ANALYST, MARCH 1986, VOL. 111 323 Spectrophotometric and Fluorimetric Methods for the Determination of lndomethacin C. S. P. Sastry, D. S. Mangala and K. Ekambareswara Rao Foods and Drugs Laboratories, School of Chemistry, Andhra University, Waltair 530 003, A. P., India Three spectrophotometric methods and a fluorimetric method are described for the determination of indomethacin in bulk samples and pharmaceutical preparations based on the formation of coloured species with m-aminophenol - chloramine-T, resorcinol - sodium hypochlorite or phloroglucinol - hypochlorite reagent and a fluorescent species with m-aminophenol - chloramine-T reagent, respectively, under specified experimental conditions. All the methods are simple, sensitive and reproducible to within + I . ~ O / O .Keywords: lndomethacin determination; spectrophotometry; fluorimetry Indomet hacin , [ 1 - (4-chloro benzoyl)-5-methoxy-2-methyl- indol-3-yllacetic acid, is well known for its antipyretic anal- gesic action and is extensively used in rheumatoid arthritis. The methods reported for the determination of indomethacin include spectrophotometric~-3 and fluorimetric methods4 and the official BP procedure.5 The existing spectrophotometric methods are time consuming, tedious and require some preliminary treatment. This paper describes three simple spectrophotometric (visible region) methods and one fluor- imetric method using pairs of reagents, rn-aminophenol - chloramine-T, resorcinol - hypochlorite, phloroglucinol - hypochlorite or rn-aminophenol - chloramine-T, under speci- fied conditions.Experimental Apparatus A Systronics Model 105 (Mk. 1) spectrophotometer with 1-cm path length cuvettes, a Perkin-Elmer Model 203 fluorescence spectrophotometer with the sensitivity setting adjusted to 10 and a Systronics Model 305 pH meter were used for absorbance, fluorescence and pH measurements, respec- tively. Reagents All the reagents were of analytical-reagent grade and all solutions were prepared in doubly distilled water. All the pharmaceutical preparations used were available commer- cially. Aqueous solutions of m-aminophenol (mAP, 0.1% in 0.025 M HCl), phloroglucinol (0.2%), resorcinol (0.270)~ chlor- amine-T (CAT, 0.03 M), sodium hypochlorite (OC1-, 0.05 M), HC1 (2 M) and potassium acid phthalate (0.05 M) were prepared. Standard drug solution.A 50-mg amount of BP-grade indomethacin (supplied by Themis Chemicals Ltd. , Bombay, India) was initially dissolved in 10 ml of 1.25 M NaOH solution and then diluted to 250 ml in a calibrated flask. Working solutions for methods A and D were prepared by suitable dilution of the stock standard solution. The solutions were stable for 2 h. Sample solution. Twenty capsules were emptied and pul- verised and an amount equivalent to 50 mg of indomethacin was taken, dissolved as described above and filtered. The solution was stable for 2 h. Spectrophotometric Procedures A. With mAP - CAT reagent A 15-ml volume of potassium hydrogen phthalate, 3 ml of CAT and 3 ml of mAP solution were placed in a 25-ml calibrated flask. A 1.04.0-ml portion of indomethacin (100 pg ml-1) solution and the requisite volume of distilled water were added to make the total volume 25 ml.The pH of the resulting solution was between 4 and 5.0. The absorbance of the coloured species was measured at 490 nm within 3 rnin against a reagent blank prepared in a similar manner. The indomethacin concentration was calculated from a calibration graph prepared with a standard solution under identical conditions. B or C. With resorcinol or phloroglucinol - OCI- reagents Aliquots of 1.0-4.0 ml of indomethacin solution (200 pg ml-1) were placed in a 25-ml calibrated flask containing 1 ml of 2 M HC1, mixed well (for 1 min), 1 ml of OC1- was added and the mixture was allowed to stand for 3 min for resorcinol or 2 rnin for phloroglucinol. A 3-ml volume of resorcinol or phloroglu- cinol was added after this period and the absorbance of the coloured species was measured at 460 nm after 10 rnin (the stability periods were as follows: B, resorcinol - OC1-, 5-20 min; C, phloroglucinol - OC1-, 10-35 min) against the corresponding reagent blanks prepared in a similar manner.The indomethacin concentration was obtained from calibra- tion graphs obtained under identical conditions. Fluorimetric Procedure D. With mAP - CAT reagent A 0.2-3.0-ml portion of indomethacin (10 pg ml-1) was placed in a 10-ml calibrated flask containing 0.5 ml of 2 M HCl, 2 ml of CAT and 2 ml of mAP solution and diluted to the mark with distilled water. The fluorescence of the solution was measured between 10 and 120 min at excitation and emission wave- lengths of 465 and 490 nm, respectively, against the reagent blank prepared in a similar manner.The concentration of indomethacin was calculated from a calibration graph. Results and Discussion Of the various combinations of phenols (phenol, catechol, resorcinol, pyrogallol and phloroglucinol) , aminophenols (0-, m-, p- and p-N-CH3) or phenylenediamines [o-, rn-, p - , and p-N7N-(CH3)2] and oxidising agents [CAT, OC1-, 104- , 103-, Cr(VI), Fe(III), S208*- , N-bromosuccinimide and324 ANALYST, MARCH 1986, VOL. 111 Table 1. Optical characteristics, precision and accuracy Method Parameter Beer’slawlimits/pgml-1 . . . . . . Molar absorptivity/l mol-1 cm-1 . . , . Sandell’s sensitivity/pg cm-2 per 0.001 absorbance unit . . . . Intercept . . . . . .. . . . . . Correlationcoefficient . . . . . . Relative standard deviation, % . . . . Range of error, % (95% confidence limits) . . . . . . Slope . . . . . . . . . . . . A 4.0 - 16 5.72 x 103 0.063 6.32 x 10-4 -3.49 x 10-3 0.999 1.39 k 1.46 B 8.0 - 24 2.27 x 103 0.16 2.41 x 10-4 3.00 x 10-3 0.998 1.65 k1.73 C D 5.5 - 24 3.18 x 103 0.2 - 3 - 0.11 - 3.42 x 10-4 0.3186 0.1698 2.03 x 10-3 0.999 0.999 1.55 1.32 k 1.62 k 1.39 Table 2. Assay and recovery of indomethacin in dosage forms Amount found/mg Labelled Proposed method Recovery, Yo amount/ Reported Capsules mg A BorC D method A B or C* D Hyderabad) . . . . 25 23.44 23.50 (B) 23.52 23.45 97.8 98.4 (B) 98.5 Idicin(IDPL, . . . . Microcid Inobid (Thomis, (Microlabs, Madras) 25 24.23 24.18 (C) 24.30 24.21 98.2 98.2 (C) 98.7 Bombay) .. . . 75 73.32 73.30 (B) 73.36 73.29 97.6 98.6 (B) 99.1 H2021 tried for developing the colour or fluorescence under acidic or alkaline conditions at laboratory temperature, mAP - CAT was found to be superior. Of the other combinations, resorcinol or phloroglucinol and OC1- were found to be suitable as chromogenic reagents. The optimum conditions for each method (A-D) were established after a thorough systematic study of the para- meters such as acid strength (pH), reagent concentration and order of addition of the reagents. Potassium hydrogen phthalate solution was found to be necessary only in method A for maintaining the resulting pH between 4 and 5. Even trace amounts of miscible alcohols such as methanol and ethanol were found to quench the fluores- cence intensity.The chromophore or fluorophore in methods A-D was partially extractable into butan-1-01 and not extractable into chloroform. The excitation spectra of the fluorescent species showed three maxima, at 245,305 and 465 nm. The peaks in the UV region were not considered as the blank interferes. The optical characteristics such as Beer’s law limits, molar absorptivity and Sandell’s sensitivity for each method are given in Table 1. The slopes, intercepts and correlation coefficients obtained by linear least-squares treatment6 of the results for the systems involving indomethacin with the mentioned reagents are also presented in Table 1. The reproducibility of the methods was found by measuring the absorbances (in methods A, B and C) or fluorescence intensity (in method D) of six replicate samples containing a known amount of drug (400 pg per 25 ml in A, B and C and 20 pg per 10 ml in D) and the results obtained are given in Table 1.The accuracy of the methods was further confirmed by adding known amounts of indomethacin to previously analysed H I OH- CH&OOH H3C0 CHZCOOH H3C0 I R‘ II I CHzCOOH 0 CHZCOOH IV I l l rn-Aminophenol: R = NH; R’ = H Phloroglucinol : R = 0; R’ = OH Resorcinol : R = 0; R’ = H Scheme 1. Reaction mechanismANALYST, MARCH 1986, VOL. 111 samples and the recoveries obtained are given in Table 2. The results obtained by the proposed and reported1 methods for indomethacin in dosage forms are also included in Table 2. The ingredients usually present in pharmaceutical prepara- tions of indomethacin, such as glucose, lactose, sodium metabisulphite, sodium chloride, magnesium stearate, starch, talc, sodium citrate and other analgesics such as paracetamol, phenacetin and analgin, did not interfere in the proposed methods.Mechanism The species reacting with the proposed reagents (Scheme 1) appears to be 5-methoxy-2-methylindol-3-ylacetic acid (11), the hydrolysis product of indomethacin (I) ,’ as the indo- methacin sample dissolved only in alkali and not in ethanol produces colour or fluorescence. The failure of tryptophan and indole-3-acetic acid to develop colours with the proposed reagents indicates the necessity to have a 5-OMe group in the indole moiety. Compound I1 reacts initially with the oxidant (CAT or OC1-) to produce the highly reactive and less stable p-benzoquinone monoimine derivative (111), as with p-phene- tidin (p-ethoxyaniline) .8 Compound I11 may react further with coupler (m-aminophenol, resorcinol or phloroglucinol) to give 7-substituted-I11 such as p-N-acetylbenzoquinone monoimine and cysteine.9 In conclusion, the proposed methods are simple, sensitive, selective and can be used for the routine determination of indomethacin in pharmaceutical preparations.325 The authors are grateful to the authorities of Andhra University and also to the Council of Scientific and Industrial Research, New Delhi, for awarding a fellowship to K. E. R. References 1. 2. 3. 4. 5. 6. 7. 8. 9. Baggi, T. R., Mahajan, S. N., and Rao, G. R., Indian 1. Pharm. Sci., 1976,38, 101. Sanghavi, N. M., and Kamala, S . , Indian J. Pharm. Sci., 1978, 40, 71. Peterkova, M., Kakac, B., and Matousova, O., Cesk. Farm., 1980, 29, 73. Garcia, C. R., Lopez, A. A., and Benet, L. Z . , Rev. SOC. Quim., 1980, 24, 68. “British Pharmacopoeia 1980,” Pharmaceutical Press, London, 1980, p. 239. Pattergill, M. D., and Sands, D. E., J. Chem. Educ., 1979,58, 244. Hajratwala, B. R., and Dawson, J. E., J. Pharm. Sci., 1977,66, 27. Davis, D. R., Fogg, A. G., Thorburn Burns, D., and Wragg, J . S . , Analyst, 1974, 99, 12. Blair, 1. A., Boobis, A. R., Davis, D. S., and Cresh, M., Tetrahedron Lett., 1980, 4947. Paper A51254 Received July 15th, 1985 Accepted September 4th, 1985