METHOD OF DETERMINING NOVOCAINE IN BIOLOGICAL MATERIAL Russian patent published in 2015 - IPC A61K31/245 G01N33/50 G01N30/02 

FIELD: biotechnology.

SUBSTANCE: method is implemented as follows: the biological material containing novocaine is crushed, treated three times with acetone, containing 0.2-0.4% water, the liquid extract is separated, the acetone from the liquid extract is evaporated together with water in the air stream at a temperature of 18-22°C until complete removal of the solvent, the residue obtained as a result of evaporation is repeatedly treated with acetone containing 0.2-0.4% water, the acetone extract is separated, combined, and the solvent from the combined extract is evaporated, the residue is dissolved in diethyl ether, the resulting solution is diluted in hexane in a ratio of 1:1 by volume, extracted with buffer solution with pH 1-2, the acid-water extract is separated, neutralized with 10% ammonia solution, alkalized with ammonium buffer solution to pH 9.0-9.5, the resulting aqueous alkaline solution is saturated with ammonium sulphate, extracted twice with portions of the organic extractant which is used as a 30% solution of camphor in methyl acetate, at a ratio of aqueous and organic phases as 1:1 by volume, the organic extracts are separated, combined, and the solvent from the combined extract is evaporated in a stream of air at a temperature of 18-22°C to dry residue, the residue is dissolved in a mixture of dichloromethane and ethanol taken in a volume ratio of 1:1, the resulting solution is applied to a column of silica gel CRA No. 80/120 mcm, chromatographed using two-phase movable phase dichloromethane-ethanol in a ratio of 1:1 by volume, the fractions of eluate containing the analyte, combined, the eluent is evaporated, the residue is dissolved in methanol and determining is performed by gas-liquid chromatography coupled with mass spectrometric detection in a capillary column DB-5 MS EVIDEX with the length of 25 m and an inner diameter of 0.2 mm with the stationary phase, which is 5%-phenyl-95%-methylpolysiloxane using helium carrier gas supplied at a rate of 0.6 ml/min and a mass selective detector operating in electron impact mode, the initial temperature of the column oven is 80°C, this temperature is maintained for 1 minute, then the temperature is raised from 80°C to 200°C with the rate of 40°C per minute, then from 200°C to 300°C with the rate of 12.5°C per minute, the final column temperature is maintained for 16 minutes, the temperature of the injector is 200°C, the temperature of quadrupole is 150°C, the temperature of the detector interface is 300°C, the intensity of the signal is recorded, due to charged particles produced by bombarding of the analyte emerged from the capillary column and entered into the ion source, with ionizing electron beam with energy of 70 eV, a mass spectrum is recorded on total ion current and the amount of novocaine is calculated based on the chromatogram peak area.

EFFECT: increased sensitivity of determining.

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