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A Note on the Assay of Digitoxin Preparations*
A Note on the Assay of Digitoxin Preparations’ By DANIEL BANES, ALBERT E. H. HOUK, and JACOB WOLFF
T
TABLE I.-ANALYSIS OF DIGITOXIN PREPARATIONS
HE METHOD recently adopted for the analysis of digitoxin preparations by the Association of Official Agricultural Chemists (1) modifies U. S. P. XV assay procedures for the drug (2) in several respects. It simplifies preparation of samples, chromatographic columns, and solvents, eliminates the isooctane wash, requires a smaller volume of eluent, and provides a test for detecting extraneous digitoxosides. We have analyzed several samples of digitoxin powders and twenty-one lots of digitoxin tablets by both the modified method and the official assay procedures. Results of these analyses (Table I ) show that the two methods yield concordant assay values. To test the efficiency of the chromatographic column in separating digitoxin from gitoxin and more polar digitoxosides, we added varying quantities of gitoxin to two of the crystalline powders, and reanalyzed the mixtures. Digitoxin was recovered in the first 200 ml of eluate. The following 50-ml portion contained less than 0 4% of the total digitoxin when determined by means of the alkaline picrate reagent. All of the added gitoxin was recovered in the proposed test for other digitoxosides (1). When the digitoxin eluates from tablet samples were similarly tested. the last 50 ml. of solution were practically devoid of digitoxin. The prescribed volume of 250 ml. includes a safety factor to ensure complete separation of digitoxin from gitoxin and more polar digitoxosides
Sample
D-1 D-la D-lb D-2 D-3 D -4 D-4a D-4b T- 1 T-2 T-3 T-4 T-5 T-6 T-7 T-8 T-9 T-10 T-11 T-12 T-13 T-14 T-15 T-16 T-17 T-18 T-19 T-20 T-21
REFERENCES (1) J . Assoc. Ofic.Agr. Chemists, 41, 56(1958). (2) “United States Pharmacopeia XV,” Mack Publishing Co., Easton, Pa., 1955, pp. 217-218. *Received December 27, 1957, from the Division of Pharmaceutical Chemistry, Bureau of Biological and Physical Sciences, U. S. Food and Drug Administration, Department of Health, Education, and Welfare, Washington 25, D. C.
Description
Crystalline powder D-1 plus 4% added gitoxin D-1 plus 10% added gitoxin Crystalline powder Crystalline powder Crystalline powder D-4 plus 470 gitoxin added D-4 plus 10% gitoxin added Tablet (mg.) 0.1 0.1 0.2 0.1 0.2 0.1 0.1 0.2 0.1 0.2 0.2 0.1 0.1 0.1 0.2 0.15 0.2 0.1 0.1 0.1 0.1
Digitoxin Found, Per Cent of Declared Modified Official Method Method
96.5
95.9
95.7
...
96.3
...
89.5
87.6
86.5
84.2
83.3
82.4
82.6
...
82.1
...
105.6 103.9 99.4 97.2 93.5 93.3 92.5 92.0 91.9 91.5 90.3 88.4 88.3 86.5 83.8 83.5 79.3 77.3 60.0 45.5 113.3
103.0 100.7 95.3 95.9 91 .o 92.8 88.9 91.7 90.0 94.0 88.7 86.3 87.6 85.2 85.7 85.0 80.1 77.2 56.3 46.6 113.0
A Note on a Simple Small Animal Respirometer* By S. T. COKER
V
ARIOUS METHODS have been used to record respiration in small animals (1-5). Most of the existing methods are not satisfactory for animals as small as rats or lack adequate sensitivity. Also, many of these methods are rather complex, or involve an elaborate system of valves. The respirometer described below is based on the principle originally used by Gaddum (1) for recording respiration in rabbits. A modification of Gaddum’s procedure by Haley (2) is similar to the one described below except a different system of valves i s used. This led to the development of a small, simple
* Received February 12, 1958, from the Department of Pharmacology, University of Kansas City, School of Pharmacy, Mo. The author is indebted t o John R . Lyon, senior laboratory assistant, for his valuable technical assistance.
respirometer which could be built completely and inexpensively in any laboratory and have sufficient sensitivity to record accurately respiration of rats as well as larger animals with proper modifications as to chamber volume and diameter of the air inlets and outlets. The apparatus is quite suitable for use in laboratory teaching a s well as drug screening on rats. The materials needed are: Plexiglas tubing one inch outside diameter by two inches in length for the chamber, and ‘/,-inch outside diameter tubing for the necessary connections, a No. 4 rubber stopper, rubber tubing, medium rubber dam, ethylene dichloride for cementing the Plexiglas, screw clamp, tracheal cannula, sensitive tambour, and a recording lever (Fig. 1, A ) .
686
T
TABLE I.-ANALYSIS OF DIGITOXIN PREPARATIONS
HE METHOD recently adopted for the analysis of digitoxin preparations by the Association of Official Agricultural Chemists (1) modifies U. S. P. XV assay procedures for the drug (2) in several respects. It simplifies preparation of samples, chromatographic columns, and solvents, eliminates the isooctane wash, requires a smaller volume of eluent, and provides a test for detecting extraneous digitoxosides. We have analyzed several samples of digitoxin powders and twenty-one lots of digitoxin tablets by both the modified method and the official assay procedures. Results of these analyses (Table I ) show that the two methods yield concordant assay values. To test the efficiency of the chromatographic column in separating digitoxin from gitoxin and more polar digitoxosides, we added varying quantities of gitoxin to two of the crystalline powders, and reanalyzed the mixtures. Digitoxin was recovered in the first 200 ml of eluate. The following 50-ml portion contained less than 0 4% of the total digitoxin when determined by means of the alkaline picrate reagent. All of the added gitoxin was recovered in the proposed test for other digitoxosides (1). When the digitoxin eluates from tablet samples were similarly tested. the last 50 ml. of solution were practically devoid of digitoxin. The prescribed volume of 250 ml. includes a safety factor to ensure complete separation of digitoxin from gitoxin and more polar digitoxosides
Sample
D-1 D-la D-lb D-2 D-3 D -4 D-4a D-4b T- 1 T-2 T-3 T-4 T-5 T-6 T-7 T-8 T-9 T-10 T-11 T-12 T-13 T-14 T-15 T-16 T-17 T-18 T-19 T-20 T-21
REFERENCES (1) J . Assoc. Ofic.Agr. Chemists, 41, 56(1958). (2) “United States Pharmacopeia XV,” Mack Publishing Co., Easton, Pa., 1955, pp. 217-218. *Received December 27, 1957, from the Division of Pharmaceutical Chemistry, Bureau of Biological and Physical Sciences, U. S. Food and Drug Administration, Department of Health, Education, and Welfare, Washington 25, D. C.
Description
Crystalline powder D-1 plus 4% added gitoxin D-1 plus 10% added gitoxin Crystalline powder Crystalline powder Crystalline powder D-4 plus 470 gitoxin added D-4 plus 10% gitoxin added Tablet (mg.) 0.1 0.1 0.2 0.1 0.2 0.1 0.1 0.2 0.1 0.2 0.2 0.1 0.1 0.1 0.2 0.15 0.2 0.1 0.1 0.1 0.1
Digitoxin Found, Per Cent of Declared Modified Official Method Method
96.5
95.9
95.7
...
96.3
...
89.5
87.6
86.5
84.2
83.3
82.4
82.6
...
82.1
...
105.6 103.9 99.4 97.2 93.5 93.3 92.5 92.0 91.9 91.5 90.3 88.4 88.3 86.5 83.8 83.5 79.3 77.3 60.0 45.5 113.3
103.0 100.7 95.3 95.9 91 .o 92.8 88.9 91.7 90.0 94.0 88.7 86.3 87.6 85.2 85.7 85.0 80.1 77.2 56.3 46.6 113.0
A Note on a Simple Small Animal Respirometer* By S. T. COKER
V
ARIOUS METHODS have been used to record respiration in small animals (1-5). Most of the existing methods are not satisfactory for animals as small as rats or lack adequate sensitivity. Also, many of these methods are rather complex, or involve an elaborate system of valves. The respirometer described below is based on the principle originally used by Gaddum (1) for recording respiration in rabbits. A modification of Gaddum’s procedure by Haley (2) is similar to the one described below except a different system of valves i s used. This led to the development of a small, simple
* Received February 12, 1958, from the Department of Pharmacology, University of Kansas City, School of Pharmacy, Mo. The author is indebted t o John R . Lyon, senior laboratory assistant, for his valuable technical assistance.
respirometer which could be built completely and inexpensively in any laboratory and have sufficient sensitivity to record accurately respiration of rats as well as larger animals with proper modifications as to chamber volume and diameter of the air inlets and outlets. The apparatus is quite suitable for use in laboratory teaching a s well as drug screening on rats. The materials needed are: Plexiglas tubing one inch outside diameter by two inches in length for the chamber, and ‘/,-inch outside diameter tubing for the necessary connections, a No. 4 rubber stopper, rubber tubing, medium rubber dam, ethylene dichloride for cementing the Plexiglas, screw clamp, tracheal cannula, sensitive tambour, and a recording lever (Fig. 1, A ) .
686