CS209335B1 - A method of dividing and determining the ratio of dihydro-alpha-ergocryptine and dihydro-beta-ergocryptine - Google Patents

Abstract

Method for separating and determining the ratio of dihydro-alpha-ergocryptine and dihydro-beta-ergocryptine. The invention solves the separation and determination of the ratio of dihydro-alpha-ergocryptine and dihydro-beta- -ergocryptine in a mixture of dihydroergocryptine and dihydroergocornine. The determination method is based on the separation of dihydro-alpha-ergocryptine, dihydro-beta- -ergocryptine, dihydroergocornine and dihydroergochristine by chromatography on a thin layer of silica gel Polygram SIL-HHR using double development with an elution system of water-diethylamine 100t4 and methanol-solution with a pH of 9 50j40, detection of the separated substances with a solution of p-dimethylaminobenzaldehyde and evaluation of the ratio of the intensity of the spots by densitometry

Description

(54) Method of separation and determination of the ratio of dihydro-alpha-ergocryptine and dihydro-beta-ergocryptine

Method for separating and determining the ratio of dihydro-alpha-ergocryptine and dihydro-beta-ergocryptine.

The invention solves the separation and determination of the ratio of dihydro-alpha-rergo cryptine and dihydro-beta-ergocryptine in a mixture with dihydroergocrlatin and dihydroergocornine.

The method is based on separating dihydro-alpha-ergocryptine, dihydro-beta-ergocryptine, dihydroergooornine and dihydroergocristine by thin-layer chromatography on Polygram SIL-HHR silica gel using two times the elution with water-diethylamine 100t4 and methanol solution at pH 9 50j40. separation of p-dimethylaminobenzaldehyde solution and densitometric evaluation of stain intensity ratio.

2Ο9 335

The invention relates to the separation and determination of the ratio of dihydro-alpha-ergocryptine and dihydro-beta ergocryptine in admixture with dihydroergocristine and dihydroergocornine (in dihydroergotoxin). According to the latest findings, it is important from the point of view of pharmacological effects that the exact ratio of the non-dihydroergocristine / dihydroergocornine and dihydroergocryptine, but also the precise ratio of dihydro-alpha-ergocryptine and dihydro-beta-ergocryptine is important. Therefore, a suitable method for monitoring this ratio is very important, both in the in-process operational control of intermediates and in the final adjustment of the desired ratio in the final product.

To date the ratio of dihydro-alpha-ergocryptine to dihydro-beta-ergocryptine has been limited to apparatus-intensive liquid chromatography. To date, dihydro-alpha-ergocryptine and dihydro-beta-ergocryptine and dihydroergocristine and dihydroergocornine have not been separated by other methods.

Recently, a simple method has been elaborated which consists in ‘separation of all four components of dihydroergotoxin by newly developed thin layer chromatography of silica gel, using new elution systems, in detection of solution of β-diiauthylaminobenzaldehyde with final densitometric evaluation.

As can be seen, the assay method of the present invention is based on the chromatographic separation of all four dihydroergotoxin components by silica gel thin layer chromatography. A prerequisite for successful separation is the use of choreography plates, POLYGRAM S1L-NHR by Macherey Nagel, Germany, because there is no separation on the other layers or the layers break apart. Using a 100: 4 water-diethylamine elution system, an excellent R 1 difference in the dihydro-alpha-ergocryptine, dihydro-beta-ergocryptine, and dihydroergocornine (Rj = 0.125 → 0.23; 0.31) was achieved. Dihydroergocristin has the same R _? such as dihydro-beta-ergocryptine. By using a second methanol-solution solution at pH 9 50:40, the dihydroergocristin is separated and thus all four ergotoxin alkaloids are separated. Separated alkaloids form round spots well densitometrically evaluable. The accompanying alkaloids of other groups or degradation products have a higher R _p and do not interfere with the assay. Disadvantages include the time of chromatographic separation, since separation by each system takes about 3 to 3 1/2 hours. Separation of all four alkaloids with less difference in Rf values compared to the two evolution can also be achieved using a single methanol-ammonium acetate solution of 0.1 M solution-diethylamine 20: 20: 1. However, due to smaller differences in R _p values, densitometric evaluation cannot be performed, but only a semi-quantitative visual estimate.

Thus, the method is primarily a new, simple method for identifying dihydroalpha-ergocryptine and dihydro-beta-ergocryptine and making available the identity and semiquantitative ratio of dihydro-alpha-ergocryptine and dihydro-beta-ergocryptine to all average laboratories. A densitometric evaluation of the ratio can be performed with sufficient accuracy up to a ratio of dihydro-alpha-ergocryptine: dihydro-beta-ergocryptine of 20: 1 or 1: 1. 1:20.

Further details resulting from the following examples:

Examples:

Example 1 p

Prepare such a solution of the test substance in chloroform-methanol 9: 1 so that, depending on the anticipated amount of dihydroergocryptin, there is at most 2 µg of each dihydroergocryptin in 5 µl. For example: for dihydroergotoxin mesylate with an alkaloid ratio of 1: 1: 1 and a dihydroergocryptine ratio of 2: 1 is <? 0.18% solution. Polygram SIL NH-R (Macherey Nagel, Germany) was then applied to the thin layer of silica gel from this solution at 3 starts at a distance of at least 2 cm, three times 5 µl. It develops in a naayetet chamber with a 100: 4 water-diethylamine elution system at a distance of 10-12 Cm. The plate was then vented in a cold air stream to dry and developed in a saturated chamber eluting with a methanol-solution of pH 9 / preparation: 1.24 g of boric acid was dissolved in 100 mL of 1 N NaOH and 1 N HCl was added until pH 9 / at a ratio of 50: 40 to a distance of 2 cm larger than the previous system (12 - 14 cm). After development, the chromatogram is dried in a stream of warm air until it is dry and sprayed with a detection reagent / 1% solution of p-dimethylaminobenzaldehyde in methanol-conc. HCl 1: 1). After spraying, warm air is applied to the plate immediately and, if necessary, the spraying and drying are repeated until optimal staining of the spots. Immediately after detection, the plate is exposed to UV radiation of 366 + 254 nm for 1 minute (e.g., performed in a Pluotest Universal from Hanau, Germany). Individual ergotoxin alkaloids have these R i values;

dihydroergocristine 0.31 dihydro-beta-ergocryptine 0.38 dihydro-alpha-ergocryptine 0.46 dihydroergocornine 0.58

The chromatogram is evaluated densitometrically. The following conditions apply to densitometers OPTON:

wavelength 580 mm shift plate - 50 mm / min recorder speed 30 cm / min slot width .0.02 mm aperture 10 mm amplification 17

Calculation of the ratio of dihydro-alpha-ergocryptine and dihydro-beta-ergocryptine is performed from the densitometric record according to the formula:

P alpha x = --- P beta where,: P alpha = mean area of dihydro-alpha-ergocryptine from densitometric records from three determinations *

P beta »mean value of the area of dihydro-beta-ergocryptine from densitometric intent from three determinations.

. (

Example 2:

The procedure is as in Example 1, except that a methanol-0.1 M ammonium acetate-diethylamine solution 20: 20: 1 is used as a developing system. Individual ergotoxin alkaloids have these Ry values and dihydroergocristin 0.21 dihydro-beta-ergocryptine 0.28 dihydro-alpha-ergocryptine 0.33 dihydroergocornine '0.39

It is not a density evaluation, but only a semi-quantitative visual estimate.

Example 3:. .

The procedure is suitable only for samples in which dihydroergocristin is not present.

The procedure is as in Example 1 except that the water-diethylamine 100: 4 mixture is used as the developing system. Individual ergotoxin alkaloids have the following values.Hg,:

dihydroergocristine 0.125 dihydro-beta-ergocryptine 0.125 dihydro-ralpha-ergocryptine 0.23 dihydroergocornin 0.31

Claims (3)

SCOPE OF THE INVENTION: > ··. Method for separating and determining the ratio of dihydro-alpha.ergocryptine and dihydro-beta-ergocryptine, characterized in that the mixture of hydrogenated ergotoxin alkaloids is dissolved, applied to a thin layer of Polygram SIL N-HR silica gel starting, separated into dihydroergocristine, dihydro-alpha -ergocryptine, dihydro-beta-ergocryptine and dihydroergocornine chromatographies using either two-fold elution with water-diethylamine 100: 4 and methanol solution pH 9 50:40, or with methanol-0.1% acetate solution . ammonium-diethylamine 20.20: 1, then detected with a solution of p-dimethylaminobenzaldehyde and the ratio of dihydro-alpha-ergocryptine and dihydro-beta-ergocryptine was evaluated. 2. Method according to claim 1, characterized in that the evaluation is performed by densitometry. 3. The method of claim 1, wherein the evaluation is performed visually.