Estimation of sulphanilamide in biological fluids

Summary

1
A study has been made of the application of paper partition chromatography in the ergot field.

2
A technique for the separation and identification of ergometrine and ergometrinine, when present in mixtures of total ergot alkaloids, has been described.

3
The method has been extended so that approximately quantitative results may be obtained and, in this way, the ergometrine contents of samples of ergot have been estimated.

4
The changes which take place in injection of ergometrine maleate B.P. 1948, during manufacture and storage have been studied chromatographically. It was found that during heat sterilisation some conversion of ergometrine to ergometrinine occurs. On storage some hydrolysis giving rise to lysergic and iso-lysergic acids takes place.

5
By preparing chromatograms from the hydrolysates of water-insoluble ergot alkaloids, identification tests for individual alkaloids have been developed.

We wish to thank Mr. H. M. Hood, B. Sc., for the photograph of a chromatogram, Mr. R. L. Grant, M. Sc., for a sample of ergometrinine and Dr. S. Smith for specimens of lysergic and iso-lysergric acids. We are also indebted to the Directors of The Wellcome Foundation for permission to publish this paper.

The separation and identification of ergot alkaloids by paper partition chromatography.

REFERENCES to the separation of alkaloids on columns by partition chromatography have been made by Catch et d.' and by Partridge et aI.a*3. The theory and use of buffered columns have been previously desc~ibed*~~~~. In the separation of the penicillins, buffered filter paper has been used to obtain suitable paper chromatograms' while the difficulty of obtaining absolute RF values for such separations has been noted*. The separation of water-soluble alkaloids of ergot has been achieved by paper chromatography using unbuffered paperg. Conditions for the separation of certain alkaloids and mixtures of alkaloids on unbuffered paper have also been describedlO. The present writers have successfully used buffered filter paper for the separation of certain of the strongly basic solanaceous alkaloids and of the feebly basic water-insoluble alkaloids of ergot. The method of separation on buffered paper should prove to be a general method for the separation of micro amounts of mixed acids or bases, provided the substances do not dimerise in either phase. Separations are only to be expected where the dissociation constants and /or partition coefficients differ from each other.

The separation of alkaloids by partition paper chromatography

THE separation of certain water-insoluble alkaloids by partition paper chromatography has been described in a previous paper in this series1, and this present communication describes an extension of the method to determine quantitatively both the water-insoluble and water-soluble alkaloids of ergot in the crude drug. Up to the present time there have been no assay methods available for the determination of the individual alkaloids of ergot, with the exception of ergometrine which is referred to later. The physiologically active and inactive alkaloids and also some deterioration products such as the lysergic acids give the same colour with p-dimethylaminobenzaldehyde reagent used for the colorimetric assay in the British Pharmacopaeia, 1948. This may be satisfactory for the estimation of total water-soluble and waterinsoluble alkaloids, but does not give a true indication of the physiological activity. The object of this present paper was to show how the active and inactive alkaloids may be separated from each other by partitition chromatography and then assayed individually. This is, of course, the ideal case and although some difficulties still remain, the author claims that the method in this paper is a considerable advance on previous assay methods. Ergots from different sources vary greatly in their alkaloidal content-both qualitatively and quantitatively ; for instance Spanish and Russian ergots contain the ergotoxine group of alkaloids together with ergometrine, whilst Central European ergots are often rich in ergotamine2. The author’s method of assay determines these individual alkaloids and also ergometrine and should be of importance to the manufacturer who wishes to purchase ergot for the preparation of ergotamine or ergometrine. Chemical assay methods which have been devised for the physiologically active alkaloids in ergot have been limited to ergometrine, which is perhaps the most important ergot alkaloid. Grove3 and Powell et aZ4 devised methods by which the ergometrine was extracted and determined directly by colorimetric assay. Further collaborative work in the United States extending over several years has been published in a report on the assay of ergot issued by the American National Formulary Committee5. It is interesting to note that the specific assay for ergometrine devised by the American workers was finally not adopted for ergot itself owing to its complexity, and thus the final assay of the crude drug determines ergometrine together with ergometrinine. Foster et aP, separated ergometrine in ergot-by paper chromatography and obtained semi-quantitative results, the error being approximately 20 per cent. Ergotamine has been separated

The separation of alkaloids by paper partition chromatography. III. The assay of ergot.

Asa N. Stevens

Papers

The Biologic and Colorimetric Assay of Ergonovine in Ergot and its Fluid Extract

The Standardization of Ergot—A Modification of Smith's Quantitative Colorimetric Assay * , 1

Para‐aminobenzenesulfonamide *: Notes on the colorimetric assay

VIII. The standardization and stabilization of ergot preparations

VIII. The standardization and stabilization of ergot preparations**Scientific Section, A. Ph. A., Miami meeting, 1931.