Hu Qiufen 1,2,3, Li Yinke 2£¬Yang Xianjun 3, Wei Qunyan 3, Huang Zhangjie 1,3
(1 Faculty of Materitals and Metallurgical Engineering, Kunming University of Science and Technology, Kunming 650031; 2 Department of Chemistry, Yunnan Institute of the Nationalities, Kunming 650031; 3 Department of Chemistry, Yunnan University, Kunming 650091)
Abstract A simple, sensitive and reproducible spectrophotometric method for the determination of etodolac was described. This method based on the etodolac can reduce Fe3+ to Fe2+ in the presence of 2,2′-bipyridyl (Bpy) and pH 3.5 ~ 6.0 acetate buffer medium. The Fe2+ can reacts with Bpy to form a Fe2+-Bpy colored complex. The maximum absorbance of the colored complex is at 500 nm. Beer’s law is obeyed in the range of 0.5 – 25 m g/mL for etodolac in this method. The method was applied to the determination of etodolac in tablets without any interference from common excipients. The relative standard deviations were £ 0.82% with recoveries 97% – 102%. Results are satisfactory.
Etodolac (ETD) is nonsteroidal anti-inflamatory antirheumatic drugs . A survey of the literature revealed that there have been very few methods for the determination of ETD in biological fluids, tablets and in presence of its enantiomer. The techniques used in this connection include only HPLC, GC, spectrofluorimetric and spectrophotometric methods [2-4]. Extensive literature survey revealed that no method is available for determination of ETD in pure form and tablets by redox reaction.
2,2′-bipyridyl (Bpy) have been used frequently in the field of pharmaceutical analysis. It is a color reagent for Fe2+. The aim of the present study was apply redox reactions to develop simple, accurate, sensitive and reproducible methods of analysis of etodolac in pure form and in tablets. The etodolac can reduce Fe3+ to Fe2+, and the Fe2+ can reacts with Bpy to form a Fe2+-Bpy colored complex. A good linearity was obtained between the absorbance of the complex at 500 nm and the etodolac concentration, and the etodolac was determined by this method with good results. This spectrophotometric method can be used in laboratories where modern and expensive apparatus, such as that required for GC or HPLC, are not available.
A UV-160 A spectrophotometer (Shimidzu Corporation, Tokyo, Japan) equipped with 1 cm cells was used for all absorbance measurements. The pH values were determined with a Beckman F-200 pH meter (Beckman Instruments, Fullerton, CA, USA).
2.2. Materials and Reagents
All chemicals and materials were of analytical grade and all solutions were freshly prepared in bidistilled water. Etodolac (ETD) pure grade supplied by Fluka Corporation and its tablets (Napilac capsules, 200 mg ETD/Capsule) and (Etodine capsules, 300 mg ETD/Capsule) was provided by Kunming Pharmacy Corporation. Stock standard solution of ETD was prepared by dissolving 100 mg pure drug in methanol and completed to 100 mL with the same solvent to obtain a standard solution of 1.0 mg/mL. Working solutions were prepared by an appropriate dilution of the stock standard solution. The iron(III)- 2,2′-bipyridyl was prepared by mixing 0.198 g of 2,2′-bipyridyl (Fluka, Swiss), 2.0 mL of 1.0 mol/L HCl and 0.16 g of ferric ammonium sulphate dodecahydrate (Fluka, Swiss) and diluted with bidistilled water to the mark in 100 mL calibrated flask. The acetate buffer solutions, buffers in pH range from (2.0 -6.0) were prepared by mixing appropriate quantities of 0.2 mol/L sodium acetate with 0.2 mol/L acetic acid to get the desired pH as recommended previously.
2.3. Recommended Analytical Procedure
Transfer aliquots (0.05-2.5 mL) of standard solutions (100 m g/mL) in a series of 10 mL calibrated flasks. To which, 1.0 mL Fe3+-Bpy or reagent solutions and 4.0 mL acetate buffer solution of pH 4.5 were added. The mixture was heated on a water bath at 80¡ãC for 10 min. Then, the mixture was cooled to room temperature (25 ¡À 1¡ãC) and the volume was made up to the mark with bidistilled water. The colored complex formed was measured at 500 nm against a reagent blank treated similarly.
2.4. Analysis of etodolac in tablets
Ten etodolac tablets were accurately weighed and powdered. An accurately weighed quantity equivalent to 20 mg ETD was dissolved in 20 mL methanol and transferred to a 100 mL calibrated flask. The contents of the flask was shaken for 10 min, and then made up to the mark with methanol. The general procedure was then followed in the concentration ranges already mentioned above.
3. Results and Discussion
3.1. Absorption Spectrum
This method is based on the formation of Bpy-Fe2+ complex upon the reaction of ETD with the Fe3+-Bpy reagent. The reaction proceeds through the reduction of Fe3+ to Fe2+ and the subsequent formation of an intensive orange-red coloration of the complex. The absorption spectra of the colored complex species in the proposed methods at the optimum conditions was scanned in the double beam mode against a reagent blank in the range 400-600 nm and recorded in the general procedures show a characteristic l max at 500 nm (Fig.1).
Fig.1 Absorption spectra
(1) Reagent blank against water
(2) Fe(III)-(2,2′-bipyridyl) with ETD (5.0 mg/mL) against reagent blank
3.2. Effect of Acidity
An acetate buffer solution was the optimal one of those examined (universal, phosphate, thiel, borate and acetate). The pH adjustment is necessary especially in acidic medium because the reaction was affected by the change of the pH in the range of (2.0 -6.0). The optimum pH value was 2.6-5.5 for this method. A acetate buffer solutions of pH 4.5 was recommended to control pH. As the use of 3.5 – 6.0 ml of the buffer solution (pH 4.5) was found to give a maximum and constant absorbance. The use of 4.0 ml buffer solution was recommended.
3.3. Effect of reagent concentration
The addition of 1.0 mL Fe3+-Bpy reagent solutions was sufficient to obtain the maximum and reproducible absorbance for 20 m g/mL-1 of ETD. Smaller amounts give incomplete complex formation. Whereas a larger concentration had no effect on complex formation, although the absorbance increased slightly due to the background of the reagent used. Accordingly, 1.0 ml of Fe3+-Bpy reagent solutions solution was added in all further measurements.
3.4. Effect of Temperature and Heating Time
The effect of temperature and heating time on the formation of the colored complex were studied. The reaction of ETD with the reagent proceeds very slowly at room temperature. Higher temperature was used to accelerate the reaction. Maximum absorbance was obtained after heating for about 10 min with Fe2+-Bpy colored complexes on a water bath at 80oC. Further heating caused no appreciable change in the color. The obtained complex was very stable for at least more than 12 h.
3.5. Calibration Curve and Sensitivity
The calibration curve shows that Beer’s law is obeyed in the concentration range of 0.5 – 25 m g/mL. The linear regression equation obtained was: A = 0.00615 + 0.0572 C (r = 0.9999). The molar absorptivity was calculated to be 1.79¡Á104 L.mol-1.cm-1. The detect limit, based on (LOD = 3s/k) is 0.06 m g/mL.
3.6. The method precision and recovery
The intra-day precision and inter-day precision were calculated from data obtained during a 7-day validation, solutions containing four different concentrations of ETD were prepared and analyzed in seven replicates. Precision of the assay was determined by repeatability (intraday) and intermediate precision (inter-day). To assess intraday variation (repeatability), calibration curve was prepared seven times on the same day. Intermediate precision was assessed by comparing the assays on different days (7 days, n = 7 at each concentration). The results shown that the relative standard derivation of overall intra-day variations were less than 0.68%, and the relative standard derivation of inter-day variations were less than 0.82 %. This method is high precision.
The recovery test of the proposed method was prepared by adding a known amount of standard at three different levels (1.0 m g/mL, 2.0 m g/mL, 8.0 m g/mL) to the pre-analysed sample. The results shown that the recoveries (n=7) were ranged from 97% – 102%. This method is high recovery.
3.7. Effects of Interference
The criterion of interference was an error of not more than ¡À 3.0% in the absorbance. To test the efficiency and selectivity of the proposed analytical method to tablets, a systematic study of additives and excipients (e.g. lactose, glucose, dextrose, talc, calcium hydrogen phosphate, magnesium stearate and starch) that usually present in dosage forms. Experimental showed that there was no interference from additives or excipients for the examined method as shown in Table 1.
Table 1. Determination of ETD in presence of additives or excipients
|Material||Amount (mg)||Recovery a %¡À SD b|
|Lactose||50||99.6 ¡À 0.8|
|Glucose||50||98.8 ¡À 0.6|
|Dextrose||50||99.3 ¡À 0.7|
|Magnesium stearate||30||99.2 ¡À 0.7|
|Calcium hydrogen phosphate||50||99.5 ¡À 0.9|
|Talc||40||99.8 ¡À 0.6|
|Starch||50||100.0 ¡À 1.1|
|a 6.0 mg/mL of ETD was taken; b Average of five determinations; SD : Standard deviation.|
3.8. Analytical Applications
The proposed methods were successfully applied to determine ETD in its tablets. Therefore, they could be used easily for the routine analysis of pure ETD and its dosage forms. Moreover, to check the validity of the proposed methods, dosage form [Napilac capsules (200 mg ETD per capsule) and Etodine capsules 300 mg ETD per capsule)] were tested for possible interference with standard addition method. The performance of the proposed methods was assessed by calculation of the t-test (for accuracy) and a variance ratio F-value (for precision) compared with the reference method (potentiometric titrate with tetrabutylammonium hydroxide) (for 95% confidence level with five degrees of freedom. The results showed that the t- and F-values were less than the critical value, indicated that there was no significant difference between the proposed and reference method for ETD. Because the proposed methods were more reproducible with high recoveries than the reference method, they can be recommended for the routine analysis in the majority of drugs quality control laboratories.
The proposed method is simpler, less time consuming and more sensitive than the published method. The proposed method was advantageous over other reported visible spectrophotometric method with respect to their higher sensitivity, simplicity, reproducibility, precision, accuracy and stability of the colored species for ³ 12 h. The proposed method is suitable for the determination of ETD in pure form and in tablets without interference from excipients such as starch and glucose or from common degradation products, suggesting applications in bulk drug analysis.