CS200720B1 - Mucidermin determination method - Google Patents

Description

The invention relates to a method for the determination of mucidermin, an antifungal antibiotic obtained from a production strain of Oudemansiella mucida by a fermentation process, in intermediate products or in the final substance. Chemically, muoidermine is 6-phenyl-3-methyl-2-methoxymethylene 3,5-hexanedioic acid methyl ester. Other substances with similar physico-chemical properties, eg muoidermine II, decomposition products, accompanying substances, etc. are still present in the mucidermin I preparation intermediates. Knowledge of the exact mucidermin content of the pure mucidermin preparation intermediates is important from a technological point of view, final dosage forms.

The mucidermin determination is carried out, in addition to the less accurate and time-consuming biological method (til% rel./), which determines only the sum of biologically active substances, by the spectrophotometric, densitometric method and the gas chromatography method. The method of direct spectrophotometry, without separation of ballasts, is practically only possible for pure substances. The sum of the substances absorbing at the measured maximum (mucidermin II, fission products, ballast substances, etc.) is actually determined for intermediates or poorly pure mucidermin I substances. The densitometry method, after thin layer chromatography, is feasible for all intermediates, but requires careful preparation of the chromatogram, use of a standard substance and, last but not least, expensive special equipment. The determination error at all conditions is up to 110% rel. Determination by gas chromatography is quantitative

200,720 by the destruction of mucidermin by high temperature (220 ° C) to 5-phenyl-2-methylbenzoic acid methyl ester. However, it is probably not possible to distinguish muoidermin I from mucidermin II by this method.

Recently, a simple, instrument-less method has been developed based on the separation of mucidermin I and II and their separation from ballast substances, accompanying metabolites, color pigments or decomposition products, by thin layer silica gel chromatography with UV 254 am fluorescence indicator, preferably Silufol UV 254, using a reliable 95: 5 benzene-ethyl acetate eluent, in detecting the z'n mucidermins below UV 254 nm, extracting them into an organic solvent, preferably methanol, separating the dispersed ailica gel, eg by filtration through glass wool, and measuring the absorbances of the thus obtained solutions on a suitable spectrophotometer at the maximum absorption of mucidermin I at 293 nm, against a solution prepared by the same sequence of operations from the same blank band area. Using the known mucidermin I extinction coefficient I, the mucidermin I or XI content is then calculated, preferably without the use of a standard, in percent.

Advantages of the assay method of the invention are the rapid separation of ballasts, fission products, color pigments and the like from muoidermins by thin layer chromatography, simple and efficient extraction into organic solvent (methanol) and rapid spectrophotometric evaluation of the solutions obtained, preferably without using standard mucidermin. Especially preferred is the use of finished films with a fluorescent indicator eilica gel (e.g.: Silufol UV 254 - silica gel with fluorine, UV 254 nm indicator on an aluminum support), as the detected and further processed zones can be simply cut from the chromatogram and Since the silica gel adheres very firmly to the aluminum support, the contamination of the extract obtained from these zones by the released silica gel particles is very low. The determination can be carried out not only for mucidermin I, but also equally accurately for mucidermin II or decomposition products, which can be separated by ohromatography in the final calculation calculated as muoidermin I.

Further details are given in the example:

The thin layer of silica gel (Silufol UV 254) is separated by grooves in the direction of development into strips 2.2 cm wide. The two starts of the treated film are then applied in 1.5 cm increments of 50 .mu.l of sample solution (sample amount corresponding to 40 mg of pure substance in 10 ml of 1: 1 isopropanol / methanol) to a further start of 50 .mu.l of the test substance solution. (40 mg of the verified mucidermin substance in 10 ml of 1: 1 isopropyl alcohol - methanol) and leave one strip free. It develops in a saturated chamber with a 95: 5 benzene-ethyl acetate mixture at a front distance of at least 18 cm. After development, the plate is dried in a stream of cold air (about 5 minutes - until solvents are removed). Below the UV 254 nm, the quenching bands of mucidermin X are then identical with the position and intensity with the main quenching band of the verified substance of Ry > 0.61, respectively the quenching bands of mucidermin II coinciding with the secondary quenching band of the verified substance of Rj, 0.44. The bands of mucidermin I and II are quickly marked with a pencil and scraped (in the case of Silufol with scissors) so that the areas of the same Rj, including the area from the free lane, are of equal size. The scraped silica gel was loaded quantitatively (cut out rectangles) into ground-glass flasks, added with 50 ml of methanol (for UV or pa), capped and shaken for 1/2 hour on a mechanical shaker. The solutions are then freed of dispersed particles of ailica gel in a suitable manner, preferably with a pipette having a tip coated with purified glass wool / 1 g of glass wool, boiled with 100 ml of methanol, decanted methanol, placed in a funnel and rinsed with another 100 ml of methanol. Vata se. it is then freed of methanol residues by extrusion and drying in an oven, taking a suitable quantity of the solution into a 1 cm quartz cuvette and measuring the absorbance against methanol as a blank test at 293 to 1 l with a suitable spectrophotometer. Mucidermin I content, rasp. mucidermine IX in the sample in percentedh, calculated as mucidermin I, is calculated using the extinction coefficient of mucidermin I, according to the formula% / mass / mass / * / - ^A / / ^{100 100} ,

100 ALIGN! n where A is the absorbance of the sample solution with mucidermin I / resp. with mucidermin II / - the average in ^{w of} two bands, ^ sl * ^ ^{e the} absorbance of a blank solution of the same R? as mucidermin I / resp. as mucidermin II /, n is the sample weight in grams a

100 is the extinction coefficient a of mucidermin I in methanol at 293 nm.

Claims (1)

subject of the invention Method for the determination of mucidermins, characterized in that ballast substances are separated on a thin layer of silica gel, mucidermin I and II are separated, mucidermin zones are extracted into methanol and, after filtration through clarified glass wool, spectrophotometric evaluation is carried out. respectively. of the mucidermin II in the sample, calculated as mucidermin I, is calculated using the extinction coefficient of mucidermin I, according to the formula And. dilution. 100% /w / w / ......... - -.....——— ’ 1 100. weighing (in g) where A is the absorbance of the mucidermin solution corrected for the absorbance of the blank with the same Rp and 1100 is the extinction coefficient a of mucidermin I in methanol at 293 nm.