ANALYST. APRIL 1989, VOL. 113 513 Extractive Spectrophotometric Determination of Some Anti-inflammatory Agents with Methylene Violet C. S. P. Sastry, A. S. R. Prasad Tipirneni and M. V. Suryanarayana Foods and Drugs Laboratory, School of Chemistry, Andhra University, Waltair 530 003, India A fairly sensitive spectrophotometric method for the determination of ibuprofen, ketoprofen, piroxicam, diclofenac sodium, mefenamic acid or enfenamic acid in bulk samples and pharmaceutical preparations is described, based on the formation of a chloroform-solu ble, coloured ion-association complex between the drug and Methylene Violet a t pH 7.6. Keywords: Anti-inflammatory agents determination; extractive spectrophotometry; Meth ylene Violet Many of the reported visible spectrophotometric methods for the determination of anti-inflammatory agents such as ibu- profen (IB).] ketoprofen (KT),2.3 piroxicam (PX),".' diclo- fenac sodium (DS),b mefenamic acid (MA)'-9 and enfenamic acid (EA)lO suffer from lack of sensitivity or simplicity.In contrast, the extractive spectrophotometric technique pro- vides a highly sensitive method for the determination of pharmaceuticals." This technique has not yet been used for the determination of these drugs with the exception of MA.9 CH3 I CH-COOH R' -3 COOR' / I MV NH2 As the six anti-inflammatory agents studied in this work are acidic in nature, attempts were made to utilise the basic dye Methylene Violet (MV; CI No. 50210) for their extractive spectrophotometric determination. Experimental Apparatus A Systronics Model 105 (MK 1) spectrophotometer with 1-cm matched glass cells was used for all absorbance measurements.All pH measurements were made with an Elico Model LI-120 digital pH meter. Reagents All chemicals were of analytical-reagent grade. Buffer solution (PH 7.6). Prepared by mixing 88.5 ml of 0.067 M disodium hydrogen phosphate solution and 11.5 ml of 0.067 M potassium dihydrogen phosphate solution. Methylene Violet, 0.068% mlV (1.95 x 10-3 M) solirtion. Prepared in buffer solution. Standard drug solutions. Stock solutions containing 500 pg ml-1 of the anti-inflammatory agents were prepared by dissolving 50 mg of each drug separately in the minimum volume of 0.1 M sodium hydroxide solution and diluting to 100 ml with distilled water. Working standard solutions were prepared as required (IB, 100 pg ml-1; KT, 50 pg ml-1; PX and EA, 40 pg ml-1; and DS and MA, 20 pg ml-1) by suitable dilution of the stock solutions with distilled water.Procedure Aliquots of the standard drug solution (0.5-3.0 ml for IB or PX; and 0.5-4.0 ml for KT, EA, MA or DS) were transferred into a series of 125-ml separating funnels and then 8 ml of buffer (pH 7.6) and 1 ml of MV solution were added to each separating funnel. The total volume of the aqueous phase was adjusted to 15 ml by the addition of distilled water. Chloro- form (10 ml) was added to each separating funnel and the contents were shaken for exactly 1 min. The absorbance of the separated chloroform layer was measured at 540 nm against a reagent blank. The amount of each drug present was calculated from its calibration graph.Procedure for the assay of dosage forms An amount of the tablet powder, capsule powder or suspen- sion equivalent to 50 mg of the anti-inflammatory agent was weighed accurately (or measured) and treated as described above for the standard drug solution. Filtration was per- formed in instances where insoluble matter remained during preparation of the sample solutions.5 14 ANALYST, APRIL 1989. VOL. 114 Results and Discussion Figs. 1 and 2 show the absorption spectra of the MV - anti-inflammatory agent ion-association complexes extracted into chloroform and of the reagent blank. All these spectra have an absorption maximum at 540 nm, hence this wavelength was used for all subsequent measurements. In order to establish the optimum pH range, each anti- inflammatory agent was allowed to react with MV in aqueous solutions buffered to pH 6.C8.0 and the complex formed was extracted into chloroform for measurement.Constant ab- sorbances were obtained over the pH range 7.4-8.0 in phosphate buffer. Hence a pH of 7.6 was used in all subsequent work. The extent of the extraction of the ion-association complex was found to be affected by the concentration of MV used. To establish the optimum amount, 1-ml aliquots of 3.5 X 10--4-2.8 x l o - 3 ~ MV solutions were used under the conditions given above. The calibration graphs obtained using 1 ml of a 3.5 x 10-4 or 7.1 x 1 0 - 4 ~ solution were not linear, although the reagent bianks had low absorbances. The graphs obtained with 9.2 x 10-4-2.8 x 1 0 - 3 ~ MV solutions were linear, but the use of more MV resulted in higher absorbances for the reagent blanks.In subsequent work 1 ml of a 1.95 X 1 0 - 3 ~ MV solution was employed. Other organic solvents were tested. but chloroform was found to be the most suitable. Shaking times of 0.5-4 min produced a constant absorb- ance, hence a shaking time of 1 min was used throughout. The zbsorbances of the separated extracts were stable for more than 1 h; some data are presented in Table 1. Regression plots showed that there was a linear dependence of absorbance on concentration over the Beer’s law ranges given in Table 1. The optimum conditions were those used in the procedure. The molar absorptivities, slopes, intercepts and correlation coefficients obtained by a linear least-squares 400 440 480 520 560 600 Wavelengthinm Fig.1. Absorption spectra of (A) MV - DS; (B) MV - MA; and ‘C) MV - EA systems. [MV] = 1.95 x 10-3; DS] = 6.2 x 10 h ; [MA) = 8.2 x 10-6: and [EA] = 1.65 x 1 0 ~ ~ ~ . [D) Reagent blank treatment of the results are also given in Table 1. The precision of the method was tested by analysing six replicate samples of each anti-inflammatory agent (60 yg of DS or MA; 100 yg of EA; 150 yg of KT; 200 pg of IB; and 80 pg of PX); the relative standard deviation and range of error obtained are given in Table 1. The stoicheiometric ratio of drug to MV in each of the coloured complexes was determined using the slope-ratio method.12 It is apparent from the data that ion-association complexes with varying drug to dye ratios (2 : 1 for IB, PX, DS, MA or EA; and 1 : 1 for KT) are formed by the reaction of these drugs with MV.Other components normally found in combination with anti-inflammatory agents, such as paracetamol (PT), diaze- pam (DI) and dextropropoxyphene hydrochloride (DP), and commonly used excipients, diluents and acidic analytes (e.g., benzoic acid, ascorbic acid, and citric acid) do not interfere with the proposed method even when present in a ten-fold excess. Some pharmaceutical dosage forms were analysed by both the proposed and official methods (DS, as for sodium benzoate,13 viz., titration with hydrochloric acid using Bromo- phenol Blue as indicator; MA, titration with sodium hydrox- ide solution using Phenol Red as indicator”; EA, a visible spectrophotometric method using sodium nitroprusside and hydroxylamine hydrochloridelfl; PX, a UV spectrophoto- metric methods; IB, titration with sodium hydroxide solution using phenolphthalein as indicator14; and KT, a UV spectro- photometric method13) (Table 2) in order to confirm the accuracy of the proposed method for the determination of the six anti-inflammatory agents. As can be seen from Table 2 the proposed method has the advantage of being virtually free from interferences; it may, therefore, be of value for the determination of trace amounts of anti-inflammatory agents in other samples.0.6 a, 0.4 -e :: Q 0.2 0 ([I a t 0 400 440 480 520 560 600 Wavelengthhm Fig. 2. MV - KT systems. [MV] = 1.95 X l(1-3; [IB] = 4.84 X 1.96 X 10-5; and [PX] = 1.2 X 10-SM. (D) Reagent blank Absorption spectra of (A) MV - IB; (B) MV - PX; and C) [KT\ = Table 1.Optical characteristics and precision data IB KT PX DS MA EA Beer’slawlimits/ugml-l . . . . Molar absorptivityil mol-1 cm--’ Sandell’s sensitivity/ pgcm-2per0.001A . . . . Correlationcoefficient . . . . Regression equation (A)* Slope ( b ) . . . . . , . . Intercept(a) . . . . . . Relative standard deviation, Yo Range of error (95% confidence limit). % . . . . . . . . Stability of drug - dye complexih . . 5-30 . . 4.74 x 103 . . 0.044 . . 0.9999 . . 2 . 2 8 9 ~ 10- . . 2.0 x 10-3 . . 1.20 k1.23 1 2.5-20 2-12 1-8 1-8 2-16 6.10 x 10’ 1.72 x 104 3.10 x 104 2.29 x 104 9.64 x 103 0.042 0.019 0.01 0.01 1 0.025 0.9997 0.9998 0.9999 0.9999 0.9998 * 2.365 X 10 2 5.186 x 10-2 9.912 x lop2 9.45 x 10-2 3.941 x 10-2 5.196 x 10 5.333 x 10-3 5.196 x lo-’ 3.119 x lo-’ 3.873 x 10-3 1.38 1.18 1.30 1.37 1.08 k1.42 L1.21 i1.34 21.41 k1.11 2 6 4 4 4 * A = u + bc, where c is the concentration in pg m1-I.ANALYST, APRIL 1989, VOL.114 515 Table 2. Determination of anti-inflammatory agents in pharmaceutical preparations using the proposed and reference methods Composition of Amount foundimg pharmaceutical preparation/ Labelled amount/ Recovery, “10 Sample mg mg Proposed method Reference method* (proposed method)t Tablets- S1 , . . . IB200 200 192.8 194.6 99.6 S2 . . . . IB400 400 386.6 389.3 99.4 S3 . . . . IB600 600 582.0 585.7 99.8 S4 . . . . IB400,PT325 400 388.6 390.2 99.6 S5 . . . . IB400,PT325,DI2 400 389.4 391.3 99.5 S6 . . . . IB 200, PT 250, D P 32.5 200 194.7 196.0 99.9 s7 .. . . PX10 10 9.8 9.9 99.8 S8 . . . , PX20 20 19.2 19.3 99.6 S9 . . . . DS25 25 24.3 24.4 99.7 S10 . . . DS50 50 48.3 48.6 99.3 S11 . . . . MA250,PT250 250 246.6 247.5 99.7 S12 . . . . EAS00,PT500 500 484.3 486.9 99.6 S13 . . . . EA300 400 389.7 391.3 99.5 S13 . . . . IB200 200 192.4 193.2 99.4 S15 . . . . IB300 300 291.6 292.4 99.8 S16 . . . . IB400 400 388.7 390.2 99.7 S17 . . . . IB200, DP32.5 200 192.5 193.2 99.9 S18 . . . . IB400,DP65 400 384.8 386.3 99.8 S19 , . . . KTSO 50 48.6 48.7 99.9 S20 . . . . KT100 100 96.4 96.7 99.8 S21 . . . . KT50, PT500 50 47.7 47.9 99.6 s22 . . . . PX10 10 9.80 9.84 99.7 S23 . . . . PX20 20 19.3 19.4 99.8 S23 . , . . MA50 50 48.6 48.9 99.4 S25 . . . , MA250 250 242.8 244.1 99.9 S26 . . . . MA500,PT500 500 486.7 488.8 99.6 S27 .. . . MA500,D12 500 484.6 487.2 99.2 S28 . . . . IB50ml 100 98.4 99.1 99.6 S29 . . . . MA50ml 10 9.86 9.89 99.9 f After adding 10 mg; each value is the average of three replicate determinations. Capsules- Susperzsion (mg ml-1)- Reference methods: BPI1 for DS, MA and KT; IPl4 for IB; Guoguanys for PX; and Sastry and RaoI0 for EA. The proposed procedure is simple, sensitive, rapid, precise and accurate compared with many of the reported methods and can be used for the routine determination of anti- inflammatory agents in their dosage forms. 7. 8. 9. Khier, A. A . , El-Sadek, M.. and Baraka, M., Analyst, 1987, 112, 1399. Hassib, S. T., Safwat, H. M., and El-Bagry, R . I., Egypt. J. Pharm. Sci., 1987, 28 (1-4), 203. Issa, A. S . , Beltagy, Y.A . , Gabr Kassem, M., and Daabees, The authors thank the Quality Control Managers of Torrent Pharmaceuticals, India. and Unichem Laboratories, India, for providing the reference samples and the UGC, New Delhi, for the award of a Junior Research Fellow to one of us (A. S. R. P. T.). References 1. Matsuda. R., Takeda, Y . , Ishibashi, M., Vchiyama. M., Suzuki. M., and Takitami, S . , Bunseki Kagaku, 1986,35, 151. 2. Vachek. J., Cesk. Furm., 1987, 36, 168. 3. Emmanuel, J . , and Fernandes. N. N . , East. Pharm.. 1988, 31, 366. 139. 4. Sastry, C. S. P., Prasad Tipirneni. A. S. R . , Suryanarayana, M. V., and Prasad, T. N . V., East. Pharm., 1988,31,367,131. 5. Guoguany. P., Yayao Gongge, 1985, 16. 248. 6. Agarwal, Y. K., Upadhyay, V. P., and Menon, S. K., Indian J . Pharm. Sci., 1988, 50, 58. H. G . , Talanta, 1%5, 32, 209. Sastry, C. S . P., and Rao, A. R. M., Indian Drugs, 1987, 24, 303. Das Gupta, V., Indian J. Pharm., 1973, 35, 77. Irving, H., Rossotti, F. J . C., and Williams, R. J . P.. J. Chem. Soc., 1955, 1906. “British Pharmacopoeia 1980,” HM Stationery Office. Lon- don, 1980, DS: p. 406; and MA: p. 273; Addendum 1983, KT: p. 130. “Pharmacopoeia of India,” Ministry of Health and Family Welfare. Government of India, New Delhi, 1985, IB: p. 251. 10. 11. 12. 13. 14. Paper 8103770A Received September 26th, 1988 Accepted November 23rd, I988