METHOD FOR DETERMINATION OF 2,6-BIS-[BIS-(BETA-OXYETHYL)-AMINO]-4,8-DI-N-PIPERIDINO-PYRIMIDO(5,4-D)PYRIMIDINE IN BIOLOGICAL MATERIAL Russian patent published in 2017 - IPC G01N30/00 

FIELD: biotechnology.

SUBSTANCE: invention relates to biology and toxicologic chemistry, namely to methods for determination of 2,6-bis-[bis-(beta-oxyethyl)-amino]-4,8-di-N-piperidino-pyrimido(5,4-d)pyrimidine in a biological material, and can be used in the practice of sanitary hygienic service, chemical-toxicological, criminalistic and veterinary laboratories. The method is implemented as follows. A biological material containing 2,6-bis [bis-(β-oxyethyl)-amino]-4,8-di-N-piperidino-pyrimido(5,4-d)pyrimidine is twice infused with acetone, each time within 30 minutes. The obtained extracts are combined. The combined extract is filtered. Then the filtrate is evaporated to obtain solids. The residue is repeatedly treated with acetone. The acetone extracts are separated and united. Then the solvent from the united extraction is evaporated. The residue is dissolved in chloroform, 0.1 N is extracted by a hydrochloric acid solution. The chloroform layer is removed and the resulting acid extract is washed by an organic solvent, which is a mixture of ether and hexane at a ratio of 1:1 by volume. The layer of organic solvent is removed and the aqueous layer is alkalized by 10% solution of sodium hydroxide to reach a pH value of 8-10 and saturated by sodium chloride. Then it is extracted with ethyl acetate. The resulting extraction is separated and dehydrated. The extractant is evaporated and the residue is dissolved in a mixture of acetonitrile and 1 N sulfuric acid solution at a volume ratio of 9:1, chromatographed in a macro column with the sorbent Silasorb C-18 having a particle size of 15 mcm by using a mobile phase consisting of acetonitrile and 1 N sulphuric acid solution in a volume ratio of 9:1. The eluate fractions, containing the test substance, are united and the test substance is determined by a physico-chemical method that the HPLC is. Then the eluate is chromatographed in a column of sorbent Zorbax SB C8 which is thermostatically controlled at 40°C using the mobile phase of acetonitrile - 0.025 M potassium-dihydrogen-phosphate solution in a volume ratio of 3:2 by volume and a detector based on photodiode array.

EFFECT: increased sensitivity.

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