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The Effect of Various Sugars and Polyols on The Stability of Vitamin B12*
The Effect of Various Sugars and Polyols on the Stability of Vitamin B,,* By MARTIN BARR, S. ROBERT KOHN, and LINWOOD F. TICE The stability of vitamin BIZwas studied i n aqueous solutions of sorbitol,. glycerin, dextrose, and sucrose. Solutions were kept at 2 5 O , 4 5 " , and 100' for periods up t o one hundred and forty days. Vitamin BIZ content was determined spectrophotometrically using a n absorption maximum of 361 m p . It was found that vitamin Blzis compatible with sorbitol and glycerin at 2 5 ' and 45 Dextrose and sucrose solutions were found to cause the loss of vitamin BIZ i n solution. Dextrose was more destructive to the vitamin than was sucrose.
'.
dextrose, and sucrose have used in oral liquid preparations containing vitamin Blz. It has been reported that sorbitol and glycerin are compatible with vitamin BIZ, while reducing agents such as dextrose and sucrose (after hydrolysis) are not (1,2). However, there is little information available indicating the degree t o which dextrose and sucrose cause the loss of vitamin BIZ. This led t h e authors t o carry out studies on the stability of vitamin Blz in solutions containing sorbitol, glycerin, dextrose, a n d sucrose over a period of time at different temperatures. ORBITOL, GLYCERIN,
S been
EXPERIMENTAL Sterile solutions containing approximately 30 mcg. of vitamin Blz in 50% w/v solutions of sorbitol,' glycerin.* dextrose,s and sucrose4 were prepared in the following manner. One hundred grams of sorbitol, glycerin, dextrose, and sucrose were dissolved in sufficient purified water t o make 1,985 ml., using a 2000-ml. volumetric flask. The solutions were then sterilized in an autoclave by heating at 15 pounds pressure (121') for twenty minutes. A stock solution containing 60 mg. of vitamin B1z6in purified water was prepared and sterilized by passing through a Selas filter.6 Fifteen milliliters of the stock vitamin B12 solution were then transferred aseptically with a sterile pipet into each of the 2,000-ml. volumetric Basks containing the polyol or sugar solutions. The finished volumes of these solutions were then 2,000 ml. and they contained approximately 30 mcg. of vitamin Blz per ml. Ten-milliliter portions of each solution were then transferred into 10-ml. glass ampuls using a sterile, automatic filler.? Sterile solutions Containing only vitamin BIZ,sorbitol, glycerin, *Received March 21, 1957, from the Department of Pharmacy, Philadelphia College of Pharmacy and Science. Work carried out with a grant from the Atlas Powder Co., Wilmington, Del. Presented to the Scientific Section, A.PH.A., New York meeting, May 1957. 1 Crystalline D-Sorbitol, Atlas Powder Co. * Glycerin, U. S. P. XV, Colgate-Palmolive-Peet Co. 3 Dextrose, U. S . P. XV, New York Quinine and Chemical Works Inc. 4 Su&ose, Analytical Reagent, J. T. Baker Chemical Co. 5 Crystalline Vitamin BIZ,Merck and Co., Inc. 0 03 Selas Filter, Selas Corporation of America. 7 Ampul Filling Machine, Baltimore Biological Laboratory, Inc., Baltimore, Md.
dextrose, and sucrose in purified water were also prepared and transferred into 10-ml. glass ampuls. These solutions served as blanks and controls in the study. The ampuls were sealed and a representative number of each solution was tested for sterility using a fluid thioglycollate medium. All were found to be sterile. The effects of the polyols and sugars on the stability of vitamin Blz were studied at three different temperatures. One series was maintained a t 100" in an oven for forty-eight hours. A second series was kept at 45". using a thermostat-controlled room for this purpose. A third series was maintained a t room temperature (approximately 25"). The solutions which were studied a t 45" and 25" were observed for a total of one hundred and forty days. Enough ampuls containing the solutions are available to permit the continuation of this study for a t least one year. The solutions in the ampuls were assayed periodically in order to determine the extent of deterioretion of the vitamin BIZin the presence of the polyols and sugars. The solutions were assayed for vitamin BIZcontent using the spectrophotometric assay in U. S. P. X V (3), which is based on an absorption maximum a t 361 mp. The sorbitol, glycerin, dextrose, and sucrose solutions were used as blanks and the vitamin BIZsolution was used as a control. All solutions for which a vitamin BIZcontent is reported were found t o exhibit ultraviolet absorption characteristics typical of the vitamin (3). The results of the stability studies are reported in Tables I and 11, and in Figs. 1 and 2.
RESULTS AND DISCUSSION The results indicate that, under accelerated test conditions of loo", there was a marked loss in vitamin Blz content in all solutions containing the vitamin. There was a loss of nearly 70% in vitamin content in the dextrose solutions in eight hours and a deterioration of nearly 40y0 in the sucrose solutions in the same period of time. It was not possible t o determine the vitamin content in the dextrose and sucrose solutions after eight hours because of discoloration of the solutions due to caramelization. It was found that these solutions no longer exhibited the ultraviolet absorption characteristics of vitamin BIZ. It is interesting t o note that the loss of vitamin BIZ in 50% w/v sorbitol and glycerin solutions was not as great as in purified water alone at 100". This
SCIENTIFIC EDITION
November 1957 TABLE I.-EFFECTS
O F POLYOLS AND
Storage at 25". days -c
Sorbitol Glycerin Dextrose Sucrose Purified water Storage at 45O, days
Sorbitol Glycerin Dextrose Sucrose Purified water Storage at
loo",
-
hours
Sorbitol Glycerin Dextrose Sucrose Purified water
-
651
SUGARSa ON VITAMIN Biz (INITIAL CONCENTRATION-29.4 MCG./ML.)
10
17
24
31
45
63
84
112
140
29.4 29.4 28.9 29.4 29.4
29.4 29.4 28.5 29.4 29.4
29.4 29.4 27.7 29.4 29.4
29.4 29.4 27.3 29.4 29.4
29.4 29.4 26.7 29.4 29.4
29.4 29.4 25.8 29.4 29.4
29.4 29.4 24.4 29.4 29.4
29.4 29.4 23.4 29.4 29.4
29.4 29.4 22.1 29.4 29.4
10
17
24
31
45
63
84
112
140
29.4 29.4 28.0 28.5 29.4
29.4 29.4 27.1 28.0 29.4
29.4 29.4 26.1 27.3 29.4
29.4 29.4 25.1 27.0 29.4
29.4 29.4 23.9 25.0 29.4
29.4 29.4 22.0 24.7 29.4
29.4 29.4 19.7 23.1 29.4
29.4 29.4 17.4 21.4 29.4
29.4 29.4
2
4
8
16
24
48
29.3 29.1 23.7 25.2 29.0
29.0 28.5 16.4 22.4 28.4
28.5 27.9 9.1 17.9 27.2
27.6 26.4
26.8 25,. 5
24.9 22.7
b
b
22.9
19.0
b b
25.1
b b
29.4
b
Polyols and sugars are present in a concentration of 50% w/v. b I t was impossible to analyze these solutions as they were discolored due to the caramelization of the sugars.
a
TABLE11.-Loss, Storage at 25O, days
Sorbitol Glycerin Dextrose Sucrose Purified water Storage at 45O, days
Sorbitol Glycerin Dextrose Sucrose Purified water
-
Storage at looo, hours
Sorbitol Glycerin Dextrose Sucrose Purified water a b
%, OF SUGARS'
IN
-
VITAMINBlz CONTENTIN SOLUTIONS CONTAINING VARIOUSPOLYOLS AND (INITIAL CONCENTRATION-29.4 MCG./ML. VITAMIN B12)
10
17
24
31
45
0.0 0.0 1.7 0.0 0.0
0.0 0.0 3.1 0.0 0.0
0.0 0.0 5.8 0.0 0.0
0.0 0.0 7.1 0.0 0.0
0.0 0.0 9.2 0.0 0.0
63
a4
112
140
0.0 0.0 12.3 0.0 0.0
0.0 0.0 17.0 0.0 0.0
0.0 0.0 20.4 0.0 0.0
0.0 0.0 24.8 0.0 0.0 140
10
17
24
31
45
63
84
112
0.0 0.0 4.8 3.1 0.0
0.0 0.0 7.8 4.7 0.0
0.0 0.0 11.2 7.1 0.0
0.0 0.0 14.6 8.2 0.0
0.0 0.0 18.7 11.9 0.0
0.0 0.0 25.2 16.0 0.0
0.0 0.0 33.0 21.4 0.0
0.0 0.0 40.8 28.2 0.0
2
4
8
16
24
48
0.3 1.0 19.3 14.0 1.4
1.4 3.1 44.4 23.8 3.3
2.7 5.1 69.0 39.1 7.5
6.1 10.2
8.5 13.3
15.8 22.4
14.6
b
b b
22.1
35.2
0.0 Ob.0 b
0.0
Polyols and sugars are present in concentrations of 50% w/v. It was impossible to analyze these solutions as they were discolored due to the caramelization of the sugars.
may be due to the fact that there is a lower concentration of water in these solutions which reduces the rate of hydrolysis, which is known t o be involved in the deterioration of vitamin BIZ. Dextrose and sucrose were the only substances which caused a reduction in vitamin BI2content a t 45". There was a reduction of about 40% caused by dextrose in one hundred and twelve days and a loss of nearly 30Oj, in the sucrose solutions during the same period of time. It was not possible t o determine the vitamin B12 content of the dextrose and sucrose solutions stored a t 45" for one hundred and forty days because of the decided discoloration of the solutions due to caramelization. These solutions no longer exhibited the ultraviolet absorption characteristics of vitamin BIZ. Dextrose was the only substance which produced a loss in vitamin content in one hundred and forty
days at 25". The deterioration at this temperature was slow, but it did amount to about a 25% loss in vitamin content during this time. Spot microbiological determinations for vitamin B12 activity were carried out on a number of the vitamin solutions stored at 25' and 45". The results revealed that only dextrose and sucrose caused a reduction in vitamin BIZactivity. I n this respect, the results were in general agreement with those obtained by the spectrophotometric method. The pH of the vitamin Blz solutions was between 5.2-6.5. All solutions became more acid during storage. Those showing the greatest loss in vitamin content were also found to be reduced the most in pH. For example, the p H of the sucrose-vitamin BIZsolutions initially was 6.5 but, after storage a t 25' and 45', their pH values were 4.3 and 3.6, respectively. The initial p H of sorbitol-vitamin BIZ
652
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION Vol. XLVI. No. 11
solutions was 5.2, falling to 4.5 and 4.2 after storage a t 25" and 45O, respectively. In this latter instance, the drop in pH was not accompanied by a loss in BH content.
CONCLUSIONS Based on one hundred and forty days of observation, it is possible to reach certain conclusions concerning the stability of vitamin Bls in the presence of various polyols and sugars. Dextrose is a reducing agent and sucrose, after hydrolysis, becomes one.
o t 25. and 45. b- 5 0 % */v Sucrose o t 25' c-Purified Water a t 25. and 45. d - 5 0 % V v Dextrose o t 25. e - 5 0 % V v Sucrose a t 45. f S O % */v Dextrose o t 45.
-
I
I
I
I
20
40
60
eo
d a y s 01 Z5.C.
Fig. 2.-Effect
b c d
- 5 0 % w/v - Purified
-
Glycerin Water 50V0 w/v Sucrose
I
100
I
120
1 140
ond 4J.C.
of various polyols and sugars on BI* a t 25' and 45".
one hundred and forty days of study, it appcars that both sorbitol and glycerin are compatible with vitamin Biz.
SUMMARY
10
Fig. 1.-Effect
20 30 40 hours at 100°C
50
of various polyols and sugars on BIZa t 100".
Both sugars caused a reduction in the vitaniiii Bla content of solutions. I n this respect, the results reported here are in agreement with those of earlicr workers (1, 2). Dextrose was more active in reducing the vitamin B12 content in the solution than sucrose for the probable reason that the latter must first undergo hydrolysis. Sorbitol and glycerin did not produce a loss in vitamin content except a t a temperature of 100". At this temperature, although there was a reduction of vitamin BIncontent in sorbito1 and glycerin solutions, it was less than the loss observed in the aqueous control. On the basis of
1. Studies have been carried out for a period of one hundred and forty days on the stability of vitamin BIZ in sorbitol, glycerin, dextrose, and sucrose solutions. 2. Vitamin BIZ was found to be compatible in sorbitol and glycerin solutions at 25O and 45". 3. Dextrose and sucrose were found t o cause the decomposition of vitamin B12 in solution at 25' and 45". Dextrose was more destructive in its effect than sucrose.
REFERENCES (1) "Vitamin BIZ," Part I, Merck Service Bulletin. Merck & Co., Inc., Rahway, N. J., 1953, pp. 8-9. (2) Gakenheimer, W. C., Drug & Cosmetic Ind., 72, 320
(1953). (3) United States Pharmacopoeia, Fifteenth Revision, Mack Printing Co., Easton, Pa., 1955, pp. 186-187.
'.
dextrose, and sucrose have used in oral liquid preparations containing vitamin Blz. It has been reported that sorbitol and glycerin are compatible with vitamin BIZ, while reducing agents such as dextrose and sucrose (after hydrolysis) are not (1,2). However, there is little information available indicating the degree t o which dextrose and sucrose cause the loss of vitamin BIZ. This led t h e authors t o carry out studies on the stability of vitamin Blz in solutions containing sorbitol, glycerin, dextrose, a n d sucrose over a period of time at different temperatures. ORBITOL, GLYCERIN,
S been
EXPERIMENTAL Sterile solutions containing approximately 30 mcg. of vitamin Blz in 50% w/v solutions of sorbitol,' glycerin.* dextrose,s and sucrose4 were prepared in the following manner. One hundred grams of sorbitol, glycerin, dextrose, and sucrose were dissolved in sufficient purified water t o make 1,985 ml., using a 2000-ml. volumetric flask. The solutions were then sterilized in an autoclave by heating at 15 pounds pressure (121') for twenty minutes. A stock solution containing 60 mg. of vitamin B1z6in purified water was prepared and sterilized by passing through a Selas filter.6 Fifteen milliliters of the stock vitamin B12 solution were then transferred aseptically with a sterile pipet into each of the 2,000-ml. volumetric Basks containing the polyol or sugar solutions. The finished volumes of these solutions were then 2,000 ml. and they contained approximately 30 mcg. of vitamin Blz per ml. Ten-milliliter portions of each solution were then transferred into 10-ml. glass ampuls using a sterile, automatic filler.? Sterile solutions Containing only vitamin BIZ,sorbitol, glycerin, *Received March 21, 1957, from the Department of Pharmacy, Philadelphia College of Pharmacy and Science. Work carried out with a grant from the Atlas Powder Co., Wilmington, Del. Presented to the Scientific Section, A.PH.A., New York meeting, May 1957. 1 Crystalline D-Sorbitol, Atlas Powder Co. * Glycerin, U. S. P. XV, Colgate-Palmolive-Peet Co. 3 Dextrose, U. S . P. XV, New York Quinine and Chemical Works Inc. 4 Su&ose, Analytical Reagent, J. T. Baker Chemical Co. 5 Crystalline Vitamin BIZ,Merck and Co., Inc. 0 03 Selas Filter, Selas Corporation of America. 7 Ampul Filling Machine, Baltimore Biological Laboratory, Inc., Baltimore, Md.
dextrose, and sucrose in purified water were also prepared and transferred into 10-ml. glass ampuls. These solutions served as blanks and controls in the study. The ampuls were sealed and a representative number of each solution was tested for sterility using a fluid thioglycollate medium. All were found to be sterile. The effects of the polyols and sugars on the stability of vitamin Blz were studied at three different temperatures. One series was maintained a t 100" in an oven for forty-eight hours. A second series was kept at 45". using a thermostat-controlled room for this purpose. A third series was maintained a t room temperature (approximately 25"). The solutions which were studied a t 45" and 25" were observed for a total of one hundred and forty days. Enough ampuls containing the solutions are available to permit the continuation of this study for a t least one year. The solutions in the ampuls were assayed periodically in order to determine the extent of deterioretion of the vitamin BIZin the presence of the polyols and sugars. The solutions were assayed for vitamin BIZcontent using the spectrophotometric assay in U. S. P. X V (3), which is based on an absorption maximum a t 361 mp. The sorbitol, glycerin, dextrose, and sucrose solutions were used as blanks and the vitamin BIZsolution was used as a control. All solutions for which a vitamin BIZcontent is reported were found t o exhibit ultraviolet absorption characteristics typical of the vitamin (3). The results of the stability studies are reported in Tables I and 11, and in Figs. 1 and 2.
RESULTS AND DISCUSSION The results indicate that, under accelerated test conditions of loo", there was a marked loss in vitamin Blz content in all solutions containing the vitamin. There was a loss of nearly 70% in vitamin content in the dextrose solutions in eight hours and a deterioration of nearly 40y0 in the sucrose solutions in the same period of time. It was not possible t o determine the vitamin content in the dextrose and sucrose solutions after eight hours because of discoloration of the solutions due to caramelization. It was found that these solutions no longer exhibited the ultraviolet absorption characteristics of vitamin BIZ. It is interesting t o note that the loss of vitamin BIZ in 50% w/v sorbitol and glycerin solutions was not as great as in purified water alone at 100". This
SCIENTIFIC EDITION
November 1957 TABLE I.-EFFECTS
O F POLYOLS AND
Storage at 25". days -c
Sorbitol Glycerin Dextrose Sucrose Purified water Storage at 45O, days
Sorbitol Glycerin Dextrose Sucrose Purified water Storage at
loo",
-
hours
Sorbitol Glycerin Dextrose Sucrose Purified water
-
651
SUGARSa ON VITAMIN Biz (INITIAL CONCENTRATION-29.4 MCG./ML.)
10
17
24
31
45
63
84
112
140
29.4 29.4 28.9 29.4 29.4
29.4 29.4 28.5 29.4 29.4
29.4 29.4 27.7 29.4 29.4
29.4 29.4 27.3 29.4 29.4
29.4 29.4 26.7 29.4 29.4
29.4 29.4 25.8 29.4 29.4
29.4 29.4 24.4 29.4 29.4
29.4 29.4 23.4 29.4 29.4
29.4 29.4 22.1 29.4 29.4
10
17
24
31
45
63
84
112
140
29.4 29.4 28.0 28.5 29.4
29.4 29.4 27.1 28.0 29.4
29.4 29.4 26.1 27.3 29.4
29.4 29.4 25.1 27.0 29.4
29.4 29.4 23.9 25.0 29.4
29.4 29.4 22.0 24.7 29.4
29.4 29.4 19.7 23.1 29.4
29.4 29.4 17.4 21.4 29.4
29.4 29.4
2
4
8
16
24
48
29.3 29.1 23.7 25.2 29.0
29.0 28.5 16.4 22.4 28.4
28.5 27.9 9.1 17.9 27.2
27.6 26.4
26.8 25,. 5
24.9 22.7
b
b
22.9
19.0
b b
25.1
b b
29.4
b
Polyols and sugars are present in a concentration of 50% w/v. b I t was impossible to analyze these solutions as they were discolored due to the caramelization of the sugars.
a
TABLE11.-Loss, Storage at 25O, days
Sorbitol Glycerin Dextrose Sucrose Purified water Storage at 45O, days
Sorbitol Glycerin Dextrose Sucrose Purified water
-
Storage at looo, hours
Sorbitol Glycerin Dextrose Sucrose Purified water a b
%, OF SUGARS'
IN
-
VITAMINBlz CONTENTIN SOLUTIONS CONTAINING VARIOUSPOLYOLS AND (INITIAL CONCENTRATION-29.4 MCG./ML. VITAMIN B12)
10
17
24
31
45
0.0 0.0 1.7 0.0 0.0
0.0 0.0 3.1 0.0 0.0
0.0 0.0 5.8 0.0 0.0
0.0 0.0 7.1 0.0 0.0
0.0 0.0 9.2 0.0 0.0
63
a4
112
140
0.0 0.0 12.3 0.0 0.0
0.0 0.0 17.0 0.0 0.0
0.0 0.0 20.4 0.0 0.0
0.0 0.0 24.8 0.0 0.0 140
10
17
24
31
45
63
84
112
0.0 0.0 4.8 3.1 0.0
0.0 0.0 7.8 4.7 0.0
0.0 0.0 11.2 7.1 0.0
0.0 0.0 14.6 8.2 0.0
0.0 0.0 18.7 11.9 0.0
0.0 0.0 25.2 16.0 0.0
0.0 0.0 33.0 21.4 0.0
0.0 0.0 40.8 28.2 0.0
2
4
8
16
24
48
0.3 1.0 19.3 14.0 1.4
1.4 3.1 44.4 23.8 3.3
2.7 5.1 69.0 39.1 7.5
6.1 10.2
8.5 13.3
15.8 22.4
14.6
b
b b
22.1
35.2
0.0 Ob.0 b
0.0
Polyols and sugars are present in concentrations of 50% w/v. It was impossible to analyze these solutions as they were discolored due to the caramelization of the sugars.
may be due to the fact that there is a lower concentration of water in these solutions which reduces the rate of hydrolysis, which is known t o be involved in the deterioration of vitamin BIZ. Dextrose and sucrose were the only substances which caused a reduction in vitamin BI2content a t 45". There was a reduction of about 40% caused by dextrose in one hundred and twelve days and a loss of nearly 30Oj, in the sucrose solutions during the same period of time. It was not possible t o determine the vitamin B12 content of the dextrose and sucrose solutions stored a t 45" for one hundred and forty days because of the decided discoloration of the solutions due to caramelization. These solutions no longer exhibited the ultraviolet absorption characteristics of vitamin BIZ. Dextrose was the only substance which produced a loss in vitamin content in one hundred and forty
days at 25". The deterioration at this temperature was slow, but it did amount to about a 25% loss in vitamin content during this time. Spot microbiological determinations for vitamin B12 activity were carried out on a number of the vitamin solutions stored at 25' and 45". The results revealed that only dextrose and sucrose caused a reduction in vitamin BIZactivity. I n this respect, the results were in general agreement with those obtained by the spectrophotometric method. The pH of the vitamin Blz solutions was between 5.2-6.5. All solutions became more acid during storage. Those showing the greatest loss in vitamin content were also found to be reduced the most in pH. For example, the p H of the sucrose-vitamin BIZsolutions initially was 6.5 but, after storage a t 25' and 45', their pH values were 4.3 and 3.6, respectively. The initial p H of sorbitol-vitamin BIZ
652
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION Vol. XLVI. No. 11
solutions was 5.2, falling to 4.5 and 4.2 after storage a t 25" and 45O, respectively. In this latter instance, the drop in pH was not accompanied by a loss in BH content.
CONCLUSIONS Based on one hundred and forty days of observation, it is possible to reach certain conclusions concerning the stability of vitamin Bls in the presence of various polyols and sugars. Dextrose is a reducing agent and sucrose, after hydrolysis, becomes one.
o t 25. and 45. b- 5 0 % */v Sucrose o t 25' c-Purified Water a t 25. and 45. d - 5 0 % V v Dextrose o t 25. e - 5 0 % V v Sucrose a t 45. f S O % */v Dextrose o t 45.
-
I
I
I
I
20
40
60
eo
d a y s 01 Z5.C.
Fig. 2.-Effect
b c d
- 5 0 % w/v - Purified
-
Glycerin Water 50V0 w/v Sucrose
I
100
I
120
1 140
ond 4J.C.
of various polyols and sugars on BI* a t 25' and 45".
one hundred and forty days of study, it appcars that both sorbitol and glycerin are compatible with vitamin Biz.
SUMMARY
10
Fig. 1.-Effect
20 30 40 hours at 100°C
50
of various polyols and sugars on BIZa t 100".
Both sugars caused a reduction in the vitaniiii Bla content of solutions. I n this respect, the results reported here are in agreement with those of earlicr workers (1, 2). Dextrose was more active in reducing the vitamin B12 content in the solution than sucrose for the probable reason that the latter must first undergo hydrolysis. Sorbitol and glycerin did not produce a loss in vitamin content except a t a temperature of 100". At this temperature, although there was a reduction of vitamin BIncontent in sorbito1 and glycerin solutions, it was less than the loss observed in the aqueous control. On the basis of
1. Studies have been carried out for a period of one hundred and forty days on the stability of vitamin BIZ in sorbitol, glycerin, dextrose, and sucrose solutions. 2. Vitamin BIZ was found to be compatible in sorbitol and glycerin solutions at 25O and 45". 3. Dextrose and sucrose were found t o cause the decomposition of vitamin B12 in solution at 25' and 45". Dextrose was more destructive in its effect than sucrose.
REFERENCES (1) "Vitamin BIZ," Part I, Merck Service Bulletin. Merck & Co., Inc., Rahway, N. J., 1953, pp. 8-9. (2) Gakenheimer, W. C., Drug & Cosmetic Ind., 72, 320
(1953). (3) United States Pharmacopoeia, Fifteenth Revision, Mack Printing Co., Easton, Pa., 1955, pp. 186-187.