A New Visible Spectrophotometric Approach for Mutual Determination of Allopurinol drug in Pharmaceuticals after Cloud Point Extraction

A new spectrophotometric methodology for allopurinol (ALLO) drug determination in pharmaceuticals was a proved. The method is based on formation of allopurinol-CoII complex in alkaline medium. A non-ionic surfactant Triton X-114 was used for the extraction of this complex at 586 nm. High extraction for complex was maintained under optimized conditions. The linearity was performed form (5.0-35.0) μg mL-1 with correlation coefficient (R2=0.9995). Limit of detection (LOD) and limit of quantification (LOQ) were 0.393 and 1.308μg mL-1, respectively. This new method was applied successfully for allopurinol determination in pharmaceuticals and maintained acceptable accuracy and precision.


Drug and Materials
The chemicals used for this work are of high purity and used as received. Distilled water was used in the preparation of all solutions and for final rinsing of glass wares.

Recommended CPE Procedure for ALLO Drug
Aliquots 10 ml of a solution containing known amount of allopurinol drug mixed with Co(II) ions, then pH was adjusted by using 0.1M NaOH , followed by addition of 1mL of 10% (v/v) Triton x-114 and the mixture was diluted to 10 mL with distilled water. The mixture was shaken for 1 min and left to stand in a thermo-stated bath at 50 o C, for 10 min. Separation of the phases was achieved by centrifugation at 4000 rpm for 10 min, then putted in 5°C ice bath for 5 minute. The aqueous phase was removed and the remaining of micellar phase was dissolved by ethanol for spectrophotometric measurement at λmax 586nm for ALLO-Co II complex against blank which was prepared in the same way but without drug.

Statistical Analysis
Excel 2010 (Microsoft Office R) was employed to carry out all statistical calculations

Absorption Spectra
The absorption spectrum of the complex product formed was recorded against the corresponding metal blank from 200 to 1100 nm before obtaining optimum conditions according to the recommended CPE procedure. It was observed that the absorption maximum of the colored product complex of allopurinol in 1.0 mL of 10% TX-114 occurred at 586nm, giving the molar absorpitivity of (1.203×10 -4 L.mol -1 .cm -1 ) for allopurinol-CoII ,thus the wavelength maximum at 586 nm for the complex product was chosen throughout this study.

Optimization of CPE Methodology
A group of experiments has been conducted to study the effect of several variables that affect the extraction efficiency of the CPE and maximize the sensitivity of the detection system for drug under study using a classical optimization. The variables such as the concentration of metal ion , best of pH ,best of buffer ,best of volume buffer , Triton X-114 amount, equilibration temperature and incubation time Figure (3) shows the effect of Cobalt ion concentrations upon the absorbance values of the extracted complexe using ( 500μg mL -1 ) of drug solution .The optimum concentration of the metal ions that gave maximum absorbance was( 50μg mL -1 ) of Co(II) for the drug. The absorbance results are shown in table (1). Plotting of the absorbance values versus the concentration of metal ion is shown in figure (3 Table 1 : Data of Absorbance to Optimum Volume of (0.8μg ml -1 ) metal ion.

Effect of pH
Figure (4) showed the value of absorbance intensity for the ( Allo-CoII ) complex against the value of pH , the best values of pH recorded for the highest absorbance values , then absorbance values were found to decrease with increasing pH which may be attributed to the formation of metal hydroxides. The absorbance results are illustrated in table ( 2). Table 2. Data of Absorbance to value of pH Plotting of the absorbance values versus the value of pH is shown in figure (4). Cloud point extraction yield plays unique role on metal a set of similar experiments in the pH range of 1.0 the described procedure The maximum sensitivity for CPE was obtained at pH 11. In more acidic solutions, deteriorate ion of the signal occurs due to the ligand protonation,

Effect of buffer solutions
The best values of buffer pH 11 were recorded for the highest absorbance values. Results of the measured absorbance for (CoII-allopurinol) complex are shown in table (3). Table 3. Type of buffer pH 11 preparation buffer pH 11 Absorbance Sodium bicarbonate buffer solutions 1.227 Sodium hydrogen ortho phosphate 1.004

Effect Type of Surfactant
The type of surfactant plays very substantial role in cloud point extraction process where each surface owns spectral properties depend on practical basis of micelles. A liquots of 10ml solution contains[1mL allopurinol , 0.8ml Co(II) ion, 0.6 mL buffer pH 11 and Triton X-114] in 10ml volumetric flask and use different surfactant for drug [Tween 20, Tween80, CTAB, SDS, Triton X-100, Triton X-114] at 50 o C for 10 min for metal incubation , then it centrifugated at 4000 rpm for 10min , separated the surfactant-rich phase and dissolved in 1mL ethanol and measured at λmax 586 nm. Results were illustrated in table (5).    From results obtained, the non ionic surfactant Triton X-114 is of high absorbance and this surface increases the efficiency of the extraction process in cloud point extraction

Effect of Triton X-114 Volume
Amount of 10ml solution are prepared[0.5ml allopurinol , 0.8ml CoII ,0.6 ml buffer pH 11 and mlTriton ×114] in 10ml volumetric flask and uses varying volumes of 10%(v/v) Triton X-114 (0.2-2.0)ml for complex then it is completed to the mark by distilled water ,are mixed , heated at 50 0 C for 10 min for ( Allo-CoII ) complex to form cloud point then centrifugation at 4000 rpm for 10min , 1ml ethanol is added to the surfactant-rich phase to dissolve it then it is measured by UV-Vis at λmax 586 nm for ( Allo-CoII ) complex results shown in table (6). Plotting the absorbance values of the cloud point versus the volume of Triton X-114 is shown in Figure (    It is clear from the result that the absorbance increases with the increase volume of Triton X-114 but suddenly decreases at higher amount. Effect the amount of surfactant on the efficiency of extraction and improve the enrichment factor (98) .These represent the optimum volume to reach equilibrium extraction process that give highest efficiency with smaller size and higher density in cloud layer. The decrease in absorbance below the optimum volume is due to insufficient micelles to entrap the hydrophobic product quantitatively . Therefore the optimum volume of Triton X-114 ( 0.2-2) ml for ( Allo-CoII ) complex fixed in subsequent experiments to achieve high extraction efficiency .

Effect of the incubation Time
Amount of 10ml solution is prepared in volumetric flask containing [1mLl allopurinol , 0.8mL Co(II ),0.6 mL buffer pH 11 and 1.2mL Triton X-11410%(v/v)] then it is completed to the mark by distilled water, are mixed and the temperature is set at 50 o C for CoII-ALLO complex and the incubation time varies from (5.0-35.0) min. to form cloud point extraction, then centrifugation at 3000 rpm for 10min , 1mL ethanol is added to the surfactant-rich phase and measured at λmax 586 nm for Allo-CoII complex. Plotting the absorbance values of the cloud point versus the time ( min.) is shown in Figure ( figure 8 showed the effect of reaction time on the complex formation, by following the variation of absorbance values. Maximum absorbance for all extracted ( Allo-CoII ) complex were observed after 15 min Heating for longer time resulted in decreased absorbance values results shown in table (7).Cloud point extraction requires enough time to get equilibrium between aqueous phase and surfactant-rich phase by more aggregation the micelles. This time represents the amount of heat accumulated in the solution that allows Micelles lose water molecules in order to give small size hydrophobic with high viscosity easily entrap the product in it. It is clear that the optimum incubation time is 15 min for ( Allo-CoII ) complex

Effect of Temperature
The complexation reactions of the studied (Allo-CoII ) complex were very slow at room temperature, while the highest absorbance of complex (Allo-CoII) was maintained at 50°C as explained in Figure (9). Heating the solutions to higher temperatures decreases the absorbance and this may be due to the decomposition of complexes ( table 8). Plotting the absorbance values of the cloud point versus the temperature is shown in Figure (9). Table 8. Data of Absorbance to Temperature / 0 C We need to heat the aqueous solution at a certain temperature to form a layer of the cloud point that smaller size and high viscosity due to aggregation the micelles this called (Cloud point temperature -CPT). The results show that the highest absorbency and extraction efficiency of the drug at temperature 50 0 C for ( Allo-CoII ) complex then decreases in absorbance at higher temperature due to decomposition of product which reduces the extraction efficiency. This temperature is fixed in subsequent experiments .

Effect of extraction time
Results in Figure 10 explained that the centrifugation time does not have a considerable effect on the analytical characteristics of the CPE method. This parameter was examined in the range of 5.0-25.0 min at 4000 rpm. A time of 10min was selected as optimum, since complete phase separation occurs in this time and no appreciable improvements were results shown in table (9).   Table 9. Data of Absorbance to extraction time Figure 11. Extraction time effect on absorbance values of ( Allo-CoII ) complex

Effect of Order Additions
The effect of order for additions of the metal on the absorbance of each analyte by the general CPE was tested. Fig (12 )shows that the best order of addition is the number 1 for target analytes due to giving a highest absorption signal among the others. The absorbance is measured and the absorbance results are shown in table (10)  Plotting of the absorbance values versus the order additions is shown in figure ( 12 )

Figure-12: Effect of Order Additions For ( Allo-CoII ) complex
It is noted that the best addition is the first order of( Allo-CoII ) complex and the best addition is the four order of ( Allo-CoII ) complex because if it's another order gets lost in the intensity of color and this order fixed in subsequent experiment

Effect of Solvents
The absorbance is measured and the absorbance results are shown in table (11). It has been shown that water is the optimum solvent , economically , sensitivity method ,cheap price, to provide and nontoxic. This solvent is fixed in subsequent experiment 3.

The Effect of Interference
The effect of interference expected present in ( Allo-CoII ) complex has been studied to know method selectivity under study by added 1ml (100 ppm) from each interference [Lactose, Starch, Arabic Gum, Glucose, Talc, Ca3(PO4)2, CaCO3] with 1ml(10 ppm) from each drug and the rest of addition are optimal conditions then diluted with distilled water in 10ml volumetric flask then measured. The interference experiment is performed to estimate the systematic error caused by other materials that may be present in the specimen being analyzed. It must be the size of interference is small for a sample to limit the dilution of sample anduse the maximum concentration expected in the sample. The results are shown in Table (  From the previous tables we notice that there is no expected interference to be present with drug in pharmaceuticals

Selected Optimum Conditions
The optimization conditions method that was used above to study the effect of variables on the absorbance intensity After the study of the effect of different physical and chemical conditions on the absorbance intensity of the colored product,that gave the optimum conditions shown in Table (13).   The optimum conditions for the proposed procedure were summarized in Table  ( 13) and were used in all subsequent experiments.

Preparation of Calibration Curve in CPE
Amount of 10ml solution is prepared containing increasing concentration of drug allopurinol by taking [(5.0-35.0) μg mL -1 allopurinol ,0.8 mL Co(II) ,0.6 mL buffer pH 11, and 1.2mL10%(v/v)Triton X-114] then it is completed to the mark by distilled water. Solutions were mixed and heated at optimum temperature in the thermostat water bath at optimum incubation time to maintain cloud point. The aqueous phase is separated from the rich phase layer by centrifuging at 4000 rpm for 10min ,1mL ethanol is added to the surfactant-rich phase and measured at λmax 586 nm for ALL-CoII complex. Plotting the absorbance values of the cloud point versus the concentration of ALL-CoII complex is explained in Figure (

Calculation of the stability constant (K) of complex
The conditional or apparent stability constant of the 1:1 ( Drug and metal ) product was evaluated and described as shown Complete founding the stability constant [K] colored product Formed imputation of ( metal :drug ) as followed: A series of solution were prepared containing three different concentration of metal and allopurinol (1:1) and the concentration (1.2 ×10 -5 ) molL -1 for (Cobalt -allopurinol) when Formed imputation under this Condition easily to Hydrolysis and the Intensity Absorption was very low . Another series of solution was prepared containing three deferent concentration of metal and allopurinol but with abundance of the metal (the best concentration ) The complex was prepared with no decomposition .express of the intensity absorption Am and application the relationship the value degree of decomposition can be calculated as follows