99.16

0.8346

0.8417

0.3407

20

Method-C

100.32

1.270

1.266

0.5185

NTZ

NTZ is Nitazoxanide, Method-A is the calibration curve method, method-B is the first derivative method and method-C is AUC method. * Here mean is the average of (n = 6) determinations. Table 3 Assay Results of Nitazoxanide in Commercial Formulation by Method-‘A’, ‘B’ & ‘C’ Name of Component

Amount Present (g/ml)

Method

Mean*

Standard Deviation

% Co-efficient of Variation

Standard Error

20

Method-A

100.05

1.372

1.371

0.5602

20

Method-B

100.29

1.255

1.251

0.5123

20

Method-C

99.96

1.468

1.469

0.5991

NTZ

NTZ is Nitazoxanide, Method-A is the calibration curve method, method-B is the first derivative method and method-C is AUC method. * Here Mean is the average of (n = 6) results. Table 4 Recovery Studies of Tablet Formulation for Nitazoxanide Level of % Recovery

Method

Amount Present (mg/tab)

A

500

B

500

C

Amount of Standard added

% Recovery* ± SD

% Co-efficient of Variance

Standard Error

400

99.59 ± 0.7301

0.7331

0.4215

400

100.75 ± 0.5956

0.5912

0.3439

500

400

99.47 ± 0.8743

0.8790

0.5048

A

500

500

100.44 ± 1.032

1.027

0.5961

B

500

500

100.92 ± 1.296

1.284

0.7485

C

500

500

99.98 ± 1.222

1.222

0.7057

A

500

600

101.09 ± 0.6813

0.6740

0.3934

B

500

600

100.09 ± 1.217

1.216

0.7026

C

500

600

99.05 ± 0.5335

0.5386

0.3080

(mg/tab) 80

100

120

* Here % recovery is average of three results at each level.

International Journal of Pharmaceutical Formulation and Analysis, September 2010

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K. V. Shah, D. R. Bhimani, B. L. Thumar & T.R.Desai Giardiasis with Nitazoxanide in a Patient with Acquired Immunodeficiency Syndrome”, Clin. Infect. Dis., 2001, 32, 1792-1794.

Method-A is the calibration curve method, method-B is the first derivative method and method-C is AUC method.

Figure 1: Spectrum of NTZ in Methanol : Water (50:50).

(2)

Abbound, P.; Lemee, V.; Gargal, G., et al., “Successful Treatment of Metronidazole and Albendazole-resistant Giardiasis with Nitazoxanide in a Patient with Acquired Immunodeficiency Syndrome”, Clin. Infect. Dis., 2001, 32, 1792-1794.

(3)

Adagu, I.S.; Nolder, D.; Warhurdt, D.C. ; Rossignol, J.F. “In Vitro Activity of Nitazoxanide and Related Compounds Against Isolates of Giardia Intestinalis, Entamoeba Histolytica and Trichomonas Vaginalis”, J.Antimicrob.Chemother., 2002, 49, 103-111.

(4)

Bailey, J.M.; Erramouspe, “J. Nitazoxanide Treatment for Giardiasis and Cryptosporidiosis in Children”, Ann. Pharmocother., 2004, 38, 634-640.

(5)

Beckett, A.H.; Stanlake, J.B., Eds., “Practical Pharmaceutical Chemistry”, 4th Edn, Part Two, CBS Publishers and Distributors, New Delhi, 2007, 278-314.

(6)

Budavari, S., Eds., in; The Merck Index. 13th Edn.; Merck & Co., Inc., Whitehouse Station, New Jersey, USA, 2001, 1152.

(7)

Favennec, L.; Jave - Ortiz, J.; Gargal, G., et al., “Doubleblind, Randomized, Placebo-controlled Study of Nitazoxanide in the Treatment of Fascionalis in Adults and Children from Northern Peru. Aliment”, Pharmacol.Therm., 2003, 17, 265-270.

(8)

Rossignol, J. F.; Ayoub A.; Ayers, “M.S. Treatment of Diarrhea Caused by Cryptosporidium Parvum: A Prospective Randomized, Double-blind, Placebocontrolled Study of Nitazoxanide”, J. Infect. Dis., 2001, 184, 103-106.

(9)

Rossignol, J.F.; Hidalgo, H.; Feregrino, M. et al., “A Double Blind Placebo-controlled Study of Nitazoxanide in the Treatment of Cryptosporidial Diarrhea in AIDS Patients in Mexico”, Trans. R. Soc. Trop. Med. Hyg., 1998, 92, 663-666.

Figure 2: Derivative Spectrum of NTZ in Methanol: Water (50:50).

Figure 3: Spectrum of NTZ in Methanol: Water (50:50) Showing AUC

References (1)

60

Abbound, P.; Lemee, V.; Gargal, G., et al., “Successful Treatment of Metronidazole and Albendazole-resistant

(10) Rossignol, J.F.; Maisonneuve, “H. Nitazoxanide in the Treatment of Taenia Saginata and Hymenolepis Nana Infections”, Am.J.Trop.Med. Hyg., 1984, 33, 511-512. (11) Sweetman, S.C., “In; Martindale: The Complete Drug Reference”; Pharmaceutical Press, London, UK; 34th Edn., 2005, 964.

International Journal of Pharmaceutical Formulation and Analysis, September 2010