Analyst, October, 1979, Vol. 104, PP. 919-927 919 Spectrop hotomet ric Determination of Acetaminophen and Salicylamide Through Nitrosation and Subsequent Chelation Saied F. Belal, M. Abdel-Hady Elsayed, A. Elwalily and H. Abdine Deflartment of Pharmaceutical Analytical Chemistry, Faculty of Phavmacy, Uvzivevsity o f A lexandvia, Alexandria, Egypt A nitrosation reaction has been adopted for the spectrophotometric deter- mination of acetaminophen and salicylamide. The selectivity of the reaction is increased through utilisation of the nitroso derivatives as chelating agents for cobalt(I1) and copper(I1) ions. The optimum experimental conditions for the application of nitrosation and nitrosation with subsequent chelation were established. The proportions of reactants in each method and the instability constants for the products were determined.The nitroso deriva- tives and their chelates obey Beer’s law and their absorbances were used for the determination of acetaminophen and salicylamide in pharmaceutical formulations. The proposed methods gave more accurate results than the official methods. Keywords : Spectrofilzotovtzetvy ; acetaminophen determination ; salicylavtzide determination ; nitroso derivatives ; chelates The official compendia describe an ultraviolet spectrophotometric assay for the determination of acetaminophen1 (N-acetyl-9-aminophenol) and non-aqueous titration for the determina- tion of salicylamide.2 The former is highly sensitive to diverse co-existing substances that are often present in pharmaceutical formulations, while the latter is not specific.Various colour reactions have been utilised for the determination of acetaminophen through its hydrolysis and subsequent reaction with alkaline 2-naphth01,~ diazotisation and ~oupling,~ or condensation with ani ill in,^ anisaldehyde,6 p-dimethylamin~benzaldehyde~ or $-dimethylaminocinnamaldehyde.8 Other spectrophotometric methods were based on the reaction of acetaminophen with phenol together with an oxidising ion such as hypobr~mite,~ hypochlorite in the presence of meta-arsenitelO or hexacyanoferrate(1II)ll; nitration of acetaminophen has also been utilised for its spectrophotometric determination.l21l3 How- ever, these methods lack the simplicity needed for routine analysis. The fluorimetric methods were developed either by oxidation of acetaminophen with alkaline he~acyanoferrate(III)~*J~ or hydrolysis followed by reaction with benzylamine.16 These methods are subject to interferences, the materials present in syrup formulations causing the most interference. Acetaminophen has also been determined by cerimetric titration,17,18 gravimetry,lg titra- tion of the hydrolytic product with nitrite20 or polarography.21 These methods are not sufficiently sensitive.The different spectrophotometric methods proposed for the determination of salicylamide were based on its reaction with iron(II1) potassium hexacyanoferrate(II1) ,24 potassium hexacyanoferrate(II1) plus dimethyl-fl-phenylenediamine,25,26 and 3,5-dichloro-P- benzoquinone ~ h l o r i m i n e . ~ ~ ~ ~ 8 Non-aqueous t i t r a t i ~ n , ~ , ~ ~ iodimetric titration30 and complexometric titration31 have been employed for the assay of salicylamide.Inamdar and Kadji32 utilised the nitrosation of acetminophen for its determination in dosage forms. They reported that the stability of the chromogen produced in an acidic medium was increased by the addition of alcohol. claimed that a nitro rather than nitroso derivative was produced on treatment of acetaminophen with nitrous acid. In this work, the chromogen was adapted to the determination of acetaminophen in some commercial preparations purchased locally. More important, the nitroso derivative was used to form chelates with cobalt(I1) and copper(I1) ions. The formation of metal chelates Other920 BELAL et al. : SPECTROPHOTOMETRIC DETERMINATION OF Analyst, VoZ. 104 of the reaction product indicates that the formation of a nitroso derivative of acetaminophen is more probable than the nitro derivative.Furthermore, application of this type of reaction was extended to the determination of salicylamide. Experimental Apparatus and Reagents All reagents were of analytical-reagent grade. Acetaminophen (BP 1973). Chemical Industries, Egypt. Salicylamide. Spectrophotometer. p H meter. Pye, Model 79. Supplied by the Alexandria Company for Pharmaceutical and Supplied by CID Company, Cairo, Egypt. A Prolabo photoelectric spectrophotometer with 1-cm silica or glass cells. General Procedure for Nitrosation (Colour Development and Preparation of Standard Calibration Graphs) A cetaminophen Transfer 5.0 ml of the assay solution (containing 0.5 mg of acetaminophen) into a 25-ml calibrated flask, add 2 ml of 3% sodium nitrite solution and 1 ml of 1 N hydrochloric acid.Mix and leave to stand for 5 min. Render alkaline with 3 ml of 1 N sodium hydroxide solution. Dilute to volume with water and measure the absorbance at 430nm using a blank prepared in the same way, but omitting the drug substance. Calculate the concentration of acetaminophen using the linear equation in Table I, which describes the calibration graph prepared by applying the same procedure to solutions of acetaminophen reference standards, in the concentration range 0.004- 0.036 mg ml-l. Mix and leave to stand for a further 5 min. TABLE I EXPERIMENTAL PARAMETERS FOR THE UTILISATION OF THE NITROSO DERIVATIVES AND THEIR CHELATES IN QUANTITATIVE ANALYSIS Concentration Regression equation Percentage Compound Derivative Medium A,.range/mg-% A = a + b C fits Acetaminophen . . . . o-Sitroso Alkaline 430 0.4-3.6 A = 0.0056 + 0.2411C 99.68 Cobalt(11) chelate Chloroform 400 0.4-3.6 A = 0.0044 + 0.02105C 99.97 Copper(I1) chelate Aqueous 535 10.0-80.0 A = 0.005 8 + 0.021 22C 99.97 Salicylamide . . . . p-IVjtroso Acidic 398 0.2-2.0 A = 0.0503 + 0.4515C 99.91 p-h I troso Alkaline 393 0.1-1.0 A =z 0.0147 + 0.9766C 99.74 Copper(I1) chelate Aqueous 520 4.0-40.0 A = 0.0080 + 0.0257C 99.47 Salicylamide Add 2 ml of 3% sodium nitrite solution followed by 0.1 ml of 1 N hydrochloric acid. Heat the mixture over a boiling water-bath for 5 min and then cool. Next, either transfer directly into a 25-ml calibrated flask (acidic method) or add 1 ml of 1 N sodium hydroxide solution and then transfer into a 25-ml calibrated flask (alkaline method).Dilute to volume with water and measure the absorbance at A,,,. 398 nm using a blank prepared in the same way, but omitting the drug substance. Calculate the concentration of salicylamide using the linear equations (see Table I), which describe the calibration graphs prepared by applying the same procedure to salicylamide reference standards in the concentration ranges 0.002-0.02 and 0.001-0.01 mg ml-l for the acidic and alkaline methods, respectively. Molecular ratio of reactants solution with the reagents, according to the volumes and concentrations given in Table 11. Transfer 5.0 ml, containing 0.3 mg of salicylamide, into a 25-ml beaker.In a 25-ml calibrated flask mix the standard solutions of the drugs and sodium nitrite General Procedure for the Formation of Cobalt( 11) Chelate of the Nitroso Derivative of Acetaminophen and Preparation of Standard Calibration Graph Into a 100-ml separating funnel transfer 5.0 ml of the assay solution (containing 0.5 mg of drug), and add 2 ml of 3% sodium nitrite solution and 1 ml 1 N hydrochloric acid. MixOctober, 1979 ACETAMINOPHEN AND SALICYLAMIDE BY NITROSATION AND CHELATION 921 and leave to stand for 5 min. Extract the chelate three times with a total volume of 25 ml of chloroform. Measure the absorbance of the chloroform extract at 400 nm against a blank, prepared in the same way but omitting the drug substance.Calculate the concentration of acetaminophen using the linear equation (Table I), which describes the calibration graph prepared by applying the same procedure to solutions of acetaminophen reference standards in the concentration range 0.004-0.036 mg ml-l. Add 5 ml of a 0.125 M solution of cobalt(I1) bromide. TABLE I1 REAGENTS AND THEIR AMOUNTS USED IN THE DETERMINATION OF THE MOLAR RATIO These reagents are added to 25-ml calibrated flasks along with 2 ml of 304 sodium nitrite solution and 1 N hydrochloric acid (0.3 ml for acetaminophen and 0.5 ml for salicylamide), and made up to 25 ml with water. No. of flask r A Reagents 1 2 3 4 5 6 7 8 9 V,/ml of drug solution* . . . . . . 0.5 1.0 1.5 2 2.5 3 3.5 4 4.5 V,/ml of copper acetate solutionf . . . . 4.5 4 3.5 3 2.5 2 1.5 1 0.5 Acetic acid solution (2% V/V)/ml .. . . - 0.5 1.0 1.5 2 2.5 3.0 3.5 4.0 * The molar concentration of acetaminophen is 4 x lo-" M and of salicylamide is 2 x lo-" M. t The copper acetate is solubilised in acetic acid solution ( 2 ml of glacial acetic acid diluted to 100 ml with water). The molar concentration of copper acetate for acetaminophen is 4 x lo-" M and for salicylamide is 2 x M. General Procedure for the Formation of Copper( 11) Chelate of the o-Nitroso Derivatives of Acetaminophen and Salicylamide and Preparation of Standard Calibration Graphs Into a wide test-tube transfer 10.0 ml of the assay solution containing 10 mg of acetamino- phen (or 5 mg of salicylamide). Add 2 ml of 3% sodium nitrite solution and 2 ml of 1% copper(I1) acetate solution.Mix well and add 0.3 ml of 1 N hydrochloric acid (0.5 ml with salicylamide). Cool and quantitatively transfer into a 25-ml calibrated flask. Dilute to volume with water and measure the absorbance at the corresponding A,,,. (535 nm for acetaminophen and 520 nm for salicylamide) against a blank, prepared in the same way but omitting the drug substance. Calculate the concentration of acetaminophen and salicylamide using the linear equation (Table I), which describes the calibration graphs prepared by applying the same procedure to solutions of acetaminophen and salicylamide reference standards in the concentration ranges 0.1-0.8 and 0.04-0.4 mg ml-l for acetaminophen and salicylamide, respectively. Molecular ratio of reactants solution with the reagents, according to the volumes and concentrations given in Table 11.Place the reaction mixture in a boiling water-bath for 25 min. In a 25-ml calibrated flask mix the standard solutions of the drugs and sodium nitrite Preparation of Assay Solutions for Real Analysis Tablets one tablet, into a 100-ml calibrated flask. dilute to volume with water. filtrate, after a suitable dilution, as mentioned under the general procedure. Weigh and powder 20 tablets. Transfer an accurately weighed amount, equivalent to Dissolve as completely as possible in water and Treat the Filter and discard the first portion of the filtrate. Syrups or drops general procedure. Dilute an aliquot volume with water to attain a concentration suitable for use in the922 BELAL et a,?. : SPECTROPHOTOMETRIC DETERMINATION OF A?Zab.St, VOz.104 Calculation of the concentration in any of the above formulations was made using the Re-standardisation for such equations was occasionally regression equations given in Table I. checked. Wavelengthhm Fig. 1. Absorption curves of nitroso derivatives of: A, acetaminophen (1.75 mg-yo) in acidic medium (-) and alkaline medium (- - - -) ; B, salicylamide ( 1 mg-yo) in acidic medium (. . . .) and in alkaline medium (-.-.-)- Results and Illiscussion Nitrosation Reaction Acetaminophen and salicylamide, owing to the presence of aromatic hydroxy groups, are capable of forming nitroso derivatives (yellow chromogen) by reaction with sodium nitrite in an acidic medium. To investigate the molecular ratio of the reactants, Job’s method of continuous variation34 was employed.Lines A in Figs. 2 and 3 show that the maximum absorption occurs when the ratio of the reagent to the drug is 1 : 1. This is confirmed qualitatively by carrying out a test (coupling with a diazonium salt) €or the presence of the second free position ortho to the hydroxy group; Absorption curves are shown in Fig. 1. 0.6 Q) 0 (CI * 0.4 0, 2 0.2 0 0.2 0.4 0.6 0.8 1 .o 0 0.2 0.4 0.6 0.8 1.0 VM VM -t vL VM V 7 Fig. 2. Continuous variation graph for : A, nitrous acid - acetaminophen ; Fig. 3. Continuous variation graph for: B, o-nitroso-p-acetamidophenol - copper( I I) A, nitrous acid - salicylamide; B, o-nitro- chelate. sosalicylamide - copper(I1) chelate.October, 1979 ACETAMINOPHEN AND SALICYLAMIDE BY NITROSATION AND CHELATION 923 therefore, an 0- or $-nitroso derivative is indicated.( K J , calculated by the Harvey and Manning equation,35 are 1.01 x which indicate the high stability of these nitroso derivatives : Their respective instability constants and 3.68 x 10-5, (.'C) (ba'C)b c (1 - a') .Ki = where a' = degree of dissociation, C = molar concentration and b = number of molecules of the reagent. Compound I1 is unstable in acidic medium but can be stabilised by addition of methan01.3~ It was found that alkalinisation of the medium resulted in the formation of a highly stable compound and a bathochromic shift, together with hyperchromic effect. This is probably due to the contribution of an extra pair of unshared electrons in the interaction with the aromatic nucleus (Scheme A, compounds I11 and 111').OH OH 0- NHCOCH, NHCOCH, NHCOCH, &n ax. 370 nm hrnax.430 nm (1) ( 1 1 ) ( 1 1 1 1 5.-.- NHCOCH, ( I l l ' ) hmax. 400 nrn NHCOCH3 Scheme A $-Nitrososalicylamide (VI) attains its maximum colour intensity and stability when 0.1 ml of 1 N hydrochloric acid is used (as higher or lower values resulted in a decrease in the colour intensity) and when the reaction mixture is heated for 5 min on a boiling water- OH OH 1 N=O CONH;! @ CONH;! N-0 ( V I I ' ) Scheme B924 BELAL et at. : SPECTROPHOTOMETRIC DETERMINATION OF Analyst, VoZ. 104 bath. As compound VI is stable in either acidic or alkaline medium, it is assumed that the chromogen exists in the most stable tautomeric form, the p-quinonoidal structure, Scheme K (compounds VI' and VII'), in both media.This may explain why the absorbance maxi- ma of chromogens of this type do not exhibit a bathochromic shift on alkalinisation of the medium. Nitrosation with Subsequent Chelation In order to increase the selectivity of the nitrosation reaction, our investigation was extended to test for the chelating power of the nitroso derivatives. Nitroso-9-acetamido- phenol is capable of forming a chelate with cobalt(I1) ion, as the chelating agent contains a phenolic OH group (a group with an easily replaceable proton) ortho to the N=O group; the latter offers a lone pair of electrons to the cobalt(I1) ion. This chelate (Scheme A, IV), is sparingly soluble in water but easily extractable into chloroform to give a coloured solution, which can be measured at 370 nm.This is evidence that nitrosation occurs favourably in the para position to the phenolic OH group. Meanwhile, introduction of the nitroso group in the ortho position to the phenolic OH group is accomplished by the action of nitrous acid in the presence of a copper(I1) salt. This copper(I1) salt is essential for stabilising the nitrosyl radical and for ensuring that the ovtho (but not the pava) nitroso derivative is Under such conditions the copper chelates of the ovtho derivative of salicylamidle (with A,,,. 520 nm) and acetaminophen (with A,,,, 535 nm) are formed. The stoicheiometric ratio [o-nitroso derivative to copper( 11)] determined by Job's method of continuous variation is 2 : 1 (lines B in Figs. 2 and 3). The instability constants, calculated using the Harvey and Manning equation, are 4.813 x for acetaminophen and salicylamide, respectively.The maximum intensity of the chelates were obtained by the addition of 1 N hydrochloric acid in volumes of 0.3 ml for acetaminophen and 0.5 ml for salicylamide (Table 111), and by heating the reaction mixture in a boiling water-bath for 25min. Fig. 4 shows their absorption curves. This method is less sensitive than the two other recommended methods, yet it has the advantage of measuring the coloured chelate in aqueous solution without the need for its solvent extraction. On the other hand, p-nitrososalicylamide fails to give a chelate with cobalt(I1) ion. and 2.17 x TABLE 111 EFFECT OF THE VOLUME OF 1 N HYDROCHLORIC ACID ON THE COLOUR INTENSITY OF THE COPPER(II) CHELATES OF THE DRUGS" Volume of 1.0 N HCl/ml 0.05 0.I 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Measured pH* of the final diluted solution 4.8 4.4 4.1 3.78 3.00 2.48 2.40 2.34 2.13 2.00 1.79 Acetaminophen chelate, Amax. 535 nm 0.300 0.470 0.780 0.960 t t t t 0.000 0.000 0.000 Salic ylamide chelate, Amax. 520 nm 0.086 0.186 0.280 0.332 0.440 0.468 0.400 0.372 0.214 0.133 0.000 * Concentration of acetaminophen 80 mg-?(, and salicylamide 20 mg-%. Precipitate formed. Methods of Assay The high stability of the nitroso derivatives of acetaminophen and salicylamide, and their chelates, stimulated further investigation to develop spectrophotometric methods for the assay of these drugs.October, 1979 ACETAMINOPHEN AND SALICYLAMIDE BY NITROSATION AND CHELATION 925 0.6 C *-‘ .- 0.4 0 2 a 0.2 \ \-- - 480 520 560 600 Wavelengthhm Fig.4. Absorption curves of copper- (11) chelates of o-nitroso derivatives of acetaminophen (-) and salicyl- amide (- - - -) Standard calibration graphs for different derivatives were prepared by making serial dilutions of acetaminophen or salicylamide and applying the conditions specified under the corresponding General Procedure. Beer’s law is valid within the concentration ranges of acetaminophen or salicylamide calculated in the final dilution, given in Table I. Using the method of least squares3’ the regression equations for the different calibration graphs were derived and utilised for the calculation of unknown concentrations in dosage forms. The percentage fits38 for different calibration graphs were calculated (Table I).The validity of the regression equations was tested by analysing laboratory-made tablets. The results obtained had good accuracy and high precision (Tables IV and V). These results, including the precision of replicates, the agreement between the three versions of the recommended spectrophotometric methods and the good recovery from synthetic mixtures, encourage the application of the proposed methods in the routine analysis of pharmaceutical preparations of acetaminophen and salicylamide. Subjecting the results of the proposed method and pharmacopoeia1 methods1p2 to statistical analysis, calculated t exceeds theoretical t ; therefore, the null hypothesis is rejected and there is a significant difference between the results of the two methods,37 the proposed method giving more accurate results (Tables IV and V).TABLE IV APPLICATION OF THE NITROSATION REACTION TO THE ASSAY OF DIFFERENT DOSAGE FORMS O F ACETAMINOPHEN The results for the laboratory-made tablets are the percentage recoveries and the results for the commercial preparations are a percentage of the labelled claim. Mean (yo) and coefficient of variation* Pharmacopoeia1 Nitroso derivative Cobalt(I1) Copper(1i) method, (alkaline method), chelation, chelation, Dosage form n = 12 n = 16 n = 16 n = 16 Laboratory-made tablet . . . . - 100.5 100.5 100.5 Paracetamol tablet . . . . .. 101.0 99.6 99.4 99.5 ‘:;3”.)5 103.5 Paracetamol syrup .. .. 104.7 103.4 Pyral syrup . . .. . . .. 107.5 K . ) 4 105.5 105.4 101.5 Pyral syrup . . .. . . .. 102.3 101.5 101.5 (0.38) (0.3) (0.3) (0.3) (0.3) (0.3) (0.2) (0.3) (1.1) (0.2) (0.2) (0.4) (0.7) (0.2) (0.3) (0.3) (0.2) * The figures in parentheses are the coefficients of variation and n is the number of experiments.926 BELAL et al.: SPECTROPHOTOMETRIC DETERMINATION OF Analyst, VoZ. 104 TABLE V APPLICATION OF THE NITROSATION REACTION TO THE ASSAY OF DIFFERENT DOSAGE FORMS OF SALICYLAMIDE The results for the laboratory-made tablets are the percentage recoveries and the results for the commercial preparations are a percentage of the labelled claim. Mean (yo) and coefficient of variation* , 1 Nitroso derivative Pharmacopoeia1 A , Copper(I1) method, Acidic method, Alkaline method, chelation, n = 12 Dosage form n = 5 n = 12 n = 12 Laboratory-made tablet . . - (0.7) Cidal fort tablet .. . . 99.6 * The figures in parentheses are the coefficients of vaxiation and n is the number of experiments. Although the official ultraviolet spectrophotometric assay for acetaminophen1 is simple, its accuracy is greatly influenced by the interferences from diluents and binders in tablets or colouring matter, sweetening agents and preservatives in syrups and drops. The official non-aqueous titration for salicylamide2 suffers from non-selectivity and low sensitivity. The recommended methods can be applied only to formulations containing either acetamino- phen or salicylamide singly in unit dose preparations. However, the application of the proposed methods for the assay of formulations containing both acetaminophen and sali- cylamide is under investigation.References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. “British Pharmacopoeia 1973,” HM Stationery Office, London, 1973, p. 340. “The Pharmacopoeia of Japan 197 1 ,” Eighth Edition, Society of Japanese Pharmacopoeia, Tokyo, Kos, J., Favmaceutski Glasn., 1966, 22, 51; AnalLyt. Abstr., 1967, 14, 3529. Agarwal, S. P., and Walash, M. I., Indian J . Pharrn., 1974, 36, 47. Vaughan, J . B., J . Pharm. Sci., 1969, 58, 469. D’Sauza, A. A., and Shenoy, K. G., Can. J . Phavm. Sci., 1968, 3, 90. Vishwasrao, D. R., Indian J . Pharm., 1973, 35, 172. Inoue, T., Tatsuzawa, M., Lee, S.-C., and Ishii, T., Eisei Kagaku, 1975, 21, 313; Analyt. Abstr., Welch, R. M., and Conney, A. H., Clin. Chem., 1965, 11, 1064.Davis, D. R., Fogg, A. G., Thorburn Burns, D., and Wragg, J. S., Analyst, 1974, 99, 12. Deodhar, R. D., Shastri, M. R., and Mehta, R. C., Indian J . Pharrn., 1973, 35, 120. Hanegraaff, C., and Chastagner, N., Annls Pharm. Fr., 1969, 27, 663. le Perdriel, F., Hanegroaff, C., Chatcugner, N., and DeMontetry, E., Annls Pharm. Fr., 1968, 26, Kaito, T., Sagara, K., Yoshida, T., and Ito, Y., Yakugaku Zasshi, 1974, 94, 633 and 639; Analyt. Kaito, T., Kasuya, K., and Inoue, T., Bunseki Kagaku, 1971, 20, 801; Analyt. Abstr., 1972, 23, Kaito, T., and Sagara, K., Yakugaku Zasshi, 1974, 94, 639; Analyt. Abstr., 1976, 30, 2E33. Kalinoswka, 2. E., and Hasztar, H., Farrnacja Pol., 1965, 21, 570; Analyt. Abstr., 1965, 13, 7092. Kalinowska, Z. E., and Hasztar, H., Farmacja Pol., 1967, 23, 447; Analyt.Abstr., 1968, 15, 4978. Poethke, W., and Kohne, H., Pharm. Zentralhalle Dtl., 1965, 104, 630; Analyt. Abstr., 1967, 14, Inamdar, M. C., Saboo, J. C., Kamdar, C. N., and Sanghavi, N. M., Indian J . Pharm., 1973, 35, 187. Brockelt, G., Pharmazie, 1965, 20, 136. Zoltai, E., Acta Pharm. Hung., 1967, 37, 74; Analyt. Abstr., 1968, 15, 3551. Ruttkowski, R., Arzneimittel-Forsch., 1954, 4, 209. Ensor, P. C., J, Ass. Publ. Analysts, 1972, 10, 56. Murai, K., Arch. Proc. Pharm., Japan, 1961, 21, 58; Analyt. Abstr., 1963, 10, 3870. Tatsuzawa, M., and Hashiba, S., Bunseki Kagaku, 1968, 17, 478; Analyt. Abstr., 1970, 18, 1218. Joy, J,, and Szekeres, L., Mikrochirn. Acta, 1975, 11, 125; Analyt. Abstr., 1976, 30, 2E30. Szekeres, L., Harmon, R. E., and Gupta, S. K., Microchem. J., 1973, 18, 101; Analyt. Abstr., 1973, 1971, p. 674. 1976, 31, 1E29. 227. Abstr., 1976, 30, 2E33. 2776. 370. 25, 4046.OCtObe?’, 1979 ACETAMINOPHEN AND SALICYLAMIDE BY NITROSATION AND CHELATION 927 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. Grabowska, I., and Weclawska, K., Farmacja Pol., 1976, 32, 363; Analyt. Abstr., 1977, 31, 5E42, Miszczuk-Lucka, B., and Taborska, H., Przem. Chena., 1955, 11, 706; Analyt. Abstv., 1957, 4, 1016. Gagewska, M., and Ciszewsk, M., Acta Pol. Pharm., 1975, 32, 607; Analyt. Absti.., 1976, 31, 1E27. Inamdar, M. C., and Kadji, N. N., Indian J . Pharm., 1969, 31, 79. Chafetz, L., Daly, R. E., Schriftman, H., and Lomner, J . J., J . Pharm. Sci., 1971, 60, 463. Rose, J., “Advanced Physico-Chemical Experiments,” Pitman, London, 1964, p. 54. Harvey, A. E., and Manning, D. L., J . A m . Chew. Soc., 1950, 72, 4488. Finar, I. L., “Organic Chemistry, The Fundamental Principles,” Volume 1, Fifth Edition, Long- Spiegel, M. R., “Theory and Problems of Probability and Statistics,” McGraw-Hill, New York, Davies, 0. L., and Goldsmith, P., “Statistical Methods in Research and Production,” Fourth Edition, mans, Green, London, 1967, p. 676. 1975, pp. 259 and 215. Oliver and Boyd, Edinburgh, 1972, p. 178. Received April 24th, 1978 Amended December 1 lth, 1978 Accepted February 14th, 1978