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A Note on the Ultraviolet Absorption Spectra of Commercial Heparins*
442
JOURNAL OF THE
AMERICANPHARMACEUTICAL ASSOCIATION
quantity of nondigitoxin cardioactive glycosides to be permitted by the definition is not clear, it is improbable that an amount as high as the 37% contained in sample 19 was contemplated. In no part of the monograph is there a test which would regularly exclude such a product. Identification Test “B” insures a certain degree of biological activity in preparations which meet its requirements, but a preliminary exploration of its discriminatory ability in this laboratory has resulted in the conclusion that samples of digitoxin diluted with inert material to the extent of 23% would be expected to pass the test half the time. No attempt has been made by this laboratory to determine how much gitoxin would have to be present in a digitoxin sample before it would fail to pass Identification Test “B.” Since gitoxin has some cardiac activity, it is obvious that appreciably more than the 23% mentioned above would have to be present before there would be a reasonable expectation of rejection by this test. The absence of a rigorous definition of digitoxin, the wide tolerance of biologic activity permitted by Identification Test “B”. and the lack of specificity of the colorimetric assay procedure have combined to produce the unsatisfactory status in which materials with a considerable range of biological activity can legally be distributed as U. S. P. digitoxin.
Vol. XLIII, No. 7
SUMMARY
1. Comparative assays were carried out by the U. S. P. XI11 and the U. S. P. XIV methods on 31 digitoxin preparations consisting of 29 tablets and two samples of crystalline digitoxin. 2. Of 31 samples examined, eight or 25 per cent, were found t o yield results b y the U. S. P XIV method which were more than 25 per cent higher than those obtained b y the U. S. P. XI11 biological method. 3. While a chemical method is t o be preferred from the standpoint of practicability and cost, the lack of specificity of the method described in the U. S. P. XIV makes it inadequate t o assure a uniform potency of digitoxin preparations.
REFERENCES (1) Bell F K and Krantz, J. C., Jr., J . Pharmacol. Erpfl Thcrap’ 87: 198(1946). (2)‘Vos B.’j and Welsh L. H. ibid. 85 346(1945). (3) Allmark.’M. G., and Bachin’ski, G., &v. can. biol., 5, 570(1946). (4) Danow, H. G., Mathieson, D., and Hayes, H. W., Federation PYOC.5. 178(1946). (5) Personal eommunication Allmark M. G. (6) Banes, D., and Carol, J.. ‘ h s JoANAL, 42,674(1953).
Notes
A Note on the Ultraviolet Absorption Spectra of Commercial Heparins * By KATHERINE A. LATHROP and J. GARROTT ALLEN
T
of members of the heparin family can be detected by the inhibition of their anticoagulant activities which, however, sheds no light upon the specific heparin that may be present. Moreover, considerable variation in the anticoagulant activity exists between heparins of different origin. Therefore, if the physiology or biochemistry of this group of compounds is to be studied extensively, improved analytic methods must be developed. Because the ultravidlet absorption spectrum of a particular heparin studied showed a high specific extinction in the region of 270 mp (1).this appeared HE PRESENCE
* Received October 14 1953 from the Division of Biological and Medical Research, ’Argoine National Laboratory, Lemont, Ill., and the Department of Surgery of the University of Chicago, Chicago, Ill.
a possible lead for the development of a simple measurement of heparin concentration. More recently Bell and Krantz (2) studied the problem and concluded that this method held promise. This paper reports additional data on this problem. METHOD
Fourteen sources of powdered sodium heparin were prepared as separate aqueous solutions, concentrations of 5 mg./ml. unless otherwise stated. Ultraviolet absorption spectra between 216 and 350 mp were determined, using silica cells 1 cm. in depth in the Beckman model DU spectrophotometer with a compensating blank of distilled water. Readings were taken at 5 mp intervals and at greater frequency when indicated.
SCIENTIFIC EDITION
July, 1954
1.000
-
0.900
-
> 0.000
-
0.700
-
0.600
-
c E
443
A
I?
:
0.500
-
0.400
-
0.300
-
0.200
-
I
0.100
0
z
0.500
-
0.400
-
0.300
-
0.e00
-
0.100
-
0
-
O L
210
230
rrso
270 WAVE
ego
310
330
350
WAVE
370
LENQTH l a p ) ,
Fig. 1.-Absorption spectra for heparins of known anticoagulant activity obtained from various pharmaceutical companies. I 127 unitdmg., I1 114 units/mg., I11 110 units/mg., IV 94.5 units m g , V 60.0 units/mg., VI 110 units/mg., VII 100 unrts/mg..
RE.UXTS
In Fig. 1 are plotted the ultraviolet spectra of seven bovine heparin preparations of differingpotencies obtained from five commercial sources. These reveal no common pattern either in selective absorption or in optical density. Four bovine heparins prepared by the same company also failed to demonstrate any agreement in absorption pattern. Likewise, no particular characteristic in the spectra of bovine, canine, or porcine heparins was evident. The patterns that did appear in agreement were restricted to liquid heparins containing phenol as a preservative. In Fig. 2 are demonstrated absorption spectra of a 0.01% aqueous heparin solution containing 0.0045% phenol and of a 0.0045% phenol solution. DISCUSSION
The chemical structure of heparin (3 and 4)is incompletely understood. It now appears that similar anticoagulant activity is possessed by a variety of sulfonated polysaccharides. Common to all of this family of anticoagulants has been their inactivation by protamine sulfate which is the more accurate procedure currently used in the identity of these substances. Although heparins react metschromatically with most of the dyes of the thiazine series, this reaction is not limited t o the heparins and therefore these methods cannot be considered specific for heparin (5).
I
L
el0
LENQTH I m p )
530
Fig. 2.-Absorption spectra of (A ) clinical heparin preparation containing 0.01% ' heparin and 0.0045% phenol and (B) 0.0045% phenol.
The deficiencies in heparin identification and assay create a need for simple and reliable techniques to this end. The results of Bell and Krantz on ultraviolet spectrum analysis seemed to offer a possible means for heparin identity. While our results confirm theirs, it must be recognized that both their results and ours represent primarily impurities in heparin preparations. In particular, the high specific extinction for clinical heparin is produced by its phenol content. Excluding this artifact there remains no residual characteristic in the absorption spectrum that can be attributed to heparin or to its anticoagulant activity.
SUMMARY The ultraviolet absorption spectrum described for liquid clinical heparins was not observed in a variety of dried sodium heparins. The pattern previously described appears to be the result of impurities, including the phenol preservative which many of these . products contain.
x EFERENCI s (1) Lathrop K. A and Allen J. G. ANL-4227 Argonne
National Lab&atory,'Lemont, Iil., pp.' 130-135(1648). (2) Bell, F. C., and Krantz, J. C., Jr., THISJOURNAL, 39,
- - ~
Rlll9fiOl
{3) Jorpes, E., J . B i d . Chem., 183, 607(1950). 4) Wolfrom, M. L., Montgomery, R., Rarabinos, J. V., and Rathgeb, P., J . Am. Chem. Sac.. 72,5796(1960). (5) Jaques, L. B., Bruce-Mitford, M., and Ricker, A. G., Rev. canad. dc b i d . , 6, 740(1947).
JOURNAL OF THE
AMERICANPHARMACEUTICAL ASSOCIATION
quantity of nondigitoxin cardioactive glycosides to be permitted by the definition is not clear, it is improbable that an amount as high as the 37% contained in sample 19 was contemplated. In no part of the monograph is there a test which would regularly exclude such a product. Identification Test “B” insures a certain degree of biological activity in preparations which meet its requirements, but a preliminary exploration of its discriminatory ability in this laboratory has resulted in the conclusion that samples of digitoxin diluted with inert material to the extent of 23% would be expected to pass the test half the time. No attempt has been made by this laboratory to determine how much gitoxin would have to be present in a digitoxin sample before it would fail to pass Identification Test “B.” Since gitoxin has some cardiac activity, it is obvious that appreciably more than the 23% mentioned above would have to be present before there would be a reasonable expectation of rejection by this test. The absence of a rigorous definition of digitoxin, the wide tolerance of biologic activity permitted by Identification Test “B”. and the lack of specificity of the colorimetric assay procedure have combined to produce the unsatisfactory status in which materials with a considerable range of biological activity can legally be distributed as U. S. P. digitoxin.
Vol. XLIII, No. 7
SUMMARY
1. Comparative assays were carried out by the U. S. P. XI11 and the U. S. P. XIV methods on 31 digitoxin preparations consisting of 29 tablets and two samples of crystalline digitoxin. 2. Of 31 samples examined, eight or 25 per cent, were found t o yield results b y the U. S. P XIV method which were more than 25 per cent higher than those obtained b y the U. S. P. XI11 biological method. 3. While a chemical method is t o be preferred from the standpoint of practicability and cost, the lack of specificity of the method described in the U. S. P. XIV makes it inadequate t o assure a uniform potency of digitoxin preparations.
REFERENCES (1) Bell F K and Krantz, J. C., Jr., J . Pharmacol. Erpfl Thcrap’ 87: 198(1946). (2)‘Vos B.’j and Welsh L. H. ibid. 85 346(1945). (3) Allmark.’M. G., and Bachin’ski, G., &v. can. biol., 5, 570(1946). (4) Danow, H. G., Mathieson, D., and Hayes, H. W., Federation PYOC.5. 178(1946). (5) Personal eommunication Allmark M. G. (6) Banes, D., and Carol, J.. ‘ h s JoANAL, 42,674(1953).
Notes
A Note on the Ultraviolet Absorption Spectra of Commercial Heparins * By KATHERINE A. LATHROP and J. GARROTT ALLEN
T
of members of the heparin family can be detected by the inhibition of their anticoagulant activities which, however, sheds no light upon the specific heparin that may be present. Moreover, considerable variation in the anticoagulant activity exists between heparins of different origin. Therefore, if the physiology or biochemistry of this group of compounds is to be studied extensively, improved analytic methods must be developed. Because the ultravidlet absorption spectrum of a particular heparin studied showed a high specific extinction in the region of 270 mp (1).this appeared HE PRESENCE
* Received October 14 1953 from the Division of Biological and Medical Research, ’Argoine National Laboratory, Lemont, Ill., and the Department of Surgery of the University of Chicago, Chicago, Ill.
a possible lead for the development of a simple measurement of heparin concentration. More recently Bell and Krantz (2) studied the problem and concluded that this method held promise. This paper reports additional data on this problem. METHOD
Fourteen sources of powdered sodium heparin were prepared as separate aqueous solutions, concentrations of 5 mg./ml. unless otherwise stated. Ultraviolet absorption spectra between 216 and 350 mp were determined, using silica cells 1 cm. in depth in the Beckman model DU spectrophotometer with a compensating blank of distilled water. Readings were taken at 5 mp intervals and at greater frequency when indicated.
SCIENTIFIC EDITION
July, 1954
1.000
-
0.900
-
> 0.000
-
0.700
-
0.600
-
c E
443
A
I?
:
0.500
-
0.400
-
0.300
-
0.200
-
I
0.100
0
z
0.500
-
0.400
-
0.300
-
0.e00
-
0.100
-
0
-
O L
210
230
rrso
270 WAVE
ego
310
330
350
WAVE
370
LENQTH l a p ) ,
Fig. 1.-Absorption spectra for heparins of known anticoagulant activity obtained from various pharmaceutical companies. I 127 unitdmg., I1 114 units/mg., I11 110 units/mg., IV 94.5 units m g , V 60.0 units/mg., VI 110 units/mg., VII 100 unrts/mg..
RE.UXTS
In Fig. 1 are plotted the ultraviolet spectra of seven bovine heparin preparations of differingpotencies obtained from five commercial sources. These reveal no common pattern either in selective absorption or in optical density. Four bovine heparins prepared by the same company also failed to demonstrate any agreement in absorption pattern. Likewise, no particular characteristic in the spectra of bovine, canine, or porcine heparins was evident. The patterns that did appear in agreement were restricted to liquid heparins containing phenol as a preservative. In Fig. 2 are demonstrated absorption spectra of a 0.01% aqueous heparin solution containing 0.0045% phenol and of a 0.0045% phenol solution. DISCUSSION
The chemical structure of heparin (3 and 4)is incompletely understood. It now appears that similar anticoagulant activity is possessed by a variety of sulfonated polysaccharides. Common to all of this family of anticoagulants has been their inactivation by protamine sulfate which is the more accurate procedure currently used in the identity of these substances. Although heparins react metschromatically with most of the dyes of the thiazine series, this reaction is not limited t o the heparins and therefore these methods cannot be considered specific for heparin (5).
I
L
el0
LENQTH I m p )
530
Fig. 2.-Absorption spectra of (A ) clinical heparin preparation containing 0.01% ' heparin and 0.0045% phenol and (B) 0.0045% phenol.
The deficiencies in heparin identification and assay create a need for simple and reliable techniques to this end. The results of Bell and Krantz on ultraviolet spectrum analysis seemed to offer a possible means for heparin identity. While our results confirm theirs, it must be recognized that both their results and ours represent primarily impurities in heparin preparations. In particular, the high specific extinction for clinical heparin is produced by its phenol content. Excluding this artifact there remains no residual characteristic in the absorption spectrum that can be attributed to heparin or to its anticoagulant activity.
SUMMARY The ultraviolet absorption spectrum described for liquid clinical heparins was not observed in a variety of dried sodium heparins. The pattern previously described appears to be the result of impurities, including the phenol preservative which many of these . products contain.
x EFERENCI s (1) Lathrop K. A and Allen J. G. ANL-4227 Argonne
National Lab&atory,'Lemont, Iil., pp.' 130-135(1648). (2) Bell, F. C., and Krantz, J. C., Jr., THISJOURNAL, 39,
- - ~
Rlll9fiOl
{3) Jorpes, E., J . B i d . Chem., 183, 607(1950). 4) Wolfrom, M. L., Montgomery, R., Rarabinos, J. V., and Rathgeb, P., J . Am. Chem. Sac.. 72,5796(1960). (5) Jaques, L. B., Bruce-Mitford, M., and Ricker, A. G., Rev. canad. dc b i d . , 6, 740(1947).