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A new spectrophotometric method for the determination of primary aromatic amines
T&ma, Vol. 26, pp. 861 to 865 0 Pergamon Press Ltd 1979. Printed
0039.9140/79/0901-0861SO2.00/0 in Great
Britain
A NEW SPECTROPHOTOMETRIC DETERMINATION OF PRIMARY
METHOD FOR THE AROMATIC AMINES
R. RAMAKRISHNAand C. S. PRAKASASASTRY Department of Chemistry, Andhra University, Waltair-3, India (Received 20 September 1978. Accepted 8 January 1979) new spectrophotometric method for the assay of primary aromatic amines with metolchromium(V1) reagent has been developed. The method is simple, reproducible and accurate within + 1.0%. It has been extended to the determination of drugs (in pure samples and pharmaceutical prepSummary-A
arations) which either contain a free primary aromatic amine or release it through hydrolysis or reduction. The method is also applicable to the assay of p-aminobenzenesulphonamides in rat urine and blood. All the sulphonamide derivatives tested, as well as other aniline derivatives with an electron-withdrawing group in the para-position, afford nearly the same molar absorptivity. Depending on the absorptivity, 0.32pmole of amine will give an absorbance of -0.1.
Several spectrophotometric and calorimetric methods of assay for primary aromatic amines are cited in the literature.‘-” All these methods suffer from the disadvantages that (a) they cannot be applied for all types of primary arylamines and (b) the compound of interest or one closely related to it structurally must necessarily be chosen for preparation of the calibration curve. We have now developed a rapid, sensitive and general method for the determination of primary aromatic amines. It is based on our observation that metol (p-N-methylaminophenol sulphate) undergoes oxidation, probably to p-N-methylquinone-imine (&, 520 mn) with potassium dichromate at pH 3.0 only in the presence of primary aromatic amines, and that the concentration of the oxidation product is directly proportional to the concentration of primary aromatic amine initially taken. This method has wide applicability in purity assays of pharmaceutical compounds which possess either a free arylamino group or release it through hydrolysis or reduction. Preliminary experiments show that this method has biological application, in that p-aminobenzenesulphonamides in rat urine and blood have been assayed successfully. EXPERIMENTAL Instrumentation
Spectral and absorbance measurements were made with a Unicam SP600 spectrophotometer with lo-mm glass cells. Time vs. absorbance measurements were recorded with a Shimadzu Double Beam Spectrophotometer (UV-140) and an automatic recorder. Reagents
All solutions were prepared in doubly distilled water. A freshly prepared 0.2% aqueous solution of metol was always used. Potassium dichromate (O.OlM) and sulphanilamide (O.OlM) solutions were prepared. The sulphanilamide was LP. grade, recrystallized and dried. Bu&r solutions. A mixture of 250 ml of 0.2M potassium hydrogen phthalate and 234 ml of O.lM hydrochloric acid was diluted to 1 litre with water (pH 2.90).
Primary aromatic amines. All the arylamines used were of commercially available G.R., LP., or B.P. grade. Their stock solutions were prepared in distilled water, the compounds insoluble in water being dissolved initially in the minimum amount of dilute hydrochloric acid or sodium hydroxide solution necessary. Working solutions were prepared by appropriate dilution of the stock solutions after neutralization of the excess of acid or alkali. The pH of the final diluted solution was in the range 6.CM.O. All the other reagents used were of analytical grade. General procedure
A l-5 ml portion of primary aromatic amine solution (concentration range as indicated in Table 1 and 0.013-0.102 mmole for the compounds in Table 2) was taken. After addition of 15 ml of buffer solution, 2 ml of 0.2% metol solution (freshly prepared) and 3 ml of O.OlM potassium dichromate, the solution was diluted to 25 ml in a standard flask and the absorbance was measured at 520 nm (after 15 min) against a reagent blank prepared in a similar manner. The primary aromatic amine concentration was read from a standard calibration curve prepared with the same compound under identical conditions (the pH of the coloured solutions was found to be 3.0 + 0.03). The pH can also be adjusted to 3.0 with dilute hydrochloric acid instead of buffer solution. Procedure for compounds which yield a primary arylamino group on hydrolysis
About 10 mg of sample were boiled with 10 ml of dilute hydrochloric acid for 1 hr under reflux, cooled, neutralized with sodium hydroxide and made up to 100 ml in a standard flask. The general procedure was then followed. Procedure for compounds which yield primary arylamino group on reduction
About 10 mg of sample were accurately weighed and treated with 10 ml of dilute hydrochloric acid and 0.25 g of zinc dust added in portions. After standing for 1 hr at room temperature, the solution was filtered through cotton wool, the residue was washed with three 5-ml portions of water and the filtrate was neutralized with sodium hydroxide and diluted to 100 ml in a standard flask. The general procedure was then applied. Procedure for pharmaceutical preparations
The sampling and preliminary treatment of pharmaceutical preparations such as tablets, capsules, injections and eye-drops were performed according to the reported pro861
862
R. RAMA KRISHNA and C. S. PRAKASA SASTRY Table 1. Spectrophotometric
Amine Aniline o-Toluidine m-Toluidine p-Toluidine o-Anisidine m-Anisidine m-Chloroaniline p-Chloroaniline p-Bromoaniline o-Aminobenzoic acid p-Aminosalicylic acid p-Aminobenzenesulphonic acid p-Aminobenzoic acid p-Aminoacetophenone Benzocaine Procaine hydrochloride Sulphanilamide Sulphafurazole Sulphamethoxazole Sulphaphenazole
assay of primary aromatic amines Molar absorptivity at 520 nm,
Beer’s law limits, ~125 ml
1 moleC’.cm-’
Recovery, %
x 10m3
75-500 100-600 75-800 100-900 75-800 50-750 50-450 50-500 50-500 50-600 100-700
2.21 1.48 1.85 1.19 2.55 4.05 5.96 4.95 5.65 3.53 2.50
100.8 99.1 100.7 100.9 100.8 99.0 99.5 99.3 99.4 99.4 98.9
60-425 45-350 45-350 50-425 85-675 55425 85-675 80-650 100-800
7.15 7.25 7.30 7.15 7.20 7.35 7.25 7.14 7.18
99.8 99.7 99.4 99.5 99.4 99.8 100.2 99.7 100.4
cedures’,22.23 and the assays were carried out as above, depending on the nature of the compound. Procedure for biological j7uids
A l-ml sample of rat urine or blood was taken and protein was precipitated with trichloroacetic acid (0.5 ml of lo”/, solution). To, the clear centrifugate, a few drops of cont. sodium hydroxide solution were added to adjust the pH to 6.G8.0. Then the proposed method was followed for the assay of free sulphonamide (unmetabolized). To determine the total amount (metabolized and unmetabolized) of the sulphonamide present, the protein-free blood or urine sample was hydrolysed by boiling with 4M hydrochloric acid for 2 hr. The general procedure was then followed after neutralization of the hydrolysate sodium hydroxide. These estimations were carried out simultaneously with Bratton-Marshallz3 assays of the same pooled samples of blood and urine, for comparison. RESULTS AND DISCUSSION
product of met01 obtained under the present experimental conditions and that obtained with iodine monochloride oxidation have been found to have the same maximum, indicating their identity (Fig. 1). At 520 nm the oxidation product has a reasonably high absorbance in comparison with that of Cr(V1). Beer’s law was found to be valid over the concentration range given in the general procedure and Table 1. The results of recovery experiments are also listed in Table 1. The values obtained by the proposed method and the Bratton-Marshall method for pharmaceutical preparations and biological fluids are compared in Tables 2 and 3. To test the reproducibility of the method, 8 replicate determinations were 06
0-0
with
Cr(VI) in
ogoinst
The spectrum (Fig. 1) of the oxidation product of metol with potassium dichromate in the presence of a primary aromatic amine at pH 3.0 has been found to have an absorption maximum at 520 run. The maximum intensity of the colour is developed within 15 min and is stable for at least 1 hr for all the amines studied. The spectrum is identical with that of the product obtained by direct oxidation of met01 by Cr(VI) at higher pH values. Fieserz4 studied the redox behaviour of metol by mixing it with potassium molybdocyanide and deduced rather reasonably that the oxidized species was the p-N-methylquinoneimine. Cihalik and Vavrejnova, who estimated metol by titrating it potentiometrically with iodine monochloride in weakly acidic or neutral media, also assumed the oxidation product of metol to be p-Nmethylquinone-imine, on the basis of their mole-ratio study.Z5*26 The absorption spectrum of the oxidation
^
with
presence
reagent
ICI
against
of
sulphocetomide
blank water
blank
05-
001 400
I
440
480
520 wavelength
Fig. 1. Absorption
560
I
600
640
(nm)
spectra of the oxidation product metol.
of
Determination
863
of primary aromatic amines
Table 2. Assay of pharmaceutical
aromatic primary amines in dosage forms*
Nominal content, g
Dosage form
proposed method
Found, g Bratton-Marshall method
Tablets
Sulphadimidine I.P. Sulphaguanidine I.P. Madribon-Sulphadimethoxine B.P. Lederkyn-Sulphamethoxypyridazine B.P. Sulphapyridine B.P. Sulfuno-Sulphamoxole B.P. Elkosin-Sulphasomidine B.P. Thalazole-Phthalylsulphathiazole 1.P.t Serepax-Oxazepam N.F.t Librium-Chlordiazepoxide 1.P.t Folic acid I.P.g,f
0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.015 0.0100 0.0050
0.500 0.498 0.497 0.508 0.492 0.509 0.505 0.490 0.014 0.0104 0.0048
0.498 0.497 0.497 0.506 0.488 0.493 0.501 0.488 0.0146 0.0105 0.0047
Capsules Chloramphenicol
0.250
0.247
0.248
1.420 1.000
1.40 1.04
1.40 1.01
I.P.9
Eye-drops and injections
Albucid-Sulphacetamide Sulphadiazine B.P.
sodium I.P.
* The results are means of duplicate determinations. t After carrying out hydrolysis. p After carrying out reduction. $ Applying suitable correction for free amine and using p-aminobenzoic acid as standard.
made with a typical primary arylamine (sulphacetamide) and the relative standard deviation was found to be 0.5%. From the results presented in Table 1, it appears that the effects (inductive, mesomeric and steric) of substituents in the positions ortho, meta and para to the amino group of aniline lead to marked variation in the catalytic oxidation of metal. It can be seen that electron-withdrawing groups have greater catalytic activity than electron-donor substituents. However, the low molar absorptivity values for o-aminobenzoic acid and o-nitroaniline (3.53 x 10” and ~2.00 x 10’ l.mole-l.cm-l) when compared to those for the corresponding meta and para compounds may be due to steric hindrance and intramolecular hydrogen bonding. It is interesting that primary aromatic amines bearing electron-acceptor groups (-SOaH, -S02NH2, -SO*NHR, -COOH, -COOR and -COCHJ para to the amino group give almost the same molar absorptivity C(7.20 f 0.25) x lo3 l.mole-’ .cm-‘1. Hence an important advantage is that the calibration curve prepared with such a primary aromatic amine can Table 3. Assay of sulphonamides
Sulphonamide Sulphanilamide Sulphacetamide
be used for the determination of any primary arylamine giving a molar absorptivity within the same range, since the oxidation product of metol is the same irrespective of the primary arylamine used and the error is less than 2%. Both p- and m-nitroaniline can also be estimated at lower concentrations (100 pg/25 ml). At higher concentrations the colour of the sample solution is quite intense and interferes in the determination. The method has the advantage over the Bratton-Marshall and most other methods that it permits the determination of sulphonamides such as sulphafurazole, sulphaphenazole, sulphamoxole etc., by use of the calibration curve obtained with any sulphonamide listed in Tables 1 and 2. Further, p-aminosalicylic acid can also be estimated by this method, which is not possible with the Bratton-Marshall method. a-Naphthylamine, o- and m-aminophenols, o-, mand p-phenylenediamines, benzidine, methylaniline, dimethylaniline and diphenylamine react directly with Cr(VI) at pH 3.0 and so cannot be determined. p-Aminophenol and 1-amino-2-naphthol-4-sulphonic acid do not respond to the method. The other nitro-
in rat urine and blood--comparison
with Bratton-Marshall
method
Urine sample Proposed B.M. method, method, &J/25 ml KG5 ml free total free total
Blood sample Proposed method, B.M. Method, n&5 ml ncg/25 ml free total free total
54.3 54.8 61.4 61.9
62.6 61.9 69.8 69.1
91.9 92.9 109.8 109.3
53.6 53.9 62.1 61.9
91.7 91.6 109.4 109.0
116.5 116.1 126.4 125.9
61.9 62.4 69.3 69.9
117.1 116.2 125.6 126.1
864
R. RAMA KRISHNA and C. S. PRAKASA SASTRY
1
4
20 8 12 16 [SULPHANILAMIDE]x IO 4
Fig. 2. Effect of concentration
of sulphanilamide
gen-bearing compounds such as methylamine, ethylenediamine, dimethylamine, trimethylamine, ethanolamine, carbamic acid derivatives, glycine, alanine, arginine, p-nitro derivatives of N-methylaniline and NJ-dimethylaniline, benzylamine, cyclohexylamine, thiamine and non-nitrogeneous compounds such as methyl and ethyl alcohols, phenol, hydroquinone, aldehydes, ketones and excipients commonly present in dosage forms, which do not undergo appreciable reaction with Cr(V1) at pH 3.0 under the experimental conditions, are found to have no influence on the accuracy of the results even when they are present in twentyfold concentration with respect to the primary aromatic amine. Evidently this method is highly specific for the determination of primary aromatic amines. Before the effect of aromatic primary amines on the metolLchromium(V1) reaction was examined, the reactions between Cr(V1) and aromatic primary amines and between Cr(V1) and metol were studied separately at pH 3.0. It was found that metol or aromatic primary amine does not react separately with Cr(V1) to a detectable extent. However, the colour of the potassium dichromate is gradually replaced by the purple-red colour of the metol oxidation product, probably p-N-methylquinone-imine, in the presence of an aromatic primary amine. For studying the catalytic effect of a primary arylamine on the oxidation of metol with Cr(VI), kinetic runs were performed at pH 3.0, keeping the concentration of metol and Cr(V1) constant and varying the sulphanilamide concentration. The effect of the concentration of the sulphanilamide on the initial rate of the reaction is illustrated in Fig. 2. After an initial increase of rate, addition of further quantities of primary aromatic amine causes no change in rate. The limiting rate is obtained at an arylamine concentration almost exactly the same as that of Cr(V1). Since the presence of aromatic primary amine produces an acceleration of the metol oxidation, it is believed that the primary amine must be involved in the ratedetermining step, and that both Cr(V1) and metol are essential in this as oxidant
24
28
on the initial rate.
and reductant. It is tentatively suggested that the reaction mechanism involves some form of ternary intermediate involving aromatic primary amine, Cr(V1) and metol in which the electron transfer between Cr(V1) and metol is facilitated by the presence of amine. Such ternary intermediates with Cr(VI), EDTA and hydrazine have been reported by Beck and Durham2’ who also observed a limiting rate (cf. the present case) as the EDTA concentration is increased. These authors believe that EDTA facilitates the reaction between hydrazine and Cr(V1). If Cr(II1) formed in situ forms a complex with the aromatic primary amine, this could increase the Cr(VI)/ Cr(II1) potential. Thus the reaction can proceed as long as there is free amine and hence the absorbance of the oxidation product of metol is proportional to the concentration of amine. Such Cr(II1) complexes with aromatic primary amines have already been reported in the literature. 2s These complexes are reportedly stable and easily formed only at higher pH values; decrease in pH reduces the stability. In the present reaction the rate also decreases with decreasing pH and this parallelism supports our conclusions about the complex formation with Cr(II1). Reports are also available that the complexes of metal ions with aromatic secondary and tertiary amines are less stable, owing to steric hindrance, than those with primary aromatic amines and this perhaps explains the fact that aromatic secondary and tertiary amines cannot be determined by this method. Presumably aliphatic amines do not form strong complexes with Cr(II1) because they are more easily protonated even at pH 3.0. Acknowledgements-The authors are indebted to Dr. D. Visweswarm and Sri S. Satyanarayana, Department of Pharmaceutical Sciences, Andhra University for giving the pooled samples of rat blood and urine. Thanks are also due to Messrs. Dolphin Laboratories and Smith, Stainstreet Co., Calcutta: Roche Products, Ciba-Geigy, German Remedies and Cynamid (India) Ltd., Bombay; IDPL, Warner-Hindustan, Hyderabad; May and Baker, Madras, for generous gifts of the sulphonamides used in these investigations. We are also highly grateful to the referee for his
Determination
of primary aromatic amines
very helpful and constructive suggestions and to the authorities of Andhra University for awarding a UGC research fellowship to RRK.
REFERENCES
1. T. Higuchi and E. Brochmann-Hanssen, Pharmaceutical Analysis, p. 137. Interscience, New York, 1961. 2. S. Patia, The Chemistry of the Amino Group, pp. 81, 160, 323. Interscience, New York, 1968. 3. N. D. Cheronis and T. S. Ma, Organic Functional Group Analysis, p. 229. Interscience, New York, 1964. 4. F. D. Snell and C. T. Snell, Co[orimetric Methods of Analysis, (a) vol. IV, p. 197. Robert E. Krieger, Huntington, New York, 1976; (b) Vol. IVA, p. 373. Van Nostrand, Princeton, 1967. 5. J. F. Whitaker, A. Stenhens and L. Naftalin. Clin. Chim. Actu, 1965,12,466. 6. G. J. Parariello and M. A. M. Janish. Anal. Chem.. 1965, 37, 899. 7. S. Hantzsch and K. E. Prescher, Z. Anal. Chem., 1965, 213, 408. 8. G. Popa, E. RadulescuJercan and F. M. Albert, Revue Roum. Chim. 1966, 11, 1449. 9. G. Boda and C. Varhelyi, Studia Univ. Babes-Bolyai, Ser. Chem., 1967, 12, 81. 10. W. Pawlowski, Chem. Anal. Warsaw, 1969, 14, 59. 11. J. T. Stewart, T. D. Shaw and A. M. Ray, Anal. Chem., 1969, 41, 360. 12. S. I. Obtemperanskaya, V. N. Likhosherstova and A. P. Terent’ev, Zh. Analit. Khim., 1970, 25, 2033.
865
13. Y. Tashima, H. Hasegawa, H. Yuki and K. Takiura, Bunseki Kagaky 1970, 19, 43. 14. Yu. I. Mushkin and M. G. Ivanov, Trudy Khim. Khim. Tekhnol. (Gor’kii), 1969, 1, 155. 15. J. Bartos and M. Pesez. Bull. Sot. Chim. France. 1970. 1627. 16. S. I. Obtemperanskaya, A. P. Terent’ev and Yu. N. Tikhonov, Vest. Mosk. Gos. Univ., Ser. Khim., 1971, 12, 369. 17. D. N. Kramer and L. U. Tolentino, Anal. Chem., 1971, 43, 834. 18. L. V. Kuritsyn and V. M. Kuritsyna, Izo. Vyssh. Ucheb. Zaved., Khim. Khim. Tekhnol., 1972, 15, 461. 19. A. SpevBk, Z. Signer and M. Matrka, Collection Czech. Chem. Commun., 1972,37,4016. 20. Ya. I. Korenman and N. G. Sotnikova, Zh. Analit. Khim., 1973, 28, 2063. 21. J. P. Rawat and J. P. Singh, Anal. Chem., 1975, 47, 738. 22. A. C. Bratton and E. K. Marshall, Jr., J. Biol. Chem., 1939, 128, 537. 23. M. M. Amer and M. I. Walash, Bull. Fat. Pharm., Cairo, Cairo Uniu., 1973, 2, 161. 24. L. F. Fieser. J. Am. Chem. Sot.. 1930.52.4915. 25. A. Berka, j. Vulterin and i Ziia, -‘Newer Redox Titrations, p. 62. Pergamon, Oxford, 1965. 26. J. Cihalik and D. Vavrejnovk Chem. L&y, 1955, 49, 1176; Collection Czech. Chem. Commun., 1956, 21, 192. 27. M. T. Beck and D. A. Durham, J. Inorg. Nucl. Chem., 1971, 33, 461. 28. J. C. Traft and M. M. Jones, J. Am. Chem. Sot., 1960, 82, 4196. 29. A. F. Trotman-Dickenson, .I. Chem. SOL, 1949, 1293.
0039.9140/79/0901-0861SO2.00/0 in Great
Britain
A NEW SPECTROPHOTOMETRIC DETERMINATION OF PRIMARY
METHOD FOR THE AROMATIC AMINES
R. RAMAKRISHNAand C. S. PRAKASASASTRY Department of Chemistry, Andhra University, Waltair-3, India (Received 20 September 1978. Accepted 8 January 1979) new spectrophotometric method for the assay of primary aromatic amines with metolchromium(V1) reagent has been developed. The method is simple, reproducible and accurate within + 1.0%. It has been extended to the determination of drugs (in pure samples and pharmaceutical prepSummary-A
arations) which either contain a free primary aromatic amine or release it through hydrolysis or reduction. The method is also applicable to the assay of p-aminobenzenesulphonamides in rat urine and blood. All the sulphonamide derivatives tested, as well as other aniline derivatives with an electron-withdrawing group in the para-position, afford nearly the same molar absorptivity. Depending on the absorptivity, 0.32pmole of amine will give an absorbance of -0.1.
Several spectrophotometric and calorimetric methods of assay for primary aromatic amines are cited in the literature.‘-” All these methods suffer from the disadvantages that (a) they cannot be applied for all types of primary arylamines and (b) the compound of interest or one closely related to it structurally must necessarily be chosen for preparation of the calibration curve. We have now developed a rapid, sensitive and general method for the determination of primary aromatic amines. It is based on our observation that metol (p-N-methylaminophenol sulphate) undergoes oxidation, probably to p-N-methylquinone-imine (&, 520 mn) with potassium dichromate at pH 3.0 only in the presence of primary aromatic amines, and that the concentration of the oxidation product is directly proportional to the concentration of primary aromatic amine initially taken. This method has wide applicability in purity assays of pharmaceutical compounds which possess either a free arylamino group or release it through hydrolysis or reduction. Preliminary experiments show that this method has biological application, in that p-aminobenzenesulphonamides in rat urine and blood have been assayed successfully. EXPERIMENTAL Instrumentation
Spectral and absorbance measurements were made with a Unicam SP600 spectrophotometer with lo-mm glass cells. Time vs. absorbance measurements were recorded with a Shimadzu Double Beam Spectrophotometer (UV-140) and an automatic recorder. Reagents
All solutions were prepared in doubly distilled water. A freshly prepared 0.2% aqueous solution of metol was always used. Potassium dichromate (O.OlM) and sulphanilamide (O.OlM) solutions were prepared. The sulphanilamide was LP. grade, recrystallized and dried. Bu&r solutions. A mixture of 250 ml of 0.2M potassium hydrogen phthalate and 234 ml of O.lM hydrochloric acid was diluted to 1 litre with water (pH 2.90).
Primary aromatic amines. All the arylamines used were of commercially available G.R., LP., or B.P. grade. Their stock solutions were prepared in distilled water, the compounds insoluble in water being dissolved initially in the minimum amount of dilute hydrochloric acid or sodium hydroxide solution necessary. Working solutions were prepared by appropriate dilution of the stock solutions after neutralization of the excess of acid or alkali. The pH of the final diluted solution was in the range 6.CM.O. All the other reagents used were of analytical grade. General procedure
A l-5 ml portion of primary aromatic amine solution (concentration range as indicated in Table 1 and 0.013-0.102 mmole for the compounds in Table 2) was taken. After addition of 15 ml of buffer solution, 2 ml of 0.2% metol solution (freshly prepared) and 3 ml of O.OlM potassium dichromate, the solution was diluted to 25 ml in a standard flask and the absorbance was measured at 520 nm (after 15 min) against a reagent blank prepared in a similar manner. The primary aromatic amine concentration was read from a standard calibration curve prepared with the same compound under identical conditions (the pH of the coloured solutions was found to be 3.0 + 0.03). The pH can also be adjusted to 3.0 with dilute hydrochloric acid instead of buffer solution. Procedure for compounds which yield a primary arylamino group on hydrolysis
About 10 mg of sample were boiled with 10 ml of dilute hydrochloric acid for 1 hr under reflux, cooled, neutralized with sodium hydroxide and made up to 100 ml in a standard flask. The general procedure was then followed. Procedure for compounds which yield primary arylamino group on reduction
About 10 mg of sample were accurately weighed and treated with 10 ml of dilute hydrochloric acid and 0.25 g of zinc dust added in portions. After standing for 1 hr at room temperature, the solution was filtered through cotton wool, the residue was washed with three 5-ml portions of water and the filtrate was neutralized with sodium hydroxide and diluted to 100 ml in a standard flask. The general procedure was then applied. Procedure for pharmaceutical preparations
The sampling and preliminary treatment of pharmaceutical preparations such as tablets, capsules, injections and eye-drops were performed according to the reported pro861
862
R. RAMA KRISHNA and C. S. PRAKASA SASTRY Table 1. Spectrophotometric
Amine Aniline o-Toluidine m-Toluidine p-Toluidine o-Anisidine m-Anisidine m-Chloroaniline p-Chloroaniline p-Bromoaniline o-Aminobenzoic acid p-Aminosalicylic acid p-Aminobenzenesulphonic acid p-Aminobenzoic acid p-Aminoacetophenone Benzocaine Procaine hydrochloride Sulphanilamide Sulphafurazole Sulphamethoxazole Sulphaphenazole
assay of primary aromatic amines Molar absorptivity at 520 nm,
Beer’s law limits, ~125 ml
1 moleC’.cm-’
Recovery, %
x 10m3
75-500 100-600 75-800 100-900 75-800 50-750 50-450 50-500 50-500 50-600 100-700
2.21 1.48 1.85 1.19 2.55 4.05 5.96 4.95 5.65 3.53 2.50
100.8 99.1 100.7 100.9 100.8 99.0 99.5 99.3 99.4 99.4 98.9
60-425 45-350 45-350 50-425 85-675 55425 85-675 80-650 100-800
7.15 7.25 7.30 7.15 7.20 7.35 7.25 7.14 7.18
99.8 99.7 99.4 99.5 99.4 99.8 100.2 99.7 100.4
cedures’,22.23 and the assays were carried out as above, depending on the nature of the compound. Procedure for biological j7uids
A l-ml sample of rat urine or blood was taken and protein was precipitated with trichloroacetic acid (0.5 ml of lo”/, solution). To, the clear centrifugate, a few drops of cont. sodium hydroxide solution were added to adjust the pH to 6.G8.0. Then the proposed method was followed for the assay of free sulphonamide (unmetabolized). To determine the total amount (metabolized and unmetabolized) of the sulphonamide present, the protein-free blood or urine sample was hydrolysed by boiling with 4M hydrochloric acid for 2 hr. The general procedure was then followed after neutralization of the hydrolysate sodium hydroxide. These estimations were carried out simultaneously with Bratton-Marshallz3 assays of the same pooled samples of blood and urine, for comparison. RESULTS AND DISCUSSION
product of met01 obtained under the present experimental conditions and that obtained with iodine monochloride oxidation have been found to have the same maximum, indicating their identity (Fig. 1). At 520 nm the oxidation product has a reasonably high absorbance in comparison with that of Cr(V1). Beer’s law was found to be valid over the concentration range given in the general procedure and Table 1. The results of recovery experiments are also listed in Table 1. The values obtained by the proposed method and the Bratton-Marshall method for pharmaceutical preparations and biological fluids are compared in Tables 2 and 3. To test the reproducibility of the method, 8 replicate determinations were 06
0-0
with
Cr(VI) in
ogoinst
The spectrum (Fig. 1) of the oxidation product of metol with potassium dichromate in the presence of a primary aromatic amine at pH 3.0 has been found to have an absorption maximum at 520 run. The maximum intensity of the colour is developed within 15 min and is stable for at least 1 hr for all the amines studied. The spectrum is identical with that of the product obtained by direct oxidation of met01 by Cr(VI) at higher pH values. Fieserz4 studied the redox behaviour of metol by mixing it with potassium molybdocyanide and deduced rather reasonably that the oxidized species was the p-N-methylquinoneimine. Cihalik and Vavrejnova, who estimated metol by titrating it potentiometrically with iodine monochloride in weakly acidic or neutral media, also assumed the oxidation product of metol to be p-Nmethylquinone-imine, on the basis of their mole-ratio study.Z5*26 The absorption spectrum of the oxidation
^
with
presence
reagent
ICI
against
of
sulphocetomide
blank water
blank
05-
001 400
I
440
480
520 wavelength
Fig. 1. Absorption
560
I
600
640
(nm)
spectra of the oxidation product metol.
of
Determination
863
of primary aromatic amines
Table 2. Assay of pharmaceutical
aromatic primary amines in dosage forms*
Nominal content, g
Dosage form
proposed method
Found, g Bratton-Marshall method
Tablets
Sulphadimidine I.P. Sulphaguanidine I.P. Madribon-Sulphadimethoxine B.P. Lederkyn-Sulphamethoxypyridazine B.P. Sulphapyridine B.P. Sulfuno-Sulphamoxole B.P. Elkosin-Sulphasomidine B.P. Thalazole-Phthalylsulphathiazole 1.P.t Serepax-Oxazepam N.F.t Librium-Chlordiazepoxide 1.P.t Folic acid I.P.g,f
0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.015 0.0100 0.0050
0.500 0.498 0.497 0.508 0.492 0.509 0.505 0.490 0.014 0.0104 0.0048
0.498 0.497 0.497 0.506 0.488 0.493 0.501 0.488 0.0146 0.0105 0.0047
Capsules Chloramphenicol
0.250
0.247
0.248
1.420 1.000
1.40 1.04
1.40 1.01
I.P.9
Eye-drops and injections
Albucid-Sulphacetamide Sulphadiazine B.P.
sodium I.P.
* The results are means of duplicate determinations. t After carrying out hydrolysis. p After carrying out reduction. $ Applying suitable correction for free amine and using p-aminobenzoic acid as standard.
made with a typical primary arylamine (sulphacetamide) and the relative standard deviation was found to be 0.5%. From the results presented in Table 1, it appears that the effects (inductive, mesomeric and steric) of substituents in the positions ortho, meta and para to the amino group of aniline lead to marked variation in the catalytic oxidation of metal. It can be seen that electron-withdrawing groups have greater catalytic activity than electron-donor substituents. However, the low molar absorptivity values for o-aminobenzoic acid and o-nitroaniline (3.53 x 10” and ~2.00 x 10’ l.mole-l.cm-l) when compared to those for the corresponding meta and para compounds may be due to steric hindrance and intramolecular hydrogen bonding. It is interesting that primary aromatic amines bearing electron-acceptor groups (-SOaH, -S02NH2, -SO*NHR, -COOH, -COOR and -COCHJ para to the amino group give almost the same molar absorptivity C(7.20 f 0.25) x lo3 l.mole-’ .cm-‘1. Hence an important advantage is that the calibration curve prepared with such a primary aromatic amine can Table 3. Assay of sulphonamides
Sulphonamide Sulphanilamide Sulphacetamide
be used for the determination of any primary arylamine giving a molar absorptivity within the same range, since the oxidation product of metol is the same irrespective of the primary arylamine used and the error is less than 2%. Both p- and m-nitroaniline can also be estimated at lower concentrations (100 pg/25 ml). At higher concentrations the colour of the sample solution is quite intense and interferes in the determination. The method has the advantage over the Bratton-Marshall and most other methods that it permits the determination of sulphonamides such as sulphafurazole, sulphaphenazole, sulphamoxole etc., by use of the calibration curve obtained with any sulphonamide listed in Tables 1 and 2. Further, p-aminosalicylic acid can also be estimated by this method, which is not possible with the Bratton-Marshall method. a-Naphthylamine, o- and m-aminophenols, o-, mand p-phenylenediamines, benzidine, methylaniline, dimethylaniline and diphenylamine react directly with Cr(VI) at pH 3.0 and so cannot be determined. p-Aminophenol and 1-amino-2-naphthol-4-sulphonic acid do not respond to the method. The other nitro-
in rat urine and blood--comparison
with Bratton-Marshall
method
Urine sample Proposed B.M. method, method, &J/25 ml KG5 ml free total free total
Blood sample Proposed method, B.M. Method, n&5 ml ncg/25 ml free total free total
54.3 54.8 61.4 61.9
62.6 61.9 69.8 69.1
91.9 92.9 109.8 109.3
53.6 53.9 62.1 61.9
91.7 91.6 109.4 109.0
116.5 116.1 126.4 125.9
61.9 62.4 69.3 69.9
117.1 116.2 125.6 126.1
864
R. RAMA KRISHNA and C. S. PRAKASA SASTRY
1
4
20 8 12 16 [SULPHANILAMIDE]x IO 4
Fig. 2. Effect of concentration
of sulphanilamide
gen-bearing compounds such as methylamine, ethylenediamine, dimethylamine, trimethylamine, ethanolamine, carbamic acid derivatives, glycine, alanine, arginine, p-nitro derivatives of N-methylaniline and NJ-dimethylaniline, benzylamine, cyclohexylamine, thiamine and non-nitrogeneous compounds such as methyl and ethyl alcohols, phenol, hydroquinone, aldehydes, ketones and excipients commonly present in dosage forms, which do not undergo appreciable reaction with Cr(V1) at pH 3.0 under the experimental conditions, are found to have no influence on the accuracy of the results even when they are present in twentyfold concentration with respect to the primary aromatic amine. Evidently this method is highly specific for the determination of primary aromatic amines. Before the effect of aromatic primary amines on the metolLchromium(V1) reaction was examined, the reactions between Cr(V1) and aromatic primary amines and between Cr(V1) and metol were studied separately at pH 3.0. It was found that metol or aromatic primary amine does not react separately with Cr(V1) to a detectable extent. However, the colour of the potassium dichromate is gradually replaced by the purple-red colour of the metol oxidation product, probably p-N-methylquinone-imine, in the presence of an aromatic primary amine. For studying the catalytic effect of a primary arylamine on the oxidation of metol with Cr(VI), kinetic runs were performed at pH 3.0, keeping the concentration of metol and Cr(V1) constant and varying the sulphanilamide concentration. The effect of the concentration of the sulphanilamide on the initial rate of the reaction is illustrated in Fig. 2. After an initial increase of rate, addition of further quantities of primary aromatic amine causes no change in rate. The limiting rate is obtained at an arylamine concentration almost exactly the same as that of Cr(V1). Since the presence of aromatic primary amine produces an acceleration of the metol oxidation, it is believed that the primary amine must be involved in the ratedetermining step, and that both Cr(V1) and metol are essential in this as oxidant
24
28
on the initial rate.
and reductant. It is tentatively suggested that the reaction mechanism involves some form of ternary intermediate involving aromatic primary amine, Cr(V1) and metol in which the electron transfer between Cr(V1) and metol is facilitated by the presence of amine. Such ternary intermediates with Cr(VI), EDTA and hydrazine have been reported by Beck and Durham2’ who also observed a limiting rate (cf. the present case) as the EDTA concentration is increased. These authors believe that EDTA facilitates the reaction between hydrazine and Cr(V1). If Cr(II1) formed in situ forms a complex with the aromatic primary amine, this could increase the Cr(VI)/ Cr(II1) potential. Thus the reaction can proceed as long as there is free amine and hence the absorbance of the oxidation product of metol is proportional to the concentration of amine. Such Cr(II1) complexes with aromatic primary amines have already been reported in the literature. 2s These complexes are reportedly stable and easily formed only at higher pH values; decrease in pH reduces the stability. In the present reaction the rate also decreases with decreasing pH and this parallelism supports our conclusions about the complex formation with Cr(II1). Reports are also available that the complexes of metal ions with aromatic secondary and tertiary amines are less stable, owing to steric hindrance, than those with primary aromatic amines and this perhaps explains the fact that aromatic secondary and tertiary amines cannot be determined by this method. Presumably aliphatic amines do not form strong complexes with Cr(II1) because they are more easily protonated even at pH 3.0. Acknowledgements-The authors are indebted to Dr. D. Visweswarm and Sri S. Satyanarayana, Department of Pharmaceutical Sciences, Andhra University for giving the pooled samples of rat blood and urine. Thanks are also due to Messrs. Dolphin Laboratories and Smith, Stainstreet Co., Calcutta: Roche Products, Ciba-Geigy, German Remedies and Cynamid (India) Ltd., Bombay; IDPL, Warner-Hindustan, Hyderabad; May and Baker, Madras, for generous gifts of the sulphonamides used in these investigations. We are also highly grateful to the referee for his
Determination
of primary aromatic amines
very helpful and constructive suggestions and to the authorities of Andhra University for awarding a UGC research fellowship to RRK.
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