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Serum levels of tetracycline after different administration forms
185
CLINICA CHIMICA ACTA
SERUM
LEVELS
OF TETRACYCLINE
ADMINISTRATION
A. E. ALTMANN, A. H. J. GIJZEN
AFTER
DIFFERENT
FORMS
H. BEEUWKES, P. J. BROMBACHER, F. P. V. MAESEN
H. J. BUYTENDIJK,
AND
St. Josefih
Hospital, Heerlen (The Netherlands)
(Received
December
8, 1967)
SUMMARY
Serum levels of tetracycline after oral administration were studied by different methods. A generally used microbiological dilution method is of no value; there is agreement between an agar plate diffusion technique and a fluorimetric procedure. It seems that addition of chelating compounds has a good effect on intestinal resorption while the use of coated tablets gives less favourable results.
INTRODUCTION
The widespread use of tetracycline as a drug in the treatment of different forms of chronic bronchitis and the fact that so many commercial preparations are available, has been our motive to perform a comparative investigation on the subject. The drug is not only distributed in several forms for oral administration but some makers also propagate addition of chelating compounds to obtain higher blood levels. To compare the effect of the various confections we determined blood levels obtained after dosage of eleven different commercial preparations under standardized conditions; the drugs had previously been checked for quality in &fro. MATERIALS
AND METHODS
Commercial specimens of the drugs were coded and distributed by the hospital pharmacist. All experiments were done “double blind”. Purity and amount of tetracycline per drug unit (capsule, coated tablet, etc.) were compared with the British Pharmaco$aea (1963) criteria. Tetracycline was administered in 5oo-mg doses to nine volunteers for each preparation. They were all in fairly good health, not confined to bed rest, and had no defects of the liver, kidney, gastrointestinal or circulatory functions. Although it has not been possible to check all preparations in the same group of people, there was a good deal of overlapping in order to minimize biological differences between groups. Tetracycline (500 mg) was administered per OS go min after a standardized Chin. Chim. Acta, 20 (1968) 185-188
186
ALTMANN
et 81.
breakfast. Not more than so ml of water was given with the drug. After 2 and 4 hours, venous blood was taken and the serum was divided in equal portions for microbiological and chemical analyses.
Two methods were used, viz. a dilution method of Rammelkampr and a diffusion method, which is described here. ~~~e~~~~~~z: 8 g nutrient broth (Difco, powder) and 20 g Bacto-Agar (Difco) were dissolved in I 1 0.015 ;I$phosphate buffer pH 7.2. The medium is sterilized during 15 min at 115"; 70 ml medium was dispensed in a Petri dish (flat bottom, diameter 14 cm), and the agar was allowed to dry without cover during 3 h at 37’, whereupon the medium was inoculated with 0.75 ml diluted (1 in 1000) 18-h old S~~~~~~~~ococc~~s culture in tryptose phosphate broth (Difco). After 30 min drying at 37O, wells were made (0.8 cm) to contain serum and standard solutions of tetracycline hydrochloride in water. All d~terIllinations were duplicated with 0.15 ml serum per well; every Petri dish contained standards of O.I-0.5-r.o-s.o-10.0 k>etracycline per well. All standards through the whole experiment originated from the same batch of tetracycline hydrochloride. A Staphy1ococu.u culture was used which has been known for years in our laboratory to show a constant growth inhibition in response to 0.2-0.3 ,ug tetracycline per ml. After 20 h, growth inhibition zones were measured and plotted semi-logarithmically against standards.
&elate compounds of tetracycline and turates, which can be extracted from serum, of the tetracycline is possible. This technique bolites and decay products do not disturb the in the drug is determined spectrophotometrically; tage of the toxic compound anhydrotetracycline powder form a long time-is deiermined.
calcium form complexes with barbiwhereupon fluorimetric determination is specific for the tetracyclines; metareaction %.The amount of tetracycline as a criterion of purity the percen“-formed on keeping tetracycline in
RESULTS
The commercial
preparations
investigated
were marked
from A. to L.
From each preparation, 10 tablets (or capsules) were thoroug~~Iy l~o~~ogenized, the tetracycline content determined and expressed as a percentage of the declared content. In general, anhydrotetracycline was not present in amounts over 1% of tetracycline, which is the maximum permissible of impurity*. Results are given in TabIe I.
From Ta.bfe II we see that administration of preparation II gives only very small amounts of tetracycline in the serum. Although there is no statistically significant difference between all values, the number of patients per preparation being Cl&. ChGn. Acda, 20 (rg68) r85-188
TETRACYCLINE TABLE
187
IN SERUM
I
TETRACYCLINECONTENTAND
PURITYOFTHE
Tetracycline %
Preparation A. B. C. D. E. F. G. H. I. K. L.
Capsules with metaphosphate Tablets Capsules Capsules Capsules Tablets with bitartrate Capsules Coated tablets Capsules Tablets Tablets with bitartrate
* The tetracycline TABLE
PREPARATIONS
content*
Anhydrotetracycl. %
co&e&
0.1 1.3 0.0 0.5 0.4 0.6 0.3 1.0 1.1 0.9 0.8
100.5 92.9 96.2 108.7 99.3 94.7 107.8 91.2 108.8 102.5 99.5
content of the preparation is expressed as a percentage of the declared content.
II
TETRACYCLINE
IN SE~u~(pg/ml);
Preparation
After
1.8
0.4 <0.6 0.9 1.4 <0.6 1.8 0.0
VALUES
After
2 h
dilution method A. Capsules with metaphosphate B. Tablets C. Capsules D. Capsules E. Capsules F. Tablets with bitartrate G. Capsules H. Coated tablets I. Capsules K. Tablets L. Tablets with bitartrate
MEAN
diffusion mkthod
chemical method
difTusion method
chemical method I.93
1.0
1.81
0.8 0.6 1.0 0.8
1.54 0.95 1.85 1.09
co.4 <0.6 2.4 1.8
I.0 0.8 0.6 1.4 1.1
1.26 2.31 1.47
1.85 1.88 0.04
1.2 0.8 0.2 1.0 0.7
2.00 2.02 0.33 I .o9 0.81
1.3
1.91
2.0
0.4
0.8
0.84
<0.6 1.9 0.0 I.2 0.4
0.6
I.0
1.72
1.4
1.2
4 h
dilution method
1.1
1.10
I.52
it seems that best results are obtained with preparations A, B, D, F, G, and I. It is also seen from the table that microbiologically obtained values are generally lower than the results of the chemical determination. Nevertheless, even in micronine,
biological tests, the same preparations appear to be better than the others. Higher values by chemical analysis can be explained by the fact that in the microbiological diffusion method part of the tetracycline may be bound to serum proteins, which inhibits diffusion. Is has been reported6 that under certain circumstances 559/o of tetracycline hydrochloride can be bound to protein. To check this, a series of experiments was performed taking tetracycline hydrochloride standards in human serum instead of in water. The differences obtained were however, the same as with the chemical method. It must be mentioned that the standard deviation of the chemical method is 2% and that of the diffusion method 16%. The results obtained with Rammelkamp’s dilution method are rather irregular. We think that this method has a very restricted value and should not be used for accurate determinations. Clin. Chim. Acta.
20
(1968) 185-188
In order to study the discrepancy between the diffusion method and the chemical method we endeavoured to further standardize the diffusion method used. The constancy of diffusion was measured by the straightness of the semi-logarithmic plot of growth inhibition zones VS. known quantities af tetracycline hydrochloride in standard solution and in serum respectively. With the method used, diffusion in the agar broth was sometimes more or less inconstant. Optimal resuhs were obtained by substituting agarose (BDH) for agar. ii fuh survey of different diffusion techniques will be ~u~~ljshed elsewhere. L)ISCUSSION
It can be concluded that some commerciali preparations of tetracycline hydrochloride administered orally give higher serum Ieveb than others. AIthaugh the difference is not significant, it seems that addition of a complex-forming compound, e.g. bitartrate or lneta~hus~hate, favourably sequences the uptake of tetracycline in the blood. Coated tablets are less effective than capsules or non-coated tablets. Although the chemical analysis is far more accurate for the determination and can be used for serum levels of o.oa pg/ml and higher, it should be emphasized that the microbiological method (from 0.5 pgjml) does not only indicate the amount of substance present, but also gives infarmation on the anti-microbial activity oi the drug. Hence, a more standardize microbioIogical diffusion technique would be the method of choice. Nevertheless, it should be borne in mind that activity & V&O against a certain micro-orgallisn~ is not necessarily identical with activity in V&O. However, we did not find any growth inhibition by serum alone.
CLINICA CHIMICA ACTA
SERUM
LEVELS
OF TETRACYCLINE
ADMINISTRATION
A. E. ALTMANN, A. H. J. GIJZEN
AFTER
DIFFERENT
FORMS
H. BEEUWKES, P. J. BROMBACHER, F. P. V. MAESEN
H. J. BUYTENDIJK,
AND
St. Josefih
Hospital, Heerlen (The Netherlands)
(Received
December
8, 1967)
SUMMARY
Serum levels of tetracycline after oral administration were studied by different methods. A generally used microbiological dilution method is of no value; there is agreement between an agar plate diffusion technique and a fluorimetric procedure. It seems that addition of chelating compounds has a good effect on intestinal resorption while the use of coated tablets gives less favourable results.
INTRODUCTION
The widespread use of tetracycline as a drug in the treatment of different forms of chronic bronchitis and the fact that so many commercial preparations are available, has been our motive to perform a comparative investigation on the subject. The drug is not only distributed in several forms for oral administration but some makers also propagate addition of chelating compounds to obtain higher blood levels. To compare the effect of the various confections we determined blood levels obtained after dosage of eleven different commercial preparations under standardized conditions; the drugs had previously been checked for quality in &fro. MATERIALS
AND METHODS
Commercial specimens of the drugs were coded and distributed by the hospital pharmacist. All experiments were done “double blind”. Purity and amount of tetracycline per drug unit (capsule, coated tablet, etc.) were compared with the British Pharmaco$aea (1963) criteria. Tetracycline was administered in 5oo-mg doses to nine volunteers for each preparation. They were all in fairly good health, not confined to bed rest, and had no defects of the liver, kidney, gastrointestinal or circulatory functions. Although it has not been possible to check all preparations in the same group of people, there was a good deal of overlapping in order to minimize biological differences between groups. Tetracycline (500 mg) was administered per OS go min after a standardized Chin. Chim. Acta, 20 (1968) 185-188
186
ALTMANN
et 81.
breakfast. Not more than so ml of water was given with the drug. After 2 and 4 hours, venous blood was taken and the serum was divided in equal portions for microbiological and chemical analyses.
Two methods were used, viz. a dilution method of Rammelkampr and a diffusion method, which is described here. ~~~e~~~~~~z: 8 g nutrient broth (Difco, powder) and 20 g Bacto-Agar (Difco) were dissolved in I 1 0.015 ;I$phosphate buffer pH 7.2. The medium is sterilized during 15 min at 115"; 70 ml medium was dispensed in a Petri dish (flat bottom, diameter 14 cm), and the agar was allowed to dry without cover during 3 h at 37’, whereupon the medium was inoculated with 0.75 ml diluted (1 in 1000) 18-h old S~~~~~~~~ococc~~s culture in tryptose phosphate broth (Difco). After 30 min drying at 37O, wells were made (0.8 cm) to contain serum and standard solutions of tetracycline hydrochloride in water. All d~terIllinations were duplicated with 0.15 ml serum per well; every Petri dish contained standards of O.I-0.5-r.o-s.o-10.0 k>etracycline per well. All standards through the whole experiment originated from the same batch of tetracycline hydrochloride. A Staphy1ococu.u culture was used which has been known for years in our laboratory to show a constant growth inhibition in response to 0.2-0.3 ,ug tetracycline per ml. After 20 h, growth inhibition zones were measured and plotted semi-logarithmically against standards.
&elate compounds of tetracycline and turates, which can be extracted from serum, of the tetracycline is possible. This technique bolites and decay products do not disturb the in the drug is determined spectrophotometrically; tage of the toxic compound anhydrotetracycline powder form a long time-is deiermined.
calcium form complexes with barbiwhereupon fluorimetric determination is specific for the tetracyclines; metareaction %.The amount of tetracycline as a criterion of purity the percen“-formed on keeping tetracycline in
RESULTS
The commercial
preparations
investigated
were marked
from A. to L.
From each preparation, 10 tablets (or capsules) were thoroug~~Iy l~o~~ogenized, the tetracycline content determined and expressed as a percentage of the declared content. In general, anhydrotetracycline was not present in amounts over 1% of tetracycline, which is the maximum permissible of impurity*. Results are given in TabIe I.
From Ta.bfe II we see that administration of preparation II gives only very small amounts of tetracycline in the serum. Although there is no statistically significant difference between all values, the number of patients per preparation being Cl&. ChGn. Acda, 20 (rg68) r85-188
TETRACYCLINE TABLE
187
IN SERUM
I
TETRACYCLINECONTENTAND
PURITYOFTHE
Tetracycline %
Preparation A. B. C. D. E. F. G. H. I. K. L.
Capsules with metaphosphate Tablets Capsules Capsules Capsules Tablets with bitartrate Capsules Coated tablets Capsules Tablets Tablets with bitartrate
* The tetracycline TABLE
PREPARATIONS
content*
Anhydrotetracycl. %
co&e&
0.1 1.3 0.0 0.5 0.4 0.6 0.3 1.0 1.1 0.9 0.8
100.5 92.9 96.2 108.7 99.3 94.7 107.8 91.2 108.8 102.5 99.5
content of the preparation is expressed as a percentage of the declared content.
II
TETRACYCLINE
IN SE~u~(pg/ml);
Preparation
After
1.8
0.4 <0.6 0.9 1.4 <0.6 1.8 0.0
VALUES
After
2 h
dilution method A. Capsules with metaphosphate B. Tablets C. Capsules D. Capsules E. Capsules F. Tablets with bitartrate G. Capsules H. Coated tablets I. Capsules K. Tablets L. Tablets with bitartrate
MEAN
diffusion mkthod
chemical method
difTusion method
chemical method I.93
1.0
1.81
0.8 0.6 1.0 0.8
1.54 0.95 1.85 1.09
co.4 <0.6 2.4 1.8
I.0 0.8 0.6 1.4 1.1
1.26 2.31 1.47
1.85 1.88 0.04
1.2 0.8 0.2 1.0 0.7
2.00 2.02 0.33 I .o9 0.81
1.3
1.91
2.0
0.4
0.8
0.84
<0.6 1.9 0.0 I.2 0.4
0.6
I.0
1.72
1.4
1.2
4 h
dilution method
1.1
1.10
I.52
it seems that best results are obtained with preparations A, B, D, F, G, and I. It is also seen from the table that microbiologically obtained values are generally lower than the results of the chemical determination. Nevertheless, even in micronine,
biological tests, the same preparations appear to be better than the others. Higher values by chemical analysis can be explained by the fact that in the microbiological diffusion method part of the tetracycline may be bound to serum proteins, which inhibits diffusion. Is has been reported6 that under certain circumstances 559/o of tetracycline hydrochloride can be bound to protein. To check this, a series of experiments was performed taking tetracycline hydrochloride standards in human serum instead of in water. The differences obtained were however, the same as with the chemical method. It must be mentioned that the standard deviation of the chemical method is 2% and that of the diffusion method 16%. The results obtained with Rammelkamp’s dilution method are rather irregular. We think that this method has a very restricted value and should not be used for accurate determinations. Clin. Chim. Acta.
20
(1968) 185-188
In order to study the discrepancy between the diffusion method and the chemical method we endeavoured to further standardize the diffusion method used. The constancy of diffusion was measured by the straightness of the semi-logarithmic plot of growth inhibition zones VS. known quantities af tetracycline hydrochloride in standard solution and in serum respectively. With the method used, diffusion in the agar broth was sometimes more or less inconstant. Optimal resuhs were obtained by substituting agarose (BDH) for agar. ii fuh survey of different diffusion techniques will be ~u~~ljshed elsewhere. L)ISCUSSION
It can be concluded that some commerciali preparations of tetracycline hydrochloride administered orally give higher serum Ieveb than others. AIthaugh the difference is not significant, it seems that addition of a complex-forming compound, e.g. bitartrate or lneta~hus~hate, favourably sequences the uptake of tetracycline in the blood. Coated tablets are less effective than capsules or non-coated tablets. Although the chemical analysis is far more accurate for the determination and can be used for serum levels of o.oa pg/ml and higher, it should be emphasized that the microbiological method (from 0.5 pgjml) does not only indicate the amount of substance present, but also gives infarmation on the anti-microbial activity oi the drug. Hence, a more standardize microbioIogical diffusion technique would be the method of choice. Nevertheless, it should be borne in mind that activity & V&O against a certain micro-orgallisn~ is not necessarily identical with activity in V&O. However, we did not find any growth inhibition by serum alone.