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Colorimetric determination of amoxycillin in pure form and in pharmaceutical preparations
Talanta,Vol. 41, No. 5, pp. 691-694, 1994 Copyright0 1994Elscvier Science Ltd Printedin GreatBritain.All rightsreserved oo39-914lV94 s7.00 + 0.00
Peqpmon
COLORIMETRIC DETERMINATION OF AMOXYCILLIN IN PURE FORM AND IN PHARMACEUTICAL PREPARATIONS A. S. AMIN,’ A. L. EL-ANSARY~and Y. M. 1s~’ ‘Chemistry Department, Faculty of Science, Benha University, Benha, Egypt 2Cairo University, Cairo, Egypt (Received 15 April 1993. Revised 5 September 1993. Accepted 6 September 1993) Summary-A simple and selective method for the determination of amoxycillin in pure form and in pharmaceutical preparations is described. The procedure is based on the reaction of amoxycillin with 4nitrophenol (I), 2,4_dinitrophenol (II), 3$dinitrobenxoic acid (III) or 3,5_dinitrosalicylic acid (IV) in alkaline medium. The method has been used for the determination of l-24 pg/ml of amoxycillin trihydrate in solution. The method is selective for the determination of amoxycillin in the presence of its degradation products, other antibiotics and different amines that are normally encountered in dosage forms.
Several methods have been reported in comprehensive reviews for the determination of amoxycillin’” (6R)-6-(a -D-p -hydroxyphenyl glycylamino) penicillanic acid [61336-70-71, a semisynthetic fi-lactam antibiotic. Most of these methods are based on the spectrophotometric determination of amoxycillin with paramolybdate anion,6 2,3-dichloro-5,6-dicyano-p-benzoquinone’ and chloranil.’ Various iodometric, alkalimetric,’ calorimetric,” potentiometric,” microbiologicali and high-performance liquid procedures have been rechromatographic’“” ported for the determination of the drug. The aim of this work was to demonstrate a simple and selective method for determination of amoxycillin in pure form and in pharmaceutical preparations.
Borate buffer solutions of different pH-values (5-l 1) were prepared as recommended.”
EXPERIMENTAL
Determination of amoxycillin in dosage forms
A Perkin-Elmer 13B spectrophotometer was used for all spectral measurements. The pH of solutions was checked using an Orion Model 60 1 A/digital Ionalyzer. All the nitroaromatics (I-IV) were Aldrich reagents, 10e3M solutions of the nitro derivatives were prepared by dissolving appropriate weight of the reagent in 100 ml of acetone. The solutions were prepared daily fresh protected from direct light. Standard solution of amoxycillin trihydrate: A 200 pg/ml solution was prepared by dissolving 0.05 g of pure amoxycillin trihydrate (amox.) in 5 ml of methanol and diluting the solution to 250 ml with water.
Capsules. Determine the average content weight of a capsule, dissolve 0.05 g amox. in the least amount of methanol and complete the volume to 250 ml with water. Transfer a volume containing 200 ug of amox. in a lo-ml measuring flask. Add 5 ml of pH 9 buffer solution and 1 ml of acetone and complete to the mark with water. Determine the concentration of amox. by following the general procedure. Syrup and ampoules. Transfer a volume of syrup or ampoule contents containing 0.05 g of amox. and complete the volume to 250 ml with water. Transfer a volume of this solution containing 200 pg of amox. into a lo-ml measuring flask, and complete as described under general
General procedure
Pipette 2 ml of 10m3M of reagent III into a IO-ml measuring flask. Add 5 ml of pH 9 buffer solution and 1 ml of acetone, a volume containing 200 pg amox. and complete to the mark with water. The reaction mixture is left to stand at room temperature (25 + SC) for 5 min. Measure the absorbance at 336 nm against a blank solution prepared simultaneously. If one of the reagents I, II and IV is to be used, mix well, place the flasks in a water bath at 50 + 2°C for 30 min and allow to stand at room temperature for 10 min.
691
A. S.
692
hlN
et aI.
Table 1. Spectral characteristics of the coloured reaction oroducts of amoxvcillin and nitroaromatics Quantitative parameters Reagent
I mar (nm 1
GwiX lo4 l/mol/cm
Linear range PRImI
Intercept
Sloue
Correlation coefficient
I II III IV
445 438 336 429
1.9 2.2 2.8 2.6
I-18 l-20 1-24 1-22
-0.0125 0.0050 0.0025 -0.0035
0.055 0.065 0.125 0.115
0.986 0.992 0.998 0.983
the concentration
of
procedure amox.
to determine
RESULTS AND DISCUSSION
Optimization
The precision values (eight replicates) were calculated for solutions containing 15 pg/ml amoxycillin. Relative standard deviations (RSD) of 0.51, 0.63, 0.37 and 0.89% were obtained for I, II, III and IV, respectively.
of reaction variables
Investigations were carried out to establish the most favourable conditions for the complexation reaction of amoxycillin with some nitroaromatics to achieve maximum colour development in the determination of amoxycillin. The influence of some variables on the reaction has been tested. The results of these studies are summarized in general procedure and Table 1. The coloured species may be formed through s-electron donation (nadduct); regarding the drug as a donor and the nitroaromatics as acceptor to form charge transfer complex. The coloured reaction product can be represented taking 3,5-dinitrobenzoic acid as example by the following structure.
Interferences
Phenylacetic acid, phenylacetamide, 6-amino penicillanic, ampicillin and cloxacillin may be present as impurities in amoxycillin as they are used as synthetic precursors. The effect of the presence of the above compounds and common co-existing additives and bases were studied. The components were added in excess of the amount used in the preparations to a solution containing 15 pg/ml of amoxycillin with 3,5dinitrobenzoic acid solution. Phenylacetic acid, phenylacetamide, 6arninopenicillanic acid, benzylpenicillin, ampicillin, flucloxacillin, cloxacillin, dicloxacillin, calcium carbonate, sodium acetate, magnesium stearate, talc powder, starch
coo
I
NH;
I CH-
Quan @cation
A linear correlation was found between absorbance and concentration in the ranges given in Table 1. Correlation coefficient, intercept and slope values for the calibration data of amoxycillin are calculated using the least-squares method.
and lactose do not interfere in the determination of amoxycillin indicating that complexation does not occur with these drugs under the reaction conditions used. Also there is no interference of penicilloic acid, penilloic acid, penillic acid, penaldic acid, penicillamine and ultimately penilloaldehyde which is the degradation products of amoxycillin*O resulting from thermal and
101.00 132.00
59.00 81.08
141.00 158.08
60 80
140 160 139.00 161.00
57.50 79.20
99.00 131.08
Found? 1 2
100 130
Taken mg
160 200
158.00 202.00
91.00 119.00
90 120
2
159.00 199.00
89.08 120.50
151.00 179.00
Found?
148.08 183.00
1
150 180
Taken mg
II
*:Manufacturer. t:Average of six determinations. t:Calculated f-value; tabulated r-value for eight degrees of freedom and P = 0.05 is 2.306. #Calculated F-value; tabulated F-value for f, =fi = 4 and P = 0.05 is 6.39. I:By the proposed method. 2:By the British Pharmacopoeia (1988) procedure. l/Egyptian International Pharmaceutical Industries Company (Egypt). ?Chemical Industries Development Company (Egypt). **:Amoun Pharmaceutical Industries Company (Egypt).
Capsules E-mox. (250 mg amox.)t E-mox. (500 mg amox.)t 8YruP Amoxicid (125 mg amox.)” Amoxicid (250 mg amox.)s Ampoules Ibiamox (250 mg amox.)** Ibiamox (500 mg amox.)**
Drug*
I
180 240
178.00 238.00
179.00 242.00
2.08 2.23
2.18 1.79
119.00 221.50
122.00 218.00
120 220
rt 1.69 2.14
2 169.00 201.00
Found?
171.00 198.00
1
170 200
Taken mg
III
2.61 2.78
2.67 3.17
3.11 2.53
Fo
Table 2. Determination of amoxycillin in different pharmaceutical preparations using nitroaromatics chromogens
140 200
160 180
220 230
Taken mg 2
161.08 178.00 141.00 198.00
158.00 178.00 139.00 201.00
218.00 231.08
Found?
222.00 233.00
1
IV
A. S. AMINet al.
694
hydrolytic degradates forms on the determination of amoxycillin.
Results of the analysis of amoxycillin capsules, syrups and ampoules by the proposed method show a good agreement with those of the British Pharmacopoeia (1988) procedure.” The relative standard deviation of the proposed method is better than 0.9% (six determinations) and the percentage recoveries of the proposed procedures were Pharmacopoeia1 and 100 f 1.67 and 98.8 zf 0.9 (six experiments), respectively, for different examined samples (Table 2). The results obtained were compared statistically by the student’s t-test and the variance ratio F-test with those obtained by Pharmacopoeial method on samples of the same batch (Table 2). The student’s t-test values obtained at the 95% confidence level and eight degrees of freedom did not exceed the theoretical tabulated value indicating no significant difference between the methods compared. The P-test also showed that there is no significant difference between the precisions of the proposed method and the official method. CONCLUSIONS
It is clear that 3,Sdinitrobenzoic acid is highly superior compared to other reagents. Very rapid fo~ation of the chromophore (5 min on cold), highest stability of colour (24 h) and lower RSD (0.37%).
REFERENCES 1. H. Bundgaard and C. Larsen, J. Pharm. Biomed. Anal.,
1983, 1, 29. 2. M. L. Lana, V. Girona, J. De Bolos and M. Castillo, Cienc. Znd. Farm., 1985,4, 13. 3. E. M. Abdel-Moety, M. A. Abounassif, M. E. Mohamed and N. A. Kbattab, Talanta, 1989,36, 683. 4. J. A. Murillo, J. Rodriguez, J. M. Lemus and A. Al&on, Analyst, 1990, 115, 1117. 5. E. M. Abdel-Moety, J. Pharm. Biomed. Anal., 1991, 9, 187. 6. P. B. lssopouios, J. Pharm. Biomed. Anal., 1988,6,97. I. H. F. Askal, G. A. Saleh and N. M. Omer, Analyst, 1991, 116, 387. 8. T. S. Al-Ghabsha, T. N. Al-Sabha and G. A. Al-Iraqi, Mcrochem. J., 1987, 36, 323. 9. S. Arora and J. P. Sharma, Nati. Acad. Sri. Len., 1990, 13, 15. 10. J. Talegaonkar and K. S. Boparai, Talanta, 1982, 29, 525. 11. D. M. Shingbal and R. R. Naik, Indian Drugs, 198522, 271. 12. H. Hopkala, Chem. Anal., 1987, 32, 929. p. 32. H. M. S. O., London, 13, British Ph~~copoe~ 1980. 14. G. W. K. Fong, D. I. Martin, R. N. Johnson and B. T. Kho, J. Chromatogr., 1984, 298, 459. 15. J. Martin, R. Mendez and A. Negro., J. Liq. Chromatogr., 1988, 11, 1707. 16, J. Haginak and J. Wakai, Analyst., 1985, 110, 1277. 17. H. Mascher and C. Kikuta, J. Chromatogr., 1990,506, 417. 18. G. R. Erdmann, K. Walker, G. S. Giebink and D. M. Canafax, J. Liq. Chromatogr., 1990, 13, 3339. 19. H. T. S. B&ton, Hydrogen Ions, 4th Ed Chapman and Hall, London, 19.52. 20. K. Florey, Analytical Profiles of Drug Sufrstances, Vol. 5, p. 34. Academic Press, London, 1978. 21. British Pharmacopoeia, p. 37. H. M. S. O., London, 1988.
Peqpmon
COLORIMETRIC DETERMINATION OF AMOXYCILLIN IN PURE FORM AND IN PHARMACEUTICAL PREPARATIONS A. S. AMIN,’ A. L. EL-ANSARY~and Y. M. 1s~’ ‘Chemistry Department, Faculty of Science, Benha University, Benha, Egypt 2Cairo University, Cairo, Egypt (Received 15 April 1993. Revised 5 September 1993. Accepted 6 September 1993) Summary-A simple and selective method for the determination of amoxycillin in pure form and in pharmaceutical preparations is described. The procedure is based on the reaction of amoxycillin with 4nitrophenol (I), 2,4_dinitrophenol (II), 3$dinitrobenxoic acid (III) or 3,5_dinitrosalicylic acid (IV) in alkaline medium. The method has been used for the determination of l-24 pg/ml of amoxycillin trihydrate in solution. The method is selective for the determination of amoxycillin in the presence of its degradation products, other antibiotics and different amines that are normally encountered in dosage forms.
Several methods have been reported in comprehensive reviews for the determination of amoxycillin’” (6R)-6-(a -D-p -hydroxyphenyl glycylamino) penicillanic acid [61336-70-71, a semisynthetic fi-lactam antibiotic. Most of these methods are based on the spectrophotometric determination of amoxycillin with paramolybdate anion,6 2,3-dichloro-5,6-dicyano-p-benzoquinone’ and chloranil.’ Various iodometric, alkalimetric,’ calorimetric,” potentiometric,” microbiologicali and high-performance liquid procedures have been rechromatographic’“” ported for the determination of the drug. The aim of this work was to demonstrate a simple and selective method for determination of amoxycillin in pure form and in pharmaceutical preparations.
Borate buffer solutions of different pH-values (5-l 1) were prepared as recommended.”
EXPERIMENTAL
Determination of amoxycillin in dosage forms
A Perkin-Elmer 13B spectrophotometer was used for all spectral measurements. The pH of solutions was checked using an Orion Model 60 1 A/digital Ionalyzer. All the nitroaromatics (I-IV) were Aldrich reagents, 10e3M solutions of the nitro derivatives were prepared by dissolving appropriate weight of the reagent in 100 ml of acetone. The solutions were prepared daily fresh protected from direct light. Standard solution of amoxycillin trihydrate: A 200 pg/ml solution was prepared by dissolving 0.05 g of pure amoxycillin trihydrate (amox.) in 5 ml of methanol and diluting the solution to 250 ml with water.
Capsules. Determine the average content weight of a capsule, dissolve 0.05 g amox. in the least amount of methanol and complete the volume to 250 ml with water. Transfer a volume containing 200 ug of amox. in a lo-ml measuring flask. Add 5 ml of pH 9 buffer solution and 1 ml of acetone and complete to the mark with water. Determine the concentration of amox. by following the general procedure. Syrup and ampoules. Transfer a volume of syrup or ampoule contents containing 0.05 g of amox. and complete the volume to 250 ml with water. Transfer a volume of this solution containing 200 pg of amox. into a lo-ml measuring flask, and complete as described under general
General procedure
Pipette 2 ml of 10m3M of reagent III into a IO-ml measuring flask. Add 5 ml of pH 9 buffer solution and 1 ml of acetone, a volume containing 200 pg amox. and complete to the mark with water. The reaction mixture is left to stand at room temperature (25 + SC) for 5 min. Measure the absorbance at 336 nm against a blank solution prepared simultaneously. If one of the reagents I, II and IV is to be used, mix well, place the flasks in a water bath at 50 + 2°C for 30 min and allow to stand at room temperature for 10 min.
691
A. S.
692
hlN
et aI.
Table 1. Spectral characteristics of the coloured reaction oroducts of amoxvcillin and nitroaromatics Quantitative parameters Reagent
I mar (nm 1
GwiX lo4 l/mol/cm
Linear range PRImI
Intercept
Sloue
Correlation coefficient
I II III IV
445 438 336 429
1.9 2.2 2.8 2.6
I-18 l-20 1-24 1-22
-0.0125 0.0050 0.0025 -0.0035
0.055 0.065 0.125 0.115
0.986 0.992 0.998 0.983
the concentration
of
procedure amox.
to determine
RESULTS AND DISCUSSION
Optimization
The precision values (eight replicates) were calculated for solutions containing 15 pg/ml amoxycillin. Relative standard deviations (RSD) of 0.51, 0.63, 0.37 and 0.89% were obtained for I, II, III and IV, respectively.
of reaction variables
Investigations were carried out to establish the most favourable conditions for the complexation reaction of amoxycillin with some nitroaromatics to achieve maximum colour development in the determination of amoxycillin. The influence of some variables on the reaction has been tested. The results of these studies are summarized in general procedure and Table 1. The coloured species may be formed through s-electron donation (nadduct); regarding the drug as a donor and the nitroaromatics as acceptor to form charge transfer complex. The coloured reaction product can be represented taking 3,5-dinitrobenzoic acid as example by the following structure.
Interferences
Phenylacetic acid, phenylacetamide, 6-amino penicillanic, ampicillin and cloxacillin may be present as impurities in amoxycillin as they are used as synthetic precursors. The effect of the presence of the above compounds and common co-existing additives and bases were studied. The components were added in excess of the amount used in the preparations to a solution containing 15 pg/ml of amoxycillin with 3,5dinitrobenzoic acid solution. Phenylacetic acid, phenylacetamide, 6arninopenicillanic acid, benzylpenicillin, ampicillin, flucloxacillin, cloxacillin, dicloxacillin, calcium carbonate, sodium acetate, magnesium stearate, talc powder, starch
coo
I
NH;
I CH-
Quan @cation
A linear correlation was found between absorbance and concentration in the ranges given in Table 1. Correlation coefficient, intercept and slope values for the calibration data of amoxycillin are calculated using the least-squares method.
and lactose do not interfere in the determination of amoxycillin indicating that complexation does not occur with these drugs under the reaction conditions used. Also there is no interference of penicilloic acid, penilloic acid, penillic acid, penaldic acid, penicillamine and ultimately penilloaldehyde which is the degradation products of amoxycillin*O resulting from thermal and
101.00 132.00
59.00 81.08
141.00 158.08
60 80
140 160 139.00 161.00
57.50 79.20
99.00 131.08
Found? 1 2
100 130
Taken mg
160 200
158.00 202.00
91.00 119.00
90 120
2
159.00 199.00
89.08 120.50
151.00 179.00
Found?
148.08 183.00
1
150 180
Taken mg
II
*:Manufacturer. t:Average of six determinations. t:Calculated f-value; tabulated r-value for eight degrees of freedom and P = 0.05 is 2.306. #Calculated F-value; tabulated F-value for f, =fi = 4 and P = 0.05 is 6.39. I:By the proposed method. 2:By the British Pharmacopoeia (1988) procedure. l/Egyptian International Pharmaceutical Industries Company (Egypt). ?Chemical Industries Development Company (Egypt). **:Amoun Pharmaceutical Industries Company (Egypt).
Capsules E-mox. (250 mg amox.)t E-mox. (500 mg amox.)t 8YruP Amoxicid (125 mg amox.)” Amoxicid (250 mg amox.)s Ampoules Ibiamox (250 mg amox.)** Ibiamox (500 mg amox.)**
Drug*
I
180 240
178.00 238.00
179.00 242.00
2.08 2.23
2.18 1.79
119.00 221.50
122.00 218.00
120 220
rt 1.69 2.14
2 169.00 201.00
Found?
171.00 198.00
1
170 200
Taken mg
III
2.61 2.78
2.67 3.17
3.11 2.53
Fo
Table 2. Determination of amoxycillin in different pharmaceutical preparations using nitroaromatics chromogens
140 200
160 180
220 230
Taken mg 2
161.08 178.00 141.00 198.00
158.00 178.00 139.00 201.00
218.00 231.08
Found?
222.00 233.00
1
IV
A. S. AMINet al.
694
hydrolytic degradates forms on the determination of amoxycillin.
Results of the analysis of amoxycillin capsules, syrups and ampoules by the proposed method show a good agreement with those of the British Pharmacopoeia (1988) procedure.” The relative standard deviation of the proposed method is better than 0.9% (six determinations) and the percentage recoveries of the proposed procedures were Pharmacopoeia1 and 100 f 1.67 and 98.8 zf 0.9 (six experiments), respectively, for different examined samples (Table 2). The results obtained were compared statistically by the student’s t-test and the variance ratio F-test with those obtained by Pharmacopoeial method on samples of the same batch (Table 2). The student’s t-test values obtained at the 95% confidence level and eight degrees of freedom did not exceed the theoretical tabulated value indicating no significant difference between the methods compared. The P-test also showed that there is no significant difference between the precisions of the proposed method and the official method. CONCLUSIONS
It is clear that 3,Sdinitrobenzoic acid is highly superior compared to other reagents. Very rapid fo~ation of the chromophore (5 min on cold), highest stability of colour (24 h) and lower RSD (0.37%).
REFERENCES 1. H. Bundgaard and C. Larsen, J. Pharm. Biomed. Anal.,
1983, 1, 29. 2. M. L. Lana, V. Girona, J. De Bolos and M. Castillo, Cienc. Znd. Farm., 1985,4, 13. 3. E. M. Abdel-Moety, M. A. Abounassif, M. E. Mohamed and N. A. Kbattab, Talanta, 1989,36, 683. 4. J. A. Murillo, J. Rodriguez, J. M. Lemus and A. Al&on, Analyst, 1990, 115, 1117. 5. E. M. Abdel-Moety, J. Pharm. Biomed. Anal., 1991, 9, 187. 6. P. B. lssopouios, J. Pharm. Biomed. Anal., 1988,6,97. I. H. F. Askal, G. A. Saleh and N. M. Omer, Analyst, 1991, 116, 387. 8. T. S. Al-Ghabsha, T. N. Al-Sabha and G. A. Al-Iraqi, Mcrochem. J., 1987, 36, 323. 9. S. Arora and J. P. Sharma, Nati. Acad. Sri. Len., 1990, 13, 15. 10. J. Talegaonkar and K. S. Boparai, Talanta, 1982, 29, 525. 11. D. M. Shingbal and R. R. Naik, Indian Drugs, 198522, 271. 12. H. Hopkala, Chem. Anal., 1987, 32, 929. p. 32. H. M. S. O., London, 13, British Ph~~copoe~ 1980. 14. G. W. K. Fong, D. I. Martin, R. N. Johnson and B. T. Kho, J. Chromatogr., 1984, 298, 459. 15. J. Martin, R. Mendez and A. Negro., J. Liq. Chromatogr., 1988, 11, 1707. 16, J. Haginak and J. Wakai, Analyst., 1985, 110, 1277. 17. H. Mascher and C. Kikuta, J. Chromatogr., 1990,506, 417. 18. G. R. Erdmann, K. Walker, G. S. Giebink and D. M. Canafax, J. Liq. Chromatogr., 1990, 13, 3339. 19. H. T. S. B&ton, Hydrogen Ions, 4th Ed Chapman and Hall, London, 19.52. 20. K. Florey, Analytical Profiles of Drug Sufrstances, Vol. 5, p. 34. Academic Press, London, 1978. 21. British Pharmacopoeia, p. 37. H. M. S. O., London, 1988.