RU2379686C2 - Method of biological fluid analysis for concentration of benzylpenicillin preparations - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method refers to medicine, namely to microbiological methods of biological material analysis, and can be used in laboratory practice to detect antibiotics of penicillin group in substrata and studying of their pharmacokinetics within therapy. The biological fluid analysis for concentration of benzylpenicillin preparations is ensured by using the microbiological agar diffusion method involving preparing of a test microbe preparation, creating a uniform agar layer of thickness 1-2 mm, dimpling in solidified agar of diametre 2 mm. The standard solutions concentrated 0.5; 1.5; 3; 4; 5; 6 mcg/ml and preliminary dissolved test samples are introduced therein. As a test microbe, Staphylococcus aureus ATCC 25923 is used in number 3·10⁶ microbial bodies per ml of the medium. The standard sample values are used for plotting a calibration curve on the semilogarithm paper. Said curve expresses a dependence of a dose and growth inhibition area diametre and used to determine concentration of the test samples.

EFFECT: lower labour intensiveness and materials consumption of the method.

Description

The invention relates to medicine, namely to microbiological methods for studying biological material, and can be used in laboratory practice to detect penicillin group antibiotics in substrates and study their pharmacokinetics during therapy.

Known methods for determining the concentration of benzylpenicillin preparations using paper chromatography methods (Alekseev V.G., Nersesova A.F., Khalyapina Y.M. Determination of benzylpenicillin, ampicillin and carbenicillin by paper chromatography. // Journal of Applied Chemistry, 2005, v. 48 , No. 4, p.613-615) and ionometry (Granzhan A.V., Charykov A.K. Use of ion-selective electrodes in pharmaceutical analysis (review). // Chemical-Pharmaceutical Journal, 1993, v.27, No. 7, p. 51-56). However, the implementation of these methods requires special expensive equipment, the acquisition of which is not practical for solving private research problems.

Closest to the claimed one is a method for determining the concentration of benzylpenicillin in biological fluids and tissues by microbiological diffusion into agar using one layer of a nutrient medium (Navashin S.M., Fomina I.P. "Rational antibiotic therapy (reference book)." - 4th ed. ., rev. and add. - M .: Medicine, 1982, p. 56-72).

The method is based on comparing the degree of inhibition of growth of the test microbe with certain concentrations of the antibiotic in the test material with the inhibition of its growth with known concentrations of the standard antibiotic.

The growth of the test microbe is suppressed by diffusion of the antibiotic from the test material into a solid nutrient medium (agar). The test material is introduced into the holes cut in the thickness of the agar. To obtain reproducible results, strict standardization of the diffusion process conditions is necessary.

In the prototype, as a test microbe for the study of drugs of the penicillin group, Staphylococcus aureus strain 209 P in a seed dose of 40 · 10 ⁶ microbial bodies per ml of medium is proposed, and the agar layer is formed by adding 10 ml of molten agar medium to a Petri dish mounted on a horizontal spirit level adjusted surface. After solidification of the agar, six holes with a diameter of 8 mm are cut out in its layer.

With this method, there are factors that violate the conditions for strict standardization of the diffusion process:

- the difficulty of creating a uniform agar layer due to objective reasons in the form of features of the shape of the Petri dishes and the physical phenomenon of wetting, leading to curvature of the surface;

- a small number of holes located on one cup, leading to the separation of standard and test samples.

The authors correct the errors that occur by the following methods:

- the presence on each plate of the control concentration of the standard, according to which amendments are made to the data obtained;

- mandatory re-studies for each sample (at least three);

- bringing the concentration of the test sample to the limits corresponding to the interval of a linear dependence of the diameter of the zone of growth inhibition on the concentration of the antibiotic (from 0.5 to 2 μg / ml).

This determines the significant complexity and material consumption of the method:

- 12 cups are required to create a standard calibration curve;

- 1 cup is consumed for each test sample;

- only multiple selection of the dilution of the test sample allows you to get the necessary antibiotic content in it, which increases the number of studies.

The objective of the invention is to improve the known method.

The technical result that will be achieved from the use of the invention is to reduce the complexity and material consumption of the method.

The technical result is achieved by the fact that in the method for determining the concentration of benzylpenicillin preparations in biological fluids by using the microbiological method of diffusion in agar, including the preparation of a test microbe preparation, creating an agar layer on glass, cutting holes in the frozen agar, introducing standard solutions into them and preliminary dilution of the tested samples, as a test microbe use Staphylococcus aureus ATCC 25923 in the amount of 3 · 10 ⁶ microbial bodies per ml of medium, a uniform layer of agar is created on the glass 1-2 mm thick. In solidified agar, holes with a diameter of 2 mm are cut into which standard solutions are added in concentrations of 0.5; 1.5; 3; four; 5; 6 μg / ml and test samples.

The essence of the invention is to change the strain of the test microbe and the technology of pouring agar.

When using Staphylococcus aureus ATCC 25923 as test microbes in an amount of 3 · 10 ⁶ microbial bodies per ml of medium, a ratio between the sizes of growth inhibition zones and doses of antibiotics is achieved, in which it is possible to increase the range of the standard calibration curve from 0.5 to 6 μg / ml

Formation of a uniform agar layer on the glass surface with a thickness of 1-2 mm allows to reduce the amount of material used and contributes to the adequate visualization of growth inhibition zones of the test culture (by increasing contrast), and also ensures that the conditions for strict standardization of the diffusion process from all wells are met, which significantly increases the accuracy of the method and eliminates the need for repeated testing of samples.

Cutting holes in solidified agar with a diameter of 2 mm reduces the area of growth inhibition zones of the test culture and allows you to place calibration and test samples on the same agar layer, which eliminates the use of control samples.

The use of solutions of benzylpenicillin standard in concentrations of 0.5; 1.5; 3; four; 5; 6 μg / ml is necessary and sufficient to create a calibration curve that allows you to determine the presence of an antibiotic in whole or diluted 1: 1 test samples, which eliminates the multiple selection of dilution of the test sample and helps to reduce the number of studies.

A significant reduction in the number of analyzed samples (due to a reduction in the number of studies, the absence of control samples and selection of dilution of the test sample) while maintaining the necessary accuracy of the method leads to a significant reduction in material and labor costs.

The method is as follows.

Prepare the required amount of molten and cooled to 50 ° C 1.5% agar, which contribute Staphylococcus aureus ATCC 25923 in the amount necessary to obtain a concentration of 3 · 10 ⁶ microbial bodies per ml of medium.

The agar was poured using a system of two glass plates fastened with clamps, between which a U-shaped frame 1-2 mm thick was installed. The approximate size of the plates is 9 × 12 cm.

After solidification of the agar, the clamps, the upper plate and the frame are removed. In an agar layer on the bottom plate, rows of 2 mm diameter holes are cut with a punch.

Standard solutions of benzylpenicillin at concentrations of 0.5 are added to the wells of one of the rows; 1.5; 3; four; 5; 6 μg / ml, in the wells of other rows - tested biological fluids (either whole or diluted 1: 1).

The plate is placed in a wet chamber and incubated at a temperature of 37 ° C for 16-18 hours.

After the indicated time, the growth inhibition zones of the test culture are measured with standard and test samples. Using the values of the standard samples, a calibration curve is constructed on semilogarithmic paper, which expresses the relationship between the dose and the diameter of the growth inhibition zones, and the concentration of the tested samples is determined from it.

The method was used to study the levels of antibacterial activity created in the blood of patients with syphilis in the first hours after the administration of benzylpenicillin preparations in the course of specific therapy. 60 studies were conducted.

When using the proposed method to obtain the results, it was necessary to prepare 10 plates (100 ml of agar) and perform 130 measurements.

In the case of applying the method specified in the prototype, for a given number of studies would require the manufacture of at least 102 cups (1020 ml of agar) and 612 measurements. In addition, additional time would be spent on performing additional dosing procedures, on calculating the average values of indicators and adjusting the results obtained for control concentrations.

This invention allows to reduce the complexity of 4.7 times and material consumption by more than 10 times.

Claims (1)

A method for determining the concentration of benzylpenicillin preparations in biological fluids by using the microbiological method of diffusion into agar, including preparing a test microbe preparation, creating an agar layer, cutting holes in the frozen agar, introducing standard solutions into them and pre-diluting the test samples, characterized in that as use a test-microbe Staphylococcus aureus ATCC 25923 in an amount of 3 × 10 ⁶ microbial cells in 1 ml medium glass agar create a uniform layer of 1-2 mm thickness in a frozen agar you ezayut wells 2 mm in diameter, in which make standard solutions at concentrations of 0.5; 1.5; 3; four; 5; 6 μg / ml and the test samples, and from the values of the standard samples on a semi-log paper, a calibration curve is constructed that expresses the relationship between the dose and the diameter of the growth inhibition zones, and the concentrations of the test samples are determined from it.