SU376719A1 - METHOD OF QUANTITATIVE DETERMINATION OF HORMONAL DRUG IN MEAT TISSUES - Google Patents

Description

one

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

There is a method for quantitative determination of hormonal preparations in meat tissues by extracting the preparation from tissues, chromatographic purification and subsequent spectrophotometric and densitometric studies.

However, the known method does not provide sufficient accuracy of the analysis.

To eliminate this drawback, in the proposed method, before the chromatographic purification, the preparation is redistributed in a mixture of 70% methanol and hexane, and the chromatographic purification is carried out by an adsorption method in three stages, the first of which is carried out on an alumina column, and the second and third thin layer of silica gel.

The extraction is carried out in three stages with chloroform at 45 ° C.

In addition, for the determination of the drug in offal, hydrolysis is carried out by the method of solvolysis.

The proposed method is carried out as follows.

Take 20 g of meat tissue (muscle, liver or kidney), crushed and thrice (40 ml at

45 ° C) is extracted with chloroform (100 ml, 50 ml and 50 ml of chloroform, respectively) with constant stirring on a magnetic stirrer. The chloroform extracts are combined and filtered through a paper filter. The filtrate is washed with 0.1 and. NaOH (1/10 volume) and 2-3 times with distilled water (1/10 volume) until neutral pH, dehydrated by filtration through Na2SO4 and dried in vacuum. The dry extract is dissolved in 30 ml of a 70% aqueous solution of methanol and vigorously shaken in a separatory funnel. S ml of hexane. Hexane is washed twice with an equal volume of 70% methanol. The combined methanol solutions are evaporated in vacuo at 45 ° C. The aqueous residue is extracted 3 times with a double volume of ethyl acetate. Ethyl acetate extracts are kept for 10-12 hours (2-4 ° C). bottom layer

discarded, the ethyl acetate is distilled off in vacuo. The dry residue is dissolved in 5 g of benzene and applied onto the alumina column (the adsorbent is standardized by the addition of 10% water); use columns with sizes 0.8-7.5 ate.

The sequence of elution: 10 ml of benzene (I fraction); 10 ml of a 0.5% ethanol / benzene solution (And fraction); 15 ml of a 1% solution of ethanol in benzene (P1 fraction); 10 ml of a 1.5% ethanol solution in benzene

(IV fraction). Methandrostenolone completely

elute with 10 ml of a 1.5% ethanol-benzene solution (IV fraction), this fraction is evaporated under vacuum at 45 ° C, and thin-layer chromatography is carried out on KSK-2 silica gel in one of the solvent systems hexane-ethyl acetate or cyclohexane-ethyl acetate (with standard and standard). The adsorbent layer is cut 1 cm above and below the spot of the standard. Obtained in the form of equal-sized rectangles are areas of the layer and two kontrolnye empty areas used for elution.

Elution was carried out with 15 ml of ethanol (30 min) and 15 ml of methanol (30 min) on a magnetic stirrer at 45 ° C. Then the adsorbent is transferred to a No. 4 glass filter and under a small vacuum, a mixture of ethanol and methanol (1: 1) is passed through until the filtrate has disappeared at K 245 mmk. The combined extracts are evaporated in vacuo, the precipitate is dissolved in 4 ml of methanol and the optical density is determined at I 245 mmk (1st quantitative determination).

After the spectrophotometric study, the methanol is evaporated in vacuo; The precipitate is dissolved in a minimum volume of 30% methanol in benzene and quantitatively applied to a Silufol plate. Various amounts of the standard drug methandrostenolone are also applied to the plate. Chromatography was carried out using a solvent system: cyclohexane-ethyl acetate (1: 1). For the manifestation of a steroid, a solution (3 ml per plate) containing concentrated sulfuric acid, glacial acetic acid, formaldehyde, and water in a ratio of 10.6: 5.0: 0.5: 84.5 is used.

A plate of a silufol is heated under the lamp for 40 s three times with a five minute interval. Then the plate is kept

30 seconds over the water bath to grow the background and warm up again.

Densitometry of the spots is carried out in clear: light using an ERJ 65 extinction recording instrument. The extinction curves extranolate according to the Gauss distribution curves and calculate the total cell concentration of each component according to the integral curve. The amount of methandrostenolan is calculated taking into account its percentage and the amount of standard applied (2nd quantitative determination).

In the determination of methandrosteiolone in by-products, prior to the purification of the preparation, the hydrolysis is carried out using the solvoln method.

Subject invention

Claims (3)

1. A method for quantitative determination of a hormonal drug in meat tissues, mainly methandrostenolone, by extracting the drug from the tissue, its chromatographic purification and subsequent spectrophotometric and densitometric studies, characterized in that, in order to improve the accuracy of the analysis, before the chromatographic purification of the drug redistribution in a mixture of 70% methanol and hexane, and chromatographic purification is carried out by the adsorption method in three stages, the first of which is carried out on Lonkila alumina, and the second and third - in a thin layer on silica gel. 2. A method according to claim 1, characterized in that the extraction is carried out in three stages with chloroform at 45 ° C. 3. The method according to claim 1, characterized in that to determine the drug in the by-products before cleaning the drug, it is hydrolyzed by the solvolysis method.