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Colorimetric determination of theophylline and aminophylline with diazotized p-nitroaniline
Talanra,Vol. 36,No. 12,pp.1288-1290, 1989 Printed in Great Britain. All rights reserved
0039-9140/89$3.00+ 0.00 Copyright 0 1989Pergamon Press plc
COLORIMETRIC DETERMINATION OF THEOPHYLLINE AND AMINOPHYLLINE WITH DIAZOTIZED p-NITROANILINE S. R. EL-SHABGURI, S. A. HUSSEIN and S. E. EMARA Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Assiut University, Assiut, Egypt (Received 27 September 1988. Revised 25 April 1989. Accepted 10 July 1989)
Summary-The reactions of theophylline and aminophylline with diazotized p-nitroaniline in alkaline medium have been studied and developed into a sensitive assay for both drugs. The yellow azo-dyes formed with theophylline and aminophylline show maximum absorption at 440 and 410 nm respectively. Beer’s law is valid within the concentration range 2-16 pg/ml for theophylline and l-8 pg/ml for aminophylline. All variables which affect the reactions were studied and optimized. The proposed method has been successfully applied to determination of the drugs in their commercially available dosage forms. Statistical analysis of the results revealed that the proposed method is as precise and accurate as the official USP procedure.
Theophylline is mainly used as a diuretic, and aminophylline is used to treat diseases of the respiratory and cardiovascular systems and cardiac pulmonary and renal oedema. Techniques used for
the determination of these drugs include titrimetry,‘s6 complexometry,’ spectrophotometry,2s8 colorimetry9~‘0 phosphorimetry,” and chromatography.‘2-‘4 The proposed assay was developed by adapting the principle of the Pauly reaction,15 which is specific for imidazole derivatives. Diazotized p-nitroaniline was used as the coupling reagent and a rapid, sensitive and selective method for the determination of theophylline and aminophylline was developed.
solution and mix well. After 10 min dilute to the mark with methanol. Measure the absorbance at the appropriate wavelength for each drug, against a blank similarly prepared. Injections. Mix the contents of ten ampoules. Dilute an accurately measured volume of the mixture, equivalent to 25 mg of aminophylline, with distilled water to obtain a 125 pg/ml solution and apply the procedure above to 1 ml of it. Tablets. Weigh and powder 20 tablets. Transfer an accurately weighed amount of the powder, equivalent to 25 mg of theophylline, to a lOO-mlstandard flask, and dilute to the mark with distilled water. Shake the mixture well and filter. Discard &he first portion of filtrate. Apply the procedure above to 1 ml of this solution.
RESULTS AND
DISCUSSION
EXPERIMENTAL Apparatus
A Zeiss PM2DL spectrophotometer
was used.
Reagents
Pharmaceutical grade anhydrous theophylline and aminophylline were obtained as gifts from manufacturers and used as working standards without further treatment. All chemicals and solvents used were of analytical grade. Various commercial dosage forms, including tablets and ampoules, were purchased from the local market. Diazotizedp-nitroaniline solution. Dissolve about 40 mg of p-nitroanaline in 2 ml of concentrated hydrochloric acid in a 25ml standard flask. Cool in an ice-bath, add 2 ml of 2% sodium nitrite solution, and after 10 min dilute to volume with water. Mix well and keep in an ice-bath. This reagent solution should be used within 5 hr. Aqueous 3.3% sodium carbonate solution. Standard theophylline and aminophylline solutions. Dis-
solve 0.0250 g of theophylline or 0.0125 g of aminophylline, accurately weighed, in exactly 100 ml of distilled water. Procedures
Transfer 1 ml of an aqueous sample solution containing about 250 pg of theophylline or 125 pg of aminophylline into a 25-ml standard flask. Add 2 ml of 3.3% sodium carbonate solution and 1 ml of diazotized p-nitroaniline
The products of coupling the two drugs with diazotized p-nitroaniline in the presence of sodium carbonate are yellow, with an absorption peak at 440 nm for theophylline and 410 nm for aminophylline (Fig. 1). Beer’s law is obeyed for both drugs (Table 1). For the diazotization step, concentrated hydrochloric acid was found superior to concentrated sulphuric acid. It was found that with OS-5 ml of 2% sodium nitrite solution and 2 ml of concentrated hydrochloric acid as the diazotization mixture, constant and maximum yield of aminophylline reaction product was obtained by the recommended procedure when at least 40 mg of p-nitroaniline was diazotized and the diazonium salt solution was diluted to 25 ml, 1 ml of which was then used for the coupling reaction (Fig. 2). Removal of excess of nitrous acid was not necessary, as it did not affect the coupling reactions. The coupling was performed in alkaline medium, and it was found essential to add the alkali first and then the diazotized p-nitroaniline; this is done in
1288
1289
SHORT COMMUNICAT’IONS
330
370
410
450
490
530
Wovelength (nm)
Fig. 1. Absorption spectra of coloured product of the drugs with diazotized p-nitroaniline. (-) Theophylline. (---) Aminophylline. order to replace the reactive hydrogen atom at position 7 with sodium, otherwise, the kinetically unstable triazoderivative would be formed. Various basic solutions (sodium acetate, sodium carbonate, sodium bicarbonate and sodium hydroxide) over a range of concentrations were tested in order to find the best to use. Sodium carbonate solution (3-3.5%) was found the most suitable (Fig. 3). For the final dilution it is immaterial whether water, methanol, ethanol or propan-l-01 is used. The coupling reaction yields the same absorbance when done at any temperature in the range O-25”, so it is not necessary to cool the solution for the purpose. At 0” more than 10 min will be needed for complete colour development before dilution to final volume. At 25”, the colour is fully developed and constant in reaction times of 5-25 min, but then begins to fade. Once the solution has been diluted to volume, however, the colour is stable for at least 12 hr. A reaction time of 10 min was selected.
ments were performed for each drug in its dosage form and pharmaceutical preparations. Statistical analysis (F-test and t-test) of the results obtained by the suggested method and an official method showed no significant difference in performance. Commonly encountered excipients and additives such as lactose, starch, magnesium stearate, talc and sodium chloride did not interfere. The method proposed has the advantages of being simple and sensitive. Also the diazonium salt couples directly with theophylline, and there is no need for alkaline hydrolysis prior to coupling.
.-.-. / 81./* 0.7r
0.6
E
0.5
p” $
$
./
0.4
.
/
0.3
I 0.04
I 0.06
I 0.12
I
1
0.16
0.20
p-Nitroaniline concentration (g/100 ml)
Fig. 2. Effect of different amounts of diazotized p-nitroaniline on the intensity of coloured reaction product with theophylline (10 pg/ml).
Interferences To assess the specificity of the method for theophylline and aminophylline, related compounds such as caffeine and theobromine as well as drugs that may be found with theophylline in certain dosage forms (e.g., ephedrine hydrochloride and phenobarbitone) were tested under the proposed reaction conditions. The results showed that these drugs give zero absorbance over the wavelength range 400-450 nm. 1
Application to dosage forms
2
4
3
Sodium carbonate concentmtion (g /lo0
Commercial tablets and ampoules containing theophylline or aminophylline were successfully analysed by the proposed method (Table 2). Recovery experi-
Theophylline Aminophylline *Of regression line.
)
Fig. 3. Effect of concentration of sodium carbonate on the intensity of coloured reaction product of theophylline (10 pg/ml) with diazotized p-nitroaniline.
Table 1. Data for the reaction of theophylline and aminophylline with diazotized p-nitroaniline
Drug
5
ml
I mai1 nm
Beer’s law range, W/ml
Intercept*
440 410
l-16 I-8
0.005 0.011
Slope*, mlllrg 0.0654 0.0940
Apparent molar absorptivity, l.mole-‘.cm-’ 1.3 x lo4 4.8 x 104
Correlation coefficient 0.9990 0.9987
1290
SHORT COMMUNlCATIONS
Table 2. Determination of theophylline and aminophylline in some commercial preparations
Proposed method Nominal Found, % + SD
Added, mg
Recovery, % + SD
Recovery by other method (reference)
3OO/tablet
100.1 f 0.4 I = 0.53 F = 1.94
300
100.1 f 0.3
100*0.3
Tepdrin tabletst (Misr, Egypt)
120/tablet
99.8 f 0.5 i CO.14 F=l.90
120
99.9 + 0.4
99.9 f 0.6 (18)
Asmasone tablet@ (Nile, Egypt)
lSO/tablet
99.8 * 0.6 t =0.14 F= 1.4
150
99.9 + 0.6
99.9 + 0.6 (18)
Asmacid tablets3 (Cid, Egypt)
120/tablet
99.9 * 0.4 t = 0.74 F = 1.14
120
99.8 f 0.3
99.7 f 0.4 (19)
250/ampoule
99.8 + 0.97 t = 0.49 F = 2.65
250
99.8 f 0.4
content, mg
Formulation Theophylline Quobron-T/SR tablets (Mead Johnson U.S.A.)
Aminophylline Cidophylline ampoules (Cid, Egypt) *Average of tEach tablet $Each tablet $Each tablet
Nature
100.0 + 0.5
(1)
(1)
8 determinations f standard deviation; theoretical values at 95% confidence limit: t = 2.14; F = 3.79. also contains ephedrine.HCl 25 mg, phenobarbitone 8 mg. also contains ephedrine.HCI 30 mg, papaverine.HCl 150 mg, phenobarbitone sodium 10 mg. also contains ephedrine.HCI 15 mg, meclozine 25 mg, and phenobarbitone 10 mg.
of the coupled product
The coupling may be considered as a protoneliminating reaction of a diazonium salt with another compound possessing an active hydrogen atom.r6 Theophylline can couple at the 8-position with the diazonium salt because this position has a pronounced nucleophilic character.” The suggested reaction path is shown below.
2. British Phurmacopeia,
Press, London, 3. G. Bazsai and 103, 205. 4. H. Raber, Sci. 5. M. B. Devani,
pp. 449, 581. Pharmaceutical 1980. L. Mosonyi, Pharm. Zentralhalle, 1964, Pharm., 1966, 34, 202.
C. J. Shishoo and D. J. Bhut, J. Pharm.
Sci., 1968, 57, 1051. 6. T. Medwick and F.
Schiesswohl, ibid., 1963, 52, 843. 7. F. Pellerin and G. Leroux-Mamo, Ann. Pharm. Fr., 1971, 29, 153. 8. J. Kandrnal, V. Lipus and J. Kratochvila, Biochem. Clin. Bohemoslov., 1984, 13, 55. 9. B. M. Scheinthal and L. Chafetz, Eur. Pat. Appl., 1982, 12. 10. A. M. Aliev and B. M. Gaseinov, Farmatsiya, 1983, 32, No. 5, 75. 11. R. P. Bateh and J. D. Winefordner, Anal. Lett., 1982, 15, 373. 12. J. Miksic and B. Hodes, J. Pharm. Sci., 1979,68, 1200.
13. V. P. Shah and S. Riegelmen, ibid., 1974, 63, 1283. 14. V. Massa, F. Gal, P. Suspulgas and G. Maestre, Trav. Sot., Montpellier, 1971, 31, 167.
15. H. Pauly, Z. Physiol. Chem., 1904, 42, 508. 16. M. Pesez and J. Bartos, Calorimetric and Fluorimetric Analysis of Organic Compounds and Drugs, p. 521. Dekker, New York, 1974. 17. A. R. Katritzky and A. J. Boulton, Adv. Heterocyclic REFERENCES 1. U.S. Pharmocopeia XXI, p. 1042. US. Pharmacopeial Convention, Rockville, MD, 1985.
Chem., 1979, 24, 222.
18. A. A. Mousa, Pharmazie, 1978, 33, 296. 19. M. M. Ayad, M.S. Thesis, Faculty of Pharmacy, Cairo University, 1971.
0039-9140/89$3.00+ 0.00 Copyright 0 1989Pergamon Press plc
COLORIMETRIC DETERMINATION OF THEOPHYLLINE AND AMINOPHYLLINE WITH DIAZOTIZED p-NITROANILINE S. R. EL-SHABGURI, S. A. HUSSEIN and S. E. EMARA Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Assiut University, Assiut, Egypt (Received 27 September 1988. Revised 25 April 1989. Accepted 10 July 1989)
Summary-The reactions of theophylline and aminophylline with diazotized p-nitroaniline in alkaline medium have been studied and developed into a sensitive assay for both drugs. The yellow azo-dyes formed with theophylline and aminophylline show maximum absorption at 440 and 410 nm respectively. Beer’s law is valid within the concentration range 2-16 pg/ml for theophylline and l-8 pg/ml for aminophylline. All variables which affect the reactions were studied and optimized. The proposed method has been successfully applied to determination of the drugs in their commercially available dosage forms. Statistical analysis of the results revealed that the proposed method is as precise and accurate as the official USP procedure.
Theophylline is mainly used as a diuretic, and aminophylline is used to treat diseases of the respiratory and cardiovascular systems and cardiac pulmonary and renal oedema. Techniques used for
the determination of these drugs include titrimetry,‘s6 complexometry,’ spectrophotometry,2s8 colorimetry9~‘0 phosphorimetry,” and chromatography.‘2-‘4 The proposed assay was developed by adapting the principle of the Pauly reaction,15 which is specific for imidazole derivatives. Diazotized p-nitroaniline was used as the coupling reagent and a rapid, sensitive and selective method for the determination of theophylline and aminophylline was developed.
solution and mix well. After 10 min dilute to the mark with methanol. Measure the absorbance at the appropriate wavelength for each drug, against a blank similarly prepared. Injections. Mix the contents of ten ampoules. Dilute an accurately measured volume of the mixture, equivalent to 25 mg of aminophylline, with distilled water to obtain a 125 pg/ml solution and apply the procedure above to 1 ml of it. Tablets. Weigh and powder 20 tablets. Transfer an accurately weighed amount of the powder, equivalent to 25 mg of theophylline, to a lOO-mlstandard flask, and dilute to the mark with distilled water. Shake the mixture well and filter. Discard &he first portion of filtrate. Apply the procedure above to 1 ml of this solution.
RESULTS AND
DISCUSSION
EXPERIMENTAL Apparatus
A Zeiss PM2DL spectrophotometer
was used.
Reagents
Pharmaceutical grade anhydrous theophylline and aminophylline were obtained as gifts from manufacturers and used as working standards without further treatment. All chemicals and solvents used were of analytical grade. Various commercial dosage forms, including tablets and ampoules, were purchased from the local market. Diazotizedp-nitroaniline solution. Dissolve about 40 mg of p-nitroanaline in 2 ml of concentrated hydrochloric acid in a 25ml standard flask. Cool in an ice-bath, add 2 ml of 2% sodium nitrite solution, and after 10 min dilute to volume with water. Mix well and keep in an ice-bath. This reagent solution should be used within 5 hr. Aqueous 3.3% sodium carbonate solution. Standard theophylline and aminophylline solutions. Dis-
solve 0.0250 g of theophylline or 0.0125 g of aminophylline, accurately weighed, in exactly 100 ml of distilled water. Procedures
Transfer 1 ml of an aqueous sample solution containing about 250 pg of theophylline or 125 pg of aminophylline into a 25-ml standard flask. Add 2 ml of 3.3% sodium carbonate solution and 1 ml of diazotized p-nitroaniline
The products of coupling the two drugs with diazotized p-nitroaniline in the presence of sodium carbonate are yellow, with an absorption peak at 440 nm for theophylline and 410 nm for aminophylline (Fig. 1). Beer’s law is obeyed for both drugs (Table 1). For the diazotization step, concentrated hydrochloric acid was found superior to concentrated sulphuric acid. It was found that with OS-5 ml of 2% sodium nitrite solution and 2 ml of concentrated hydrochloric acid as the diazotization mixture, constant and maximum yield of aminophylline reaction product was obtained by the recommended procedure when at least 40 mg of p-nitroaniline was diazotized and the diazonium salt solution was diluted to 25 ml, 1 ml of which was then used for the coupling reaction (Fig. 2). Removal of excess of nitrous acid was not necessary, as it did not affect the coupling reactions. The coupling was performed in alkaline medium, and it was found essential to add the alkali first and then the diazotized p-nitroaniline; this is done in
1288
1289
SHORT COMMUNICAT’IONS
330
370
410
450
490
530
Wovelength (nm)
Fig. 1. Absorption spectra of coloured product of the drugs with diazotized p-nitroaniline. (-) Theophylline. (---) Aminophylline. order to replace the reactive hydrogen atom at position 7 with sodium, otherwise, the kinetically unstable triazoderivative would be formed. Various basic solutions (sodium acetate, sodium carbonate, sodium bicarbonate and sodium hydroxide) over a range of concentrations were tested in order to find the best to use. Sodium carbonate solution (3-3.5%) was found the most suitable (Fig. 3). For the final dilution it is immaterial whether water, methanol, ethanol or propan-l-01 is used. The coupling reaction yields the same absorbance when done at any temperature in the range O-25”, so it is not necessary to cool the solution for the purpose. At 0” more than 10 min will be needed for complete colour development before dilution to final volume. At 25”, the colour is fully developed and constant in reaction times of 5-25 min, but then begins to fade. Once the solution has been diluted to volume, however, the colour is stable for at least 12 hr. A reaction time of 10 min was selected.
ments were performed for each drug in its dosage form and pharmaceutical preparations. Statistical analysis (F-test and t-test) of the results obtained by the suggested method and an official method showed no significant difference in performance. Commonly encountered excipients and additives such as lactose, starch, magnesium stearate, talc and sodium chloride did not interfere. The method proposed has the advantages of being simple and sensitive. Also the diazonium salt couples directly with theophylline, and there is no need for alkaline hydrolysis prior to coupling.
.-.-. / 81./* 0.7r
0.6
E
0.5
p” $
$
./
0.4
.
/
0.3
I 0.04
I 0.06
I 0.12
I
1
0.16
0.20
p-Nitroaniline concentration (g/100 ml)
Fig. 2. Effect of different amounts of diazotized p-nitroaniline on the intensity of coloured reaction product with theophylline (10 pg/ml).
Interferences To assess the specificity of the method for theophylline and aminophylline, related compounds such as caffeine and theobromine as well as drugs that may be found with theophylline in certain dosage forms (e.g., ephedrine hydrochloride and phenobarbitone) were tested under the proposed reaction conditions. The results showed that these drugs give zero absorbance over the wavelength range 400-450 nm. 1
Application to dosage forms
2
4
3
Sodium carbonate concentmtion (g /lo0
Commercial tablets and ampoules containing theophylline or aminophylline were successfully analysed by the proposed method (Table 2). Recovery experi-
Theophylline Aminophylline *Of regression line.
)
Fig. 3. Effect of concentration of sodium carbonate on the intensity of coloured reaction product of theophylline (10 pg/ml) with diazotized p-nitroaniline.
Table 1. Data for the reaction of theophylline and aminophylline with diazotized p-nitroaniline
Drug
5
ml
I mai1 nm
Beer’s law range, W/ml
Intercept*
440 410
l-16 I-8
0.005 0.011
Slope*, mlllrg 0.0654 0.0940
Apparent molar absorptivity, l.mole-‘.cm-’ 1.3 x lo4 4.8 x 104
Correlation coefficient 0.9990 0.9987
1290
SHORT COMMUNlCATIONS
Table 2. Determination of theophylline and aminophylline in some commercial preparations
Proposed method Nominal Found, % + SD
Added, mg
Recovery, % + SD
Recovery by other method (reference)
3OO/tablet
100.1 f 0.4 I = 0.53 F = 1.94
300
100.1 f 0.3
100*0.3
Tepdrin tabletst (Misr, Egypt)
120/tablet
99.8 f 0.5 i CO.14 F=l.90
120
99.9 + 0.4
99.9 f 0.6 (18)
Asmasone tablet@ (Nile, Egypt)
lSO/tablet
99.8 * 0.6 t =0.14 F= 1.4
150
99.9 + 0.6
99.9 + 0.6 (18)
Asmacid tablets3 (Cid, Egypt)
120/tablet
99.9 * 0.4 t = 0.74 F = 1.14
120
99.8 f 0.3
99.7 f 0.4 (19)
250/ampoule
99.8 + 0.97 t = 0.49 F = 2.65
250
99.8 f 0.4
content, mg
Formulation Theophylline Quobron-T/SR tablets (Mead Johnson U.S.A.)
Aminophylline Cidophylline ampoules (Cid, Egypt) *Average of tEach tablet $Each tablet $Each tablet
Nature
100.0 + 0.5
(1)
(1)
8 determinations f standard deviation; theoretical values at 95% confidence limit: t = 2.14; F = 3.79. also contains ephedrine.HCl 25 mg, phenobarbitone 8 mg. also contains ephedrine.HCI 30 mg, papaverine.HCl 150 mg, phenobarbitone sodium 10 mg. also contains ephedrine.HCI 15 mg, meclozine 25 mg, and phenobarbitone 10 mg.
of the coupled product
The coupling may be considered as a protoneliminating reaction of a diazonium salt with another compound possessing an active hydrogen atom.r6 Theophylline can couple at the 8-position with the diazonium salt because this position has a pronounced nucleophilic character.” The suggested reaction path is shown below.
2. British Phurmacopeia,
Press, London, 3. G. Bazsai and 103, 205. 4. H. Raber, Sci. 5. M. B. Devani,
pp. 449, 581. Pharmaceutical 1980. L. Mosonyi, Pharm. Zentralhalle, 1964, Pharm., 1966, 34, 202.
C. J. Shishoo and D. J. Bhut, J. Pharm.
Sci., 1968, 57, 1051. 6. T. Medwick and F.
Schiesswohl, ibid., 1963, 52, 843. 7. F. Pellerin and G. Leroux-Mamo, Ann. Pharm. Fr., 1971, 29, 153. 8. J. Kandrnal, V. Lipus and J. Kratochvila, Biochem. Clin. Bohemoslov., 1984, 13, 55. 9. B. M. Scheinthal and L. Chafetz, Eur. Pat. Appl., 1982, 12. 10. A. M. Aliev and B. M. Gaseinov, Farmatsiya, 1983, 32, No. 5, 75. 11. R. P. Bateh and J. D. Winefordner, Anal. Lett., 1982, 15, 373. 12. J. Miksic and B. Hodes, J. Pharm. Sci., 1979,68, 1200.
13. V. P. Shah and S. Riegelmen, ibid., 1974, 63, 1283. 14. V. Massa, F. Gal, P. Suspulgas and G. Maestre, Trav. Sot., Montpellier, 1971, 31, 167.
15. H. Pauly, Z. Physiol. Chem., 1904, 42, 508. 16. M. Pesez and J. Bartos, Calorimetric and Fluorimetric Analysis of Organic Compounds and Drugs, p. 521. Dekker, New York, 1974. 17. A. R. Katritzky and A. J. Boulton, Adv. Heterocyclic REFERENCES 1. U.S. Pharmocopeia XXI, p. 1042. US. Pharmacopeial Convention, Rockville, MD, 1985.
Chem., 1979, 24, 222.
18. A. A. Mousa, Pharmazie, 1978, 33, 296. 19. M. M. Ayad, M.S. Thesis, Faculty of Pharmacy, Cairo University, 1971.