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Spectrofluorimetric assay of tetracycline and anhydrotetracycline in combination
~39-91~/85 33.00+ 0.00 Copyright Q 1985Pergamon Press Ltd
Tafanto,Vol. 32, No. 12, pp. 1153-1155,1985 Printed in Great Britani. All rights reserved
SPECTROFLUORIMETRIC ASSAY OF TETRACYCLINE AND ANHYDROTETRACYCLINE IN COMBINATION M. ABDEL-HADY ELSAYED, M. H. BARARY and H. MAHGDUB Department of Pharmaceutical Analytical Chemistry. Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt (Receroed 19 aired
1984. Raised 5 August 1985. Accepted 23 Augusr 1985)
Summary-Spectrofluonmetric methods are described for the assay of tetracycline (TC) and anhydrotetracycline (ATC) in combination, without prior separation. The interference from ATC in the TC assay has been corrected for by forming the aluminium complexes of both drugs and measuring the difference in fluorescence at 475 and 418 nm, with excitation at 393 nm. Similarly, measurement of the fluorescence of the magnesium complexes at 525 and 470 nm (excitation at 440 nm) nullifies TC interference in the ATC assay.
475 (F4,5) and 418 nm (F,,,) with excitation at 393 nm. Prepare and measure a blank solution in the same way. Plot F473 and F&7$ - F,,, vs. concentration. Fur ATC. Transfer 1.00,2.00. . .6.00, 7.00 ml portions of ATC into IOO-mlstandard flasks and dilute to volume with water. For each standard transfer a S.OO-mlportion into a 25-ml conical flask (initially dry) containing 5.00 ml of pH 6 buffer, and add 2.50 ml of magnesium sulphate solution. Let stand for 15 min at room temperature (- 200), then measure the fluorescence at 525 (F$&) and 470 nm (F9& with excitation at 440 nm. Apply corrections for a blank similarly prepared. Plot FTj5- FdTOvs. concentration.
Microbiological,‘” spectrophotometric4 and fluorimetric7-9 methods have been used for assay of tetracyclines. Compared with the fluorimetric methods, the m~erobiolo~cal methods lack specificity and the spectrophotometric methods are Iess sensitive. Fluorimetric determination of tetracyclines has been based on formation of the aluminium’ or magnesiumlO complexes, but there is mutual interference when tetracycline (TC) and anhydrotetracline are both present. ATC is a degradation product of TC and a limit for it is a compendia1 requirement.* The present work describes a mathematical method
for eliminating
RJ?SULTSAND DISCUSSION
the effect of the intereference.
The methods described were tested as follows. EXPERIMENTAL Apparatus
Determination
A Perkin-Elmer 650-10s fluorescence spectrometer was used, with a I x I cm cross-section quartz cuvette. The spectra1 band-width was IO nm for both excitation and emission. Before measurement of each batch of samples, the instrument was standardized with quinine sulphate solution (0.05 mg/l.) in 0.1 M sulphurtc acid to give a scale reading of 80% with excitation at 350 nm and emission measurement at 455 nm. Reagents Aluminiurn chloride solution, 0.75M. Magnesia sulp~ate solution, 0.75h4. Stirensen’s citrate buffer, pH 6.0. Made by mixing 59.5 ml
of 0.1 M disodium citrate (2 1.Og of citric acid monohydrate dissolved in 200 ml of 1M sodium hydroxide and made up to 1 litre) with 40.5 ml of O.lM sodium hydroxide, the pH being checked by pH-meter and adjusted if necessary. Standard solutions of TC and ATC. Made by dissolving 100 mg of tetracycline hydrochloride (Lederle) or 20.0 mg of anhydrotetracycline hydrochloride (prepared according to Simmons et al.“) and diluting to volume with water in a lOO-ml standard flask.
of
TC in the presence of ATC
To prepare mixtures of known composition, standard TC solution was accurately diluted tenfold with water, and 0.50, 1.OO, 1SO, 2.00, 2.50, 3.00 ml portions were mixed with l.OO-ml portions of standard ATC solution and diluted accurately to 100 ml. Five-ml portions of each solution were then treated and measured as described. Commercial capsules were also assayed for TC and ATC. The contents of 20 “Tetracid” capsules (Cid Laboratories, Egypt) were mixed and an amount equivalent to IO0 mg of TC hydrochloride was transferred to a IOO-ml standard flask, and shaken with about 40 ml of water for 3 min to ensure complete dissolution. The solution was then made up to volume with water and filtered; 10.00 ml of filtrate were diluted to volume in a 100-ml standard flask and this solution was analysed as above. The results are given in Table 1. Determination
For TC. Transfer 0.50, 1.00, 1.50, 2.00 and 2.50 ml portions of standard TC solution into loo-ml standard kasks, dilute to volume w&h water and mix. Add 5.00 ml of PH-6 buffer to 5.00 ml of each dilution. followed bv 2.50 ml bf aluminium chloride solution. Let stand for l? min at room temperature (25”). then measure the fluorescence at
Standard TC solution was accurately diluted fourfold with water and 2.00-ml portions were mixed with 1.oo, 2.00, **. 6.00,7.00 ml portions of standard ATC solution and diluted to 100 ml accurately with water. The procedure for ATC calibration graphs was then 1153
7.48.3212-E
of
ATC in the presence of TC
Calibration graphs
1154
SHORT COMMLlNICATlONS
Table 1. Fluorometric determination of TC in presence of ATC, in a laboratory-made mixture and in “Tetracid” cansules with added ATC. bv the AF method
60
Recovery, % Ratio TClATC
“Tetracid” capsules with added ATC*
Laboratory-made mixture*
114 112 I/1.30 l/l.00 l/O.80 110.67
99.8 101.5 100.8 99.8 100.1 99.8
105.2 105.8 105.2 106.1 106.3 105.4
Mean C.V., %
100.3 0.7
105.7 0.5
50 8 5 :: pj 40 2 E p 30 6 B a 20
*Each mixture contained 0.08 mg of ATC and 0.0220.12 mg of TC per 100 ml of solution. The recovery for the
10
capsule analysis is based on the nominal TC content of the capsules. Xhm)
applied to 5-ml portions of these solutions. results are given in Table 2.
The
Fluorescence characteristics
Addition of aluminium to TC and ATC in Sorensen buffer at pH 6 gives the corresponding Table 2. Fluorometric detennination of ATC in presence of TC by the AF method Ratio ATC/TC
v
Recovery, %
I/2.50 l/l.25 l/O.83 l/O.63 I/O.50 l/0.42 I/O.36
100.0 101.o 100.7 99.9 100.4 101.1 100.3
Mean C.V.. %
100.5 0.5
Fig. 2. Fluorescence emisston spectra for 1.2 pg/ml solutions of TC-AI(-) and ATC-AI (- - -) (I,, = 393 nm).
TC-AI and ATC-Al fluorophores, which have different excitation and emission maxima (Fig. 1). Excitation of both fluorophores at 393 nm gives an emission maximum at 475 nm for TC-Al but almost constant background emission at 418 and 475 nm for ATC-Al (Fig. 2). Addition of magnesium instead of aluminium changes the excitation and emission spectra (Fig. 3). Excitation of both fluorophores at 440 nm gives a maximum at 525 nm for ATC-Mg and almost constant fluorescence in the 470-540 nm region from TC-Mg (Fig. 4). Assay for TC
The difference in the fluorescence spectra of TC-Al and ATC-Al allows determination of TC without
I
I
I
I
I
260
340
420
500
560
Xtnm) Fig. 1. Fluorescence excitation (a) and emission (b) spectra for 1.2 pg/ml solutions of TC-Al (-) and ATC-A1 (---). Emission spectra for excitation at 393 nm for TC-Al and 320 nm for ATC-AI.
X(nm1
Fig. 3. Fluorescence excitation (a) and emission (b) spectra for 1.2 pg/ml solutions of TC-Mg (-) and ATC-Mg (---). Emission spectra for excitation at 380 nm for TC-Mg and 440 nm for ACT-Mg.
SHORT
1155
COMMUNICATIONS
Assay for ATC
Fig. 4. Fluorescence solutions
of
emission spectra for 1.2 pg/ml TC-Mg (-), and ATC-Mg (---) (A, = 440 nm).
The AFmethod is again applied, with measurement of fluorescence intensity of ATC-Mg at 525 and 470 nm (A,, 440 nm). A linear relationship is obtained over the concentration range 0.8-0.48 pg/ml in the final solutions. The validity of the method is confirmed by the results in Table 2. The proposed AF methods may find wide application, especially in the spectrofluorimetric assay of the pure drugs or pharmaceutical multi-component formulations containing excipients or diverse components which give linear or no background interference (since correction methods can be applied for linear background). Moreover the high sensitivity makes the methods suitable for biological fluid analysis, after an initial extraction step.9q10 REFERENCES
interference from ATC. The fluorescence intensity at 475 nm (F475) (A,, 393 nm) is the sum of that for TC-AI alone (F,,,,) and that for the fluorescence background of ATC-AI (Fa) (Fig. 2) and the fluorescence intensity at 418 nm (F4,*) of the same solution (A,, still 393 nm) is again the sum of the two fluorescence intensities, F,,,, for TC-Al and FB for ATC-Al. The difference, AF = FdT5- F4,8r is clearly equal to Fm, - F,,,,, for TC-Al. Calibration graphs for TC in the absence of ATC can be prepared by plotting AF for the TC-Al fluorophore us. [TC], a linear relationship being obtained. The method is also valid for the determination of TC alone or in the presence of ATC, as confirmed by the results in Table 1.
1.
D. C. Grove and W. A. Randall, Assay Metho& for Antibiotics, Medical Encyclopedia, New York, 1955. 2. U.S. Pharmacopoeia, 20th Rev., p. 780, U.S. Pharmacopeial Convention, Rockville, Md., 1980. 3. B&h Phnrmacopoeia, p. 448. HMSO, London, 1980. 4. 0. Hrdv and H. Bevrodtovit. Cesk. Farm.. 1974.23.122. 5. A. M. Wahbi and s. Ebel, Anal. Chim. Acta, 1974, 70, 57. 6.
A. F. Zak, G. I. Loseva, L. P. Snezhnova, V. M.
Shatrova, T. I. Navolotskaya and L. M. Yakobson, Antibiotiki, 1975, 20, 685. 7. A. I. Cherkesov and T. V. Alykova, Zh. Analit. Khim, 1973, m, 377. 8. D. Hall, J. Pharm. Pharmacol., 1976, Zs, 420. 9. R. G. Kelly, L. M. Peets and K. D. Hoyt, Anal. Biochem., 1969, 28, 222. 10. H. Poiger and Ch. Schlatter, Analysf, 1976, 101, 808.
11. D. L. Simmons, H. S. L. Woo, C. M. Koorengevel and P. Seers, J. Pharm. Sci., 1966, 55, 1313.
Tafanto,Vol. 32, No. 12, pp. 1153-1155,1985 Printed in Great Britani. All rights reserved
SPECTROFLUORIMETRIC ASSAY OF TETRACYCLINE AND ANHYDROTETRACYCLINE IN COMBINATION M. ABDEL-HADY ELSAYED, M. H. BARARY and H. MAHGDUB Department of Pharmaceutical Analytical Chemistry. Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt (Receroed 19 aired
1984. Raised 5 August 1985. Accepted 23 Augusr 1985)
Summary-Spectrofluonmetric methods are described for the assay of tetracycline (TC) and anhydrotetracycline (ATC) in combination, without prior separation. The interference from ATC in the TC assay has been corrected for by forming the aluminium complexes of both drugs and measuring the difference in fluorescence at 475 and 418 nm, with excitation at 393 nm. Similarly, measurement of the fluorescence of the magnesium complexes at 525 and 470 nm (excitation at 440 nm) nullifies TC interference in the ATC assay.
475 (F4,5) and 418 nm (F,,,) with excitation at 393 nm. Prepare and measure a blank solution in the same way. Plot F473 and F&7$ - F,,, vs. concentration. Fur ATC. Transfer 1.00,2.00. . .6.00, 7.00 ml portions of ATC into IOO-mlstandard flasks and dilute to volume with water. For each standard transfer a S.OO-mlportion into a 25-ml conical flask (initially dry) containing 5.00 ml of pH 6 buffer, and add 2.50 ml of magnesium sulphate solution. Let stand for 15 min at room temperature (- 200), then measure the fluorescence at 525 (F$&) and 470 nm (F9& with excitation at 440 nm. Apply corrections for a blank similarly prepared. Plot FTj5- FdTOvs. concentration.
Microbiological,‘” spectrophotometric4 and fluorimetric7-9 methods have been used for assay of tetracyclines. Compared with the fluorimetric methods, the m~erobiolo~cal methods lack specificity and the spectrophotometric methods are Iess sensitive. Fluorimetric determination of tetracyclines has been based on formation of the aluminium’ or magnesiumlO complexes, but there is mutual interference when tetracycline (TC) and anhydrotetracline are both present. ATC is a degradation product of TC and a limit for it is a compendia1 requirement.* The present work describes a mathematical method
for eliminating
RJ?SULTSAND DISCUSSION
the effect of the intereference.
The methods described were tested as follows. EXPERIMENTAL Apparatus
Determination
A Perkin-Elmer 650-10s fluorescence spectrometer was used, with a I x I cm cross-section quartz cuvette. The spectra1 band-width was IO nm for both excitation and emission. Before measurement of each batch of samples, the instrument was standardized with quinine sulphate solution (0.05 mg/l.) in 0.1 M sulphurtc acid to give a scale reading of 80% with excitation at 350 nm and emission measurement at 455 nm. Reagents Aluminiurn chloride solution, 0.75M. Magnesia sulp~ate solution, 0.75h4. Stirensen’s citrate buffer, pH 6.0. Made by mixing 59.5 ml
of 0.1 M disodium citrate (2 1.Og of citric acid monohydrate dissolved in 200 ml of 1M sodium hydroxide and made up to 1 litre) with 40.5 ml of O.lM sodium hydroxide, the pH being checked by pH-meter and adjusted if necessary. Standard solutions of TC and ATC. Made by dissolving 100 mg of tetracycline hydrochloride (Lederle) or 20.0 mg of anhydrotetracycline hydrochloride (prepared according to Simmons et al.“) and diluting to volume with water in a lOO-ml standard flask.
of
TC in the presence of ATC
To prepare mixtures of known composition, standard TC solution was accurately diluted tenfold with water, and 0.50, 1.OO, 1SO, 2.00, 2.50, 3.00 ml portions were mixed with l.OO-ml portions of standard ATC solution and diluted accurately to 100 ml. Five-ml portions of each solution were then treated and measured as described. Commercial capsules were also assayed for TC and ATC. The contents of 20 “Tetracid” capsules (Cid Laboratories, Egypt) were mixed and an amount equivalent to IO0 mg of TC hydrochloride was transferred to a IOO-ml standard flask, and shaken with about 40 ml of water for 3 min to ensure complete dissolution. The solution was then made up to volume with water and filtered; 10.00 ml of filtrate were diluted to volume in a 100-ml standard flask and this solution was analysed as above. The results are given in Table 1. Determination
For TC. Transfer 0.50, 1.00, 1.50, 2.00 and 2.50 ml portions of standard TC solution into loo-ml standard kasks, dilute to volume w&h water and mix. Add 5.00 ml of PH-6 buffer to 5.00 ml of each dilution. followed bv 2.50 ml bf aluminium chloride solution. Let stand for l? min at room temperature (25”). then measure the fluorescence at
Standard TC solution was accurately diluted fourfold with water and 2.00-ml portions were mixed with 1.oo, 2.00, **. 6.00,7.00 ml portions of standard ATC solution and diluted to 100 ml accurately with water. The procedure for ATC calibration graphs was then 1153
7.48.3212-E
of
ATC in the presence of TC
Calibration graphs
1154
SHORT COMMLlNICATlONS
Table 1. Fluorometric determination of TC in presence of ATC, in a laboratory-made mixture and in “Tetracid” cansules with added ATC. bv the AF method
60
Recovery, % Ratio TClATC
“Tetracid” capsules with added ATC*
Laboratory-made mixture*
114 112 I/1.30 l/l.00 l/O.80 110.67
99.8 101.5 100.8 99.8 100.1 99.8
105.2 105.8 105.2 106.1 106.3 105.4
Mean C.V., %
100.3 0.7
105.7 0.5
50 8 5 :: pj 40 2 E p 30 6 B a 20
*Each mixture contained 0.08 mg of ATC and 0.0220.12 mg of TC per 100 ml of solution. The recovery for the
10
capsule analysis is based on the nominal TC content of the capsules. Xhm)
applied to 5-ml portions of these solutions. results are given in Table 2.
The
Fluorescence characteristics
Addition of aluminium to TC and ATC in Sorensen buffer at pH 6 gives the corresponding Table 2. Fluorometric detennination of ATC in presence of TC by the AF method Ratio ATC/TC
v
Recovery, %
I/2.50 l/l.25 l/O.83 l/O.63 I/O.50 l/0.42 I/O.36
100.0 101.o 100.7 99.9 100.4 101.1 100.3
Mean C.V.. %
100.5 0.5
Fig. 2. Fluorescence emisston spectra for 1.2 pg/ml solutions of TC-AI(-) and ATC-AI (- - -) (I,, = 393 nm).
TC-AI and ATC-Al fluorophores, which have different excitation and emission maxima (Fig. 1). Excitation of both fluorophores at 393 nm gives an emission maximum at 475 nm for TC-Al but almost constant background emission at 418 and 475 nm for ATC-Al (Fig. 2). Addition of magnesium instead of aluminium changes the excitation and emission spectra (Fig. 3). Excitation of both fluorophores at 440 nm gives a maximum at 525 nm for ATC-Mg and almost constant fluorescence in the 470-540 nm region from TC-Mg (Fig. 4). Assay for TC
The difference in the fluorescence spectra of TC-Al and ATC-Al allows determination of TC without
I
I
I
I
I
260
340
420
500
560
Xtnm) Fig. 1. Fluorescence excitation (a) and emission (b) spectra for 1.2 pg/ml solutions of TC-Al (-) and ATC-A1 (---). Emission spectra for excitation at 393 nm for TC-Al and 320 nm for ATC-AI.
X(nm1
Fig. 3. Fluorescence excitation (a) and emission (b) spectra for 1.2 pg/ml solutions of TC-Mg (-) and ATC-Mg (---). Emission spectra for excitation at 380 nm for TC-Mg and 440 nm for ACT-Mg.
SHORT
1155
COMMUNICATIONS
Assay for ATC
Fig. 4. Fluorescence solutions
of
emission spectra for 1.2 pg/ml TC-Mg (-), and ATC-Mg (---) (A, = 440 nm).
The AFmethod is again applied, with measurement of fluorescence intensity of ATC-Mg at 525 and 470 nm (A,, 440 nm). A linear relationship is obtained over the concentration range 0.8-0.48 pg/ml in the final solutions. The validity of the method is confirmed by the results in Table 2. The proposed AF methods may find wide application, especially in the spectrofluorimetric assay of the pure drugs or pharmaceutical multi-component formulations containing excipients or diverse components which give linear or no background interference (since correction methods can be applied for linear background). Moreover the high sensitivity makes the methods suitable for biological fluid analysis, after an initial extraction step.9q10 REFERENCES
interference from ATC. The fluorescence intensity at 475 nm (F475) (A,, 393 nm) is the sum of that for TC-AI alone (F,,,,) and that for the fluorescence background of ATC-AI (Fa) (Fig. 2) and the fluorescence intensity at 418 nm (F4,*) of the same solution (A,, still 393 nm) is again the sum of the two fluorescence intensities, F,,,, for TC-Al and FB for ATC-Al. The difference, AF = FdT5- F4,8r is clearly equal to Fm, - F,,,,, for TC-Al. Calibration graphs for TC in the absence of ATC can be prepared by plotting AF for the TC-Al fluorophore us. [TC], a linear relationship being obtained. The method is also valid for the determination of TC alone or in the presence of ATC, as confirmed by the results in Table 1.
1.
D. C. Grove and W. A. Randall, Assay Metho& for Antibiotics, Medical Encyclopedia, New York, 1955. 2. U.S. Pharmacopoeia, 20th Rev., p. 780, U.S. Pharmacopeial Convention, Rockville, Md., 1980. 3. B&h Phnrmacopoeia, p. 448. HMSO, London, 1980. 4. 0. Hrdv and H. Bevrodtovit. Cesk. Farm.. 1974.23.122. 5. A. M. Wahbi and s. Ebel, Anal. Chim. Acta, 1974, 70, 57. 6.
A. F. Zak, G. I. Loseva, L. P. Snezhnova, V. M.
Shatrova, T. I. Navolotskaya and L. M. Yakobson, Antibiotiki, 1975, 20, 685. 7. A. I. Cherkesov and T. V. Alykova, Zh. Analit. Khim, 1973, m, 377. 8. D. Hall, J. Pharm. Pharmacol., 1976, Zs, 420. 9. R. G. Kelly, L. M. Peets and K. D. Hoyt, Anal. Biochem., 1969, 28, 222. 10. H. Poiger and Ch. Schlatter, Analysf, 1976, 101, 808.
11. D. L. Simmons, H. S. L. Woo, C. M. Koorengevel and P. Seers, J. Pharm. Sci., 1966, 55, 1313.