METHOD OF DETERMINING HYDROXYBENZENE AND ITS MONOMETHYL-SUBSTITUTED IN BIOLOGICAL MATERIAL Russian patent published in 2015 - IPC G01N33/50 G01N30/02 

FIELD: medicine.

SUBSTANCE: biological object, which contains mixture of hydroxybenzene and its monomethyl-substituted are crashed, processed twice with ethylacetate for 30 minutes with ethylacetate, ethylacetate extracts are combined, treated with ethanol solution of calcium hydroxide, solvent from combined ethylacetate extract is evaporated at 16-20°C, residue is repeatedly treated with acetone, containing hydrochloric acid in excess with respect to potassium hydroxide, present of residue, acidified acetone extracts are combined, treated with water solution of sodium hydroxide to neutralise residues of hydrochloric acid in acetone and create excess of alkaline medium, acetone is evaporated from combined extract, water-alkali residue is diluted with water, formed solution is acidified to pH 2-3, saturated with sodium sulphate, extracted with diethyl ether, extract is dehydrated, evaporated, chromatographed in column with silica gel with application of mobile phase hexane-diethyl ether with ratio 6:4 in volume, eluate fractions, containing hydroxybenzene and its monomethyl substituted, are combined, extracted with buffer solution with pH 12-13, water-alkali extract is acidified with 24% solution of hydrochloric acid to pH 2-3, saturated with sodium sulphate, extracted with dichloromethane, dichloromethane extract is dehydrated, analysed substances, contained in dichloromethane extract, are transferred into corresponding trimethylsilyl derivatives, for which purpose dichloromethane extract is treated for 20 minutes with N-methyl-N-trimethylsilyltrifluoracetamide under conditions of heating at temperature 60°C, qualitative and quantitative determination by physical-chemical method, such as chromate-mass-spectrometry, is performed in capillary column, 25 m long with internal diameter 0.2 mm with immobile phase (5%-phenyl)-methylpolysiloxane, with application of helium carrier gas, supplied at rate 0.6 ml/min, and mass-selective detector, working in electron impact mode, initial temperature of column thermostat constitutes 70°C, said temperature is maintained for 3 minutes, and then temperature is programmed from 70°C to 290°C at rate 20°C, final temperature of column is maintained for 10 minutes, injector temperature constitutes 250°C, quadrupole temperature - 150°C, temperature of ion source - 230°C, temperature of detector interface - 300°C, intensity of signal, conditioned by charged particles, formed in bombardment of analysed substance, leaving capillary column and getting into source of ions, with ionising beam of electrons with energy 70 eV, is registered, mass-spectrum is registered by full ion flow, qualitative determination of analysed substance is realised by time of exposure, set and intensity of signals of characteristic charged particles in mass-spectrum of its trimethylsilyl derivative, with quantity of determined compound being calculated by area of chromatographic peak of its trimethylsilyl derivative.

EFFECT: increasing sensitivity and reduction of determination duration.

6 tbl, 1 dwg, 5 ex