Department of Analytical Chemistry and Electrochemistry, University of Extremadura, Badajoz, Spain. Bestimmung von Oxytetracyelin in Urin und Humanserum.
Frcsenius Z Anal Chem (1989) 335:1002-1004
Determination of oxytetracycline in urine and human serum by differential pulse polarography E. Pinilla Gil, L. Calvo Blazquez, R. M. Garcia-Monco Carra, and A. Sanchez Misiego Department of Analytical Chemistry and Electrochemistry, University of Extremadura, Badajoz, Spain Bestimmung von Oxytetracyelin in Urin und Humanserum durch Differential-Pulspolarography Summary. A differential pulse polarographic method for the determination of oxytetracycline in urine and human serum in acid media (HC104 of pH 2) is proposed. The effects of the amount of sample taken and the concentration of HC104 present were investigated. The detection limit was 5.5 x 10 -6 mol/1. The standard deviation of the determination of 5.5 x 10- s mol/1 of oxytetracycline in 2 ml of urine was 1.7x10-6mol/1 and that of the determination of 5.5 x 10-s mol/1 of oxytetracycline in 2 ml of human serum was 1.9 x 10 .6 tool/1.
Introduction The tetracyclines are bacteriostatic agents that act by blocking the attachment of transfer R N A to ribosomes, thus interfering with the protein synthesis. The tetracyclines have a wide range of activity, inhibiting Gram-positive and Gramnegative organisms. Due to their wide antibacterial activity, they are widely used in the treatment of infections of the respiratory tract, infections of the urinary tract and in the treatment of syphilis, gonorrhoea, antrax and actinomicosis. Structure (I) illustrates the tetracycline ring structure, and the numbering system employed here. An important tetracycline derivative is oxytetracycline (II). RI
I~2 R3 H
H N(CH3) 2
Offprint requests to: L. Calvo Blazquez
The polarographic reducibility of tetracyclines has been investigated [1, 2]. The polarographic waves were complex and largely dependent upon pH and nature and concentration of the buffer used. The reduction of oxytetracycline appeared to take place in two 2-electron waves which were attributed to the reduction of the two unsaturated carbonyl systems in the molecule. Doskocilova  was the first to attempt to interpret the reduction waves and to coordinate these with specific functional groups. A survey of the polarographic methods used for the determination of tetracyclines shows that it is a good analytical tool for such analysis [ 4 - 9 ] ; because, in most the cases, the time expended for sample manipulation is very low and the measure process is very fast. This work is carried out, using acid medium, in order to study the applicability of differential pulse polarography to the rapid determination of oxytetracycline in urine and human serum. The acid medium is generally used for separation of proteins in the treatment and determination of organic compounds from related fluids.
Experimental Apparatus. A Metrohm Herisau E-506 Polarecord assembly was used with a polarographic Stand Metrohm 663-VA provided with the following electrodes: working electrode DME, reference electrode Ag/AgC1/saturated KC1, and a glassy carbon auxiliary electrode. Reagents. A 10 -4 mol/1 methanolic stock solution of oxytetracycline was prepared by dissolving an appropiated amount of oxytetracycline in methanol (A. R.). All the other reagents were of analytical grade. Procedure O.~cytetracycline in urine. A sample of 1 to 7 ml of urine was accurately measured. Then it was treated with different amounts of HC104 in order to precipitate interferents. The precipitate was separated by centrifugation. After this previous treatment, the sample was transferred into a 20 ml calibration flask and then diluted to the mark with 0.05 mol/1 NaC104. This solution was transferred into a polarographic cell, dissolved air was removed with nitrogen and the polarogram was recorded. The same procedure was applied to human urine samples containing an adequate amount of oxytetracycline, previously added.
1003 Table 1. Results of the determination of oxytetracycline in urine Sample No.
Oxytetracycline (mol/1) x 10 5 A a
5.3 5.6 5.2 5.4 5.5 5.5 5.3 5.4 5.3 5.5 5.3 5.3
5.5 5.6 5.2 5.6 5.7 5.7 5.3 5.6 5.3 5.6 5.3 5.4
2 3 4 5 6 7 8 9 10 11 Average
" A: Determined by using the calibration graph b B: Determined by the standard addition method
of oxytetracycline was varied. It was found that the optimum HC104 concentration for the determination of oxytetracycline in urine was 0.2 tool/1.
0,'84 -E (Volt 0,'72 0,'60 s)
Fig. 1. Differential pulse polarogram of a sample of urine. A 2 ml of urine; B 2 ml of urine plus 1.0 x 10 .7 mol of oxytetracycline. Conditions: td = 1.0 S; E = 50 mV; scan rate = 6 mVs -1 and HC104 = 0.2 mol/l
Oxytetracycline in human serum. A sample between 1 and 4 ml of human serum was accurately measured. Then it was treated with different amounts of conc. HC104. The solution was thoroughly mixed and the coagulated proteins were filtered through fluted filter paper. The precipitate was washed with 5 ml of 0.05 tool/1 NaC104. The solution was transferred into a 25 ml volumetric flask and diluted to the mark with 0.05 tool/1 NaC104. This solution was transferred into a polarographic cell, dissolved air was removed with nitrogen and the polarogram was recorded. The same procedure was applied to human serum samples containing an adequate amount oxytetracycline, previously added.
Results and discussion
Influence of the volume of urine. The influence of the volume of urine was studied with samples of I to 7 ml of urine containing 5.5 x 10 -5 of oxytetracycline. For samples of more than 3 ml of urine, severe interferences in the determination of oxytetracycline were observed, owing to the large amount of organic material present.
Variation of peak current with concentration of oxytetracycline added to urine. Using 0.2 mol/1 HC104 and working at room temperature, the influence of the concentration of oxytetracycline in 2 ml o f urine was studied over the range 6.5 x 1 0 - 6 9.8 x 10-s mol/1. A linear regression coefficient (r) of 0.9992 was obtained.
Determination of oxytetracycline in urine. For this determination 2 ml samples of urine containing 0.2 mol/1 HC104 were taken, the method of standard additions and the calibration graph were applied. The results are shown in Table I. The standard deviation of 5.5 x 10- ~ mol/1 of oxytetracycline in urine was 1.7 x 1 0 - 6 tool/1 and the recovery of oxytetracycline was 80%, comparing the amounts of oxytetracycline added and the amounts of oxytetracycline found.
The utilisation o f HC104 is recommended for good definition of the polarograms and precipitation of proteins, as shown by results of studies o f the differential pulse polarographic behaviour o f oxytetracycline in aqueous solution at a D M E .
Human serum. H u m a n serum was obtained from various
Urine. Figure 1 shows the polarograms obtained for samples of urine (A) and spiked urine (B) using the above procedure. Two reduction peaks appear at potentials less cathodic than the reduction potential of oxytetracycline, which under the above working conditions is - 0 . 8 6 4 V.
Influence of HCI04 concentration. The amount of HC104
subjects. When different amounts of oxytetracycline were added to serum, the reduction peak of oxytetracycline was observed at the same potential as that found for urine samples (Fig. 2).
added to 2 ml samples of serum containing 5.5 x 10 -5 tool/1 of oxytetracycline was varied. The optimum concentration range of HC104 was 0 . 5 - ].25 mol/1.
Influence of the volume of serum. The influence of the Influence of HCI04 concentration. The amount of HC104 added to 2 ml samples of urine containing 5.5 x 10-5 tool/1
volume of serum was studied with samples of 1.0 to 4.0 ml of serum containing 5.5 x 10- s mol/1 of oxytetracycline. The
1004 Table 2. Results of the determination of oxytetracycline in human
serum Sample No.
Oxytetracycline (mol/1) x 10-5
1 2 3 4 5 6 7 8 9 10 11 Average
5.2 5.0 5.5 5.4 5.4 5.2 5.4 5.2 5.3 5.1 5.4 5.2
5.2 5.0 5.2 5.0 4.9 5.0 5.1 4.9 5.0 5.1 5.2 5.0
A: Determined by using the calibration graph b B: Determined by the standard additions method
15 nA Ip
Variation of peak current with the concentration of oxytetracycline added to serum. Using I tool/1 HC104 and 2 ml of serum at r o o m temperature, the influence o f the concentration o f oxytetracycline was studied over the range 9.5 x 1 0 - 6 _ 1.2 x 10-4 tool/1. A linear regression coefficient (r) o f 0.9993 was obtained.
Determination of oxytetracycline in serum. F o r this determination 2 ml samples o f h u m a n serum containing 1 tool/1 HC104 were taken, the m e t h o d o f standard additions and the calibration graph were applied. The results are shown in Table 2. The standard deviation o f the determination o f 5.5x10-Smol/1 o f oxytetracycline in serum was 1.9 • 10 -6 mol/1 and the recovery o f oxytetracycline was 85%. The p o l a r o g r a p h i c method, described in this paper, permits the determination o f oxytetracycline, with a suitable exactness, in h u m a n serum and h u m a n urine, in the range studied. However, the cleaning o f samples is m o r e effective in the h u m a n serum than in the h u m a n urine; this is p r o b a b l y due to a more quantitative precipitation o f proteins and macromolecules from the h u m a n serum. F o r this reason, the h u m a n serum p o l a r o g r a m s are clearer than those registered for h u m a n urine. The detection limit was 5.5 x 10-6 tool/1 for both fluids, using the signal-noise characteristics (s/n = 3), a usual procedure for that kind o f analysis. This m e t h o d was not applied to samples from patients, but it is the first study o f the p o l a r o g r a p h i c determination o f oxytetracycline in these fluids.
Acknowledgement. This research was supported by the Central Nuclear Almaraz, Caceres, Spain.
0,72 -E (Volts)
Fig. 2. Differential pulse polarogram of a sample of human serum. A 2 ml of human serum. B 2 ml of human serum plus 1.9 x 10- 7 mol of oxytetracycline. Conditions: td = 1.0 S; E = 50 mV; scan rate = 6 mVs-1 and HC10~ 1 mol/1
o p t i m u m volume o f serum was less than 2 ml under the conditions used.
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