METHOD OF DETERMINING THE CONTENT OF HELLEBORE LOBEL ALKALOIDS IN MEDICINES (VERSIONS) Russian patent published in 2023 - IPC A61K31/395 A61K36/88 G01N30/00 G01N30/90 G01N33/15 

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions relates to methods of qualitative determination of steroidal alkaloids of hellebore Lobel by high-performance thin-layer chromatography. A method of qualitative determination of the content of hellebore Lobel alkaloids in a medicinal product, including the stage of sample preparation, namely the preparation of the test solution of the medicinal product — hellebore Lobel rhizomes with roots in the following way: 1.0 g hellebore Lobel rhizomes with roots are crushed to the size of particles passing through a sieve with 1 mm holes, placed in a 100 ml flask, 10 ml of 96% alcohol are added and heated on a water bath for 15 minutes, cooled and filtered, 10 ml of water, 1 ml of concentrated 25% ammonia solution are added and extracted with ether twice in 15 ml, the ether extracts are combined and filtered into a 100 ml round-bottom flask through a paper filter pre-moistened with ether containing 2 g of anhydrous sodium sulfate, the filter is washed with 10 ml of ether, the ether is distilled off on a rotary evaporator at a temperature not exceeding 40°C until dry, the dry residue is dissolved in 2.0 ml of 96% alcohol, then the test solution is chromatographed by the ascending high-performance thin-layer chromatography method as follows: 10 μl of the test solution is applied to the start line of the HPTLC plate with a layer of silica gel in the form of a strip 10 mm long and not more than 2 mm wide, then the plate is placed in a chamber, previously saturated for 1 hour with a mixture of solvents butanol: acetic acid: water 4:1:1, when the solvent front passes 80–90% of the length of the plate from the start line, it is removed and dried until removed traces of solvents are examined under UV light at a wavelength of 365 nm, then the chromatographic plate is treated with a modified Dragendorff reagent and viewed in daylight in a UV cabinet, the content of hellebore Lobel alkaloids in the medicinal plant raw material of hellebore Lobel rhizomes with roots is determined by detecting on the chromatogram of the test subject solution in the middle third of the plate at least three adsorption zones of orange to red-orange colour. A method of qualitative determination of the content of hellebore Lobel alkaloids in a medicinal product, including the stage of sample preparation, namely the preparation of the test solution of the medicinal product — the pharmaceutical substance "Hellebore tincture" as follows: 25 ml of "Hellenic hellebore tincture" are placed in a 250 ml separating funnel, 25 ml of water and 1 ml of concentrated 25% ammonia solution are added and extracted with ether twice in 15 ml, the ether extracts are combined and filtered into a 100 ml round-bottom flask through a paper filter pre-moistened with ether containing 2 g of anhydrous sodium sulfate, the filter is washed with 10 ml of ether, ether distilled on a rotary evaporator at a temperature not exceeding 40°C until dry, the dry residue is dissolved in 2.0 ml of 96% alcohol, then the test solution is chromatographed by the ascending high-performance thin layer chromatography method as follows: 5 μl of the test solution is applied to the start line of the HPTLC plate with a layer of silica gel in the form of a strip 10 mm long and not more than 2 mm wide, then the plate is placed in a chamber, previously saturated for 1 hour with a mixture of solvents butanol: acetic acid: water 4:1:1, when the solvent front passes 80–90% of the length of the plate from the start line, it is removed and dried until removed traces of solvents are examined under UV light at a wavelength of 365 nm, then the chromatographic plate is treated with a modified Dragendorff's reagent and viewed in daylight in a UV cabinet, the content of hellebore Lobel alkaloids is determined by detecting at least three zones on the chromatogram of the test solution in the middle third of the plate adsorption of orange to red-orange colour. A method of qualitative determination of the content of hellebore Lobel alkaloids in a medicinal product, including the stage of sample preparation, namely the preparation of the test solution of the medicinal product — the medicinal product “Hellenic water” in the following way: 25 ml of “Hellenic water” is placed in a 250 ml separating funnel, 25 ml are added water, 1 ml of concentrated 25% ammonia solution and extracted with ether twice in 15 ml, the ether extracts are combined and filtered into a 100 ml round-bottom flask through a paper filter pre-moistened with ether containing 2 g of anhydrous sodium sulfate, the filter is washed with 10 ml of ether, ether distilled on a rotary evaporator at a temperature not exceeding 40°C until dry, the dry residue is dissolved in 2.0 ml of 96% alcohol, then the test solution is chromatographed in an ascending high-performance thin-layer chromatography method as follows: 10 μl of the test solution is applied to the HPTLC start line — a plate with a layer of silica gel in the form of a strip 10 mm long and not more than 2 mm wide, then the plate is placed in a chamber, previously saturated for 1 hour with a mixture of solvents butanol: acetic acid: water 4:1:1, when the solvent front passes 80–90% of the length of the plate from the start line, it is removed and dried until removed traces of solvents are examined under UV light at a wavelength of 365 nm, then the chromatographic plate is treated with a modified Dragendorff's reagent and viewed in daylight in a UV cabinet, the content of hellebore Lobel alkaloids is determined by detecting at least three zones on the chromatogram of the test solution in the middle third of the plate adsorption of orange to red-orange colour.

EFFECT: above methods expand the arsenal for determining the content of steroidal alkaloids hellebore Lobel in medicines, the methods are highly specific.

3 cl, 3 dwg, 1 tbl, 2 ex