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The Separation of the Ergot Alkaloids by Paper Partition Chromatography†
354
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
says are more rapid than the official assay and require less material for the assay.
SUMMARY A comparison of two colorimetric assays with the official assay of belladonna and stramonium was made. One colorimetric assay was’based on the Vitali color reaction; the other colorimetric assay was based on a n alkaloidal reineckate precipitate which gives a color when dissolved in acetone. Both assays were found to be rapid and simple t o perform. T h e reineckate assay was t h e more accurate, whereas t h e Vitali assay was more sensitive and more rapid. The Vitali as-
Vol. XLI, No. 7
say is accurate enough t o show coniparativc results when only a small amount of material t o be assayed is at hand. A research project is now i n progress in which a cornparison of these methods on a larger scale is being undertaken.
REFERENCES (1) “United States Pharmacopeia,” Fourteenth Revision, Mack Publishing Co.,Easton, Pa., 1950, pp. 71, 282. (2) “The National Formulary,” 9th ed., Mack Publishing C o . , Easton, Pa., 1950, p. 504. (3) Allport. N. L., and Wilson, E., Quart. J . Phavm. Phavmacol., 12, 399(1939). (4) Morin, M., J. Phavm. chim., 23, 545(1936). (5) Bandelin, F. J . , THISJOURNAL, 39. 493(1950). (6) Guth. E. P., Cosgrove, F. P . , and Bogg, W., A Study of the Characteristics of Ammonium Reineckate as a Precipitant of Atropine Sulfate,” unpublished.
The Separation of the Ergot Alkaloids by Paper Partition Chromatography*>t By V. E. TYLER, Jr. and A. E. SCHWARTING T h e problem of separation of ergot alkaloids i n solvent systems is complicated by the varied solubility of the alkaloids and by their instability. T h e conventional procedure of paper partition chromatography, in which water is the stationary phase, does not separate the ergotoxine and ergotamine alkaloids since they follow the solvent front of the wash liquid. A partition coefficient, favoring the stationary phase, was achieved by substituting an organic-aqueous system for the water. T h e stability of the alkaloids was assured by using acidic wash liquids.
of the investigation of t h e biosynthesis of ergot alkaloids by Claviceps Fur#urea i n submerged culturc, i t became necessary t o be able t o detect minute quantities of material. This objective, together with a method for t h e separation of all the alkaloids, has been achieved by the process of paper partition chroniatography. Ergonovine and ergometrinine have been separated by paper partition chromatography (1). A recent note (2) indicated a method for the separation of the water-insoluble alkaloids on buffered filter paper. A detailed report of this investigation has appeared (3). T h e impregnation of filter paper with glycols (4) a n d procedures for reverse phase partition chromatography have been described (5-7). N THE COURSE
* Received January 25, 1952, from the University of Connccticut College of Pharmacy, Storrs, Conn. t This investigation was supported i n part by a grant from E l i Lilly and Co., Iadianapnlis, Ind.
MATERIALS Alkaloids.-Ergotoxine ethanesulforiate B. P., ergonovinc maleate U. S. P., and crgotamine tartrate U. S.P. were used in the preparation of the alkaloids. Ergotoxine and ergotamiiie wcrc each prepared by dissolving 100 mg. of each salt in 25 cc. of water and 5 cc. of acetone. Sodium bicarbonate was added to saturation. The base was collected, washed with water, and dried over phosphorous pentoxide. Ergonovine was prepared by dissolving 100 mg. of the maleate in 2 cc. of ethanol and 3 cc. of stronger ammonia T. S. Fifty cubic ceutimeters of an aqueous, saturated sodium chloride solution was added. The base was extracted with ether. The solution was dried and the solvent was evaporated. The product was dried over phosphorous pentoxide. Ergornetrinine was prepared by dissolving 25 mg. of the base in 0.3 cc. of glacial acetic acid. Three cubic centimeters of water was added and the mixture was refluxed for one hour. The solution, made alkaline with ammonium hydroxide, was extracted with ether. The residue after evaporation was collected. Ergotininc and ergotaminine wcrc each prepared by refluxirig 25 mg. of the 1-base of each cornpout~dill
SCIENTIFIC EDUIUN
July, 1952
2 cc. of methanol for one hour. The reaction products, each containing the isomeric pairs, were dissolved in ethanol for usc. Lysergic acid, equivalent to 128 6% of ergonoviue maleate by colorimetric assay, was used. The fluorescence of the alkaloids served as a means for their localization on paper. The zones of distribution of all alkaloids were detectable when 5 p g . was chromatographed, although the water-insoluble alkaloids are more readily detectable when 10-20 pg. are used, since the wash liquids used in this investigation caused a damping of their fluorescence. The levorotatory alkaloids and their salts were used in ethanol solutions containing 5 pg./20 MI. Twenty-microliter volumes were chromatographed. Forty microliters each of the ergonovine-ergometrinine mixture in cther and the ergotoxine-ergotinine and ergotaminine mixtures in cthanol were used. Each volumc of the isomeric pairs represented approximately 10 pg. of alkaloids. Lysergic acid was applied in 20-pI. volumes containing 5 pg. of the substance. Wash Liquids.-A n-bUtaIIOl-ghCkdl acetic acidwater (4: 1 :5) mixture was prepared. The saturated butanol layer and the saturated water layer were uscd as wash liquid3 in separate experiments. The aqueous layer had a p H of 2.1. Phosphate citric acid buffer systems ( 8 ) , 6 volumes, a t p H levels of 3.0, 4 5, and 6.0 were each equilibrated with 4 volumes of n-butanol. The aqueous plyase of each was used a? a wash liquid.
EXPERIMENTAL The procedure of paper partition chromatography eniploying the descending method was used in all procedures except those reported in Table I. Strips of Whatinan No. I paper, 1 x 22l/? in., or sheets, 18L/4x 221/2in. were used. The solvent front was allowed to move a t least three-fourths of the length of the paper a t a temperature of 21" f 1.0". Thc RF values of the lcvorotatory alkaloids and their common salts aiid of lysergic acid were first determined by chrornatographing, by asccnding technique, on strips. The wash liquid, butanol-acetic acid-water (butanol phase), gave values reported in Table I. This mcthod did not give separation of the waterinsoluble alkaloids, but provides a mcthod for the dctcction of lysergic acid and ergonovine. Filter paper strips were impregnated with proyylene glycol, arid fonnamide, and after application of the alkaloids, the wash liquids of benzene, tolulcnc, and xylcne, each equilibrated with the stationaryphase liquid, were used to form the chromatograms. Although separations of the water-insoluble alkaloids were possible, the method was not adaptable to an unambiguous separation of all of the alkaloids. The combination of propyleiic glycol and xylene or toluene gave best results. The results of this work are not reported. Hydrophobic filter paper was prepared by dipping strips or sheets of paper in a 5% (v/v) solution of Dow-Corning Silicone No. 1107 in heptane (Bastman, practical grade). The paper, allowed to dry in air, was heated in an oven at 150" for three minutes, and aiter application of the alkaloids or alkaloid mixture, was cquilibratctl for twclvc hours with thr vapor of thc saturated butanol phase of the
355 TABLE 1
-
~~
Compoimd
Rt.
Ergotarnine Ergotoxine Ergonovine Ergotamine tartrate Ergotoxine ethanesulfonatc Ergonovine maleate Lysergic acid
0 .08 0.98 0 67 0.98 0.98 0.66
0.51
wash liquids. The chromatograms were formed with the saturated aqueous phase of the several wash liquids. The aqueous phase of the butanol-acetic acid-water system and the aqueous phase of the butanol-pH 3.0 buffer gave the broader separations. RF values of the alkaloids obtained with these two systems are reported in Table 11. These values must be considered relative, since it was observed that exact reproduction of the RF values is influenced by changes in the silicone content and by differences in the curing process.
TABLE TI
~-
-
---
-
-RFValues----
Compound
ButanolAcetic AcidWater
ButanolBuffer ( P H 3.0)
Ergonovinc Ergometrinine Ergotonine Ergotiniue Ergotarnine Ergotarninine Lysergic acid Ergonovine maleate Ergotoxine ethdnesulfonate Ergotarnine tartrate
0.62 0.78 0.85 0.87 0.75 0.57 0.52 0.64 0.86 0.78
0.52 0.72 0.67 0.68 0.54 0.72 0.34 0.50 0.67 0.54
The methods fail in the separation of crgotoxine and its isomer. This pair, ergotoxine-ergotinine, is separable when chromatographed with the butanol p H 4.5 buffer system. By this procedure, the RF of ergotoxine is 0.60 and the RF of ergotinine is 0.69. The alkaloid bases do not tail but assume rather large arcas by these processes. The wash liquids do not dissolve paper constituents, and i t was noted that extracts of crgot chromatographed with, reniarkable clarity. Two-diniensional chroniatograms of a mixture of the levorotatory alkaloid bases and lyscrgic acid have bccn prepared. The KF values obtained on strips are duplicated on silicone-treated sheets when the first wash is the aqueous phase of the butanolacetic acid-water systcm. Equilibration of the paper with the butdnol phase is necessary before each formation.
REFERENCES (11 Foster, G. E., Macdonald, J., and Jooes, T. S. G.. J . Pharm. Pharmacol., 1, WZ(1949). (2) Carless, J. E., and Woodhead, H. B., Nature, 168, 203 (19.51). (3) Brindle, H., Carless, J. E., and Woodhead. H. B . , J. Phavm. Pharmacnl. 3 , 793(1951). 14) Zaffroni, A., &ton, R . , and Keutman, R . , J . R i d Chrm., 177 I09(1949). (5) Krit:hevsky, T. H., and Tiselius, A,. Science, 114, O C L C L ? 1 CL
c
,,
L:rJ( L J D I , .
(6) Jullander, I., Nnltcve 162 300(1948). ( 7 ) Partridge S. M., a d Sw$n. T.. ibid., 166, 2i2(196U). (8) M o c I l v a i ~ e T. , C.,J . B i d . Ckem., 49, 183(1921).
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
says are more rapid than the official assay and require less material for the assay.
SUMMARY A comparison of two colorimetric assays with the official assay of belladonna and stramonium was made. One colorimetric assay was’based on the Vitali color reaction; the other colorimetric assay was based on a n alkaloidal reineckate precipitate which gives a color when dissolved in acetone. Both assays were found to be rapid and simple t o perform. T h e reineckate assay was t h e more accurate, whereas t h e Vitali assay was more sensitive and more rapid. The Vitali as-
Vol. XLI, No. 7
say is accurate enough t o show coniparativc results when only a small amount of material t o be assayed is at hand. A research project is now i n progress in which a cornparison of these methods on a larger scale is being undertaken.
REFERENCES (1) “United States Pharmacopeia,” Fourteenth Revision, Mack Publishing Co.,Easton, Pa., 1950, pp. 71, 282. (2) “The National Formulary,” 9th ed., Mack Publishing C o . , Easton, Pa., 1950, p. 504. (3) Allport. N. L., and Wilson, E., Quart. J . Phavm. Phavmacol., 12, 399(1939). (4) Morin, M., J. Phavm. chim., 23, 545(1936). (5) Bandelin, F. J . , THISJOURNAL, 39. 493(1950). (6) Guth. E. P., Cosgrove, F. P . , and Bogg, W., A Study of the Characteristics of Ammonium Reineckate as a Precipitant of Atropine Sulfate,” unpublished.
The Separation of the Ergot Alkaloids by Paper Partition Chromatography*>t By V. E. TYLER, Jr. and A. E. SCHWARTING T h e problem of separation of ergot alkaloids i n solvent systems is complicated by the varied solubility of the alkaloids and by their instability. T h e conventional procedure of paper partition chromatography, in which water is the stationary phase, does not separate the ergotoxine and ergotamine alkaloids since they follow the solvent front of the wash liquid. A partition coefficient, favoring the stationary phase, was achieved by substituting an organic-aqueous system for the water. T h e stability of the alkaloids was assured by using acidic wash liquids.
of the investigation of t h e biosynthesis of ergot alkaloids by Claviceps Fur#urea i n submerged culturc, i t became necessary t o be able t o detect minute quantities of material. This objective, together with a method for t h e separation of all the alkaloids, has been achieved by the process of paper partition chroniatography. Ergonovine and ergometrinine have been separated by paper partition chromatography (1). A recent note (2) indicated a method for the separation of the water-insoluble alkaloids on buffered filter paper. A detailed report of this investigation has appeared (3). T h e impregnation of filter paper with glycols (4) a n d procedures for reverse phase partition chromatography have been described (5-7). N THE COURSE
* Received January 25, 1952, from the University of Connccticut College of Pharmacy, Storrs, Conn. t This investigation was supported i n part by a grant from E l i Lilly and Co., Iadianapnlis, Ind.
MATERIALS Alkaloids.-Ergotoxine ethanesulforiate B. P., ergonovinc maleate U. S. P., and crgotamine tartrate U. S.P. were used in the preparation of the alkaloids. Ergotoxine and ergotamiiie wcrc each prepared by dissolving 100 mg. of each salt in 25 cc. of water and 5 cc. of acetone. Sodium bicarbonate was added to saturation. The base was collected, washed with water, and dried over phosphorous pentoxide. Ergonovine was prepared by dissolving 100 mg. of the maleate in 2 cc. of ethanol and 3 cc. of stronger ammonia T. S. Fifty cubic ceutimeters of an aqueous, saturated sodium chloride solution was added. The base was extracted with ether. The solution was dried and the solvent was evaporated. The product was dried over phosphorous pentoxide. Ergornetrinine was prepared by dissolving 25 mg. of the base in 0.3 cc. of glacial acetic acid. Three cubic centimeters of water was added and the mixture was refluxed for one hour. The solution, made alkaline with ammonium hydroxide, was extracted with ether. The residue after evaporation was collected. Ergotininc and ergotaminine wcrc each prepared by refluxirig 25 mg. of the 1-base of each cornpout~dill
SCIENTIFIC EDUIUN
July, 1952
2 cc. of methanol for one hour. The reaction products, each containing the isomeric pairs, were dissolved in ethanol for usc. Lysergic acid, equivalent to 128 6% of ergonoviue maleate by colorimetric assay, was used. The fluorescence of the alkaloids served as a means for their localization on paper. The zones of distribution of all alkaloids were detectable when 5 p g . was chromatographed, although the water-insoluble alkaloids are more readily detectable when 10-20 pg. are used, since the wash liquids used in this investigation caused a damping of their fluorescence. The levorotatory alkaloids and their salts were used in ethanol solutions containing 5 pg./20 MI. Twenty-microliter volumes were chromatographed. Forty microliters each of the ergonovine-ergometrinine mixture in cther and the ergotoxine-ergotinine and ergotaminine mixtures in cthanol were used. Each volumc of the isomeric pairs represented approximately 10 pg. of alkaloids. Lysergic acid was applied in 20-pI. volumes containing 5 pg. of the substance. Wash Liquids.-A n-bUtaIIOl-ghCkdl acetic acidwater (4: 1 :5) mixture was prepared. The saturated butanol layer and the saturated water layer were uscd as wash liquid3 in separate experiments. The aqueous layer had a p H of 2.1. Phosphate citric acid buffer systems ( 8 ) , 6 volumes, a t p H levels of 3.0, 4 5, and 6.0 were each equilibrated with 4 volumes of n-butanol. The aqueous plyase of each was used a? a wash liquid.
EXPERIMENTAL The procedure of paper partition chromatography eniploying the descending method was used in all procedures except those reported in Table I. Strips of Whatinan No. I paper, 1 x 22l/? in., or sheets, 18L/4x 221/2in. were used. The solvent front was allowed to move a t least three-fourths of the length of the paper a t a temperature of 21" f 1.0". Thc RF values of the lcvorotatory alkaloids and their common salts aiid of lysergic acid were first determined by chrornatographing, by asccnding technique, on strips. The wash liquid, butanol-acetic acid-water (butanol phase), gave values reported in Table I. This mcthod did not give separation of the waterinsoluble alkaloids, but provides a mcthod for the dctcction of lysergic acid and ergonovine. Filter paper strips were impregnated with proyylene glycol, arid fonnamide, and after application of the alkaloids, the wash liquids of benzene, tolulcnc, and xylcne, each equilibrated with the stationaryphase liquid, were used to form the chromatograms. Although separations of the water-insoluble alkaloids were possible, the method was not adaptable to an unambiguous separation of all of the alkaloids. The combination of propyleiic glycol and xylene or toluene gave best results. The results of this work are not reported. Hydrophobic filter paper was prepared by dipping strips or sheets of paper in a 5% (v/v) solution of Dow-Corning Silicone No. 1107 in heptane (Bastman, practical grade). The paper, allowed to dry in air, was heated in an oven at 150" for three minutes, and aiter application of the alkaloids or alkaloid mixture, was cquilibratctl for twclvc hours with thr vapor of thc saturated butanol phase of the
355 TABLE 1
-
~~
Compoimd
Rt.
Ergotarnine Ergotoxine Ergonovine Ergotamine tartrate Ergotoxine ethanesulfonatc Ergonovine maleate Lysergic acid
0 .08 0.98 0 67 0.98 0.98 0.66
0.51
wash liquids. The chromatograms were formed with the saturated aqueous phase of the several wash liquids. The aqueous phase of the butanol-acetic acid-water system and the aqueous phase of the butanol-pH 3.0 buffer gave the broader separations. RF values of the alkaloids obtained with these two systems are reported in Table 11. These values must be considered relative, since it was observed that exact reproduction of the RF values is influenced by changes in the silicone content and by differences in the curing process.
TABLE TI
~-
-
---
-
-RFValues----
Compound
ButanolAcetic AcidWater
ButanolBuffer ( P H 3.0)
Ergonovinc Ergometrinine Ergotonine Ergotiniue Ergotarnine Ergotarninine Lysergic acid Ergonovine maleate Ergotoxine ethdnesulfonate Ergotarnine tartrate
0.62 0.78 0.85 0.87 0.75 0.57 0.52 0.64 0.86 0.78
0.52 0.72 0.67 0.68 0.54 0.72 0.34 0.50 0.67 0.54
The methods fail in the separation of crgotoxine and its isomer. This pair, ergotoxine-ergotinine, is separable when chromatographed with the butanol p H 4.5 buffer system. By this procedure, the RF of ergotoxine is 0.60 and the RF of ergotinine is 0.69. The alkaloid bases do not tail but assume rather large arcas by these processes. The wash liquids do not dissolve paper constituents, and i t was noted that extracts of crgot chromatographed with, reniarkable clarity. Two-diniensional chroniatograms of a mixture of the levorotatory alkaloid bases and lyscrgic acid have bccn prepared. The KF values obtained on strips are duplicated on silicone-treated sheets when the first wash is the aqueous phase of the butanolacetic acid-water systcm. Equilibration of the paper with the butdnol phase is necessary before each formation.
REFERENCES (11 Foster, G. E., Macdonald, J., and Jooes, T. S. G.. J . Pharm. Pharmacol., 1, WZ(1949). (2) Carless, J. E., and Woodhead, H. B., Nature, 168, 203 (19.51). (3) Brindle, H., Carless, J. E., and Woodhead. H. B . , J. Phavm. Pharmacnl. 3 , 793(1951). 14) Zaffroni, A., &ton, R . , and Keutman, R . , J . R i d Chrm., 177 I09(1949). (5) Krit:hevsky, T. H., and Tiselius, A,. Science, 114, O C L C L ? 1 CL
c
,,
L:rJ( L J D I , .
(6) Jullander, I., Nnltcve 162 300(1948). ( 7 ) Partridge S. M., a d Sw$n. T.. ibid., 166, 2i2(196U). (8) M o c I l v a i ~ e T. , C.,J . B i d . Ckem., 49, 183(1921).