The method of determination of avermectins

 

(57) Abstract:

The use of biotechnology. The essence of the invention: method of determination of avermectins, based on the extraction of the investigated sample with an organic solvent, the dilution of the obtained extract by the method of limiting dilutions and measure the optical density of the resulting solution at a wavelength of 244-243 nm. table 1. 1 Il.

The invention relates to biotechnology and concerns the control of the production and use of avermectins, which are used in animal husbandry, agriculture and medicine for combating endo - and ectoparasite animals, plants and humans.

Known methods of determination of avermectins, based on biological activity and embryo death, in particular, he nematodes activity [1] This method is extremely tedious, time-consuming and unsuitable for the rapid analysis of avermectins.

It is also known use for the determination of avermectins method of thin-layer chromatography [2] fluorescent method [3] and HPLC method [4] the Latter is the most accurate and faster method, but requires expensive equipment.

Closest to the present invention is a method of determining that to a solution of the sample in an organic solvent, add a solution of orcine reagent in n-butanol, the mixture is heated for 15 min at 80oC and measure the optical density of the obtained colored solution at 630-640 nm. However, this method has several stages of sample preparation, requires expensive reagents and obtained reproducible results are difficult.

The aim of the invention is the simplification and acceleration of the method of determination of avermectins and improve its reproducibility.

The objective is achieved by the fact that the avermectins extracted from the analyzed sample with an organic solvent, the extract obtained is diluted by the method of limiting dilutions and measure the optical density of the obtained solution at 244-248 nm. The selected wavelengths correspond to the maximum absorption of avermectins. In the method can be used those solvents which, on the one hand, well-extracted with avermectins, and on the other, do not interfere with its spectrophotometric determination in the above mentioned wavelengths. Such solvents include, for example, acetonitrile, toluene, chloroform, ethanol, butanol, methanol, having a minimum optical density in the area 240-250 nm.

Method lim is D. while receiving a number of solutions with decreasing concentration.

The use of such serial dilutions allows you to achieve reproducible results when measuring the optical density, while other methods of dilution of the extract to a concentration suitable for UV studies give poorly reproducible results.

The optical density is measured, as a rule, for multiple consecutive dilutions (at least three). The concentration of avermectins in a sample is calculated by a calibration curve for building which is used as ivermectin or ivermectin.

Example. The calibration curve.

To construct the calibration curve, use a solution of ivermectin firm Merck (Germany) containing ivermectin 5 mg/ml Of him prepare a series of dilutions in ethanol to obtain solutions with concentrations from 10 to 40 g/cm3. The density measurement is carried out at a wavelength of 246 nm against control ethanol solution in a quartz cuvette with a long face 1 see

A calibration curve is constructed on the basis of four parallel dimensions given on the drawing.

Definition conosle 100; 140; 168 h of cultivation centrifuged 15 min at a speed of 8000 rpm

The supernatant liquid is poured, to the precipitate add 5 cm3distilled water and mix. After cooling of the solution for 10 min in a refrigerator at a temperature of 5 0,5oC, the precipitate was separated in a centrifuge (15 min at 8000 rpm) the Supernatant is poured, the precipitate is poured 5 cm3ethanol for the extraction of avermectins. The extraction was carried out with stirring on a magnetic stirrer for 20 min, the Extract is separated in a centrifuge (15 min at 8000 rpm). The precipitate discarded. From the obtained extract is taken 1 cm3transferred to a clean test tube and dissolve up to 5 cm3ethyl alcohol (breeding 5), then the newly prepared solution taken 1 cm3again transferred into a clean test tube and again diluted to 5 cm3(breeding 25). Similarly, perform the following breeding, thus obtaining solutions with dilution 125, 625 times.

Measurement of the optical density of the obtained solutions is performed on the spectrophotometer Jas. co UVIDEC-610 at a wavelength of 246 nm against a control solution of ethyl alcohol.

The results obtained for the three parallel measurements prio optical density range of 0.3 to 1.6. Via a calibration curve to determine the concentration of the standard and the average value of the concentration of the standard multiplied by the ratio of breeding and by a factor of K=0,35, calculated on the basis of the control definition concentration of avermectins parallel to the claimed method and HPLC method.

The necessity of introducing this factor is related to the fact that as a standard for calibration use a solution of embryo death.

Comparing values of concentrations and embryo death, obtained by the claimed method and the HPLC method shows that the proposed method allows to determine the concentration and embryo death quickly and without the use of special equipment and reagents.

The method of determination of avermectins, including extraction of avermectins organic solvent and measuring the optical density, characterized in that the extract is diluted by the method of limiting dilutions and measure the optical density of the obtained solution at 244 248 nm.

 

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SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

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15 cl, 4 ex

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