Simple and rapid spectrophotometric assay of albendazole in pharmaceuticals using iodine and picric acid as CT complexing agents

Two simple, rapid and inexpensive spectrophotometric methods are described for the determination of albendazole (ALB) in bulk drug and in tablets. The methods are based on charge-transfer (CT) complexation reaction involving ALB as n-donor and iodine as σ-acceptor (method A) in dichloromethane or picric acid (PA) as π-acceptor (method B) in chloroform. The absorbance of CT complexes was measured at 380 nm for method A, and 415 nm for method B. The optimization of the experimental conditions is described. Under optimum conditions, Beer’s law obeyed over the concentration ranges 8.0-240 and 2.4-42 mg mL-1 for method A and method B, respectively. The apparent molar absorptivity of CT complexes at the respective lmax are calculated to be 1.17×103 and 5.22×103 L mol-1 cm-1 respectively, and the corresponding Sandell sensitivity values are 0.2273 and 0.0509 ng cm-2. The limits of detection (LOD) and quantification (LOQ) are calculated to be (0.69 and 2.08), and (0.10 and 0.30) mg mL-1 with method A, and method B, respectively. The intra–day and inter-day accuracy expressed as % RE and precision expressed as % RSD were less than 3%. The methods were applied to the determination of ALB in tablets.

Several direct and indirect visible spectrophotometric methods based on a variety of reaction chemistries are also found in the literature.A method in which ALB was treated with a measured excess of chloramine-T (CAT) in acid medium followed by the determination of unreacted oxidant by reacting with methyl orange or indigo carmine has been described by Basavaiah and Prameela (2003a).Similar methods involving CAT-methyl-sulphanilic acid (Basavaiah, Prameela, 2003b), in situ bromine-methyl orange (Basavaiah, Prameela, 2004;Basavaiah, Prameela, 2005), N-bromosuccinimide (NBS)-methyl orange or indigo carmine (Basavaiah, Ramakrishna, Somashekar, 2006), N-chlorosuccinimide (NCS)-iron(II)-thiocyanate (Basavaiah, Nagegowda, 2004), periodate-KBr-methyl orange or indigo carmine (Basavaiah et al., 2006) and perchloric acid-crystal violet (Basavaiah, Nagegowda, Ramakrishna, 2004) have been reported for the indirect assay of ALB in its dosage forms.In two related procedures (Basavaiah et al., 2009), ALB was treated with a measured excess of NBS in HCl medium, and the unreacted oxidant was reduced by iron(II) and the resulting iron(III) was complexed with thiocyanate or chelated with tiron (4,5-dihydroxy-1,3-benzene disulfonic acid) offering two sensitive methods for ALB.
In contrast, only a few direct methods are available.Zarapkar and Deshpande (1988) have determined ALB in tablets and syrup based on the reduction of Folin-Ciocalteu reagent to a blue colored chromogen, which was measured at 700 nm.There are three reports on the application of ionpair complexation reaction based on spectrophotometry for the assay of ALB in pharmaceuticals.Sane et al. (1989b) have devised four procedures based on this reaction using bromocresol green, bromophenol blue, bromothymol blue and bromophenol red as ion-pair reagents in acidic buffer medium.The ion-pair complexes formed were extracted into chloroform and measured at 420 nm.The methods were applied to tablets and syrup.Thymol blue and five alizarin derivatives (alizarin (I); alizarin red S (II), alizarin yellow (III); quin alizarin (IV), alizarin blue fluorine (V) (Kamel, Barsoum, Sayed, 2008) are the other ion-pair reagents used for the assay of ALB.Sastry et al. (1997) have described extractive spectrophotometric methods for the estimation of some benzimidazole anthelmintics including ALB in pharmaceuticals based on ion pair reaction employing acidic dyes.
Methods based on charge-transfer (CT) complexation reactions have also been reported for ALB.The CT reaction of ALB with π-acceptor chloranilic acid (CAA) in alcoholic acetone medium was used by Zhao et al. (2001) for the determination of ALB in tablets.Based on similar reaction and employing CAA and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) as π-acceptors, determination of ALB in tablets has been reported by Refat, Mohammed and Fathi (2011).
Most of the reported indirect spectrophotometric methods described above (Basavaiah, Prameela,  2004,2003a,2003b;Basavaiah et al., 2006Basavaiah et al., , 2009;;Basavaiah, Ramakrishna, Somashekar, 2006;Basavaiah, Nagegowda, Ramakrishna, 2004) are time consuming, and cumbersome since involve multi reagents and multi step reactions, and hence prone to inaccuracy and imprecision.The methods based on ion-pair complexation reactions (Sane et al., 1989b;Sastry et al., 1997;Kamel, Barsoum, Sayed, 2008), though sensitive; suffer from such disadvantages as tedious and timeconsuming extraction step, critical dependence on pH, and aqueous to aqueous-organic phase's ratio.There are also prone to inaccuracies owing to incomplete extraction or formation of emulsion between the hydrocarbon solvent and the drug solution.The reported methods based on CT formation reaction (Zhao et al., 2001;Refat, Mohammed, Fathi, 2011) are less sensitive and applicable over narrow linear ranges.

EXPERIMENTAL Apparatus
A Systronics model 166 digital spectrophotometer (Systronics, Ahmedabad, Gujarat, India) equipped with 1cm matched quartz cells was used for all absorbance measurements.

Reagents and chemicals
All reagents used were of analytical reagent grade, and HPLC grade organic solvents were used throughout the investigation.0.3% Iodine (Merck, Mumbai, India) and 0.05% picric acid (S.D. Fine chem., Mumbai, India) solutions were prepared separately in dichloromethane (DCM) for method A, and chloroform for method B.

Standard ALB solution
A stock standard solution containing 1000 mg mL -1 ALB was prepared by dissolving 100 mg of pure drug in dichloromethane and diluting to volume with the same solvent in a 100 mL calibrated flask.This was diluted appropriately with same solvent, to get working concentration of 400 mg mL -1 in method A. In method B, a 100 mg mL -1 ALB solution was prepared by dissolving 10 mg of pure drug in chloroform and diluting to mark with the same solvent in 100 mL calibrated flask, and a working concentration of 60 mg mL -1 was prepared by diluting with chloroform.

Method A (Using iodine, I 2 )
Different aliquots of 0.1, 0.25, 0.5, 1.0, 2.0, 2.5 and 3.0 mL of standard ALB solution (400 mg mL -1 ) were accurately transferred to 5 mL standard flasks using a microburette, and the total volume was adjusted to 3.0 mL by adding adequate quantity of DCM.One mL of 0.3% iodine was added to each flask and the mixture was diluted to the volume with DCM and mixed well.The absorbance of each solution was measured at 380 nm against a reagent blank after 5 min.

Method B (Using picric acid, PA)
Aliquots of 0.2, 0.5, 1.0, 2.0, 3.0 and 3.5 mL of standard ALB solution (60 mg mL -1 ) were accurately added to 5 mL calibrated flasks using a microburette, and the total volume was adjusted to 3.5 mL with chloroform.One mL of 0.05% picric acid was added to each flask and the mixture was diluted to the volume with chloroform and mixed well.After 5 min the absorbance of each solution was measured at 415 nm against a reagent blank prepared simultaneously.

Procedure for tablets
The content of ten tablets each containing 400 mg ALB was weighed accurately and ground into a fine powder.An accurately weighed quantity equivalent to 100 mg of ALB was transferred into a 100 mL calibrated flask and dissolved in 60 mL of DCM.The content was shaken thoroughly for 15-20 min, diluted to the mark with DCM, mixed well, and filtered using a Whatman No. 42 filter paper.The first 10 mL portion of the filtrate was discarded, and a suitable aliquot of the filtrate (1000 mg mL -1 ) was diluted to get working concentration of 400 mg mL -1 with DCM in method A, and analyzed by taking an appropriate volume.
In method B, an accurately weighed quantity of tablet powder equivalent to 10 mg of ALB was transferred into a 100 mL calibrated flask and dissolved in 60 mL of chloroform, as described above using chloroform and steps described above were applied to get a working concentration of 60 mg mL -1 before it was analyzed by taking a suitable aliquot.

Procedure for selectivity study
Selectivity was evaluated by using placebo blank and synthetic mixture analysis.A placebo blank, consisting of starch (20 mg), acacia (25 mg), hydroxyl cellulose (20 mg), sodium citrate (30 mg), talc (20 mg), magnesium stearate (25 mg) and sodium alginate (20 mg) was prepared by thorough mixing and its solution was prepared as described under "Procedure for tablets", and then subjected to analysis.
A synthetic mixture was prepared by adding 10 mg of pure ALB to 8 mg of the above mentioned placebo blank, and the mixture was homogenized.Following the same procedure for tablets, the synthetic mixture solution was prepared, and a suitable aliquot was subjected to analysis by both methods (n=3), after appropriate dilution.

Reaction mechanism
The chemistry involved in the proposed method is the charge-transfer complex formed by the electronic transition to an excited state where the transfer of electronic charge from donor to acceptor moiety takes place partially.As a result, the transition in this excitation energy occurs very frequently in the visible region of the electromagnetic spectrum (Abou, 2000).This produces the usually intense color characteristic of these complexes.Therefore, ALB, a nitrogenous base acting as n-donor was made to react with iodine (σ-acceptor) and picric acid (π-acceptor) to produce colored charge-transfer complexes in dichloromethane and chloroform, respectively.
In method A, ALB reacts with the reagent and gives a wine red chromogen that exhibits a maximum absorption at 380 nm in dichloromethane medium.This can be attributed to the formation of charge-transfer complex between ALB (D Figure 2) and iodine followed by the formation of radical ion, which was probably due to the dissociation of (ALB-iodine) complex in dichloromethane.The interaction between the donor and acceptor occurs according to the Figure 4 below: Picric acid, a trinitrophenol, reacts with electron donor molecule to forms charge transfer and proton transfer complexes.It was used for the determination of some amine derivatives through formation of intense yellow colored complex (Ebeid et al., 1998;Regulska, Tarasiewicz, Puzanowska, 2002;El-Mammli, 2003;El-Yazbi et al., 2003;Abdel-Hay et al., 2004;Karuna et al., 2006;Rajendra, Basavaiah, Vinay, 2011;Johnson, Ukpe, 2011;Pinderjit, Rajanish, Harinder, 2011;Sameer, Basavaiah, 2011;Prashanth, Basavaiah, 2012;Raghu, Basavaiah, 2012).
In method B, the interaction of ALB with PA in chloroform at room temperature gave an intense yellow colored chromogen with strong absorption at 415 nm due to the formation of the free radical anion.The interaction between ALB (D Figure 3), an n-donor and picric acid (A), a π-acceptor, is a charge transfer complexation reaction followed by the formation of radical ions according to the following scheme (Figure 5).

Absorption spectra
The reaction of iodine or picric acid with ALB results in the formation of intense wine red or yellow colored intensity at the optimum wavelength was studied by adding different amounts of iodine or PA to a fixed concentration of ALB.It was found that 1 mL of 0.3% iodine and 1 mL of 0.05% PA was sufficient for the production of maximum and reproducible color intensity in method A and method B, respectively (Figures 5a, 5b).

Effect of solvent
In order to select the suitable solvent for chargetransfer complex formation, the reaction of ALB with I 2 or PA was carried out in different solvents such as DCM, chloroform, acetonitrile, methanol, 1,2-dichloroethane, 1,4-dioxane and benzene.The DCM was found to be ideal for method A and chloroform for method B (Figures 6a, 6b).Similarly, the effect of the diluting solvent was studied for both methods and the results showed that DCM and chloroform are ideal for methods A and B, respectively.product.The absorption of colored products was recorded at 360-480 nm against the corresponding reagent blank solutions.The resulting colored charge-transfer complexes showed maximum absorbance at 380 nm and 415 nm for ALB-I 2 and ALB-PA, respectively (Figures 4a, 4b).

The effect of different experimental variables
Experimental variables were optimized in order to achieve the intense color, maximum sensitivity and stability and adherence to Beer's law.

Effect of reagent concentration
The effect of the reagent concentration on the color

Effect of reaction time, and stability of color
The optimum reaction time was determined by following the color development upon the addition of reagent solution to the ALB solution at room temperature.Complete color development was attained after 5 min with I 2 while the reaction with PA was instantaneous.The absorbance of these radical anions remained stable for at least 30 and 120 min for method A (I 2 ) and method B (PA), respectively.

Stoichiometric relationship
Job's method of continuous variations of equimolar solutions was employed to establish the stoichiometry of formed CT complexes.Solutions equivalent to 1.51×10 -3 M ALB and I 2 were prepared in DCM in method A, and 2.26×10 -4 M ALB and PA were prepared in method B by dissolving the calculated quantities in chloroform.A series of solutions was prepared in which the total volume of ALB and reagent was kept at 2.5 mL in 5 mL calibrated flasks.
The solutions were mixed well; the volume was completed to the mark with respective organic solvent.The absorbance of the resulting solution was measured after 5 min at the respective wavelengths of maximum absorbance against the respective organic solvent as blanks.The resulting graph (Figures 7a, 7b) showed that the interaction occurs on an equimolar basis (1:1 reaction stoichiometry), owing to the presence of one basic nitrogen containing group.

Analytical data
The linear regression equations were obtained by the method of least squares, and the Beer's law range, molar   Variance (Sa 2 ) 9.96×10 -3 9.96×10 -3 Regression coefficient (r) 0.9991 0.9984 *Limit of determination as the weight in µg mL -1 of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm 2 and l = 1 cm.**Y=a+bX, Where Y is the absorbance, X is concentration in µg mL -1 , a is intercept and b is slope.absorptivity, correlation coefficient, confidence limits for slope and intercept for both methods are given in Table I.The detection limit (LOD) and limit of quantification (LOQ) were calculated by using the following equations (ICH Harmonised Tripartite Guideline, 2005).LOD = 3.3 S/slope and LOQ = 10 S/slope, where, S is the standard deviation of the absorbance of seven blank readings.

Accuracy and precision
In order to determine the accuracy and precision of the proposed methods, pure ALB solution at three different concentration levels (within working range) were prepared and analyzed in seven replicates during the same day (intra-day precision) and on five consecutive days (interday precision) and the results are presented in Table II.The percentage relative error (RE %) was ≤ 2.60, which indicates that the accuracy of the methods is satisfactory.
Percentage relative standard deviation (RSD %) for intraday was ≤ 0.96 and for inter-day it was ≤ 2.34 indicating repeatability and usefulness of the proposed methods in the analysis.

Selectivity
In order to evaluate the selectivity of the proposed methods for the analysis of pharmaceutical formulations, the effect of the presence of common excipients was tested for possible interference in the assay by placebo blank and synthetic mixture analyses and no significant interference was observed from these excipients.The percent recoveries of ALB were 98. 3±2.3 and 101.2±2.8 for method A and B respectively.The placebo blank absorbance was similar to reagent blank absorbance in both methods.This confirms the selectivity of the methods in the presence of the common tablet adjuvants.

Robustness and ruggedness
To determine the robustness of the proposed methods, two important experimental variables: reagent volume and reaction time were slightly varied and the effect of these changes on the absorbance of the CT complex was studied.The results of this effect are presented in Table III and indicated that the proposed methods are robust.Method ruggedness was evaluated by performing the analysis following the general procedures by three different analysts and on three different spectrophotometers by the same analyst.From the % RSD values presented in Table III, it is concluded that the proposed methods are rugged.

Recovery studies
Accuracy of the proposed methods was further ascertained via standard-addition technique.Pre-analyzed tablet powder (ABD 400, Alworm-400 and Bandy-400) was spiked with pure ALB at three different concentration levels (50, 100, and 150% of the quantity present in the tablet) and the total was found by the proposed methods.The results of the recovery study presented in Table IV indicate that the common tablet excipients did not interfere in the assay procedures.

Application for tablets
The proposed methods were applied to the determination of ALB in tablets (Table V).The results obtained were statistically compared with those of the official method (British Pharmacopoeia, 2009) by applying the Student's t-test for accuracy and F-test for precision.The official method describes a potentiometric titration with perchloric acid in formic acid-acetic acid medium.As can be seen from Table V, the calculated t and F-values at 95% confidence level did not exceed the tabulated values of 2.78 and 6.39 respectively, for four degrees of freedom.The results showed that there is no difference between the proposed methods and the official method with respect to accuracy and precision.

CONCLUSIONS
Two moderately sensitive, extraction-free, rapid and cost-effective spectrophotometric methods based on In both methods, the volumes of reagent were 1±0.1 mL.Hence, the methods can be used in routine analysis of the drug in quality control laboratories and pharmaceutical analysis.

FIGURE 2 -FIGURE 3 -
FIGURE 2 -Reaction pathway for the formation of CT complex between ALB and I 2 in dichloromethane.

TABLE I -
Sensitivity and regression parameters

TABLE II -
Evaluation of intra-day and inter-day accuracy and precision a Mean value of 7 determinations; b Relative standard deviation (%); c Relative error (%).

TABLE III -
Method robustness and ruggedness expressed as intermediate precision Simple and rapid spectrophotometric assay of albendazole in pharmaceuticals using iodine and picric acid as CT complexing agents 847 a & b The reaction times were 5±1 min.[ a Method A and b Method B]

TABLE IV -
Results of recovery study via standard addition method with tablet

TABLE V -
Results of analysis of tablets by the proposed methodsMean value of five determinations.Tabulated t-value at the 95% confidence level is 2.78.Tabulated F-value at the 95% confidence level is 6.39.charge-transfer complex reactions were developed and validated for the determination of ALB.The reagents utilized in the proposed methods are cheap, readily available, and quite stable in solution unlike many reagents used in above reported methods.The procedures do not involve any critical reaction conditions such as rigid pH control, tedious and time-consuming extraction or heating step.The methods are more sensitive than many of the reported spectrophotometric methods and applicable over wide linear dynamic ranges.The methods are free from interferences from the common excipients.The statistical parameters and the recovery data reveal good accuracy and precision of the methods.The methods have many other advantages such as reduced cost, simplicity, and speed. *