SU1693539A1 - Method of chromatographic analysis ob biologically active substances - Google Patents

Abstract

The invention relates to liquid chromatography and can be used in medicine and pharmaceuticals to conduct qualitative and quantitative analyzes of complex mixtures of biologically active natural compounds used as pharmaceuticals, mainly drugs. The purpose of the invention is to increase the reliability of the determination of narcotic substances in biological fluids during chemical and toxicological expertise. For this, chromatography of the analyzed mixture is carried out on silica gel plates and modified silica gel and used as a mobile phase for plates with a modified silica gel 1.0-1.5 M solution of sodium chloride in a mixture of CaHsOH-HaO 80:20 (determination of hashish) and a 0.1-0.3% solution of diethylamine in a mixture of C2HsOH-H20 with a volume ratio of 70:30 (determination of the alkaloids of the opi group and methadone). 3 hp ff, 3 tab. SO with

Description

The invention relates to the analysis of biologically active substances, in particular to the chromatographic analysis of drugs in a thin layer of sorbent, and can be used in control and analytical laboratories to determine the amount of complex multicomponent mixtures in order to reliably confirm positive results.

The purpose of the invention is to increase the selectivity and reliability of the results of chromatographic analysis of biologically active substances, mainly drugs, when they are determined in biological fluids.

The method is carried out as follows.

Samples of the analyzed substances are applied to a chromatographic plate with a prepared silica gel layer and onto a plate with a layer of modified silica gel, eluted with a mixture of organic solvents in a saturation chamber, the plates are inspected under UV light, treated with a color reagent, and the analytes are identified by Rf values, moreover, for the plates with unmodified silica gel, non-polar solvent systems are used, and for plates with modified silica gel, polar systems ethanol-water are used with a volume ratio of components nantov

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(70-80) :( 20-30) containing 1.0–1.5 M sodium chloride (when determining hashish or 0.1–0.3% of diethylamine when determining alkaloids of the opi group and methadone).

Example 1. 10 μl of an alcohol standard cannabinoid solution (hashish), samples of the analyzed substances are applied to the start line (1.5 cm from the edge) of the Silufol UV-254 plate (silica gel), samples of the analyzed substances, and elution is carried out with the ascending method in the petroleum system ether (40-70 ° С) - diethyl ether in a ratio of 80:20 components. At the end of the elution (the front of the eluent does not reach the edge of the plate 1 cm), the plate is removed from the chamber, dried in air for 5 minutes and spray dried with a 0.5% aqueous solution of strong blue B after drying. Red, orange and purple spots are observed on the plate on light brown background. The values of Rf for separable components of hashish are given in Table. 1. Then, 10 μl of a standard cannabinoid solution, samples of the same analytes of the samples and the plate are placed in a chamber containing 1M sodium chloride solution in a glass or Silufol-type plate with modified silica gel (silica gel Ca grafted onto silica gel). ethanol-water mixtures (80:20).

After chromatographic separation, the plate is removed from the chamber, dried in air for 20 minutes, and sprayed with a 0.5% solution of strong blue 5. Red, orange and purple spots are observed on the plate on a light brown background (Table 1).

A change in the order of yield of A9-tetrahydrocannabinol with psychotropic properties makes it possible to identify it with greater certainty.

EXAMPLE 2 A 10 µl dose of Silufol UV-254 (silica gel) plate is applied simultaneously with standard microchlorofom solutions of the opi alkaloids (morphine, codeine), and the test samples are chromatographed in an ethyl acetate – methanol –– system. ammonia (90: 10: 5). After chromatographic separation, the plate is removed from the chamber, dried in air and then viewed in UV light, the Rf spot value is measured and developed with Mark's reagent. Then, 10 µl of chloroform standard solutions of morphine and codeine and the same test samples are applied simultaneously to a glass or Silufol-type plate with a grafted alkylsilyl phase C2. Chromatograph in the system ethanol - water

(70:30) containing 0.1% vol. diethylamine. After chromatography, the plate is taken out of the chamber, dried in air for 20 minutes, and viewed in UV light, fixing the values of Rf, n, and showing Mark reagent. Data on the separation of the analyzed opiate alkaloids are presented in Table. 2

Example 3. Chloroform solutions of methadone are applied to the start line of a Silufol-type plate (silica gel), the studied samples are chromatographed in the toluene – acetone – ethanol – ammonia system (45: 45: 7.5: 7.5). After separation, the plate is removed from the chamber, dried in the air, viewed in

By UV light, the Rf spot values are measured and developed with a 5% ammonium vanadate solution. Then, 10 µl of methadone chloroform standard solutions and samples of the same test substances are applied to a glass or Silufol-type plate with a Cz grafted phase. Chromatographic in the system ethanol - water (70:30), containing 0.3% diethylamine. After chromatography, the plate is removed from the chamber, dried

exposed to air for 20 minutes, viewed under UV light, fixing Rf values, and exhibited with a 5% ammonium vanadate solution.

The results of the analysis are presented in

tab. 3

Claims (4)

1. Method of chromatographic analysis of biologically active substances, including the separation of samples of the analyzed substances on a chromatographic plate with silica gel and on a chromatographic plate with abrasive phase sorbent, processing of chromatograms with a color reagent and Identification of substances by their magnitudes of chromatographic mobility, characterized in that, in order to increase the reliability of the results of the analysis in the determination of drugs in biological samples, the separation of a sample of a substance on a plate with a reversed-phase sorbent is carried out using an ethanol-water solvent mixture as the mobile phase organic base or inorganic salt with an ethanol content in a mixture of 70-80%, and identification is carried out by comparing the values of chromatographic mobility uemogo Tilov substance on both plates. 2. A method according to claim 1, characterized in that, in order to increase the reliability of determining the main components of hashish, sodium chloride with a concentration of 1.0-1.5 M is used as an additive in the mixture. 3. A method according to claim 1, characterized in that diethylamine is used as an additive in order to increase the reliability of the determination of the opi and methadone alkaloids. 4. Method of pop, 1, characterized in that silica gel with C2 grafted phase is used as a reversed-phase sorbent. Table 1 table 2 Table 3