METHOD FOR THE DETERMINATION OF CAFFEINE IN BIOLOGICAL MATERIAL Russian patent published in 2017 - IPC G01N33/50 

FIELD: biotechnology.

SUBSTANCE: biological material is treated with acetone, the liquid extraction is separated by filtration, evaporated in an air stream at room temperature, the aqueous residue is diluted with water, saturated with ammonium sulfate, adjusted to pH 4.2-4.5, extracted once with a mixture of organic solvents of ethyl acetate and chloroform, taken in a volume ratio of 2:8, the organic extract is separated, chromatographed and physicochemically determined by calculating the quantitative content of the analyte, and characterised in that the treatment for a biological object, acetone is carried out repeatedly twice for 30 minutes, is extracted under conditions when the volume of the aqueous phase is equal to the volume of the mixture of organic solvents, before chromatography, the solvents are evaporated from the extract to obtain a dry residue, the residue is dissolved in a mixture of propanol-2-chloroform solvents taken in the ratio 5:5 by volume, chromatographed in a column of silica gel L 40/100 mcm using a mobile phase of propanol-2-chloroform in a ratio of 5:5 by volume, the eluate fractions containing the analyzed the substance is combined, the eluent is evaporated in an air current at a temperature of 18-22°C, then in a nitrogen stream until all the solvents are removed, the residue is dissolved in dichloromethane, as a physicochemical method, chromatography-mass spectrometry is used, the determination is carried out in a 25 m capillary column and an internal diameter of 0.2 mm with a fixed phase of 5% phenylmethyl polysiloxane using a helium carrier gas fed at a rate of 0.7 ml/min and a mass selective detector operating in the electron impact mode, the initial temperature of the column thermostat is 80°C, this temperature is maintained for 1 minute, then the temperature is programmed first from 80°C to 200°C at a speed of 40°C per minute, then from 200°C to 300°C at a speed of 12.5°C per minute, the temperature of the injector is 200°C, the interface temperature of the detector is 300°C, the intensity of the signal due to the charged particles formed during bombardment of the analyte that emerged from the capillary column and got to the ion source by an ionizing beam of electrons with an energy of 70 eV, the mass spectrum is recorded in full the equation of the calibration curve is calculated by the least-squares method using the method of least squares, which in this case has the form: S=5238700⋅C+43091, where S is the area of the chromatographic peak, and C is the concentration of the substance to be determined in the chromatographed sample, mcg, and the amount of caffeine is calculated by the area of the chromatographic peak.

EFFECT: increased sensitivity of caffeine detection in biological material.

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