CS195151B1 - Method for the determination of hydrogenated alkaloids ergotoxine type ratio - Google Patents

Description

The invention relates to the determination of the ratio of hydrogenated alkaloids of the ergotoxin group / dihydroergocristine, dihydroergocryptine and dihydroergocornine / present in the form of salts or bases. Knowing the exact proportions of hydrogenated ergotoxin alkaloids in dihydroergotoxin salts or bases is important in terms of their effect and hence necessary in the control of intermediates and final products in their manufacture.

To determine the ratio of hydrogenated ergotoxin alkaloids, the chromatographic separation of the individual components by either paper, thin-layer chromatography is used. gas or liquid. The content of the separated alkaloids is evaluated according to the chromatographic method used, either by extracting the alkaloids from the paper with final photometric evaluation, densitometric evaluation from a thin layer, or evaluating the records from a liquid or gas chromatograph. These methods are characterized by some drawbacks, especially the methods used for separation of paper chromatography are very time consuming (1.5 to 2 working shifts), other methods are again very instrumental, moreover, the method of gas chromatography is problematic due to partial cleavage of alkaloids in the determination . Also, the preparation of a chromatogram for densitometric purposes requires special care and the whole result of the deposititometric determination is dependent on obtaining well-separated spots of the individual components in the chromatogram, which are not always sufficiently successful.

Recently, a simple method of instrument-less work has been developed, characterized in that the mixture of hydrogenated ergotoxin alkaloids is dissolved in chloroform-methanol 9: 1, applied to the starting lane of a thin layer of cellulose impregnated with dimethyl phthalate. system containing 0.05 N HCl, the chromatogram is preferably detected by drying with a 0.5X solution of p-dimethylaminobenzaldehyde in cyclohexane and exposed to hydrochloric acid vapors without drying, then quantitatively separating the alkaloid-separated zones, extracting from cellulose into an aqueous solution acetic acid, preferably 25X, color reaction by adding van Urkov Reagent, cellulose separation by filtration or centrifugation and color evaluation of the solutions obtained on a spectrophotometer *

As can be seen, the assay method of the invention is based on the chromatographic separation of the dihydroergotoxin components on a thin layer of cellulose impregnated with dimethyl phthalate, achieving the important advantage of good and reliable separation of alkaloids even under different chromatographic conditions / plate humidity, solvent quality, temperature, humidity air, chamber value, etc./. The use of an acidic eluent system, preferably 0.05 N HCl solution, allows high sample loading per chromatogram, is advantageous in terms of alkaloid stability, and provides preferred Rp values (dihydroergocristine 0.19, dihydroergocryptine 0.35 and dihydroergocornine 0.48). After chromatography, the chromatogram is preferably detected by drying with a solution of p-dimethylaminobenzaldehyde (0.5%) in cyclohexane and without exposure to hydrochloric acid vapor for the shortest time necessary without drying and removing the dimethyl phthalate from the plate. Extraction is performed on quantitatively scraped blue bands of alkaloids with an aqueous solution of acetic acid and a solution of p-dimethylaminobenzaldehyde in dilute sulfuric acid (Van Urka reagent).

After cellulose separation, preferably by centrifugation or filtration through a sinter G 4 and a coloring reaction (30 min.), The extinction of the solutions is measured on a suitable apparatus and the percentage of the individual components ^of dihydroergotoxin is calculated.

The advantages of the assay method according to the invention are the reliable separation of the individual components, the new rapid detection and extraction method, the inexpensive instrumentation and the high-quality results, and thus represent above all an improvement in the reliability of control of the various stages of production and final products, salts or bases. The assay can be performed with sufficient accuracy up to a content of 5% of any component (e.g. at a ratio of 50: 45 or 5 or 95: 5: 0 etc./. At less than 5 Z the assay is less accurate.

Further details follow from the following examples.

Examples

Example 1

Cellulose thin film / commercial products, e.g.,; Lucefol ^R Quick from Kavalier Sázava or DC-Karten CE Cellulose fUr DC from Riedel de Haun AG, Seelze-Hanover, Germany is impregnated by immersion in a 4% solution of dimethyl phthalate in chloroform and vented in a cold air stream for 5 minutes. 40 µl of a solution / 50 mg in 10 ml of chloroform-methanol 9 are then applied to the start in a 7 cm long strip; 1 /. Foresee 1 cm per start with 0,05 N hydrochloric acid, dry in a cold air stream and then evolve again with 0,05 N hydrochloric acid as an elution system in a saturated chamber to a distance of 12 cm. The plate is then dried in a stream of cold or warm air until visual drying / 10 min. 5 min), immersed in a 0.5% solution of p-dimethylaminobenzaldehyde in cyclohexane for 10 seconds and after brief drying (1 min), the plate is exposed in a suitable chamber to hydrochloric acid vapor until the individual alkaloid bands are visible. (The air flow is realized, for example, by a hair dryer placed above the chromatographic plate at a distance of 25 cm). Ergotoxin alkaloids have the following Ry values: dihydroergocristin 0.19, di-

Claims (1)

Method for determining the ratio of hydrogenated ergotoxin group alkaloids, characterized in that the hydrogenated ergotoxin alkaloid mixture is dissolved in chloroform-methanol 9: 1, applied to the starting lane of a thin layer of cellulose impregnated with dimethyl phthalate, chromatographic separation of the individual components in the acid system 05N HCl, the chromatogram is preferably detected without drying hydroergocryptine 0.35, dihydroergocornine 0.48. Single strips are quantitatively scraped into tubes, 2 ml of 25% acetic acid are added and after thoroughly shaking 4 ml of Urkova reagent (0.125 g of p-dimethylaminobenzaldehyde in 100 ml of 65% sulfuric acid with 0.1 ml of 5% ferric chloride solution). Again mix thoroughly and filter for 5 minutes through a sinter of G 4 or centrifuge for 30 minutes at 3000 rpm, clear solutions are measured in a suitable spectrophotometer at 580 nm in 1 cm cuvettes against a blank prepared from 30 min. 2 ml 25% acetic acid and 4 ml van Urk reagent. The weight percent calculation of the individual alkaloids is calculated according to the equation; KO =% KRX = KRY ₌ Li / + / Acres  Mkry / + • + / How · /Χο / = ^{χ a} kri - ^m krx . 100 ALIGN! > X AND , M .100 KRY KRY X F , M , 100 KO ' KO where: Acrylic, Aro are the extinction values of dihydroergocristine, dihydroergocryptine and dihydroergocornine. The crystals are the molecular weights of the salts or bases of dihydroergocristine, dihydroergocryptine and dihydroergocornine. The procedure is suitable for samples with a ratio of approximately 1: 1: 1. Example 2 The procedure is suitable for dihydroergotoxin samples in which a component predominates. The procedure is as in Example 1 except that β is added to the scraped strips of the predominant component with 4 ml of 25% acetic acid + 8 ml of van Urk reagent and to the remaining two (or one) strips / strips of 2 ml of 25% acid. + 4 ml van Urk reagent. When calculating the ratio, the predominant component must then be multiplied by two. OF THE INVENTION by eluting with a 0.5% solution of para-dimethylaminobenzaldehyde in cyclohexane and exposing to hydrochloric acid vapor, thereafter quantitatively separating the alkaloid-separated zones, extracting from cellulose into an aqueous acetic acid solution, preferably 25%. of cellulose by filtration or centrifugation and color evaluation of the obtained solutions on a spectrophotometer.