SPECTROPHOTOMETRIC DETERMINATION OF ETODOLAC USING CHARGE TRANSFER COMPLEX

SPECTROPHOTOMETRIC DETERMINATION OF ETODOLAC USING CHARGE TRANSFER COMPLEX

Abdulbari Mahdi Mahood and Sura L. Alkhafaji ^*

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Kerbala, Iraq.

ABSTRACT: In this study, we developed a cheap and precise analytic method to measure Etodolac concentration in bulk and pharmaceutical formula. The modified, updated method depends on the reaction between ferric ion and Etodolac to yield ferrous ion. Then the former ion reacted with potassium ferric cyanide to form colored compound has a maximum absorbance at 700 nm. The method was validated by calculating the precision and accuracy. The method follows Beer-Lamber law so that we used the range between 1-22.5 µg/mL and the correlation coefficient at 0.997. Accuracy and precision of the proposed method were estimated and shown average of recoveries between (96.67-99.0%) with precision represented by RSD % equal to 1.02. We also calculated the detection limit (LOD) and quantitative limit (LOQ), which respectively were 0.6 µg/mL and 1.2 µg/mL. The modified suggested method was used to measure Etodolac in pharmaceutical preparation without drug additives interference and shows a good agreement with the standard method. Therefore, it can be used for routine works for quality control for the estimation of the Etodolac in bulk and pharmaceutical preparation as tablets.

Etodolac, Spectrophotometric method, Estimation, Charge transfer complex

INTRODUCTION: Etodolac is a non steroidal-inflammatory drug used for the treatment of postoperative pain and inflammation of osteoarthritis and rheumatoid arthritis ¹. The principle action of Etodolac is blocking the action of prostaglandin chemokines that play a role in the inflammatory process, which characterizes by pain and fever ². It is selective cycloxygenase COX-2 inhibitors. Etodolac recommended daily dose is 200-400 mg every 6-8 h for relieving of general pain. Recently, it is approved that Etodolac derivatives have antitumor activity ^{3, 4}. Etodolac is presented as a racemic mixture; the (+) S stereoisomer is a biologically active form ⁵.

Etodolac has a relative bioavailability of 100% and it is highly plasma protein binding. Primarily, it is oxidized in the liver to give 6-, 7-, and 8-hydroxylated metabolite that is readily eliminated in urine as glucuronide conjugate ⁶. The chemical structure of Etodolac is [1, 8-diethyl - 1, 3, 4, 9-tetrahydropyran (3, 4-B) indol 1-acetic acid) as shown in Fig. 1.

FIG. 1: CHEMICAL STRUCTURE OF ETODOLAC

Chemically, Etodolac is white crystals, water-insoluble and soluble in organic solvents like alcohols, chloroforms, dimethylsulfoxide, and aqueous polyethylene glycols.

Various analytical methods used for the determination of this drug in the body fluid and pharmaceutical preparations. These include Spectrofluorometric method ^{7, 8}, Gas Chromatography GC ⁹, Sequential injection analysis ¹⁰, High-Performance Method HPLC ^11-17, High-Performance Thin Layer Chromatography HTLC ¹⁸, Capillary electrophoresis ^{19, 20}, Voltammetry ²¹ and UV-spectro-photometric ^21-30. The current paper reports a simple, sensitive spectrophotometric method for determination of Etodolac via the reaction of the drug with ferric ion Fe³⁺ to form the corresponding ferrous ion Fe²⁺ and subsequent reaction with potassium hexacyano-ferrate to form a colored compound that is absorbed in 700 nm.

MATERIALS AND METHODS:

Instruments: All absorption measurements were carried out using A UV-1800 UV-Vis Spectro-photometer SHIMADZU-1800, (Kyoto, Japan) equipped with a 1.0 cm quartz cells. pH meter was used throughout this research. The solutions were heated using a water bath of frost instruments, Ltd.

Reagents and Chemicals: All chemicals used were of analytical reagent grade. Ammonium ferric sulfate, potassium ferricyanate, hydrochloric acid HCl and methanol solvent were supplied by Hi-Media, India. The drug was obtained in pure form from SDI- Samarra, Iraq. Etodolac (Lodine)^R (400mg) tablets were purchased from the local a market (Drogsan).

Standard Stock Solutions: (1 µg/mL) of Etodolac were prepared by dissolving 0.1000 g Etodolac in 1:9 v/v methanol: water. Then the solution was diluted to the 100 ml in a volumetric flask.

Ammonium Ferric Sulfate NH₄Fe(SO₄)₂: To prepare a 1000 µg/mL, we dissolved 0.1000 g of the NH₄Fe(SO₄)₂ in 100 ml of d H₂O.

Potassium Ferricyanate K₃Fe(CN)₆: To prepare 1000 µg/mL, we dissolved 0.1000 g of K₃Fe(CN)₆ in 100 ml dH₂O.

Recommended Procedure: In a series of 10 ml volumetric flasks, transfer increasing of different volume of Etodolac (100 µg/mL) to cover the range of calibration curve (1-22.5) µg/mL, added 2 ml (1000 µg/mL) of ammonium ferric sulfate and shook well.  A 2.5 ml (1000 µg/mL) of K₃Fe(CN)₆ was added then 1 ml of concentrated HCl was added, and the solution was left for 10 min at water bath at 30 ºC, then make the solution to the marks with distilled water. We measured the absorbance of the colored formula, which we got at 700 nm after we set up the reagent blank.

Procedure for Pharmaceutical Preparation: Ten tablets of 400 mg of the drug per tablet were accurately weight and grounded well in a mortar. An accurate weight of final powdered equivalent to 0.1000 g was dissolved in (1:9) methanol: distilled water. The prepared solution was transferred to 100ml calibrated flasks and shaken well for ten min; then, this solution was filtrated using a Whatman filter paper no. 42 to avoid any suspended particles. The general procedure was then applied in concentration ranges previously cited above.

RESULTS AND DISCUSSION: Different parameters that influence on the sensitivity and stability of the forming complex were carefully studied and optimized.

The Spectrum of the Complex: The purposed method is based on the reduction of iron Fe⁺³ III to iron Fe⁺² II by Etodolac in the presence of concentrated hydrochloric acid. Subsequently, these ions have were reacted with potassium ferric cyanate to produce blue colored products. The absorption spectrum of the complex species in this method at the optimum conditions was scanned against a reagent blank in the range 400-800 nm and show that has a particular λ_max at 700 nm Fig. 2. Whereas the reagent blank recorded negligible absorbance at the same λ_max.

FIG. 2: ABSORPTION SPECTRUM of (5 µg/mL) ETODOLAC WITH NH₄Fe(SO₄)₂, K₃Fe(CN)₆ MIXTURE

Effect of Ferric Ammonium Sulfate Concentration: The complex formation reached its maximum when 2 ml of 1000 μg/mL of ferric ammonium sulfate solution was added to a mixture of etodolac and ferric potassium cyanate. Therefore, this amount used in the procedure was suitable because it gives high and reproducible color intensity as shown in Fig. 3.

Effect of Ferric Potassium Cynate Concentration: To find a suitable concentration of ferric potassium cyanate, different concentration of 1000μg/mL was studied by adding a different volume of the reagent (0.1-5) ml. Hence, 2.5 ml of the reagent was founded as the optimum volume that gives high color intensity Fig. 4. This concentration was taken in the subsequent study.

Effect of Different Acids on the Absorbance of the Colored Compound: Different acids were tested individually to study their effect on charge transfer reaction as H₃PO₄, H₂SO₄, HNO₃, CH₃COOH, and HCl acids. It was found that all these acids give unsatisfactory results except HCl acid. The effect of volumes 0.5 - 3 ml of HCl solution on the absorbance of the colored complex was studied. Thus, 1 ml of concentrated hydrochloric acid was adequate and sufficient for completing the reaction and giving a stable complex. Moreover, a further increase in HCl volume decreases the complex absorbance.

Effect of Reaction Time: It was observed that the absorbance of the complex was increased with increasing of the time of reaction and reached a maximum at 10 min. Therefore; 10 min was chosen for further use.

Effect of Temperature: The optimum reaction temperature in a water bath that gives high absorbance was 30 ºC. At this temperature, Etodolac reacts with reagents to give colored complex that was stable for a period of more than 2h.

Order of Addition: The effect of the order of addition on the intensity of the colored compound was studied. Table 1 shows the order of addition was followed as given under the general procedure, Drug: K₃Fe(CN) ₆: NH₄Fe(SO₄)₂ because it gave the highest absorption value. So it was chosen for further study.

TABLE 1:  EFFECT OF ORDER OF ADDITION

Abs.: Absorbance

Calibration Graph: Under the optimum conditions, a linear calibration graph was established by making serial dilution of etodolac and employing the condition stipulated under the correlated recommended procedure, it obeyed Beer`s law in range of (1-22.5) µg/mL, the linear regression equation obtained was (y= 0.0314×+ 0.1006) with correlation coefficient of 0.997 Fig. 5. The molar absorptivity value was estimated (2.4 × 10⁴ L.mol^-1.cm^-1) and is indicated that the method used is sensitive as listed in Table 2. Limit of quantification LOQ and limit of detection LOD were calculated based on standard deviation and the slop of calibration graph ³¹.

Precision and Accuracy: The efficiency of the method was statistically assessed by measuring the accuracy and precision of the suggested method. The accuracy was known as percentage relative error (RE% = found concentration- taken concentration ×100/ taken concentration) and the relative standard deviation (RSD %) was shown according to the accuracy of the tested method. However, the accuracy of the modified analytical method was measured for a series of five replicates at three levels of Etodolac concentration (4, 10 and 20) µg/mL. RSD% was found to be 1.02%, and RE% was found to be 1.01%. These results indicated that this method is as accurate and precise as summarized in Table 3.

FIG. 5: CALIBRATION GRAPH FOR DETERMINATION OF ETODOLAC

TABLE 2: OPTICAL CHARACTERISTIC AND VALIDATION DATA FOR PROPOSED METHOD

TABLE 3: THE ACCURACY AND PRECISION OF THE PROPOSED METHOD

RE: Relative Error, RSD: Relative Standard Deviation

Interference: The interference effect of some excipients, which often brought pharmaceutical formulation, was examined by measuring the absorbance of a solution of 30 μg/mL that containing 100 μg/mL of the drug and variety excipients to the final volume 10 ml. Interestingly, we found that the chosen excipients did not affect the results even when used in large doses see Table 4.

TABLE 4: THE EFFECT OF EXCIPIENTS (100 μg/mL) ON THE RECOVERY

Rec.: Recovery, RE: Relative Error.

Analytical Applications: The proposed method was effectively applied to determine Etodolac in its commercial formulation obtained. The amount of tablet containing 400 mg Etodolac was determined for five determinations as in Table 5.

TABLE 5: DETERMINATION OF ETODOLAC IN PHARMACEUTICAL FORMULATION (400 mg/TABLET)

*Mean of five determinations. Rec.: Recovery.

Stoichiometric Ratio for the Formation of Colored Complex: By application of Job’s method of continuous variation ³². It was found that the selected drug form a colored complex product with the reagent in a 1:1 molar ratio as shown in Fig. 6 which represented Job’s plot of Etodolac with the reagent.

FIG. 6: STOICHIOMETRIC RATIO FOR THE FORMATION OF COLOURED COMPLEX

CONCLUSION: A soluble colored complex has been synthesized successfully via chelation of potassium hexacyanoferrate(ΙΙΙ) with iron (ΙΙ) that was formed from the reduction of iron(ΙΙΙ) by Etodolac. It is concluded that the proposed method is a precise, accurate and reproducible for determination of Etodolac in the pure and pharmaceutical formulation without the interference of any excipients. The proposed method was rapid, less time consuming than other spectrophotometric methods and linked with good accuracy and precision which rend the method appropriate for a routine assay of Etodolac in quality control in laboratories. Besides, this simple method doesn`t require any procedure of extraction, prior treatment of drug and expensive reagents.

ACKNOWLEDGEMENT: The authors would like to express special thanks to the staff of Pharmaceutical Chemistry Department, Faculty of Pharmacy, University of Kerbala, Iraq for providing the support to carry out research work. Furthermore, the authors also would like to thank the reviewers for their valuable comments and recommendations to develop the quality of this paper.

CONFLICT OF INTEREST: The authors announce no conflict of interest.

REFERENCES:

1. Veys EM: Clinical performance of Etodolac in patients with osteoarthritis and rheumatoid arthritis. Eur J Rheumatol Inflamm 1994; 14: 23-27.
2. Inoue N, Nogawa M, Ito S, Tajima K, Kume S and Kyoi T: The enantiomers of Etodolac, a racemic anti-inflammatory agent, play different roles in efficacy and gastrointestinal safety. Biological and Pharmaceutical Bulletin 2011; 34: 655-659.
3. Kummari B, Polkam N, Ramesh P, Anantaraju H, Yogeeswari P, Anireddy JS, Guggilapud S and Babu BN: Design and synthesis of 1,2,3-triazole–etodolac hybrids as potent anticancer molecules. Royal Society of Chemistry Advanced 2017; 7: 23680-23686.
4. Çıkla P, Özsavcı D, Bingöl-Özakpınar Ö, Şener A, Çevik Ö, Özbaş-Turan S, Akbuğa J, Şahin F and Küçükgüzel ŞG.: Synthesis, cytotoxicity, and pro-apoptosis activity of Etodolac hydrazide derivatives as anticancer agents. Archiv der Pharmazie-Chemistry in Life Sciences 2013; 346: 367-379.
5. Humber LG, Demerson CA, Swaminathan P and Birdt PH: Etodolac (1, 8-Diethyl-1, 3, 4, 9-tetrahydropyran 3[4-b] indole-1-acetic acid): a potent anti-inflammatory drug. Conformation and absolute configuration of its active enantiomer. Journal of Medicinal Chemistry 1986; 29: 871–874.
6. de Miranda Silva C, Rocha A, Tozatto E, da Silva LM, Donadi EA and Lanchote VL; Enantioselective analysis of etodolac in human plasma by LC-MS/MS: Application to clinical pharmacokinetics. Journal of Pharmaceutical and Biomedical Analysis 2016; 120: 120-126.
7. Ulu ST: New and sensitive spectrofluorimetric method for the determination of non-steroidal anti-inflammatory drugs, Etodolac and Diclofenac sodium in pharmaceutical preparations through derivatization with 7-fluoro-4-nitrobenzol-2-oxa-1, 3-dizaole. Journal of Food and Drug Analysis 2011; 19: 94-101.
8. Abd El-Hay SS, Colyer CL, Hassan WS and Shalaby A: Spectrofluorimetric determination of Etodolac, Moxepril HCl and Fexofenadine HCl using europium sensitized fluorescence in bulk and pharmaceutical preparations. Journal of Fluorescence 2012; 22: 247-252.
9. Giachetti C, Assandri A, Zanolo G and Brembilla E: Gas Chromatography-Mass Spectrometry determination of Etodolac in human plasma following single epicutaneous administration. Biomed Chromat 1994; 8: 180-183.
10. Garcia JB, Saraiva MLMFS and Lima JLFC: Determination and antioxidant activity evaluation of etodolac, an anti-inflammatory drug, by sequential injection analysis. Analytica Chimica Acta 2006; 573: 371-375.
11. Caccamese S: Direct High-Performance Liquid Chromatography (HPLC) separation of Etodolac enantiomers using chiral stationary phases. Chirality 1992; 5: 164-167.
12. Ammar A and Surmann P: Improvement of etodolac purity test by Reversed-Phase High-Performance Liquid Chromatography. Die Pharmazie 2008; 12: 913-914.
13. Saleh OA, El-Azzouny AA, Aboul-Enein HY, Badawey AM and Rashed MS: Development and validation of stability-indicating High-Performance Liquid Chromato-graphic (HPLC) and DD1-spectrophotometric assays for Etodolac in bulk form and the pharmaceutical dosage form. Journal of Liquid Chromatography & Related Technologies 2009; 32: 2584-2599.
14. Patel MJ, Badmanaban R and Patel CN: A Reversed Phase-High Performance Liquid Chromatographic method for simultaneous estimation of Tolperisone hydrochloride and Etodolac in combined fixed-dose oral formulations. Pharmaceutical Methods 2011; 2: 124-129.
15. Hameed ASA and Afifi SA: A validated HPLC-DAD method for simultaneous determination of Etodolac and pantoprazole in rat plasma. Journal of Chemistry 2014; 2014: 1-8.
16. Kandimalla S and Pai KV: Method development and validation of HPLC for simultaneous determination of Etodolac. Scholar Research Library 2015; 7: 317-321.
17. Lalla JK, Bhat SU, Sandy NR, Shah MU and Hamrapurkar PD: HPTLC determination of Etodolac in pharmaceutical formulations and human samples: HPTLC HPLC. Indian Drugs 1999; 36: 115-118.
18. Sane RT, Francis M and Khatri AR: High-Performance Thin-Layer Chromatographic determination of Etodolac in pharmaceutical preparations. Journal of Planar Chromatography-Modern TLC 1998; 11: 211-213.
19. Dogrukol-Ak D, Kutluk ÖB, Tuncel M and Aboul-Enein HY: Capillary electrophoretic method for the determination of Etodolac in pharmaceutical tablet formulation. Journal of Liquid Chromatography and Related Technologies 2001; 24: 773-780.
20. De Pablos RR, Garcia-Ruiz C, Crego AL and Marina ML: Separation of Etodolac enantiomers by capillary electrophoresis, validation, and application of the chiral method to the analysis of commercial formulations. Electrophoresis 2005; 26: 1106-1113.
21. Yılmaz S, Uslu B and Özkan SA: Anodic oxidation of Etodolac and its square wave and differential pulse voltammetric determination in pharmaceuticals and human serum. Talanta 2001; 54: 351-360.
22. El Kousy NM: Spectrophotometric and spectrofluorimetric determination of Etodolac and Aceclofenac. Journal of Pharmaceutical and Biomedical Analysis 1999; 20: 185-194.
23. Gouda AA and Hassan WS: Spectrophotometric determi-nation of Etodolac in pure form and pharmaceutical formulations. Chemistry Central Journal 2008; 2: 1-8.
24. Hu Q, Li Y, Yang X, Wei Q and Huang Z: Study on spectrophotometric determination of Etodolac. Chemistry Journal on Internet 2008; 10: 31-33.
25. Ye Y, Yinke L and Huang Y: Spectrophotometric determination of Etodolac. Asian Journal of Chemistry 2009; 21: 649-654.
26. Duymus H, Arslan M, Kucukislamoglu M and Zengin M: Charge transfer complex studies between some non-steroidal anti-inflammatory drugs and π-electron acceptors. Spectrochimica Acta 2006; 65: 1120-1124.
27. Amer SM, El-Saharty YS, Metwally FH and Younes KM: Spectrophotometric study of Etodolac complexes with copper (II) and iron (III). Journal of AOAC International 2005; 88: 1637-1643.
28. Alpa JV, Bhavika GA, Mital SM, Patel BA, Parmar SJ: Method development and validation for the simultaneous estimation of Paracetamol and Etodolac by derivative UV Spectroscopic method. International Journal of Pharm Tech Research 2013; 5: 1155-1160.
29. Bhalani M, Subrahmanyam EVS and Shabaraya AR: New analytical methods and their validation for the estimation of Etodolac in bulk and marketed formulations. International Journal of Pharmaceutical Sciences and Research 2015; 6: 414-416.
30. Karajgi SR, Rub TA and Kotnal RB: First derivative UV spectrophotometric method for the determination of Etodolac in solid dosage forms. RGUHS Journal of Pharmaceutical Sciences 2016; 6: 32-37.
31. Shaikh A, Singh G, Jain N and Gupta M: Development and validation of new simple, sensitive and validated UV-spectrophotometric and RP-HPLC method for the simultaneous estimation of Paracetamol and Etodolac in the marketed Journal of Drug Delivery and Therapeutics 2017; 4: 120-124.
32. Analytical Procedures and Methods Validation: Chemistry, Manufacturing, and Controls, Federal Register Notices 2000; 65: 776-7.
33. Huang CY: Determination of binding stoichiometry by the continuous variation method, the Job Plot. Methods in Enzymology 1982; 87: 509-525.
    

    ---

    How to cite this article:

    Mahood AM and Alkhafaji SL: Spectrophotometric determination of Etodolac using charge transfer complex. Int J Pharm Sci & Res 2019; 10(3): 1330-35. doi: 10.13040/IJPSR.0975-8232.10(3).1330-35.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---