Extractional-Spectrophotometric Determination of Fenbendazole and Ornidazole in Pharmaceutical Formulations

Methods for spectrophotometric determination of fenbendazole and ornidazde are described. The methods are based on the formation and extraction of the ion-pair complex formed between bromothymol blue and either fenbendazole (FBZ) or ornidazole (ORN). The extracted coloured complexes absorb at 416 and 446 nm, respectively. The effect of different factors, e.g. pH, organic solvent, reagent concentration, extraction time, shaking time and common interfering species have been investigated. Fenbendazole and ornidazole can be determined over the range 1.2 to 24.0 ppm and 5.5 to 77.0 ppm, respectively. The precision of the methods was tested for the determination of pure samples of FBZ and ORN and the mean RSD was found to be 3.0 and 2.3% for FBZ and ORN, respectively.The proposed methods were successfully applied for the determination of FBZ and ORN in commercially dosage fornis. A comparison between the suggested methods and the other reported methods was also studied.

The proposed methods were successfully applied for the determination of FBZ and ORN in coriil~iercially dosage fornis.A comparison between the suggested methods and the other reported metl~ods was also studied .ion-pair complexes formed between bromothymol blue and drugs, a new method for their determination of these two drugs.

Experimental
Apparatus : A Pye-Unicam UV-visible spectrophotometer, model UV-2 was used for spectrophotometric measurements.The cells used for measurements were 1x1 cm quartz cells.A Hanna 8521 model pH meter was used for pH measurements.

Reagents and materials
Ornidazole and febendazole were supplied from Sigma and used as working standards.Bromothymol blue (BTB), and chloroform were manufactured by Aldrich.
All chemicals were of analytical grade and manufactured by Merck and BDH.Water was doublydistilled and stored in a glass container.

Solutions
A freshly prepared 1 .ox10" M aqueous solution of pure ornidazole was used as stock solution.A 1.0x10-~ M fenbendazole was prepared by dissolving the appropriate amount of drug in chloroform.Bromothymol blue (BTB) solutions (10x10-~ M) were prepared by dissolving the appropriate amounts in about 2 ml of 0.1 M sodium hydroxide, then 20 ml of ethanol (96%) were added and the volume was completed to exactly 100 ml using double distilled water.Britton Robinson buffers were prepared according to the method listed in the literature .

Reconzntended procedure for deterntination of fenbendnzole
Into a 100-ml separatory funnel, exactly 2.0 ml of 1.0~10" M BTB were transferred, followed by 5.0 ml of buffer of the recommended pH and mixed.Then, an appropriate volume of drug containing an amount of FBZ in the range 1.2-24.0pg/ml was added and the volume completed to 12 ml with water.The aqueous solution was extracted with 20ml portions of chloroform, and shaked well to ensure complete extraction (for lmin).The extract was diluted to 25.0 ml with chloroform and the absorbance was determined in a tightly stoppered cell at 416 nm at 25OC against a reagent blank prepared similarly without the addition of FBZ.

Recomnzended procedure for deternzinntion of ORN
Exactly 7.0 ml of 1.0~10" M BTB solution were transferred into a 50-ml separatory hnnel.A volume (5.0 ml) of the desired buffer solution was added, followed by a portion of the drug solution containing 5.5-77.0p.g/ml and then 7.0 ml of chloroform.The mixture was shaken for 1 min, and then allowed to stand for 2 min.
The organic layer was collected into a 10-ml volumetric flask and the volume was completed to the mark with chloroform.The absorbance was measured at 446 nm against a reagent blank .

Deterntination of FBZ in veterinary fornzulntions
Ten tablets of Panacur drug (250mg) were weighed out and finely powdered.A weight equivalent to 250 mg FBZ (one tablet) was dissolved in 500 ml chloroform at 40°C with stirring for 15 minute.The supernatent solution was filtered through 1 .O-L volumetric flask and the volume completed with chloroform.A 1.0 ml of the final solution was used for the determination of FBZ, using the recommended procedure .

Determination of ORN in pharmaceutical formulations
Ten tablets of each amboral (500mg) and tiberal(500 mg) were weighed out and finely powdered.A weight equivalent to 500 mg ORN (one tablet) of the drug was transferred into a 500 rnl beaker and stirred with 300 ml doubly distilled water for 20 min at 40 "C .The supernatent solution was filtered through a 1.0 L volumetric flask and the volume completed with doubly distilled water.1.0 rnl of this solution was used for the determination of ORN using the recommended procedure .

Results and Discussion
Fenbendazole and ornidazole react with the n-acceptor bromothymol blue (BTB) to form ion-pair or charge transfer complexes.The yellow ion-pair complexes formed were completely extracted with chloroform and exhibited absorption maxima at 416 and 446 nm for FBZ -BTB and ORN -BTB complexes, respectively as shown in Fig. 1.Neither fenbendazole or ornidazole nor BTB alone exhibits any significant absorbance at 4 16 and 446 nm, under the same conditions.On the other hand, the absorbance of the repeated chloroform extract of the remaining of the reaction mixture was insignificant.
Investigations of several experimental conditions were carried out to establish the most favourable conditions to achieve the maximum colour development for the quantitative determination of fenbendazole and ornidazole.These experimental conditions include effects of pH, solvent, shaking time, temperature, buffer constituents and dye concentration.

Effect of pH
The effect of pH of the aqueous phase on the absorption maxima of the ionpairs formed was studied in the pH range 2-9.For ornidazole determination, at pH> 4, the two layers take a long time (-15 min) for complete separation, where it takes less than 1 min at pH 2. As shown in Fig. 2, maximum color development had been constant within the pH range 2-5 for the fenbendazole -BTB complex and the maximum absorbance was observed at pH 3 for the ornidazole -BTB complex.
Therefore, the recommended optimum pH for'further studies was found to be pH 2 and 3 for the fenbendazole and ornidazole determination, respectively.
The effect of several types of buffer solution, such as Britton-Robinson, acetate and citrate buffer having the same pH value have been also investigated,and maxmium absorbance was achieved in the presence of Britton-Robinson and acetate buffer.. On the other hand, long time was needed for complete separation of aqueous and organic layers in the presence of acetate buffer.Therefore, theBritton-Robinson buffer was chosen to be the most suitable one for the tietermination of the two ion-pairs.

Analytical applications
Under the recommended conditions described, standard calibration curves for FBZ and ORN with BTB were constructed by plotting absorbance versus concentration.Beer's law was valid over the concentration ranges 1.2-24.0and 5.5-77.0ppm for FBZ and ORN, respectively.
The precision of the methods of determination of both, FBZ and ORN, in pure raw materials was also checked.The determination of various concentrations of each drug was carried out using the recommended procedure.The mean relative standard deviations were found to be 3.0 and 2.3% for FBZ and ORN, respectively .
Table 1 summarizes the results obtained for the optimum conditions and spectral characteristics of the suggested methods for the determinations of FBZ and O W in pure raw materials.
On the other hand, the validity of the proposed methods was assessed by applying the standard addition technique and the mean percent recovery of added standard ranges from 99.8-100.5% for FBN, and from 100.7-10 1.4% for ORN.

Analysis of pharmaceutical formulations
The applicability of the suggested methods for the determination of ORN and FBZ in commercial dosage forms was examined.Amboral tablets, each containing 500 mg of ORN (Eastren Chemical Company, Palestine), Tiberal tablets, each containing 500 mg of ORN (Hoffman La Roche, Switzerland) and panacur tablets, containing 250 mg of FBZ per tablet (Hoechst AG, Germany) were analysed.The Results ofthe analyses of the above dosage forms using the suggested method compared to reference method are measured in Table 3.
Fenbendazole (I) [nietliyl-5-(phenyl-thio) benzimidazole -2-carbamate] and ornidazole (11) [ -chloromethyl-2-methyl-5nitroimidazole -1ethanol] are derivatives of benzimidazoIe and nitroimidazole, respectively.These drugs have an anti~nicrobial action : they are used in tlie treatment of susceptible protozoal infections and in the treatment of bacterial infection.Various methods have been reported for the determination of the investigated drugs in different biological samples.including high perforniance liquid chromatography [I-41, liquid chromatograpliy [5-71, voltanimetric methods (for ornidazole only) [8-101 and spectrophotometric methods [1 1-1 31.To the best of our knowledge, there is only one published study concerning the spectrophotometric deter~i~ination of fenbendazole in pharmaceutical formulations [I I].On the other hand, a little attetitioli has been paid for the determination of ornidazole [ I 2-131 using spectroscopic methods.The aim of the present work was to develop n rletv sensitive, selective and accurate spectroplioto~iietric tnethod for the determination of fenbendazole and ornidazole in pharmaceutical forn~iilations .The method is based on the extraction of tlie coloured Extractional-Spectrophotometric Determination of Fenbendazole and Ornidazole . . .111 pH

Comparison of the proposed method with other metho&
Very few spectrophotometric and chromatographic methods have been reportedfor the determination of FBZ and ORN in pharmaceutical formulations.Table2shows a comparison between the proposed methods and other reported methods for FBZ and ORN determinations.It was found that the suggested method competes well with other methods in sensitivity and precision and it is suitable for routine analysis of FBZ and ORN in the dosage forms.