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Antimolding Agents for Syrups*
438
JOURNAL OF THE
AMERICANPHARMACEUTICAL ASSOCIATION
SUMMARY 1. There is a considerable reduction in procaine content during the hPat sterilization of procaine hydrochloride solution in the presence of small quantities of sodium formaldehydesulfoxylate. 2. A condensation product of procaine and sodium formaIdehydesulfoxyIate was prepared and characterized by analysis and ultraviolet absorption. The compound exists as rt zwitterion, is stable in the dry form, and is water soluble.
Vol. XLIII, No. 7
3. The compound is of low toxicity and devoid of local anesthetic activity. REFERENCES (1) “The United States Pharmacopeia,” XIV, Mack Pubishing Co. Easton Pa. 1950 p. 491. 2) BauLr, H.,J : A k . C k e k Sac. 61 617(1939). Clemence, L. ’W.:and Kremens, A. I., [3) Raiziss, G. W., U. S. pat. 2,229,127, Jan. 21, 1941. (4) Brit. pat. 503 271 April 4 1939. (5) Ramlet, J., a i d Rbsenthal’L.,U. S. pat. 2,309,248, Jan. 26 1943. 16) Rosenthal, S. M., and Bauer, H., U. S. pat. 2,234,981, Mar. 10, 1941. (7) Raiziss, G. W.,Clemence, L. W., and Freifelder, M., U. S. pat. 2 256 575 Sept. 23 1941. (8) Riegil, E:R.,’and Buclkald, K. W.. J . A m . Chem. Sac , 51,484(1929).
Antimolding Agents for Syrups* By CLIFTON P. LORD, Jr.,t and WILLIAM‘J. HUSAt
.
The antimolding effects of more than two hundred substances were determined using mycophil agar inoculated with microorganisms from deteriorated syrup. Many of these substances were found to equal or exceed the effectiveness of benzoic acid and esters ofp-hydroxybenzoic acid. Certain flavoring and perfumery materials were found to possess antimolding pro erties. In comparison with benzoic acid which was effective at a concentration o& :1,000,cinnamic aldehyde was effective at 1 :10,000,and oxyquinoline sulfate was effective at 1:100,000.
YRUPS which have deteriorated due to growth
S of
molds, yeasts, or bacteria usually exhibit evidences of fermentation, turbidity, precipitation, moldy odor, and a change in taste. The purpose of the present investigation was to study the effect of chemical preservatives upon the microorganisms which cause deterioration of syrups. Using an accelerated test method, more than 200 chemicals, volatile oils, and synthetic flavoring agents were evaluated in regard to their preservative effects, particularly as antimolding agents. EXPERIMENTAL The materials used were obtained from reputable manufacturers or distributors. The antimolding effect of each substance w a s determined in a medium of mycophil agar which was particularly favorable for the growth of molds. The agar was seeded with molds occurring in a deterior-
*
Received November 13, 1953, from the College of Pharmacy, University of Florida, Gainesville, Fla. Presented to the Scientific Section, A. PH. A,, Salt Lake City meeting, August, 1953. This paper is based on a dissertation presented to the Graduate Council of the University of Florida by Clifton F. Lord, Jr., in partial fulfillment of the requirements for the degree of Doctor of Philosophy. t Present address: School of Pharmacy, University of Buffalo, Buffalo, N. Y. 1 Head, Department of Pharmacy, University of Florida, Gainesville, Fla.
ated syrup and comparisons were made of the growth of the molds with and without the addition of the substances which were being added for antimolding effects. The details were as follows. Mycophil agar was prepared as directed in the Baltimore Biological Laboratory Manual (1) and distributed into clean dry Pyrex culture tubes. The tubes were plugged with bleached cotton pledgets and sterilized in an eight-quart pressure cooker at 12 lb. steam pressure for twenty minutes. Ten per cent alcoholic dilutions (w/v in the case of solids and v/v for liquids) were prepared of each substance to be tested and portions of these dilutions were used to facilitate the measuring and mixing of the substances being tested. By means of a sterile pipet, 0.4 cc. of the 10% alcoholic solution was placed in a sterile Petri dish using aseptic technique. To this solution in the plate were added 16.6 cc. of melted mycophil agar cooled to approximately 40” and 3 cc. of contaminated simple syrup. The simple syrup containing microorganisms had been placed earlier in culture tubes in the desired quantity so that the syrup could be readily added to the plate at the same time as the medium. Once the three components were within the plate, they were mixed by a side-to-side and rotating motion for several seconds. The materials were considered sufficiently mixed when all evidence of striations in the mixture had disappeared. The plates were allowed to stand on a level surface until the agar congealed. When the agar had solidified the plates were placed in an incubator thermostati-
SCIENTIFIC EDITION
July, 1954
cally controlled at 28-29 O . Observations of the Petri dishes and contents were made after 48,96,and 144 hours. Certain readings were extended t o a longer period of time in those instances failing t o exhibit the growth or presence of microorganisms after 144 hours. Control tests were conducted simultaneously using the same conditions and materials from the same lots, the only exception being that in place of the 0.4cc. of alcoholic solution of the test substance, an equal volume of alcohol was added t o the Petri dish. Control tests were thus conducted t o give assurance that the syrup used t o add the mold organisms t o the plate actually contained microorganisms. The tests also indicated what effect the same volume of ethyl alcohol had by comparison with the volume of the diluted substance being tested. Sterility of the media and of the Petri dishes was also checked. A' few substances were used in aqueous solution due t o low solubility in alcohol; in such cases blank determinations were made without alcohol.
TABLE II.-sUBSTANCES FOR
INHIBITING MOLDGROWTH 144 HOURS IN THE PETRIDISH METHOD AT 1 :,500 CONCENTRATION OR LESS
Inhibited a t 1:500 but not at 1:1.000 ,---
Aldehyde C-16 Benzaldehyde Benzyl acetate Bergamot oil Borneo1 Bouquet Bluebell perfume oil' Butyric acid Cajuput oil Cetylpyridinium chloride sol. 107" Coriander" oil Coumarin .-
Cumin, synthetic) Dill weed oil Ethyl phenylacetate Isobutyl formate Jasmin We, Lavender oil
ups6
Laurel leaf oil Lemon oil Lime oil Linalyl acetate, 92% Lovage oil Mandarin oil Maple flavor, imitation' Menthyl acetate Methyl benzoate Methyl heptenone Methyl @-tertiary butyl benzoate Musk, artificial= Neroli oil, synthetice Nerolin Novoviol, alpha Novoviol, beta Novoviol-ketone Novoviol, methyl Orange oil, bitter Orange oil, sweet Origanum oil, commercial' Orris root oil, concretec Parsley oil Patchouly oil Phenylacetic acid, amyl ester Pinexapple, Pritzbro-aromec Pine needle oil, dwarf Rosemary oil Rosesol' Rose soluble, fluidd Rum ether" Saccharin sodium Sage oil, Dalmatian Santal oil Sodium benzoate Sodium caprylate Sodium citrate Sodium propionate Sodium salicylate Spruce oil Tartaric acid Terpinyl acetate Vanilla flavor base 8-fold, imitation' Vetiver oil Yerba Santa, aromatic concentrate for making syrupsb Ylang-ylang oil, syntheticc Zinc caprylate Zinc propionate
Hemlock oil Heptyl acetate Hexyl acetate Hydroxy-citronella1 Hydroxy-citronella1 dimethyl acetal Isobutyl acetate Isobutyl valerate Jasmine flower oil, syntheticc Juniper oil Lactic acid a Product of Magnus, Mabee & Reynard, Inc. b Product of Eli Lilly and Co. C Product of Fritzsche Brothers, Inc. d Product of Parke, Davis & Co.
Lime oil, terpeneless Linaloe oil Linalool Linalyl acetate 65- 70% from petitgrain oil Menthol Neroli oil Methyl salicylate Nutmeeoil Pepp&&t oil Petitgrain oil Phenylethyl alcohol Propionic acid Salicylic acid Spearmint oil Springflower Bouquet perfume oil0 Wintergreen leaf oil
Inhibited at 1:1,000 but not at 1 :2,000
Anethol Anisic aldehyde Benzoic acid Bois de rose oil femelleb Caproic acid Caraway oil Cinnamic alcohol Citronella oil Cumin oil Diacetyl salicylate TABLE ~.-SIJBSTANCES NOTPRODUCING COMPLETE Ethyl Ethyl vanillin Fennel oil INHIBITION OF MOLDGROWTH FOR 144 HOURSIN Geraniol THE PETRI DISH METHOD AT 1:500 CONCENTRATION Geranium oil, Algerian Acetophenone Acetylmethyl carbinol Amyl acetate Amyl butyrate Anisyl alcohol Balsam fir, Canada Balsam fir, Oregon Bornyl acetate Calcium propionate Camphor Camphor oil, white Cardamom oil Celery oil Chlorobutanol Citric acid Citronella1 Citronellyl butyrate Cognac oil Colonial bouquet perfume oila Copaiba oil Diphenylmethane Ethyl acetate Ethyl benzoate Ethyl butyrate Ethyl caproate Ethyl lactate Ethyl malonate Ethyl myristate Ethyl oenanthate Ethyl pelargonate Ethyl sebacate Eucalypt01 Eucalyptus oil Foenugreek solid extrnct Formic acid Geranyl acetate Geranyl butyrate Ginger fluidextract Ginger oil Glycyrrhiza, fluid for syr-
439
Heliotropin May blossom flower oil, svntheticb Peinvroval oil Methyl i-hydioxybenzoate Neutroleum alpha Piperonone Rose geranium oil Safrol Sassafras oil Sweet marjoram oil Terpineol Tolyl aldehyde Vanillin
Inhibited a t 1:2,000 but not at 1:5,000 AldehydeC-18 Aldehyde C-20 Anethol Aniseoil Bay oil Benzophenone Benzyl butyrate Carnation flower oil, syntheticb Cassia oil Citral, sample A Citronellol Clove oil
Dimethyl anthranilate Eugenol Lemon grass oil Methyl anthranilate Methyl cinnamate Myrcia oil Origanum oil, Cretic Pimento oil Propyl p-hydroxybenzoate Rosanlikb Thyme oil, red Thyme oil, white
Inhibited at 1:5,000but not at 1:10,000 Aldehyde C-14 Butyl-g-hydroxybenzoate Cinnamon oil
Citral, sample B Indol Rhodinol
Inhibited at 1:10,000 but not at 1:20,000 Cinnamic aldehyde
Inhibited at 1:100,000 but not at 1:200,000 Oxyquinoline sulfate
0
Product of Magnus, Mabee & Reynard, Inc. Product of Fritzsche Brothers, Inc. Product of Synfleur Scientific Laboratories, Inc.
Each substance evaluated for possible preservative action was retested at a later time t o determine whether or not the results were dependable and reproducible and t o establish the fact that none of the other volatile subdances in the incubator a t that time had affected the growth of the molds in a particular plate.
DISCUSSION OF RESULTS Upon mixing the mycophil agar of PH 7 with deteriorated syrup of PH 5, the resulting product showed a PH of 6, before the addition of any preservatives. Under these conditions benzoic acid was an effective preservative in a concentration of
440
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION Vol. XLIII, No. 7
1:1,000 while sodium benzoate was ineffective at 1:500. These results are in accord with previous reports (2) that sodium benzoate is effective only in acid solutions. The esters of p-hydroxybenzoic acid were effective at the following concentrations: methyl 1:1,000, propyl 1 :2,000, butyl 1:5,000. Many of the volatile oils, organic acids, and other substances tested have been previously reported as having preservative effects on the basis of different types of tests but it was deemed of interest to evaluate their antimolding properties by comparing them directly under the same conditions. In addition, tests were made on other substances such as flavoring and perfumery materials not previously reported as preservatives; these substances were tested with the thought that they might possess previously unsuspected preservative effects. Many of the substances tested showed an effectiveness equal or superior to that of benzoic acid and esters of p-hydroxybenzoic acid. However, further studies must be made before these substances can be recommended for actual use, giving careful consideration to odor, taste, toxicity, and compatibility with medicaments. Meanwhile the data will be of assistance in determining what antimolding effect, if any, can be expected from various substances which are employed in formulations primarily as flavors or medicaments. The results obtained with agar plates would not necessarily be directly applicable to other products of a different nature.
Of the substances included in the present study, the two most effective mold inhibitors were cinnamic aldehyde, which was effective at a concentration of 1: 10,000 and oxyquinoline sulfate which was effective at 1:100,000.
SUMMARY
1. Tests of antimolding properties have shown that a number of substances equal or exceed the effectiveness of benzoic acid and esters of phydroxybenzoic acid. 2. Further consideration must be given t o odor, taste, toxicity, and compatibility with medicaments before these substances can be recommended for actual use: 3. Meanwhile the data will be of assistance in determining what antimolding effect, if any, can be expected from various substances which are employed in formulations primarily as flavors or medicaments. REFERENCES (1) “Products for the Bacteriology Laboratory,” 2nd ed., Baltimore Biological Laboratory, Inc., Baltimore, Md., 1950, p. 44. (2) Goshorn. R . H . , Degering, E. F., and Tetrault, P. A., Znd. E n g . Chem., 30, 646(1938).
A Comparative Study of the Assay of Digitoxin by the U. S. P. XI11 Pigeon Method and the U. S. P. XIV Colorimetric Assay* By HERBERT A. BRAUN and LEHMAN M. LUSKY The results obtained on 31 digitoxin preparations consisting of 2 9 tablets and two samples of “purified digitoxin” were compared by the U. S. P. XI11 pigeon method and b y the U. S. P. XIV colorimetric assay. Eight of these preparations were more than 25 per cent higher by the chemical method than b the biological assay. It appears that the U. S. P. XIV colorimetric method lacks tge specificity necessary for the assay of digitoxin and its preparations. primary problems encountered in field of standardization of drugs is the selection of the proper method of assay. In the U. S . P. XIV a colorimetric method (1) supplanted the intravenous pigeon method as the method of assay for digitoxin. This colorimetric method (1) is a modification of the Baljet reaction NE OF THE
0
* Received October 14. 19.53. from the Division of Phar14, 1953, Food andand Drug Administration, administration, Department of macology, hoodHealth, Education, and Welfare, Washington, D. C. Presented in part before the American Society for Pharmacology and Experimental Therapeutics at New York City, April, 1952.
in which an orange-red color is developed by the digitalis glycosides in the presence of alkaline picrate solution. In the case of digitalis and its preparations Vos and Welsh (2) and Allmark and Bachinski (3) have shown that the chemical method yields results which do not always agree with those found b y the biological assay. They found that with some preparations the chemical method gave results which were 45-75 per cent higher than those obtained by the biological assay. Danow,
JOURNAL OF THE
AMERICANPHARMACEUTICAL ASSOCIATION
SUMMARY 1. There is a considerable reduction in procaine content during the hPat sterilization of procaine hydrochloride solution in the presence of small quantities of sodium formaldehydesulfoxylate. 2. A condensation product of procaine and sodium formaIdehydesulfoxyIate was prepared and characterized by analysis and ultraviolet absorption. The compound exists as rt zwitterion, is stable in the dry form, and is water soluble.
Vol. XLIII, No. 7
3. The compound is of low toxicity and devoid of local anesthetic activity. REFERENCES (1) “The United States Pharmacopeia,” XIV, Mack Pubishing Co. Easton Pa. 1950 p. 491. 2) BauLr, H.,J : A k . C k e k Sac. 61 617(1939). Clemence, L. ’W.:and Kremens, A. I., [3) Raiziss, G. W., U. S. pat. 2,229,127, Jan. 21, 1941. (4) Brit. pat. 503 271 April 4 1939. (5) Ramlet, J., a i d Rbsenthal’L.,U. S. pat. 2,309,248, Jan. 26 1943. 16) Rosenthal, S. M., and Bauer, H., U. S. pat. 2,234,981, Mar. 10, 1941. (7) Raiziss, G. W.,Clemence, L. W., and Freifelder, M., U. S. pat. 2 256 575 Sept. 23 1941. (8) Riegil, E:R.,’and Buclkald, K. W.. J . A m . Chem. Sac , 51,484(1929).
Antimolding Agents for Syrups* By CLIFTON P. LORD, Jr.,t and WILLIAM‘J. HUSAt
.
The antimolding effects of more than two hundred substances were determined using mycophil agar inoculated with microorganisms from deteriorated syrup. Many of these substances were found to equal or exceed the effectiveness of benzoic acid and esters ofp-hydroxybenzoic acid. Certain flavoring and perfumery materials were found to possess antimolding pro erties. In comparison with benzoic acid which was effective at a concentration o& :1,000,cinnamic aldehyde was effective at 1 :10,000,and oxyquinoline sulfate was effective at 1:100,000.
YRUPS which have deteriorated due to growth
S of
molds, yeasts, or bacteria usually exhibit evidences of fermentation, turbidity, precipitation, moldy odor, and a change in taste. The purpose of the present investigation was to study the effect of chemical preservatives upon the microorganisms which cause deterioration of syrups. Using an accelerated test method, more than 200 chemicals, volatile oils, and synthetic flavoring agents were evaluated in regard to their preservative effects, particularly as antimolding agents. EXPERIMENTAL The materials used were obtained from reputable manufacturers or distributors. The antimolding effect of each substance w a s determined in a medium of mycophil agar which was particularly favorable for the growth of molds. The agar was seeded with molds occurring in a deterior-
*
Received November 13, 1953, from the College of Pharmacy, University of Florida, Gainesville, Fla. Presented to the Scientific Section, A. PH. A,, Salt Lake City meeting, August, 1953. This paper is based on a dissertation presented to the Graduate Council of the University of Florida by Clifton F. Lord, Jr., in partial fulfillment of the requirements for the degree of Doctor of Philosophy. t Present address: School of Pharmacy, University of Buffalo, Buffalo, N. Y. 1 Head, Department of Pharmacy, University of Florida, Gainesville, Fla.
ated syrup and comparisons were made of the growth of the molds with and without the addition of the substances which were being added for antimolding effects. The details were as follows. Mycophil agar was prepared as directed in the Baltimore Biological Laboratory Manual (1) and distributed into clean dry Pyrex culture tubes. The tubes were plugged with bleached cotton pledgets and sterilized in an eight-quart pressure cooker at 12 lb. steam pressure for twenty minutes. Ten per cent alcoholic dilutions (w/v in the case of solids and v/v for liquids) were prepared of each substance to be tested and portions of these dilutions were used to facilitate the measuring and mixing of the substances being tested. By means of a sterile pipet, 0.4 cc. of the 10% alcoholic solution was placed in a sterile Petri dish using aseptic technique. To this solution in the plate were added 16.6 cc. of melted mycophil agar cooled to approximately 40” and 3 cc. of contaminated simple syrup. The simple syrup containing microorganisms had been placed earlier in culture tubes in the desired quantity so that the syrup could be readily added to the plate at the same time as the medium. Once the three components were within the plate, they were mixed by a side-to-side and rotating motion for several seconds. The materials were considered sufficiently mixed when all evidence of striations in the mixture had disappeared. The plates were allowed to stand on a level surface until the agar congealed. When the agar had solidified the plates were placed in an incubator thermostati-
SCIENTIFIC EDITION
July, 1954
cally controlled at 28-29 O . Observations of the Petri dishes and contents were made after 48,96,and 144 hours. Certain readings were extended t o a longer period of time in those instances failing t o exhibit the growth or presence of microorganisms after 144 hours. Control tests were conducted simultaneously using the same conditions and materials from the same lots, the only exception being that in place of the 0.4cc. of alcoholic solution of the test substance, an equal volume of alcohol was added t o the Petri dish. Control tests were thus conducted t o give assurance that the syrup used t o add the mold organisms t o the plate actually contained microorganisms. The tests also indicated what effect the same volume of ethyl alcohol had by comparison with the volume of the diluted substance being tested. Sterility of the media and of the Petri dishes was also checked. A' few substances were used in aqueous solution due t o low solubility in alcohol; in such cases blank determinations were made without alcohol.
TABLE II.-sUBSTANCES FOR
INHIBITING MOLDGROWTH 144 HOURS IN THE PETRIDISH METHOD AT 1 :,500 CONCENTRATION OR LESS
Inhibited a t 1:500 but not at 1:1.000 ,---
Aldehyde C-16 Benzaldehyde Benzyl acetate Bergamot oil Borneo1 Bouquet Bluebell perfume oil' Butyric acid Cajuput oil Cetylpyridinium chloride sol. 107" Coriander" oil Coumarin .-
Cumin, synthetic) Dill weed oil Ethyl phenylacetate Isobutyl formate Jasmin We, Lavender oil
ups6
Laurel leaf oil Lemon oil Lime oil Linalyl acetate, 92% Lovage oil Mandarin oil Maple flavor, imitation' Menthyl acetate Methyl benzoate Methyl heptenone Methyl @-tertiary butyl benzoate Musk, artificial= Neroli oil, synthetice Nerolin Novoviol, alpha Novoviol, beta Novoviol-ketone Novoviol, methyl Orange oil, bitter Orange oil, sweet Origanum oil, commercial' Orris root oil, concretec Parsley oil Patchouly oil Phenylacetic acid, amyl ester Pinexapple, Pritzbro-aromec Pine needle oil, dwarf Rosemary oil Rosesol' Rose soluble, fluidd Rum ether" Saccharin sodium Sage oil, Dalmatian Santal oil Sodium benzoate Sodium caprylate Sodium citrate Sodium propionate Sodium salicylate Spruce oil Tartaric acid Terpinyl acetate Vanilla flavor base 8-fold, imitation' Vetiver oil Yerba Santa, aromatic concentrate for making syrupsb Ylang-ylang oil, syntheticc Zinc caprylate Zinc propionate
Hemlock oil Heptyl acetate Hexyl acetate Hydroxy-citronella1 Hydroxy-citronella1 dimethyl acetal Isobutyl acetate Isobutyl valerate Jasmine flower oil, syntheticc Juniper oil Lactic acid a Product of Magnus, Mabee & Reynard, Inc. b Product of Eli Lilly and Co. C Product of Fritzsche Brothers, Inc. d Product of Parke, Davis & Co.
Lime oil, terpeneless Linaloe oil Linalool Linalyl acetate 65- 70% from petitgrain oil Menthol Neroli oil Methyl salicylate Nutmeeoil Pepp&&t oil Petitgrain oil Phenylethyl alcohol Propionic acid Salicylic acid Spearmint oil Springflower Bouquet perfume oil0 Wintergreen leaf oil
Inhibited at 1:1,000 but not at 1 :2,000
Anethol Anisic aldehyde Benzoic acid Bois de rose oil femelleb Caproic acid Caraway oil Cinnamic alcohol Citronella oil Cumin oil Diacetyl salicylate TABLE ~.-SIJBSTANCES NOTPRODUCING COMPLETE Ethyl Ethyl vanillin Fennel oil INHIBITION OF MOLDGROWTH FOR 144 HOURSIN Geraniol THE PETRI DISH METHOD AT 1:500 CONCENTRATION Geranium oil, Algerian Acetophenone Acetylmethyl carbinol Amyl acetate Amyl butyrate Anisyl alcohol Balsam fir, Canada Balsam fir, Oregon Bornyl acetate Calcium propionate Camphor Camphor oil, white Cardamom oil Celery oil Chlorobutanol Citric acid Citronella1 Citronellyl butyrate Cognac oil Colonial bouquet perfume oila Copaiba oil Diphenylmethane Ethyl acetate Ethyl benzoate Ethyl butyrate Ethyl caproate Ethyl lactate Ethyl malonate Ethyl myristate Ethyl oenanthate Ethyl pelargonate Ethyl sebacate Eucalypt01 Eucalyptus oil Foenugreek solid extrnct Formic acid Geranyl acetate Geranyl butyrate Ginger fluidextract Ginger oil Glycyrrhiza, fluid for syr-
439
Heliotropin May blossom flower oil, svntheticb Peinvroval oil Methyl i-hydioxybenzoate Neutroleum alpha Piperonone Rose geranium oil Safrol Sassafras oil Sweet marjoram oil Terpineol Tolyl aldehyde Vanillin
Inhibited a t 1:2,000 but not at 1:5,000 AldehydeC-18 Aldehyde C-20 Anethol Aniseoil Bay oil Benzophenone Benzyl butyrate Carnation flower oil, syntheticb Cassia oil Citral, sample A Citronellol Clove oil
Dimethyl anthranilate Eugenol Lemon grass oil Methyl anthranilate Methyl cinnamate Myrcia oil Origanum oil, Cretic Pimento oil Propyl p-hydroxybenzoate Rosanlikb Thyme oil, red Thyme oil, white
Inhibited at 1:5,000but not at 1:10,000 Aldehyde C-14 Butyl-g-hydroxybenzoate Cinnamon oil
Citral, sample B Indol Rhodinol
Inhibited at 1:10,000 but not at 1:20,000 Cinnamic aldehyde
Inhibited at 1:100,000 but not at 1:200,000 Oxyquinoline sulfate
0
Product of Magnus, Mabee & Reynard, Inc. Product of Fritzsche Brothers, Inc. Product of Synfleur Scientific Laboratories, Inc.
Each substance evaluated for possible preservative action was retested at a later time t o determine whether or not the results were dependable and reproducible and t o establish the fact that none of the other volatile subdances in the incubator a t that time had affected the growth of the molds in a particular plate.
DISCUSSION OF RESULTS Upon mixing the mycophil agar of PH 7 with deteriorated syrup of PH 5, the resulting product showed a PH of 6, before the addition of any preservatives. Under these conditions benzoic acid was an effective preservative in a concentration of
440
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION Vol. XLIII, No. 7
1:1,000 while sodium benzoate was ineffective at 1:500. These results are in accord with previous reports (2) that sodium benzoate is effective only in acid solutions. The esters of p-hydroxybenzoic acid were effective at the following concentrations: methyl 1:1,000, propyl 1 :2,000, butyl 1:5,000. Many of the volatile oils, organic acids, and other substances tested have been previously reported as having preservative effects on the basis of different types of tests but it was deemed of interest to evaluate their antimolding properties by comparing them directly under the same conditions. In addition, tests were made on other substances such as flavoring and perfumery materials not previously reported as preservatives; these substances were tested with the thought that they might possess previously unsuspected preservative effects. Many of the substances tested showed an effectiveness equal or superior to that of benzoic acid and esters of p-hydroxybenzoic acid. However, further studies must be made before these substances can be recommended for actual use, giving careful consideration to odor, taste, toxicity, and compatibility with medicaments. Meanwhile the data will be of assistance in determining what antimolding effect, if any, can be expected from various substances which are employed in formulations primarily as flavors or medicaments. The results obtained with agar plates would not necessarily be directly applicable to other products of a different nature.
Of the substances included in the present study, the two most effective mold inhibitors were cinnamic aldehyde, which was effective at a concentration of 1: 10,000 and oxyquinoline sulfate which was effective at 1:100,000.
SUMMARY
1. Tests of antimolding properties have shown that a number of substances equal or exceed the effectiveness of benzoic acid and esters of phydroxybenzoic acid. 2. Further consideration must be given t o odor, taste, toxicity, and compatibility with medicaments before these substances can be recommended for actual use: 3. Meanwhile the data will be of assistance in determining what antimolding effect, if any, can be expected from various substances which are employed in formulations primarily as flavors or medicaments. REFERENCES (1) “Products for the Bacteriology Laboratory,” 2nd ed., Baltimore Biological Laboratory, Inc., Baltimore, Md., 1950, p. 44. (2) Goshorn. R . H . , Degering, E. F., and Tetrault, P. A., Znd. E n g . Chem., 30, 646(1938).
A Comparative Study of the Assay of Digitoxin by the U. S. P. XI11 Pigeon Method and the U. S. P. XIV Colorimetric Assay* By HERBERT A. BRAUN and LEHMAN M. LUSKY The results obtained on 31 digitoxin preparations consisting of 2 9 tablets and two samples of “purified digitoxin” were compared by the U. S. P. XI11 pigeon method and b y the U. S. P. XIV colorimetric assay. Eight of these preparations were more than 25 per cent higher by the chemical method than b the biological assay. It appears that the U. S. P. XIV colorimetric method lacks tge specificity necessary for the assay of digitoxin and its preparations. primary problems encountered in field of standardization of drugs is the selection of the proper method of assay. In the U. S . P. XIV a colorimetric method (1) supplanted the intravenous pigeon method as the method of assay for digitoxin. This colorimetric method (1) is a modification of the Baljet reaction NE OF THE
0
* Received October 14. 19.53. from the Division of Phar14, 1953, Food andand Drug Administration, administration, Department of macology, hoodHealth, Education, and Welfare, Washington, D. C. Presented in part before the American Society for Pharmacology and Experimental Therapeutics at New York City, April, 1952.
in which an orange-red color is developed by the digitalis glycosides in the presence of alkaline picrate solution. In the case of digitalis and its preparations Vos and Welsh (2) and Allmark and Bachinski (3) have shown that the chemical method yields results which do not always agree with those found b y the biological assay. They found that with some preparations the chemical method gave results which were 45-75 per cent higher than those obtained by the biological assay. Danow,