Indirect Spectrophotometric Determination of Niclosamide in Pharmaceutical Preparations

ةيفيط ةقيرط ريوطت ثحبلا نمضتي  يف ديماسولكينلا ريدقتل ةساسحو ةطيسب ةرشابم ريغ ماسولكينلل ةلزتخملا ةغيصلا ةدسكأ ىلع ةقيرطلا دمتعت . ةينلاديصلا تا رضحتسملا نويا ةطاسوب دي فشاكلا عم جتانلا يئانثلا ديدحلا ةلعافمو يضماحلا طسولا يف يثلاثلا ديدحلا 10،1  نيلورثنانيف يفلا دقعم جتنيل  وكي يذلاو نيور ةفاضإب لضفأ ةيرا رقتسا ىلع لوصحلا مت( ا رقتسمو ءاملا يف ابئاذ ن لولحم EDTA يجوملا لوطلا دنع صاصتما ىلعأ يطعيو ) 510  رتيمونان  ،يروصلا لولحملا لباقم ملا ةيصاصتملاا ةميق تناكو ةيرلاو 9 2.1  × 10 4  لوم.رتل 1 .  مس 1  ريب نوناق قبطنيو .  ىدم يف نم زيكرتلا 2.5  ىلإ 225  / ما رغوركيام 10   نيب يبسنلا أطخلا حوا رت دقو .لم –  1.80  +و 1.50 % نيب يبسنلا يسايقلا فا رحنلااو ±  0.8  و ±  2.07  لا زيكرت ىلع ادامتعا % قيبطت مت دقو .ديماسولكين طلا . ةيئاودلا تا رضحتسملا يف ديماسولكينلا ريدقتل حاجنب ةقير


Introduction
Niclosamide (NIC) is an anthelmintic, BCS class II drug of low solubility and high permeability (1) . It is the first choice in the treatment of beef tapeworm (Taenia saginata), fish tapeworm (Diphyllobothrium latum) and pork tapeworm (Taenia solium). It inhibits phosphorylation in the mitochondria of Cestodes. Both in vitro and in vivo, the scolex and proximal segments are killed on contact with the drug (2) . NIC is used also as a molluscicide for the treatment of water in Schistosomiasis control programmes (3) NIC and its two novel synthesized derivatives constructed to float on the water surface were able to kill cercariae (4,5) , also possessed promising activity in vitro against an apicomplexan parasite Toxoplasma gondii (6) . NIC chemically is 5 -chloro -N -( 2-chloro -4 -nitrophenyle ) -2 -hydroxybenzamide. NIC is a yellowishwhite or yellowish , fine crystals , practically insoluble in water , slightly soluble in ethanol (7) . Niclosamide has different proprietary names such as : Yomesan; Niclocide ; Cestocide (8) , and has the following structure:

Chemical structure of niclosamide
Several methods have been reported for the determination of NIC, these methods include derivative spectrophotometric method (9) . Other spectrophotometric methods depend on reduction of NIC with zinc powder in presence of hydrochloric acid followed by reaction with different reagents such as metol and potassium dichromate to give a colored product (10) and p-benzoquinone to form a pink product which absorbs at 506 nm (11) . NIC was also determined in tablets by dissolution of the tablets in 0.1 N sodium hydroxide solution followed by measurement of the absorbance at 375 nm (12) . Also other techniques have been utilized for the determination of NIC such as liquid chromatography (13) , gas -liquid chromatography (14) , cyclic voltametry (15) and fluorometry (16) . The standard method for determination of NIC includes potentiometric titration (7) .
The present work aims mainly to develop a simple and sensitive spectrophotometric method for the determination of NIC in pharmaceutical preparations basded on the oxidation of the reduced form of NIC with iron ( III ) and the liberated iron ( II ) reacts with 1,10-phenanthroline in aqueous solution to form a colored complex that has been proved successfully for the determination of niclosamide in both pure form and in its pharmaceutical preparation .

Apparatus
All spectrophotometricmeasurements were carried out on a double beam spectrophotometer type CECIL CE 7200 and Shimadzu UV-160A UV-visible recording spectrophotometer with 1.0 cm matched quartz cells , pH measurements were performed by pH meter type TRANS BP 3001.
Thermostatic controlled water-bath type memmert was used for heating process .

Reagents
All chemicals used are of analytical reagents grade.

Standard solution of niclosamide (10000 µg/ml).
This solution was prepared by dissolving 0.5 g of pure NIC (Sigma-Italy) in 50 ml of 1 : 1 ethanol-acetone mixture in a volumetric flask.
Standard reduced form solution of niclosamide(500 µg / ml) A 5.0 ml of NIC (10000 µg / ml) solution was transferred to dry beaker, add 0.1 g of zinc powder, 5ml of 1M HCl and heat the mixture in boiling water -bath for 15 min., cool and filter the solution into a 100 ml volumetric flask, and then the volume was completed to mark with distilled . (7) water

Reduced working niclosamide (RNIC) solution (50 µg / ml )
A 10 ml of 500 µg / ml is diluted with distilled water to the mark in a 100 ml volumetric flask .

Ferric sulphate (3.74×10
This solution was prepared by dissolving 0.1499 of ferric sulphate (BDH) in distilled water in the presence of 3ml of 0.25M sulphuric acid and the volume was completed to 100 ml in a volumetric flask with distilled water.

1,10-phenanthroline monohydrate solution ( 1.68×10 -2 M )
This solution was prepared by dissolving 0.3348g of the 1,10phenanthroline monohydrate (Fluka) in 5 ml of ethanol and the solution was made up to 100 ml in a volumetric flask with distilled water.

Ethylenediaminetetraacetic acid (EDTA) Solution( 1% )
This solution was prepared by dissolving 1 g of disodium salt of the EDTA (Fluka) in 100 ml distilled water.

Tablets (Yomesan) solution ( 10000µg / ml ) Indirect Spectrophotometric Determination of Niclosamide in …
Ten tablets of yomesan (each tablet contain 500 mg of niclosamide) are finely powdered , an accurately weighed of the powder equivalent to 0.5 g of NIC is dissolved in 20 ml of ethanolacetone 1:1 mixture and the residue is filtered into 100 ml calibrated flask and then the volume was completed to the mark by repeated washing with the same solvent . This solution was treated in the same way as the standard niclosamide to prepare the reduced and the working solutions of niclosamide drug .

Procedure and calibration graph
Accurately measured volumes containing 2.5-225 µg of of RNIC were transferred into a series of 10 ml calibrated flask . To each of these flasks 0.6 ml of 3.74×10 -3 M ferric sulphate and 2.5 ml of 1.68 × 10 -2 M 1, 10phenanthroline monohydrate solutions were added , then the flasks left to stand for 30 minutes at room temperature,followed by addition of 1ml of 1% EDTA solution and diluted to the mark with distilled water , the absorbance of the red colored product was then measured at 510 nm a gainst the reagent blank solution.A linear calibration graph is obtained over the concentration range of 2.5 to 225 µg niclosamide/10ml (i.e 0.25 to 22.5µg / ml) (Fig 2). The sensitivity of the method , expressed as the molar absorptivity has been found to be 2.19×10 4 l.mol -1 .cm -1 . Then the corresponding amine of NIC undergoes oxidation -reduction reaction with Fe(III) to produce Fe(II) which subsequently reacts with 1,10-phenanthroline to produce a red colored complex of ferroin having an absorption maximum at 510 nm as shown in the following reaction : Fe +2 + 1,10-phenanphthroline Red complex

Optimum reaction conditions
The effect of various parameters on the intensity of the colored product has been studied and optimum conditions have been selected .

Effect of ferric sulphate solution
The effect of different amounts of ( oxidizing agent ) ferric sulphate solution on the absorbance has been investigated ( Table 1 ).  The results illustrated in Table 1 indicate that 0.6 ml of 3.74×10 -3 M ferric sulphate solution gives the highest absorbance over a range of determined concentration of 25 to 125µg RNIC / 10 ml , therefore it is recommended for the subsequent experiments.

Effect of 1,10-phenanthroline monohydrate reagent amount
The effect of different amounts of 1,10-phenanthroline monohydrate reagent on the absorbance of solutions containing different amounts of RNIC is studied. The results indicated that the absorbance increases with increasing reagent concentration and reached maximum on using a volume of 2.5 ml of 1.68×10 -2 M 1,10-phenanthroline monohydrate which also gives the highest value of determination coefficient ( Table 2).

Effect of pH
The effect of pH on color intensity is examined since the extent of complex formation and hence, the absorbance of the final solution is often a function of hydrogen ion concentration. Different volumes of different acid and base solutions have been used and the absorbances and pH values were measured as in Tables (3,4).

• Absorbance without addition of acid or base = 0.328 • pH without addition of acid or base = 4.42
The results shown in Tables (3) and (4)

Effect of buffer solution
The effect of buffer solution on the absorbance of produced complex was studied by addition 3 ml of three buffer solutions of pH 4.4 with different compositions (17) as in Table 5. The results in Table 5 indicate that using of these buffer solutions decrease the absorbance, therefore, it has been recommended to eliminate the use of these buffer solutions The reaction time was determined by following the color development at different temperatures in thermostatically controlled water-bath. The absorbance was measured at 5 min. intervals against reagent blank treated similarly ( Table 6).

Effect of temperature and reaction time on absorbance
The results in Table 6 indicate that the absorbance of the resulting complex increased with increasing of temperature and time reaction, probably due to side reactions at high temperatures. Also, the blank value increase tremendously, so room temperature and 30 min. as a reaction time were selected for subsequent experiments.

Order of addition
To obtain optimum results the order of addition of reagents should be followed as given under the general procedure, otherwise less absorbance intensity was observed .

Effect of surfactants
The effect of different types of surfactants on absorbance of complex has been studied . The results shown in Table 7 confirm that there is no improvement in the absorption, therefore they were excluded .

Effect of time on the complex stability
The effect of time on the development and stability of the colored complex for different amounts of RNIC is investigated under the optimum experimental conditions. The absorbance of the complex increased continuously with the time, and it is found that the stability is enhanced in the presence of masking agents such as NaF and EDTA.

Effect of masking agents on stability
The effect of masking agents on the stability and the sensitivity of the colored complex is investigated by carrying out a series of experiments using 0.5,1.0,1.5,2.0 ml of 1% of NaF or EDTA-Na 2 solutions added individually before dilution to the mark.The obtained results in Table 8 indicate that using of 1ml of 1% EDTA-Na 2 gives the highest absorbance and improve the stability of the complex up to 24 hours, so it has been recommended for the subsequent experiments.

Final absorption spectra
When reduced form of niclosamide is treated according to the recommended procedure, the absorption spectra shows a maximum absorption at 510 nm. characteristic of the ferroin chromophore, in contrast to the reagent blank which shows a low absorbance (0.053) at this wavelength, emphasizing the need for measurements to be performed against the reagent blank (Fig.3).

Stoichiometry of the reaction
The stoichiometry of the reactants was investigated by the Job ُ ُs method (18) .The experimental results indicate that the iron (III) reacts with RNIC in the ratio of 2:1 as the following suggested reaction :

RNIC
The produced ferrous ion is chelated by 1,10-phenanthroline to form the well-known red chelate (19) :

Effect of Interferences
The influence of excipients compounds ( glocose, lactose, starch and arabic gum ) which often accompanied pharmaceutical preparations were studied by adding four different amounts (100,250,500 and 1000 µg) to 50 µg of RNIC in a final volume 10 ml . The results in Table (9) indicate that the studied excipients compounds do not interfere in the determination of niclosamide using proposed method.

Application of the method
The proposed method is applied to determine niclosamide in pharmaceutical preparations on appling proposed procedure, good recovery is obtained as shown in Table (   The performance of the proposed method for the determination of niclosamide in tablets was assessed by calculation of the t-test compared with the standard method (7) at 95% confidence level with eight degrees of freedom. The results in (Table 11) showed that t-value was less than the theoretical value, indicating no significant difference between the proposed method and standard method for the determination of niclosamide in tablets .

Accuracy and precision
To check the accuracy and precision of the method, RNIC is determined at three different concentrations . The results illustrated in Table (12) indicated that the method is satisfactory .

Conclusion
The proposed method for the determination of NIC in pharmaceutical formulation is simple, sensitive, accurate and precise. The method is based on oxidation -reduction between RNIC and Fe III, then the subsequent reaction of librated Fe II with 1,10-phenanthroline to produce a ferroin complex which is water -soluble , stable , and has a maximum absorption at 510 nm against the reagent blank. The proposed method has been applied successfully to the determination of the intended compound in its pharmaceutical formulation (tablets).

Color of complex Red Yellow
Stability of the color (hr.) 24 3 Tablets  Tablets