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Use of ammonium molybdate in the colorimetric assay of cephalosporins
Talanta, Vol. 31, No. 8, pp. 635-637, 1984 Printed in Great Britain. All rights reserved
0039-9140/84s3.00+0.00 Copyright 0 1984 Pergamon Press Ltd
USE OF AMMONIUM MOLYBDATE IN THE COLORIMETRIC ASSAY OF CEPHALOSPORINS M. M. ABDEL-KHALEK and M. S. MAHROUS Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt (Received 21 October 1983. Revised 22 December
1983. Accepted 17 February 1984)
Summary-A calorimetric method for the determination of five cephalosporins (cefoxitin sodium, cefotaxime sodium, cephapirin sodium, cephalothin sodium and cephaloridine), based on the blue colour formed by reaction of the cephalosporins with ammonium molybdate, is described. The effects of reagent concentration and reaction conditions are discussed. The proposed method has been applied to the analysis of cephalosporin injections, the results of which are in good agreement with those obtained by the official method of the British Pharmacopoeia.
Cephalosporins
have been determined
as reagent. This procedure has been applied to a wide variety of cephalosporins (for structures see Table 1) in pure form and in injections. The reaction product is presumably an isopoly molybdenum blue.
iodometrically
after hydrolysis of the p-lactam ring,‘** and titrimetrically with N-bromosuccinimide3 or potassium iodate4. Cephalosporins have also been determined spectrophotometrically after treatment with hydroxylamine-nickel reagents and after preliminary acid hydrolysis.6 Cephapirin has been assayed spectrophotometrically after degradation under controlled conditions.’ An air-segmented continuousflow spectrophotometric method has been utilized for the determination of cephalosporins.s In addition, the use of high-pressure liquid chromatography,9v’0 and fluorimetric”~‘* and polarographic’3*‘4 methods of analysis have been reported. The aim of the present work was to develop a simple and accurate quantitative method for determining cephalosporins, with ammonium molybdate
EXPERIMENTAL Apparatus
A Perkin-Ehner Model 550 S spectrophotometer with matched l.OO-cm quartz cells was used for absorbance measurements. Reagents Ammonium molybdate solution, 1.5%. Sulphuric acid, OSM. Cephalosporin solutions, 0.2 mg/ml, freshly prepared
in water. Laboratory reference standards were used for calibration. A sample of cefoxitin was provided by Merck Sharp and Dohme (UK), of cefotaxime by Hoechst Orient Laboratories (Egypt), of ccphapirin by Bristol Laboratories
Table 1. Structures of cephalosporins R-CO-Nil 0 COOB 18 R'
B
_ccHj
-CCONE
R Cefoxitln CH2-
2
Cefotaxime -ccoc83
Cephapirin _R
--MER3
_H
--occcH3
--H
3’
Gephslothin CH2_
Cephaloridins
3-
\
636
SHORT COMhWMCATIONS
Table 2. Optical characteristics, accuracy and precision (10 replicates) Molar absorptivity, Cephalosporin
i -,
Cefoxitin sodium Cefotaxime sodium Cephapirin sodium Cephalothin sodium Cephaloridine
684-696 686-697 692-708 684-694 684-695
nm
Coefficient of variation,
I.mole-‘.cm-’
%
7.1 x 5.9 x 5.2 x 4.8 x 4.3 x
0.4 0.1 0.2 0.2 0.5
103 103 10) 103 10’
0.9
0.9 t
t
0.6
600
640
660
720
Wavelength
760
0.1
800
t
(nm)
0.0
Fig. 1. Absorption spectra of the coloured products from: A, cefoxitin sodium; B, cefotaxime sodium; C, cephapirin sodium; D, cephalothin sodium; and E, cephaloridine. [Cephalosporin] = 50 pg/ml in each instance. (U.S.A.) and samples of other cephalosporins by Lilly Research Centre Ltd. (U.S.A.). All chemicals and reagents used were of analyticalreagent or pharmaceutical grade. Distilled water was used throughout. General procedure and preparation of calibration graph
1
I
I
1
I
1.0
1.5
2.0
2.5
Concentration
of 1.0 ml ammonium
solution
added
RESULTS AND DISCUSSION
spectra
The absorption spectra of the reaction products in the range 600-800 nm are shown in Fig. 1. Cephalosporins and ammonium molybdate all have negligible absorbance at and near the 1,,, regions. Beer’s law is obeyed over the concentration range 20-100 pg/ml. The molar absorptivities are given in Table 2. The coefficient of variation was less than 0.4%. Choice
of analytical
conditions
A sulphuric acid concentration of about OSM is optimal (Table 3). Use of 1 ml of 1.5-2.5% ammo-
Table 3. Influence of acidity on the absorbance IWMW 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00
Cefoxitin sodium
Cefotaxime sodium
0.160 0.500 0.800 0.770 0.710 0.410 0.280 0.200 0.040
0.140 0.450 0.624 0.595 0.530 0.380 0.240 0.198 0.032
molybdote
(%w /v)
Fig. 2. Effect of ammonium molybdate concentration on the absorbance at J_ of the coloured products from: 0-O cefoxitin sodium; x - x cefotaxime sodium; 0-O cephapirin sodium; &--A cephalothin sodium; and D---O cephaloridine. [Cephalosporin] = 50 pg/ml in each instance.
Absorption
Transfer 5 ml of an aqueous solution containing from 0.2 to 1.0 mg of the sample into a test-tube. Add 1 ml of ammonium molybdate solution and 1 ml of 0.5M sulphuric acid. Prepare a corresponding blank solution with 5 ml of water. Mix well and heat the solutions in a boiling waterbath for 30 min. Cool and transfer into a lo-ml standard flask and dilute to volume with water. Measure the absorbance of the blue colour at Iz,, for the drug (Table 2). Prepare a calibration graph of absorbance us. concentration of the reference cephalosporin. For injecrions. Mix the powder from a vial, and accurately weigh a portion equivalent to about 10 mg of the drug and dissolve it in water. Dilute with water to volume in a lOO-ml standard flask. Transfer 5 ml of this solution into a test-tube and complete the assay as described above.
’ 0.5
Absorbance at &a Cephapirin Cephalothin sodium sodium 0.130 0.380 0.586 0.566 0.516 0.367 0.215 0.190 0.027
0.120 0.400 0.585 0.560 0.520 0.366 0.242 0.187 0.026
Cephaloridine 0.110 0.370 0.520 0.505 0.492 0.350 0.230 0.180 0.020
637
SHORT COMMUNlCATlONS
Table 4. Results for determination of cephalosporins in pure form and in injections Found* f s.d.,“/. Proposed method
Cephalosporin Cefoxitin sodium powder Mefoxin injection (MSD) Cefotaxime sodium powder Claforan injection (Hoechst) Cephapirin sodium powder Cephatrexyl injection (Bristol) Cephalothin sodium powder Kefin injection (Lilly) Cephaloridine powder Keflodin injection (Lilly) *Average of ten determinations, TCritical value 2.1 (p = 0.05). tCritica1 value 3.2 (p = 0.05).
BP method
&alc.t
F&S
100.4f 0.4
-
-
-
101.3 f 0.4
-
-
-
99.7 f 0.1
-
-
-
101.6 f 0.3
-
-
-
99.8 f 0.2
-
-
-
100.5 f 0.1
-
-
-
99.7 & 0.2
99.9 * 0.3
1.6
1.5
101.0 f 0.4
101.2 f 0.4
1.3
1.2
100.7 & 0.3
100.9 f 0.4
1.4
1.6
101.1 + 0.5
101.3 f 0.5
1.1
1.0
referred to nominal content.
tween the two sets of results. However, the molybdenum blue is simpler and faster than the BP method (but is not stability-indicating).
0.9 0.6 0.7
Interferences The possibility of interference by other constituents in injections cannot be overlooked. In the determination of cephalosporins in capsules, the excipients usually added, such as starch, lactose and glucose, interfere in the analysis by the proposed method.
0.6 0.5 0.4 0.3
REFERENCES 0.2
1.
0.1
2.
0.0 al Time
30
40
(min)
Fig. 3. Effect of heating time on the formation of the coloured product. 0-0 cefoxitin sodium; x - x cefotaxime sodium; O-0 cephapirin sodium; A-A cephacephaloridine. [Cephalothin sodium; and 0-n losporin] = 50 pg/ml in each instance. nium molybdate solution in the total volume of 10 ml gives maximum absorbance (Fig. 2). Figure 3 shows that heating for at least 30 min is essential for full colour development. The absorbance remains constant for about an hour after maximum colour developement. The order of addition of reagents is not CritiCill.
Table 4 gives the results obtained by application of the proposed method and the official method 1 to the determination of cephalosporins in pure form and in
injections. The results are in good agreement. Statistical comparison by the Student t-test and the variance ratio, F,15 shows no significant difference beTAL 3118-F
3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
British Pharmacopoeia 1980, H.M. Stationery Office, London, 1980. S. Okada, K. Hattori and T. Takano, Bull. Chem. Sot. Japan, 1965, 38, 2186. J. F. Alicino, J. Pharm. Sci., 1976, 65, 300. J. K. Grime and B. Tan, Anal. Chim. Acta, 1979, 105, 369. D. L. Mays, F. K. Bangert, W. C. Cantrell and W. G. Evans, Anal. Chem., 1975, 47, 2229. P. Papazova, P. R. Bontchev and M. Kacarova, Talanta, 1983, 30, 51. J. E. Bodnar, W. G. Evans and D. L. Mavs. _ J. Pharm. Sci., 1917, 66, 1108. M. A. Abdalla, A. G. Fogg, J. G. Bader and C. Burgess, Analyst, 1983, 108, 53. J. S. Wold and S. A. Tumispeed, J. Chromatog., 1977, 136. 170. P. k. Coomber, J. P. Jefferies and J. D Woodford, Analyst, 1982, 107, 1451. R. Aikawa, M. Nakano and T. Arita, Chem. Pharm. Bull., 1976,24, 2350. J. L. Fabregas and J. E. Beneyto, Analyst, 1980, 105, 813.
13. J. A. Squella, L. Nunez-Vergara and E. M. Gonzatez, J. Assoc. Off. Anal. Chem., 1979, 62, 556. 14. A. G. Fogg and M. J. Martin, Analyst, 1981,106,1213. 15. L. Saunders and R. Fleming, Mathematics and Statistics, 2nd Ed., pp. 192-197, Pharmaceutical Press, London, 1971.
0039-9140/84s3.00+0.00 Copyright 0 1984 Pergamon Press Ltd
USE OF AMMONIUM MOLYBDATE IN THE COLORIMETRIC ASSAY OF CEPHALOSPORINS M. M. ABDEL-KHALEK and M. S. MAHROUS Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt (Received 21 October 1983. Revised 22 December
1983. Accepted 17 February 1984)
Summary-A calorimetric method for the determination of five cephalosporins (cefoxitin sodium, cefotaxime sodium, cephapirin sodium, cephalothin sodium and cephaloridine), based on the blue colour formed by reaction of the cephalosporins with ammonium molybdate, is described. The effects of reagent concentration and reaction conditions are discussed. The proposed method has been applied to the analysis of cephalosporin injections, the results of which are in good agreement with those obtained by the official method of the British Pharmacopoeia.
Cephalosporins
have been determined
as reagent. This procedure has been applied to a wide variety of cephalosporins (for structures see Table 1) in pure form and in injections. The reaction product is presumably an isopoly molybdenum blue.
iodometrically
after hydrolysis of the p-lactam ring,‘** and titrimetrically with N-bromosuccinimide3 or potassium iodate4. Cephalosporins have also been determined spectrophotometrically after treatment with hydroxylamine-nickel reagents and after preliminary acid hydrolysis.6 Cephapirin has been assayed spectrophotometrically after degradation under controlled conditions.’ An air-segmented continuousflow spectrophotometric method has been utilized for the determination of cephalosporins.s In addition, the use of high-pressure liquid chromatography,9v’0 and fluorimetric”~‘* and polarographic’3*‘4 methods of analysis have been reported. The aim of the present work was to develop a simple and accurate quantitative method for determining cephalosporins, with ammonium molybdate
EXPERIMENTAL Apparatus
A Perkin-Ehner Model 550 S spectrophotometer with matched l.OO-cm quartz cells was used for absorbance measurements. Reagents Ammonium molybdate solution, 1.5%. Sulphuric acid, OSM. Cephalosporin solutions, 0.2 mg/ml, freshly prepared
in water. Laboratory reference standards were used for calibration. A sample of cefoxitin was provided by Merck Sharp and Dohme (UK), of cefotaxime by Hoechst Orient Laboratories (Egypt), of ccphapirin by Bristol Laboratories
Table 1. Structures of cephalosporins R-CO-Nil 0 COOB 18 R'
B
_ccHj
-CCONE
R Cefoxitln CH2-
2
Cefotaxime -ccoc83
Cephapirin _R
--MER3
_H
--occcH3
--H
3’
Gephslothin CH2_
Cephaloridins
3-
\
636
SHORT COMhWMCATIONS
Table 2. Optical characteristics, accuracy and precision (10 replicates) Molar absorptivity, Cephalosporin
i -,
Cefoxitin sodium Cefotaxime sodium Cephapirin sodium Cephalothin sodium Cephaloridine
684-696 686-697 692-708 684-694 684-695
nm
Coefficient of variation,
I.mole-‘.cm-’
%
7.1 x 5.9 x 5.2 x 4.8 x 4.3 x
0.4 0.1 0.2 0.2 0.5
103 103 10) 103 10’
0.9
0.9 t
t
0.6
600
640
660
720
Wavelength
760
0.1
800
t
(nm)
0.0
Fig. 1. Absorption spectra of the coloured products from: A, cefoxitin sodium; B, cefotaxime sodium; C, cephapirin sodium; D, cephalothin sodium; and E, cephaloridine. [Cephalosporin] = 50 pg/ml in each instance. (U.S.A.) and samples of other cephalosporins by Lilly Research Centre Ltd. (U.S.A.). All chemicals and reagents used were of analyticalreagent or pharmaceutical grade. Distilled water was used throughout. General procedure and preparation of calibration graph
1
I
I
1
I
1.0
1.5
2.0
2.5
Concentration
of 1.0 ml ammonium
solution
added
RESULTS AND DISCUSSION
spectra
The absorption spectra of the reaction products in the range 600-800 nm are shown in Fig. 1. Cephalosporins and ammonium molybdate all have negligible absorbance at and near the 1,,, regions. Beer’s law is obeyed over the concentration range 20-100 pg/ml. The molar absorptivities are given in Table 2. The coefficient of variation was less than 0.4%. Choice
of analytical
conditions
A sulphuric acid concentration of about OSM is optimal (Table 3). Use of 1 ml of 1.5-2.5% ammo-
Table 3. Influence of acidity on the absorbance IWMW 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00
Cefoxitin sodium
Cefotaxime sodium
0.160 0.500 0.800 0.770 0.710 0.410 0.280 0.200 0.040
0.140 0.450 0.624 0.595 0.530 0.380 0.240 0.198 0.032
molybdote
(%w /v)
Fig. 2. Effect of ammonium molybdate concentration on the absorbance at J_ of the coloured products from: 0-O cefoxitin sodium; x - x cefotaxime sodium; 0-O cephapirin sodium; &--A cephalothin sodium; and D---O cephaloridine. [Cephalosporin] = 50 pg/ml in each instance.
Absorption
Transfer 5 ml of an aqueous solution containing from 0.2 to 1.0 mg of the sample into a test-tube. Add 1 ml of ammonium molybdate solution and 1 ml of 0.5M sulphuric acid. Prepare a corresponding blank solution with 5 ml of water. Mix well and heat the solutions in a boiling waterbath for 30 min. Cool and transfer into a lo-ml standard flask and dilute to volume with water. Measure the absorbance of the blue colour at Iz,, for the drug (Table 2). Prepare a calibration graph of absorbance us. concentration of the reference cephalosporin. For injecrions. Mix the powder from a vial, and accurately weigh a portion equivalent to about 10 mg of the drug and dissolve it in water. Dilute with water to volume in a lOO-ml standard flask. Transfer 5 ml of this solution into a test-tube and complete the assay as described above.
’ 0.5
Absorbance at &a Cephapirin Cephalothin sodium sodium 0.130 0.380 0.586 0.566 0.516 0.367 0.215 0.190 0.027
0.120 0.400 0.585 0.560 0.520 0.366 0.242 0.187 0.026
Cephaloridine 0.110 0.370 0.520 0.505 0.492 0.350 0.230 0.180 0.020
637
SHORT COMMUNlCATlONS
Table 4. Results for determination of cephalosporins in pure form and in injections Found* f s.d.,“/. Proposed method
Cephalosporin Cefoxitin sodium powder Mefoxin injection (MSD) Cefotaxime sodium powder Claforan injection (Hoechst) Cephapirin sodium powder Cephatrexyl injection (Bristol) Cephalothin sodium powder Kefin injection (Lilly) Cephaloridine powder Keflodin injection (Lilly) *Average of ten determinations, TCritical value 2.1 (p = 0.05). tCritica1 value 3.2 (p = 0.05).
BP method
&alc.t
F&S
100.4f 0.4
-
-
-
101.3 f 0.4
-
-
-
99.7 f 0.1
-
-
-
101.6 f 0.3
-
-
-
99.8 f 0.2
-
-
-
100.5 f 0.1
-
-
-
99.7 & 0.2
99.9 * 0.3
1.6
1.5
101.0 f 0.4
101.2 f 0.4
1.3
1.2
100.7 & 0.3
100.9 f 0.4
1.4
1.6
101.1 + 0.5
101.3 f 0.5
1.1
1.0
referred to nominal content.
tween the two sets of results. However, the molybdenum blue is simpler and faster than the BP method (but is not stability-indicating).
0.9 0.6 0.7
Interferences The possibility of interference by other constituents in injections cannot be overlooked. In the determination of cephalosporins in capsules, the excipients usually added, such as starch, lactose and glucose, interfere in the analysis by the proposed method.
0.6 0.5 0.4 0.3
REFERENCES 0.2
1.
0.1
2.
0.0 al Time
30
40
(min)
Fig. 3. Effect of heating time on the formation of the coloured product. 0-0 cefoxitin sodium; x - x cefotaxime sodium; O-0 cephapirin sodium; A-A cephacephaloridine. [Cephalothin sodium; and 0-n losporin] = 50 pg/ml in each instance. nium molybdate solution in the total volume of 10 ml gives maximum absorbance (Fig. 2). Figure 3 shows that heating for at least 30 min is essential for full colour development. The absorbance remains constant for about an hour after maximum colour developement. The order of addition of reagents is not CritiCill.
Table 4 gives the results obtained by application of the proposed method and the official method 1 to the determination of cephalosporins in pure form and in
injections. The results are in good agreement. Statistical comparison by the Student t-test and the variance ratio, F,15 shows no significant difference beTAL 3118-F
3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
British Pharmacopoeia 1980, H.M. Stationery Office, London, 1980. S. Okada, K. Hattori and T. Takano, Bull. Chem. Sot. Japan, 1965, 38, 2186. J. F. Alicino, J. Pharm. Sci., 1976, 65, 300. J. K. Grime and B. Tan, Anal. Chim. Acta, 1979, 105, 369. D. L. Mays, F. K. Bangert, W. C. Cantrell and W. G. Evans, Anal. Chem., 1975, 47, 2229. P. Papazova, P. R. Bontchev and M. Kacarova, Talanta, 1983, 30, 51. J. E. Bodnar, W. G. Evans and D. L. Mavs. _ J. Pharm. Sci., 1917, 66, 1108. M. A. Abdalla, A. G. Fogg, J. G. Bader and C. Burgess, Analyst, 1983, 108, 53. J. S. Wold and S. A. Tumispeed, J. Chromatog., 1977, 136. 170. P. k. Coomber, J. P. Jefferies and J. D Woodford, Analyst, 1982, 107, 1451. R. Aikawa, M. Nakano and T. Arita, Chem. Pharm. Bull., 1976,24, 2350. J. L. Fabregas and J. E. Beneyto, Analyst, 1980, 105, 813.
13. J. A. Squella, L. Nunez-Vergara and E. M. Gonzatez, J. Assoc. Off. Anal. Chem., 1979, 62, 556. 14. A. G. Fogg and M. J. Martin, Analyst, 1981,106,1213. 15. L. Saunders and R. Fleming, Mathematics and Statistics, 2nd Ed., pp. 192-197, Pharmaceutical Press, London, 1971.