12 ArtaZyst, January, 1974, Vol. 99, $$. 12-18 A Colorimetric Method for the Determination of Phenacetin and Paracetamok Part 111." Studies of the Indophenol Reaction: an Alternative Manual Procedure for the Determination of Phenacetin and Paracetamol and its Application to the Determination of Other Pharmaceuticals, including Sulphonamides, Procaine and Related Compounds BY D. R. DAVIS, A. G. FOGG AND D. THORBURN BURNS AND J. S. WRAGG (Chemistry Department, University of Technology, Loughborough, Leicestershire, LE11 3T U ) ( A nalytical Research, Quality Control, The Boots Company Ltd., Pennyfoot Street, Nottingham) ,4n alternative manual procedure is described for the colorimetric deter- mination of phenacetin and paracetamol as indophenol dyes. The procedure differs from that described in Part I1 in that phenacetin and paracetamol are hydrolysed first to p-phenetidine and p-aminophenol, respectively, before reac- tion with hypochlorite to form p-quinonechlorimide, which then undergoes a reaction with phenol.By using solid p-quinonechlorimide as starting material, the molar absorptivity of the indophenol dye formed, a t its wavelength of maximum absorption (625 nm), has been shown to be 2.85 x lo4 1 mol-1 cm-1. With the introduction of the hydrolysis step, complete reaction of phenacetin and paracetamol is attained, which is expedient in any colori- metric procedure. Furthermore, the pH a t which oxidation with hypo- chlorite is effected is not so critical as in the procedure described in Part 11. The main disadvantage of the procedure compared with that in Part I1 is the increased analysis time.The potentialities of the indophenol reaction as a method of determining other pharmaceutical compounds have been investigated. The identification of the products of reaction of the hydrolysed compounds with hypochlorite has not been attempted but the apparent molar absorptivities ( 1041mol-lcm-l) a t 625 nm of the indophenol dye formed in each instance are as follows: p-aminobenzoic acid, procaine and benzocaine, 1.65 ; aniline and acetanilide, 2.11 ; sulphanilic acid, 1.01 ; sulphaguanidine, 1-07 ; sulphathiazole, 1.00; sulphanilamide, 0.49; folic acid, 0.76; o-aminobenzoic acid, 0.47; and ametho- caine, 0.29. AUTOMATIC^ and manual2 colorimetric procedures for the determination of paracetamol and phenacetin based on the indophenol reaction were described in Parts I and I1 of this series.The compound to be determined is made to react with acidified hypochlorite solution to form a quinonechlorimide, excess of hypochlorite is reduced with arsenic(II1) and the quinone- chlorimide is made to react with phenol to form an indophenol dye. The apparent molar absorptivities at the wavelength of maximum absorption (625 nm) of the indophenol dyes formed from phenacetin and paracetamol were different, being 1.79 x lo4 and 2.26 x 104 1 mol-1 cm-1, respectively.2 This difference could be due either to the formation of different indophenol dyes or to the incomplete reaction of one or both drugs. The present study was made in order to try to distinguish between these alternatives, and to extend the method to the determination of other pharmaceutical compounds. EXPERIMENTAL Absorbance measurements were made with a Uvispek 700 spectrophotometer in 1-cm Wavelength calibration was carried out with a didymium glass filter and the absorbance cells.scale was checked by means of standard neutral filters. * For details of Part I1 of this series, see reference list, p. 18. @ SAC and the authors.DAVIS, FOGG, THORBURN BURNS AND WRAGG 13 PREPARATION OF QUINONECHLORIMIDE INTERMEDIATES- p-Quinonechlorimide was prepared by the method of Gibbs.2 $-Aminophenol (5 g) was dissolved in 10 ml of concentrated hydrochloric acid P l ~ s 50 ml of water and 50 g of crushed ice was added. The cold mixture was then added slowly to 100 ml of sodium hypo- chlorite solution containing 12 to 14 per cent.of available chlorine and which also contained crushed ice. The addition of excess of $-aminophenol, indicated by the appearance of a permanent blue or dark brown colour, was avoided. 9-Quinonechlorimide separated as a yellow solid, which was washed on a filter with cold distilled water until free from the odour of chlorine. After being partially dried in air, the crystals were completely dried at 40 "C in an oven. Recrystallisation from light petroleum (boiling range 40 to 60 "C) gave yellow needles in 95 per cent. yield (m.p. 84 to 85 "C). Paracetamol and phenacetin (3 g) were refluxed separately with 100 ml of concentrated hydrochloric acid for 40 minutes. On adding the cooled solution to 150 ml of sodium hypo- chlorite solution containing crushed ice, a yellow crystalline product was obtained in each instance. The products were identified as 9-quinonechlorimide from their melting-points and infrared spectra.P-Quinonechlorimide is the expected product from paracetamol, as the latter compound is hydrolysed to @-aminophenol. 9-Phenetidine, formed by hydrolysis of phenacetin, clearly loses its ethoxy group on oxidation with hypochlorite. FORMATION OF INDOPHENOL DYE FROM 9-QUINONECHLORIMIDE- The formation of indophenol dye from 9-quinonechlorimide under the conditions of the colorimetric determination was studied. $-Quinonechlorimide (100 mg) was dissolved in 25 ml of ethanol and the resulting solution was diluted to 100 ml with water in a calibrated flask. A dilute standard solution was then prepared by a fifty-fold dilution of this solution with water.A 10-ml amount of the dilute standard solution plus 2 ml of 6 per cent. m/V phenol solution were placed in a calibrated flask and diluted to 50 ml with borate buffer (pH 10.0). The solution developed a green tinge owing to the formation of a second reaction product (Amax. = 394 nm) in addition to the indophenol dye (AmaL = 625nm). After the colour had been allowed to develop for 1 hour, the apparent molar absorptivity based on the absorbance at 625 nm was 1.62 x lo4 1 mol-l cm-1 and that at 394 nm was 1-05 x lo4 1 mol-l cm-l. When the solution was heated in a water-bath, the rate of colour development increased and the height of the peak at 625 nm was increased with a concomitant decrease in that of the peak at 394 nm (see Table I).The results of a study of the effect of the pH of the solution on colour development at room temperature are given in Table 11. Over the pH range 9.2 to 9.6, high apparent molar absorptivities were attained; a higher proportion of the indophenol colour compared with that of the second reaction product was formed at the lower pH values within this range. Absorbance measurements were made on this solution. The reaction was very slow. TABLE I fi-QUINONECHLORIMIDE (UNCATALYSED REACTION) EFFECT OF TEMPERATURE ON THE FORMATION OF INDOPHENOL DYE FROM Apparent molar absorptivity after 30 minutes/l mol-1 cm-l x 10-4 Temperature of A -l heating/'C 394 nm 625 nm 50 0.66 2.04 100 0.43 2-25 Several catalysts have been suggested for indophenol reaction^.^-^ We found mangan- ese(I1) to be effective, but the peak at 394 nm persisted and a cloudiness was formed owing to precipitation of manganese(I1) hydroxide.Ethylenediaminetetraacetic acid and acetone were both found to be ineffective under the experimental conditions used in this work. The rate of colour development in the procedure described in Part 112 was rapid compared with the results obtained so far in this work. The only reagent missing from the solutions used in this work was arsenic(II1) , which had been used in the earlier work to reduce excess of hypochlorite in the recommended procedure; arsenic(II1) had been omitted so far in this14 DAVIS et al. : A COLORIMETRIC METHOD FOR THE TABLE I1 [Analyst, Vol.99 EFFECT OF PH ON THE FORMATION OF INDOPHENOL DYE FROM 9-QUINONECHLORIMIDE AT ROOM TEMPERATURE (UNCATALYSED REACTION) Apparent molar absorptivity/l mol-1 cm-l x 10-4 r h > After 2 hours After 24 hours After 3 days --- PH 394nm 625 nm 394 nm 625 nm 394 nm 625 nm 11-74 0-74 0-44 0.67 0.22 0.56 0.05 9-62 1.00 1-69 1.02 1.70 - - 9-55 0.90 1-77 0.90 1.94 - - 9.44 0-78 1.95 0.79 1.94 - - 9.24 0.37 1.55 0.56 2.08 - - 9-58 0.00 0.08 0.18 0.57 0.22 0.95 work as no hypochlorite had been added. However, on studying the effect of arsenic(II1) on the rate of formation of indophenol dye, it was found to be an extremely efficient catalyst and that no peak was obtained at 394 nm when it was used. The effect of pH on the formation of indophenol dye at room temperature when 2 ml of 4 per cent.sodium arsenite solution was added to the solution described earlier is shown in Table 111. In the pH range 9.8 to 10.4, a constant apparent molar absorptivity a t 625 nm of 2.85 x lo4 1 mol-l cm-l was obtained after 30 minutes. This value was the highest apparent molar absorptivity obtained, and, for the purposes of this paper, has been assumed to be the true value of the molar absorptivity a t this wavelength of the indophenol dye formed. The coefficient of variation calculated from results of absorbance measurements on ten solutions at pH 10 was less than 1 per cent. TABLE I11 EFFECT OF PH ON THE FORMATION OF INDOPHENOL DYE FROM $-QUINONECHLORIMIDE AT ROOM TEMPERATURE [ARSENIC(III)-CATALYSED REACTION] Apparent molar absorptivity a t 625 nm/l mol-l cm-1 x 10-4 A r > PH After 7 minutes After 20 minutes After 30 minutes 8.8 0.64 1.21 1.49 9.16 1.45 2.44 2-67 9-50 1.74 2.52 2-68 9-83 2.30 2-82 2-85 9.96 2-52 2.83 2.85 10.46 2.64 2.83 2-85 REACTION OF PARACETAMOL AND PHENACETIN WITH ACIDIC HYPOCHLORITE- 9-Quinonechlorimide is the product of the reaction of 9-aminophenol and P-phenetidine with hypochlorite.This reaction does not prove conclusively, however, that $-quinone- chlorimide is the product of the reaction of paracetamol and phenacetin with acidified hypo- chlorite solution, as the possibility of introducing a chlorine atom into the benzene ring cannot be ruled out. The following ultraviolet study of the reaction with acidified hypochlorite was carried out in order to obtain further information on this aspect.The wavelength of maximum absorption of p-quinonechlorimide in aqueous solution a t pH 3.4 in the ultraviolet region is 287 nm and its molar absorptivity at this wavelength was found to be 2.13 x lo4 1 mol-1 cm-l. Solutions of paracetamol and phenacetin treated with acidified hypochlorite solution also developed a peak at 287 nm. At this wavelength, the molar absorptivity of both paracetamol and phenacetin is only 0.12 x l o 4 1 mol-1 cm-l. It was possible, therefore, to determine by ultraviolet spectrophoto- metry the amount of 9-quinonechlorimide formed (assuming this to be the product) during the reaction of hypochlorite with paracetamol and with phenacetin, with a negligible error due to absorption by the unreacted paracetamol and phenacetin (see Table IV).The procedure for the determination of paracetamol and phenacetin described in Part I1 was followed until just before the addition of arsenic(II1) to reduce excess of hypochlorite. These solutions were then made up to volume and absorbance measurements were made at 287 nm. The results obtained are given in Table IV, and are compared with the resultsJanuary, 19741 DETERMINATION OF PHENACETIN AND PARACETAMOL. PART 111 15 obtained using the full indophenol reaction as described in Part 11. The extents of reaction as determined by the absorption of p-quinonechlorimide at 287 nm and of the indophenol dye at 625 nm are in very good agreement and, although not conclusive, these results strongly suggest that the low apparent molar absorptivities obtained by using the procedure described in Part I1 are due to the incomplete hydrolysis of paracetamol and phenacetin, and that $-quinonechlorimide is the only product of the reaction of these compounds with hypochlorite.TABLE IV EXTENT OF REACTION OF HYPOCHLORITE WITH PARACETAMOL AND PHENACETIN Formation of p-quinonechlorimide A I 3 Formation of Apparent molar Extent of reaction, indophenol dye absorptivity a t per cent. 287 nm/l mol-l r-A-, Extent of reaction, - per cent. Compound cm-l x lo-* A* Bt p-Quinonechlorimide 2-13:: Paracetamol 1.71 Phenacetin 1.47 - 100 80 79 a2 69 67 65 - * Uncorrected for absorption by unreacted paracetamol or phenacetin. Corrected for absorption by unreacted paracetamol or phenacetin. True molar absorptivity. FORMATION OF INDOPHENOL DYE FROM $-AMINOPHENOL, AND, AFTER HYDROLYSIS, FROM PARACETAMOL AND PHENACETIN- The full procedure described in Part 11, vix., treatment with hypochlorite, removal of excess of hypochlorite with arsenic(II1) and reaction with phenol at pH 10, was carried out on $-aminophenol.As no hydrolysis reaction took place in this instance, it was expected that complete formation of the indophenol dye would be achieved and, indeed the apparent molar absorptivity at 625 nm was found to be 2.72 x lo4 1 mol-l cm-l. This value is about 3 per cent. lower than that obtained when starting from 9-quinonechlorimide but the coefficient of variation was again less than 1 per cent. Studies were next made of the time required for the complete hydrolysis of paracetamol and phenacetin by using concentrated hydrochloric acid.The hydrolysis procedure and subsequent formation and measurement of indophenol were carried out as described in the recommended procedure below. The results are shown in Table V; a hydrolysis time of 20 to 25 minutes was sufficient to obtain a molar absorptivity of 2.76 x lo4 1 mol-1 cm-1, i.e., complete reaction. TABLE V EFFECT OF TIME OF HYDROLYSIS OF PARACETAMOL AND PHENACETIN WITH CONCENTRATED HYDROCHLORIC ACID ON FORMATION OF INDOPHENOL DYE Compound Paracetamol . . Phenacetin Reflux time/ minutes 5 10 12 15 20 3 5 10 12 15 20 Apparent molar absorptivity a t 625 nm/l mol-l cm-1 2-51 2.69 2.78 2.76 2.76 2.10 2-25 2-61 2.69 2.75 2.76 x 10-4 In the procedure described in Part 11, the pH of the hypochlorite solution is highly critical (see Fig.4 in Part 11), which is to be expected if hydrolysis has to precede the reaction of paracetamol and phenacetin with hypochlorite. When the paracetamol and phenacetin16 DAVIS et al.: A COLORIMETRIC METHOD FOR THE [Analyst, Vol. 99 are completely hydrolysed before the reaction with hypochlorite, reaction with hypochlorite is complete provided that the pH of the hypochlorite solution is less than 6. This result is shown in Fig. 1, which also shows a titration curve for the acidification of a commercial sodium hypochlorite solution. 06 L i 6.0 2 3 4 5 6 7 8 9 10 11 pH of sodium hypochlorite - hydrochloric acid mixture Fig. 1. Curve A (left-hand axis) : effect of pH of hypochlorite solution on the completeness of the reaction of hypochlorite with hydrolysed paracetamol and phenacetin, as measured by the absorbance of the final indophenol dye solution.Curve B (right-hand axis) : typical curve for the titration of alkaline hypochlorite solution with 0.1 M hydrochloric acid Reaction times of 5 minutes with the hypochlorite and 10 minutes with arsenic(II1) In the present work, a reaction time of 1 minute was found were recommended in Part 11. to be sufficient in both instances. REAGENTS- Sodium hypochlorite solution containing 0.4 per cent. m/V of available chlorine-Prepare by dilution of a commercial sample containing about 16 per cent. m/V of available chlorine. Borate bzc$er solution (PH about 9-9)-Dissolve 20 g of boric acid, 24 g of potassium chloride and 11 g of sodium hydroxide in 2 litres of water.Hydrochloric acid, concentrated and 0.1 M. Sodium arsenite solution, 4 per cent. m/V. Phenol solution, 6 per cent. m/V. PROCEDURE- Pre-determine the amount of 0.1 M hydrochloric acid required to adjust the pH of 5 ml of the sodium hypochlorite solution to about pH 3.5 (the pH of the solution containing hypo- chlorite and hydrolysed sample must be less than 6). Transfer an accurately weighed amount of sample, containing up to 500 mg of paraceta- mol or 550 mg of phenacetin, into a flat-bottomed 50-ml conical flask fitted with a reflux condenser, add 10 ml of concentrated hydrochloric acid and reflux the mixture for 20 to 25 minutes on a hot-plate. Cool the solution, add 25 ml of ethanol, transfer the mixture into a 100-ml calibrated flask and dilute to volume with water.Transfer 25 ml of this solution by pipette into a 250-ml calibrated flask and dilute to volume with water. Transfer 10 ml of this solution by pipette into a second 250-ml calibrated flask and dilute to volume with water. To a 50-ml calibrated flask, add 5 ml of sodium hypochlorite solution and a pre-determined amount of 0.1 M hydrochloric acid so that the pH lies within the range 3 to 5. Add 10 ml of the diluted sample solution from a pipette held just above the surface of the hypochlorite solution, then mix and allow the solution to stand for 1 minute. Add 2 ml of sodium arsenite solution, METHODJanuary, 19741 DETERMINATION OF PHENACETIN AND PARACETAMOL. PART 111 17 mix and allow the solution to stand for 1 minute. Add 2 ml of phenol solution, dilute the solution to 50ml with borate buffer solution, then mix and allow the solution to stand for 30 minutes.Rectilinear calibration graphs are obtained when pure samples of paracetamol and phena- cetin are used. The coefficients of variation (ten determinations) obtained when this pro- cedure was applied to 300 mg of pure paracetamol and to 300 mg of pure phenacetin were 0.5 and 0.7 per cent., respectively. The molar absorptivity in both instances was 2-78 x lo4 1 mol-1 cm-l. The procedure was tested for the determination of paracetamol in an aspirin - paraceta- mol tablet mix previously analysed in this 1aborato1-y.~ The paracetamol content determined by the present method was found to be 42.5 per cent. m/m with a coefficient of variation of 0.5 per cent.(six determinations), which agrees well with the previous results (e.g., 43.5 per cent. m/m by a direct ultraviolet absorption procedure). APPLICATION OF THE INDOPHENOL METHOD TO OTHER COMPOUNDS Measure the absorbance of the solution at 625 nm against water in l-cm cells. Many other compounds are known to react with hypochlorite and to give indophenol dyes, and a brief study was made of some of these reactions. The procedures used were ana- logous to that described above, and the results obtained are shown in Table VI. Procaine and benzocaine, which are hydrolysed to $-aminobenzoic acid before reaction with hypo- chlorite, naturally give the same indophenol dye as $-aminobenzoic acid. Similarly, the reaction is equally sensitive for both acetanilide and aniline. TABLE VI APPARENT MOLAR ABSORPTIVITIES AT 725 nm OF INDOPHENOL DYES FORMED FROM OTHER PHARMACEUTICAL COMPOUNDS Hydrolysis time/ Compound minutes p-Aminobenzoic acid 0 10 Procaine .. .. 15" Benzocaine 10* Aniline .. .. Acetanilide . . .. Sulphanilic acid Sulphanilamide . . Sulphaguanidine Sulphathiazole Sulphadiazine Sulphadimidine Folic acid o-Aminobenzoic acid Amethocainet .. 0 25* 0 25 0 10 0 30 0 30 0 30 0 30 60 0 0 10" Apparent molar absorptivity/ 1.73 1.65 1.65 1.65 2.11 2.1 1 1-03 1.01 0.52 0.48 1.07 1.05 0.93 1.00 0.37 0.94 -0.2 1 0-75 0.76 0.47 0.12 0.29 1 niol-l cm-l x * Apparent molar absorptivity effectively constant at and above these hydrolysis times. t A yellow colloid that was formed on adding hypochlorite was redissolved by adding ethanol. DISCUSSION In the alternative manual colorimetric procedure for the determination of paracetamol and phenacetin described in this paper, complete formation of the indophenol dye appears to be effected, and the procedure is precise and reliable.It is not certain whether the indo- phenol dye formed is the parent indophenol or one of its derivatives, e.g., a chloro derivative. Corbett* gives the wavelength of maximum absorbance of the parent indophenol anion as 637 nm with a molar absorptivity of 3.16 x lo4 1 mol-l cm-l, which differs considerably18 DAVIS, FOGG, THORBURN BURNS AND WRAGG from the results obtained in the present work (625 nm, 2-85 x lo4 1 mol-l cm-l). On the other hand, Corbett gives the molar absorptivity of the 2-chloroindophenol anion as 2-82 x lo4 1 mol-l cm-l at the wavelength of maximum absorption (630 nm).When paracetamol and phenacetin are made to react with acidified hypochlorite solution according to the manual colorimetric procedure described in Part 11, a peak appears in the ultraviolet absorption spectrum of the resulting solutions at the same wavelength as that for +-quinonechlorimide (287 nm) . When these solutions undergo a further reaction with phenol, the ultraviolet absorption spectrum of the solution formed has a peak at the same wavelength (625nm) as that of the indophenol dye formed in the procedure described in this paper. The apparent molar absorptivities at both 287nm and 625 nm are consistent with an 82 per cent. reaction of paracetamol and a 65 per cent. reaction of phenacetin. This result is not conclusive proof, however, that the same indophenol dye is formed in this reaction, and that the lower apparent molar absorptivities obtained are caused by incomplete reaction.The same result would be obtained by the complete formation of other substituted quinone- chlorimides and indophenols with molar absorptivities in the same ratio as p-quinonechlori- mide and the indophenol dye formed here, and with the same peak wavelengths. Although this seems improbable, nevertheless, Corbett8 has shown that the spectra of substituted 9- quinonemonoimines and indophenols do not differ greatly from those of the parent compounds. Despite the greater uncertainty concerning the reactions that take place, the manual pro- cedure described in Part I1 has the major advantage over the present method that the reaction with acidified hypochlorite solution takes place in the cold and no previous refluxing with acid is necessary in order to hydrolyse the paracetamol and phenacetin. This advantage makes the procedure more convenient for routine use and it is especially suitable for use in an automated procedure, such as that described in Part I. Information on sensitivities is given with regard to the determination of other pharma- ceutical compounds, including procaine and sulphonamides, by using procedures similar to that described for the determination of paracetamol and phenacetin. Preliminary results with these reactions have been highly reproducible. The reactions could form the basis of manual procedures as indicated here, or of automated procedures similar to that described in Part I. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. NOTE-References 1 and 2 are to Parts I and I1 of this series, respectively. Murfin, J. W., Analyst, 1972, 97, 663. Murfin, J. W., and Wragg, J. S., Ibid., 1972, 97, 670. Gibbs, H. D., J . Bid. Chem., 1927, 72, 649. Russell, J. A., Ibid., 1944, 156, 457. Crowther, A. B., and Large, R. S., Analyst, 1956, 81, 64. Tetlow, J. A., and Wilson, A. L., Ibid., 1964, 89, 453. Fogg, A. G., Sausins, P. J., and Smithson. J. R., Analytica Chim. Acta, 1970, 49, 342. Corbett, J. F., J . Chem. SOC., B, 1970, 1502. Received May 31st, 1973 Accepted August 14th, 1973