INTRODUCTION:

Nitazoxanide (NTZ), chemically N-(5-nitro-2-thiozoly) salicylamide acetate¹, is anantiamoebic and anhtelmintic agent. It is indicated for amoebiasis, helminthiasis, giardiasis, fasciasis, trichomoniasis and cryptosporidiosis, including those with AIDS or HIV infections^{2, 3}. It is not yet official in any of the pharmacopoeia. Literature survey revealed that HPTLC method for its determination in bulk drug and pharmaceutical formulation⁴. Few UV spectrophotometric methods and HPLC methods have been reported for the estimation of NTZ^5-9. Literature review revealed that no HPTLC method is yet reported for the development and validation of the NTZ in tablet dosage form at single wavelength. Therefore, it was thought worthwhile to develop simple, precise, accurate HPTLC method for development and validation of NTZ in tablet dosage form.

MATERIALS AND METHODS:

Pharmaceutical grade NTZ (Batch no. SNTXO107030) was supplied as a gift sample by Ind-Swift Laboratories Limited, Samba, (Jammu and Kashmir), India. The tablet dosage form (Nizonide, Batch no. 8MKT40, mfg dt. 09/2007 and Exp. dt. 08/2009) was procured from the local market (Label claim: 500mg NTZ) marketed by Ind-Swift Laboratories Limited, Samba, (Jammu and Kashmir), India.

All chemicals used were of HPLC grade and were purchased from Loba chemicals, Mumbai, India.

System consist of Camag linomate V semiautomatic sample applicator and HPTLC plates silica gel 60 f 254. the plates were developed in Camag twin trough chamber and scan under Camag TLC scanner 3 and the software used was Win CATS software (version 1.6.41).Among the several mobile phases used for the simultaneous estimation of NTZ and OFL, chloroform: methanol (8:2 v/v) with 15 min time of saturation with filter paper with plate equilibrium was found to be most suitable.

Stock solution of NTZ 1mg/ml concentration was prepared in methanol. 100mg of NTZ was dissolved in 100 ml of methanol and concentration of 1 mg/ml was obtained. It was further diluted with methanol to obtained required concentration of 5, 10, 15, 20, 25 µg/µl. All solutions were stored at room temperature; these solutions were shown to be stable during the period of study.

Volumes of 6 µl of each standard were applied as 8mm bands on the TLC plate. All measurements were repeated five times for each concentration and respective calibration curves were constructed by plotting the peak area versus the corresponding drug concentration. The slope and correlation coefficients were determined (Table1).

To determine the content of NTZ in tablet dosage form (Label claim: 500mg of NTZ; twenty tablets were weighed; their average weight was determined and finely powdered. Then triturate tablet dosage form equivalent to 500mg of NTZ was taken and was dissolved in 40ml methanol and sonicated for 20 min and diluted to mark with methanol. The extracts were filtered through Whatman filter paper no. 42 and required dilutions were made to get the final concentration of 5µg/µl for NTZ. Each of these solutions was applied as bands ranging from 2-10 µl on TLC plate with Linomate V. The plates were developed in twin trough chamber which was already saturated with mobile phase for 15 min. The results are reported in Table 2 and the amount of both the drugs was determined.

TABLE 1: RESULTS OF VALIDATION STUDIES

SST and other parameters

NTZ

* Rf value

λmax

Linearity range

LOD

LOQ

% recovery

Slope (m) in tablet form

Intercept (b) in tablet form

Co-relation coefficient

Specificity/Selectivity

Stability of sample solution

0.63± 0.03

330 nm

5-50 µg/spot

60 µg/spot

180 ng/spot

99-101

895.33

1044.9

0.9949

No interference

≥ 24 hrs

SST: System suitability test, * calculated at 5% peak height, LOD: Limit of detection, LOQ: Limit of quantitation

TABLE 2: RESULTS OF ANALYSIS OF TABLET FORMULATION

Drug

Conc. taken

(µg/ml)

Conc. found

(µg/ml)

% RSD

Found (Label claim)

NTZ

(Label claim: 500 mg)

5.002

10.007

15.005

20.001

25.003

0.027

0.019

0.011

0.013

0.144

0.55

0.19

0.07

0.06

0.56

101.00

100.04

100.01

100.02

100.08

500.06

500.02

500.26

500.01

500.04

Conc.: concentration, SD: standard deviation, RSD: relative standard deviation, * Results are mean of five replications.

To check the accuracy of the developed methods and to study the interference of formulation additives, analytical recovery experiments were carried out by standard addition method. From the total amount of drug found, the percent recovery was calculated. The results of the analysis are reported in Table 3 and 4.

TABLE 3: RESULTS OF RECOVERY STUDY OF NTZ

Drug

Initial conc.

(µg/ml)

Conc. added (µg/ml)

% Recovery

NTZ

99.21

99.81

100.19

99.23

99.87

99.60

99.72

100.63

100.00

99.29

99.81

99.57

99.23

99.54

99.69

Conc: Concentration, Results of five replicates, % Recovery is more than 99%; hence method is accurate and precise.

Optimization of mobile phase was performed base on peak parameters such as height, area, tailing factor, Rf value, run-time and resolution. The mobile phase chloroform: methanol (8:2 v/v) with 15 min time of saturation with filter paper with plate equilibrium was found to be satisfactory and gave a symmetrical and well resolved peak for NTZ. The Rf value for NTZ was 0.63± 0.03 (Fig. 1). The wavelength 330 nm was selected for development and validation based on maximum absorption of the drug and best scanning response at this wavelength.

Fig.1: Typical Chromatogram of NTZ

Chromatogram showing Rf value, Rf= 0.85 for NTZ in tablet dosage form

The calibration curve for NTZ was obtained by plotting the peak area of NTZ versus their concentration over the range of 5-25 µg/µl for NTZ and was found to be linear with r = 0.9949. The detection limit for NTZ was 60 ng/spot. The quantitation limit for NTZ was 180 ng/spot which suggested that a nanogram quantity of the drug can be estimated accurately. The validation and system suitability parameters are shown in Table1. The recovery of NTZ was found to be in the range 99.21-100.19 % (Table3). Repeatability, accuracy and precision data are reported (Table 4).

TABLE 4: REPEATABILITY, ACCURACY, PRECISION STUDIES OF NTZ

Std. Conc.

NTZ

Mean peak area*

%RSD

12134

22384

32600.74

42864.53

73108.74

265.25

394.13

493.57

683.39

934.70

1.12

0.29

0.20

0.06

0.34

Std. Conc: Standard concentration, SD: standard deviation, RSD: relative standard deviation, * Mean of three peak areas

RESULTS AND DISCUSSION:

The thin layer chromatographic method was applied for the development and validation of NTZ in tablet dosage form. The result of analysis of tablet formulation containing 5-25 µg of NTZ revealed SD ranging from 0.011 to 0.144 and percent RSD was ranging from 0.06 to 0.56, all values being mean of five replicates (Table 2).

CONCLUSION:

Proposed method describes a new HPTLC method for the determination of NTZ in tablet dosage form. The method gives good resolution for the drug. The method was validated and found to be simple, accurate, sensitive and precise. Percentage of recovery shows that the method is free from interference of the excipient used in the formulation. Therefore the proposed method can be used for routine analysis of NTZ in tablet dosage form.

ACKNOWLEDGEMENT:

The authors are thankful to Ind-Swift Laboratories, India and Medley Pharmaceuticals Limited, India for providing drug sample of NTZ and also thankful to Anchrom Labs, Mumbai for providing necessary facilities for completion of HPTLC work.

REFERENCES:

1. Sweetman, SC, editors. Martindale: The Complete Drug Reference. 33^rd ed. London: The Pharmaceutical Press; 2002. p. 598.

2. Yoshimasa Y, Amal H, Heman F, et al. Nitazoxanide: A nitrothiazolide antidepressant drug is an antihelicobacter pylori agent with anti- vacuolating toxin activity. Antimicrobial Agents Chemotherapy 1996; 40:2266-70.

3. Cavier R. Nitazoxanide in treatment of anthelminthiasis. European Journal of Medicinal Chemistry. 1978; 13:539-49.

4. Kapse GK, Prabhakar G and Appala RS. Spectrophotometric methods for the Estimation of Nitazoxanide in Pharmaceutical formulation. Indian Journal of Pharmaceutical sciences. 2006. 403-406.

5. Sweetman SC, editors. Martindale: The Complete Drug Reference. 33^rd ed.London: The Pharmaceutical Press; 2002. p. 232.

6. Prabhakar G, Kapse GK, Appala RS. Spectrophotometric Determination of Nitazoxanide in pharmaceutical dosage form. The Indian Pharmacist, 2006; 83-84.

7. Scott, P.W., (1994), Liquid Chromatography for the Analyst, 67, Marcel Dekker, New York, 1-4.

8. United States Pharmacopoeia, Vol. 24, Supplement 1, Rockville, MD: The United States Pharmacopoeial Convention, Inc: 2000.p. 1215.

9. British Pharmacopoeia, Vol. 1, London: The British Pharmacoporia Commission: 2002. p 1247.

Received on 30.01.2010 Modified on 10.02.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 608-610