SU438928A1 - Method for quantitative determination of hormonal drug in meat tissues - Google Patents

Description

one

The invention relates to meat industry, namely to a method for quantitative determination of diethylstilbestrol in meat and by-products.

There is a method for quantitative determination of hormonal preparations in meat tissues by extracting the preparation from tissues, hydrolysis, its redistribution in solvents, chromatographic purification and subsequent densitometric study.

However, this method does not provide sufficient sensitivity and reliability of the analysis.

To eliminate this drawback in the proposed method, the extraction is carried out in 2 stages: in the field of centrifugal forces and on the adsorption column, the hydrolysis is carried out in a solution of inorganic acid with the addition of a solvent, and the chromatographic purification is carried out sequentially by distribution and adsorption methods.

The proposed method is carried out as follows.

Three samples (250 g each) of meat tissue (muscle, liver, or kidney) are taken. Extraction, hydrolysis and the first stage of purification in the CHCI3-H20-Ma2CO3-NaOH system are carried out for each of the three samples separately. Thoroughly shredded tissue is extracted twice in

centrifugal extractor with 96% ethanol (500 ml). After filtering through a paper filter, the fabric is mixed with an equal amount of celite - 545 by weight, transferred to

extraction column (0.8X6.0 cm) and again extracted with 96% ethanol (1000 ml per 250 g of tissue). To 1500 ml of the combined ethanol extract of each sample, 50 ml of 2N are added. HC1, evaporated to a volume of 100-

150 ml and cleaning in the system chloroform-water-sodium carbonate is sodium hydroxide. 100 ml of chloroform and 300 ml of water are added to the hydrolyzate. After shaking and separating the layers, the chloroform phase is transferred to a separatory funnel and washed with 100 ml of water. Extraction with chloroform and washing it is repeated twice more (50 ml of chloroform each). The aqueous phases are discarded. The chloroform phases are combined and their

further processing is successively carried out in three separatory funnels. The first funnel contains 20 ml of 10% Na2COz, the second and third funnels contain 40 ml of 1% NaOH. After shaking and phase separation

CHCl3-layer and Na2CO3 layers are discarded. The combined NaOn layers are acidified with 2N. HC1 and extracted three times with 30 ml of chloroform. In the presence of yellow color of the final chloroform extracts processing in the system

Ma2CO3-NaOH is repeated.

Subsequently, extracts of three samples (corresponding to tissue) are combined and evaporated in vacuo. The dry residue is dissolved in 30 ml of 70% methanol and vigorously shaken in a separatory funnel with 8 ml of hexane. Hexane is washed twice with an equal volume of 70% methanol and discarded. The methanol extracts are combined and evaporated in vacuo; aqueous residue after acidification with 2N. HC1 is extracted twice with chloroform (30 ml each). The aqueous phase is discarded. Chloroform is dehydrated, evaporated to a volume of 1-2 ml, and silica gel L (0.6x8.0 cm) is applied to the column. Elution is carried out

(0.5 ml / min) 20 ml of chloroform (fraction 1, not containing diethylstilbestrol) and 30 ml of 1% ethanol in chloroform (fraction 2, containing diethylstilbestrol). Fraction 2 is evaporated on a rotary evaporator to a minimum volume and applied on a silufol plate. Chromatographic in the solvent system ether-benzene (i: 4). The plate of Silucol is irradiated under UV light for 30 minutes, the chromatogram band (4X15 cm) is cut with the spots of the specific product of the photochemical reaction - 3,4,5,6,12,13 hexahydro-3, 6-dioxo - 9,10 - diethylphenanthrene; densitometry in reflected light on an extinction-recording device.

In the absence of conditions for densitometry, quantitative determination after paper chromatography (in the benzene-methanol-water system, 5: 7: 3) or in a thin layer of silica gel (ethanol-benzene, 1: 9) is carried out by spectrophotometric or fluorometric methods. Spectrophotometrically (with I

910 nm) (after UV irradiation in a mixture of 96% ethanol - 1.8% K2HP04, 1: 1) the yellow-colored product of the photochemical reaction; 3,6-dihydroxy-9,10-diethylphenanthrene (fluorescence excitation at K = 360 nm, measurement at, 400 nm) is determined fluorometrically.

Subject invention

Claims (3)

1. A method for quantifying a hormonal drug in meat tissues, such as diethylstilbestrol, by extracting the preparation from tissue, hydrolysis, redistribution in solvents, chromatographic purification and subsequent densitometric studies, characterized in that, in order to increase the sensitivity and reliability of the analysis, extraction carried out in two stages: first in the field of centrifugal forces, and then using adsorption, the hydrolysis is carried out in a solution of an inorganic acid with the addition of a solvent, and chromium ograficheskuyu cleaning are sequentially distribution and adsorption methods. 2. A method according to claim I, characterized in that the redistribution of the hydrolyzate is carried out sequentially in two solvent systems: first in the chloroform-water-carbonate sodium hydrate sodium oxide system, and then in the methanol-hexane-chloroform system. 3. The method according to claim 1, wherein the densitometric study carried out using a photochemical reaction.