Stability-Indicating LC Determination of Nitazoxanide in Bulk Drug and in Pharmaceutical Dosage Form 2008, 67, 455–459

Vipul P. Rane1,2, Jaiprakash N. Sangshetti1, Kiran R. Patil1,2, Ravindra D. Yeole2, Devanand B. Shinde1,& 1

2

Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431004, MS, India; E-Mail: [email protected] Wockhardt Research Centre, Aurangabad 431210, MS, India

Received: 23 April 2007 / Revised: 23 November 2007 / Accepted: 4 December 2007 Online publication: 31 January 2008

Introduction

Abstract A novel stability-indicating high-performance liquid chromatographic assay method was developed and validated for quantitative determination of nitazoxanide in bulk drugs and in pharmaceutical dosage form in the presence of degradation products generated from forced decomposition studies. An isocratic, reversed phase LC method was developed to separate the drug from the degradation products, using an Ace5- C18 (250 mm · 4.6 mm, 5 lm) column, and 50 mM ammonium acetate (pH 5.5 by acetic acid) and acetonitrile (55:45 v/v) as a mobile phase. The detection was carried out at a wavelength of 240 nm. The nitazoxanide was subjected to stress conditions of hydrolysis (acid, base), oxidation, photolysis and thermal degradation. Degradation was observed for nitazoxanide in base, acid and in 30% H2O2 conditions. The drug was found to be stable in the other stress conditions attempted. The degradation products were well resolved from the main peak. The percentage recovery of nitazoxanide was from (100.55 to 101.25%) in the pharmaceutical dosage form. The developed method was validated with respect to linearity, accuracy (recovery), precision, system suitability, specificity and robustness. The forced degradation studies prove the stability indicating power of the method.

Keywords Column liquid chromatography Forced degradation Stability indicating and method validation Nitazoxanide

Full Short Communication DOI: 10.1365/s10337-007-0515-0 0009-5893/08/03

Nitazoxanide is a nitrothiozole derivative. Chemically it is 2-acetyloxyl-N-(5-nitro2-thiazolyl)benzamide or N-(5-nitro-2thiazolyl) salicylamide acetate (ester). Nitazoxanide is antiparasitic which is effective against a wide variety of protozoa, helminthes and gram-negative organisms. Nitazoxanide is a novel broad-spectrum anti-parasitic agent originally discovered in the 1980s by J.F. Rossignol. Nitazoxanide is effective in a broad range of parasitic and protozoal infections including Giardia lamblia, Entameoba histolytica, and Taenia solium [1]. Nitazoxanide has good oral bioavailability and is well tolerated, with mild gastrointestinal side effects, used in Giardia intestinalis-induced diarrhea in patients [2]. The antiparasitic activity of nitazoxanide is believed to be due to interference with the pyruvate-ferredoxin oxidoreductase (PFOR) enzyme dependent electron transfer reaction which is essential for anaerobic energy metabolism of the parasites. However, interference with PFOR enzyme depen-

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dent electron reaction may not be the only pathway by which nitazoxanide exhibits its antiprotozoal activity. This drug has been approved by US FDA (in November 2002) [3]. A literature survey reveals a single sensitive high-performance liquid chromatographic method was reported for the determination of nitazoxanide in human plasma [4]. The specrophotometric method for the estimation of nitazoxanide in tablets [5] and stability indicating HPTLC was recently published in the literature [6]. TLC has limitations in the quality control department for dissolution studies, identification of degradation products and metabolites when coupled with mass spectroscopy. LC is a versatile technique. LC with UV detection is often preferred in ordinary laboratories and quality controlled laboratories because of its wide suitability and availability. According to current good manufacturing practices, all drugs must be tested with a stability-indicating assay method before release. So far, to our present knowledge, no stability-indicating LC assay method for the determination of nitazoxanide is available in the literature. It was felt necessary to develop a stability indicating LC method for the determination of nitazoxanide as bulk drug and pharmaceutical dosage form and separate the drugs from the degradation products under the ICH suggested conditions (hydrolysis, oxidations, photolysis and thermal stress) [7, 8]. Therefore, the aim of the present study was to develop and validate a stability-indicating LC assay method for nitazoxanide as bulk drug and in pharmaceutical dosage form as per ICH guidelines [9].

Experimental Material and Reagents Nitazoxanide bulk drug (purity 99.8) and tablet nizonide (500 mg) were obtained from Lupin (Mumbai, India). Ammonium acetate and hydrochloric acid were obtained from Qualigens Fine Chemicals, India. Acetonitrile, hydrogen peroxide, sodium hydroxide were obtained from Rankem laboratories, India. All chemicals and reagent were

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used as LC grades. The UV cabinet was from Kumar (India). Milli-Q-Water was used throughout the experiment.

Chromatographic Conditions

From these stock solutions 5 mL of nitazoxanide standard solution were transferred in a 50 mL volumetric flask and diluted with diluent B. This final solution contained 100-lg mL
1 of nitazoxanide.

An LC system (Shimadzu, Japan) consisting of a quaternary solvent delivery pump, a degasser, an auto-injector, a column oven and a photodiode array detector, 10A-VP series with LC-10 software were used. A chromatographic column of 250 mm length and internal diameter of 4.6 mm filled with octadecyl silane Ace5-C18 (Advance Chromatography Technology, USA) stationary phase with particle size 5 lm were used. The instrumental setting was a flow of 1 mL min
1, the injection volume was 10 lL. The peak purity was checked with the photodiode array detector from 10A-VP.

Ten tablets of nizonide (500 mg) were finely ground using agate mortar and pestle. The ground material, which was equivalent to 100 mg of the active pharmaceutical ingredient, was extracted into diluent A in a 100 mL volumetric flask by vortex mixing followed by ultra sonication and make-up of the volume by diluent B. The solution was filtered through a 0.45-lm filter and an appropriate concentration of sample (100 lg mL
1 assay concentration) was prepared in diluents at the time of analysis.

Mobile Phase

Specificity/Selectivity

The mobile phase consisted of buffer and acetonitrile in the ratio (55:45 v/v). The buffer used in the mobile phase contained 50 mM of ammonium acetate in double-distilled water. The pH of the mobile phase was adjusted to 5.5 with acetic acid. The mobile phase was premixed and filtered through a 0.45 lm nylon filter and degassed.

Specificity is the ability of the method to assess unequivocally the analyte in the presence of components, which may be expected to be present. Typically, these might include degradation products, matrix etc. [10]. The specificity of the developed LC method for nitazoxanide was carried out in the presence of its degradation products. Stress studies were performed for nitazoxanide bulk drug to provide an indication of the stability indicating property and specificity of the proposed method. Intentional degradation was attempted to stress conditions exposing it with acid (0.5 N hydrochloric acid), alkali (0.1 N NaOH), hydrogen peroxide (30%), heat (80 C) and UV light (254 and 366 nm wavelength) to evaluate the ability of the proposed method to separate nitazoxanide from its degradation products. For light study, the study period was 10 days whereas for acid, oxidation 48 h, for heat 24 h and for base 1 h. Peak purity test for nitazoxanide was by using PDA detector in stress samples. Assay studies were carried out for stress samples against nitazoxanide reference standard and the mass balance (% assay + % sum of all impuri-

Preparation of Standard Stock Solutions All solutions were prepared on a weight basis and solution concentrations were also measured on weight basis to avoid the use of an internal standard. Standard solution of nitazoxanide was prepared by dissolving the drugs in the diluents and diluting them to the desired concentration. Diluent A was composed of methanol and acetonitrile in the ratio of (50:50 v/v) and diluent B was composed of water and actonitrile in the ratios of (55:45 v/v). Approximately 50 mg of nitazoxanide was accurately weighed, transferred in a 50 mL volumetric flask, dissolved and diluted to 50 mL with the diluent A.

Sample Solution (Tablets)

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Fig. 1. a Chromatogram of nitazoxanide in acid degradation [acid degraded product (4.9) and nitazoxanide (5.68)]. b Chromatogram of nitazoxanide in base degradation [base degraded products (4.8, 4.5) and nitazoxanide (5.68)]. c Chromatogram of nitazoxanide in oxidative degradation [oxidative degraded product (4.9) and nitazoxanide (5.68)]. d. A typical chromatogram of the tablet: nitazoxanide (5.65)

ties + % sum of all degradants) was calculated. The excipient mixture present in nizonide tablets were injected in the optimized conditions to show the specificity of the method in formulation of nitazoxanide.

Results and Discussion Optimization of Chromatographic Conditions The primary target in developing this stability indicating LC method is to achieve the resolution between nitazoxanide and its degradation products. To achieve the separation of degradation products we used a stationary phase C-18 and combination of mobile phase 50 mM ammonium acetate with acetonitrile. The separation of the degradation product and nitazoxanide was achieved on Ace5 octadecyl silane C-18

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stationary phase and 50 mM ammonium acetate pH 5.5 by acetic acid; acetonitrile (55:45 v/v) as a mobile phase. The column temperature was 30 C. The tailing factor obtained was less than two and retention time was about 5.8 min for the main peak and less than 5 min for the degradation products, which would reduce the total run time and ultimately increase the productivity thus reducing the cost of analysis per sample. Forced degradation study showed the method is highly specific and entire degradation products were well resolved from the main peak. The developed method was found to be specific and method was validated as per international guidelines.

Result of Forced Degradation Experiments Degradation was not observed for nitazoxanide samples during stress condi-

tions like heat, UV and light, except in base, acid and oxidation. Nitazoxanide was degraded into acid (Fig. 1a), base (Fig. 1b) and oxidation (Fig. 1c) and forms polar impurities. The acceptance criterion for the stability of nitazoxanide is 20–80% degradation for a forced degradation study. Singh and Bakshi [11], in their article on stress testing suggested a target degradation of 20–80% for establishing the stability indicating nature of the assay method. Conditions used for forced degradation were attempted to achieve degradation in the range of 20–80%. In the acidic condition 2.5% in the basic condition 79.5% after one-hour and in the oxidative condition 5.8% degradation was observed for nitazoxanide. Peak purity results greater than 990 indicate that the nitazoxanide peak is homogeneous in all stress conditions tested. The mass balance of nitazoxanide in stress samples was close to 100% and moreover, the

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Table 1. Summary of forced degradation results

a

Stress conditions

Time

Assay of active substance %

Mass balance (% assay + % sum of impurities + sum of all degradents)

% Degradation

Peak puritya

Acid hydrolysis (0.5 N HCl) Base hydrolysis (0.1 N NaOH) Oxidation (30% H2O2) Thermal (80 C) UV 254 nm UV 366 nm

48 h 1h 48 h 24 h 10 days 10 days

96.9 21.5 79.10 99.82 99.86 99.88

99.89 98.85 99.82 99.82 99.86 99.88

2.5 79.4 5.8 No degradation No degradation No degradation

999 999 999 999 999 999

Peak purity values in the range of 990–1000 indicate the homogeneous peak

unaffected assay of nitazoxanide in the tablets confirms the stability indicating power of the method. The summary of the forced degradation studies is given in Table 1.

values for nitazoxanide ranged from 100.97 to 101.25%. The average recoveries of three levels of nine determinations for nitazoxanide were 100.5– 101.25%.

Method Validation

Linearity

Precision

The linearity of the response of the drug was verified at seven concentration levels, ranging from 10 to 200% of the targeted level (100 lg mL
1). Concentration standard solutions containing 10–200 lg mL
1 of nitazoxanide in each linearity level were prepared. Linearity solutions were injected in triplicate. The calibration graphs were obtained by plotting peak area versus the concentration data and was treated by least-squares linear regression analysis. The equation of the calibration curve for nitazoxanide obtained y = 8240.1x
386.09, the calibration graphs were found to be linear in the aforementioned concentrations (the RSD, for slopes and intercept are 1.2, 0.91, respectively. The coefficient of determination is 0.999).

Assay of method precision (intra-day precision) was evaluated by carrying out six independent assays of nitazoxanide test samples against reference standard, the percentage of RSD of six assay values obtained was calculated. The intermediate precision (inter-day precision) of the method was also evaluated using two different analysts, different LC systems and different days in the same laboratory; results are shown in Table 2.

Accuracy (Recovery Test) Accuracy of the method was studied by recovery experiments. The recovery experiments were performed by adding known amounts of the drugs in the placebo. The recovery was performed at five levels, 50, 75, 100, 125 and 150% of the label claim of the tablet (500 mg of nitazoxanide). The recovery samples were prepared in the aforementioned procedure, and then 5 mL of nitazoxanide solution were transferred into a 50 mL volumetric flask and diluted to volume with diluent B. Three samples were prepared for each recovery level. The solutions were then analyzed, and the percentage recoveries were calculated from the calibration curve. The recovery

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nitazoxanide in TLC were 15 and 45 ng/ spot, respectively. LOD and LOQ were calculated by using the following equations.

LOD ¼ Cd  Syx=b LOQ ¼ Cq  Syx=b

Limit of Detection and Limit of Quantification (LOD and LOQ) The limit of detection (LOD) and limit of quantification (LOQ) were determined by calibration curve method [12] specific calibration curve was constructed using samples containing the analytes in the range of LOD and LOQ. The LOD and LOQ for nitazoxanide in LC was 0.025 and 0.073 lg mL
1, respectively. The LOD and LOQ for

Where, Cd/Cq is coefficient for LOD/ LOQ; Syx is residual variance due to regression; b is slope. Calculations were performed by considering a value of coefficient of LOD (Cd) is 3.3 and coefficient of LOQ (Cq) is 10, respectively. Precision at limit of quantification and limit of detection was checked by analyzing six test solutions prepared at LOQ and LOD levels and calculating the percentage RSD of area, which was less than 1.9% for LOQ and 5.4% for LOD.

Robustness To determine the robustness of the developed method experimental condition were purposely altered and the resolution between nitazoxanide and acid degradation products were evaluated. The flow rate of the mobile phase was 1.0 mL min
1. To study the effect of flow rate on the resolution, it was changed by 0.2 units from 0.8 to 1.2 mL min
1. The effect of percent organic strength on resolution was studied by varying acetonitrile from –10 to +10%. The effect of column temperature on resolution was studied at 25 and 35 C instead of 30 C while the other mobile phase components were held constant as stated in chromatographic condition. The resolution in the robustness study was not less than 3.5 in all conditions.

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Stability of Analytical Solution

The stability of the standard solutions and the sample solutions was tested at intervals of 24, 48 and 72 h. The stability of solutions was determined by comparing results of the assay of the freshly prepared standard solutions. The RSD for the assay results determined up to 72 h for nitazoxanide was 0.65%. The assay values were within 1.5% after 72 h. The results indicate that the solutions were stable for 72 h at ambient temperature.

Determination of Active Ingredients in Tablets The contents of drug in tablets were determined by the proposed method using the calibration curve. The chromatogram of the tablet sample is shown in (Fig. 1d).

Conclusion The developed method is stability indicating and can be used for assessing the stability of nitazoxanide in bulk drugs and pharmaceutical dosage form. The developed method can be conveniently used for the assay determination of nitazoxanide in bulk drugs and pharmaceutical dosage form. The developed LC method was more specific, selective robust, rugged and precise than the TLC method. The coefficient of determination obtained from TLC and LC was 0.997 and 0.999, respectively. As compared to

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Table 2. Result of precision of test method Sample number

1 2 3 4 5 6 Mean RSD

Assay of nitazoxnide as % of labeled amount Analyst-1 (intra-day precision)

Analyst-2 (inter-day precision)

99.15 99.27 99.52 99.87 99.32 99.44 99.42 0.23

99.33 98.99 99.54 99.13 99.77 99.22 99.33 0.26

TLC, LC is often preferred in ordinary laboratories because of its wide suitability, sensitivity and availability. The developed stability method indicates the advantage of LC over TLC regarding the identification of degradation products. They can be easily identified and quantified by LC coupled with mass spectrometry (LC–MS–MS). The mobile phase used in LC is also mass compatible. LC is more sensitive than TLC and can be conveniently used for dissolution of tablets of the pharmaceutical dosage form containing nitazoxanide in quality control laboratories.

Acknowledgments The authors are grateful to the Lupin Ltd (Mumbai, India) for gift samples (nitazoxanide) and Head-Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathawada University, Aurangabad, India for providing laboratory facility for this research work.

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