RU2384846C1 - Method of determining use of corticotropins for during doping tests in athletes - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to analytical chemistry. A method for determining use of corticotropins during doping test in athletes by analysing body fluid samples and detecting glucocorticosteroids is proposed. The method involves taking a urine sample for high-performance liquid chromatography coupled with tandem mass spectrometry and determining content of indirect biochemical markers of intake of corticotropins - cortisol, cortisone and 6βOH-cortisol. If the value of the cortisol/cortisone ratio in the sample is higher than 0.3-1.00 and the 6βOH-cortisol/cortisol ratio is below 15.0, corticotropins have been used.

EFFECT: method cuts the time for testing a group of athletes for doping and expenses for carrying out analysis.

4 ex, 3 tbl

Description

 The invention relates to the field of research or analysis of materials, including xenobiotics, by dividing samples of materials into components using chromatography and mass spectrometry, and more specifically to methods for identifying and determining corticotropins, and can be used in doping control.

The closest to the claimed object in its technical essence and the achieved technical result is a method of doping control of corticotropin intake by direct determination of the content of glucocorticotropins - synactene in the blood sample. According to this method, the sample is subjected to "immunological purification" - electrophoretic distillation of proteins and their fractions, affinity purification and subsequent analysis by high performance liquid chromatography in combination with tandem mass spectrometry (HPLC-MS / MS) [1, 2].

The specified method is very long (two days), complicated, involves traumatic sampling (blood sampling), which certainly affects the performance of the process during a mass examination of athletes and the cost of such examination.

The technical result, the achievement of which the creation of this invention is directed, is to increase the reliability of doping control of corticotropin use by determining the level and ratio of the content of indirect biochemical markers - cortisol, cortisone and 6βOH-cortisol in the athlete’s urine while reducing the duration of the analysis and its cost reduction.

The technical result is achieved by the fact that the urine samples of athletes are subjected to high-performance liquid chromatography in combination with tandem mass spectrometry and the content of indirect biochemical markers of corticotropin intake - cortisol, cortisone and 6βOH-cortisol - is determined and when the ratio in the cortisol / cortisone sample is above 0 , 3-1.0 and the ratio 6βOH-cortisol / cortisol below the limit of 15.0 judge the administration of corticotropins.

The authors found an unexpected solution.

The kinetics of the consonant biochemical reactions taking place in the body, especially hormonal metabolism, are naturally determined by the concentration of substances involved in this process. Since there is a close relationship between the release of glucocorticosteroids from the anterior pituitary gland and the concentration of hormones in the blood of the adrenal cortex, it can be argued that when synthetic corticotropins (for example, synactene) are introduced into the body, the content and ratio of corticosteroid metabolism products in the body and its secretions should change dramatically. . These products are cortisol, cortisone and 6βOH-cortisol. This implies the possibility of indirect doping control of the illegal use of corticotropins by athletes.

As an average norm of the content of cortisol, cortisone and 6βOH-cortisol, the processed statistical data on the content of these substances in the urine of healthy individuals are taken taking into account the daily fluctuations in the content of the substances being determined.

Cortisol, cortisone, and 6βOH-cortisol with a content of the main component of 99% (Sigma-Aldrich, Germany) are used as standard determinants.

As internal standards (ISTD) use fluoxymesterone with a content of the main component of 99% (Sigma-Aldrich, Germany).

Sinactene is used as exogenous corticotropin (Sinacten DEPO Novartis Pharma AG, Austria).

Standard analyte solutions (1 mg / ml) are prepared by dissolving the exact weights in the exact volume of methanol. Working solutions are obtained by dilution. Store working solutions at -20 ° C.

The eluent is prepared using methanol "for chromatography" (Chromasolv®, Merck, Germany), ammonium acetate and formic acid (Fluka, Switzerland).

The mobile phase is prepared on the basis of deionized water obtained on a Milli-Q plus installation (Millipore, France).

High-performance liquid chromatography in combination with tandem mass spectroscopy (HPLC-MS / MS) was used using a TSQ Quantum triple quadrupole analyzer (Thermo Finnigan, USA) coupled to a Surveyor high-performance liquid chromatograph, equipped with an autosampler, high pressure pump and degasser (Thermo Finnigan, USA).

For chromatographic separation using an Eclipse XDB-C18 column, 150 × 2.1 mm, with a particle size of 5 μm, with a pore size of 100 Ǻ (company Agilent, USA).

As the mobile phase, a 0.05% solution of formic acid with a 20 mM solution of ammonium acetate (pH 3.0) (A) and methanol (B) are used. The flow rate of the mobile phase is kept constant.

Ionization at atmospheric pressure is carried out by electrospray in the registration mode of negative ions.

Detection of analytes is carried out in the mode of registration of selective reactions (SRM).

Data processing is carried out using Xcalibur software version 1.3 (Thermo Finnigan, USA).

To determine the average content of cortisol, cortisone and 6βOH-cortisol, urine samples were analyzed from 87 healthy individuals in a daily regimen in a calm state, 39 athletes, from various sports in a calm regimen and during intensive training in a competition mode, as well as volunteers after taking corticotropin — synactin — and the weighted average ratios of cortisol / cortisone and 6βOH-cortisol / cortisol were found from the values found. The indicated ratios are presented in table 1.

Table 1 The ratio of glucocorticosteroids Healthy individuals Intensive Training Athletes Individuals after receiving synactin Cortisol / cortisone 0.34-0.49 0.4-1.00 2.00-4.00 6βOH-cortisol / cortisol 14.0-19.0 10.0-30.0 1.2-5.5

The invention can be implemented as follows.

Prepare solutions of reference substances in organic solvents and a solution of the internal standard.

The chromatographic and mass spectrometric characteristics of the reference substances are recorded and recorded (at least three characteristic ions of each reference substance are detected, the retention time, molecular weight, precursor ions, characteristic ions, and the lower detection limit are determined) and the results are entered into software, for example Xcalibur version 1.3 from Thermo Finnigan, USA.

Next, sample preparation is carried out, in which an internal standard solution is introduced into the urine sample, the sample pH is adjusted to 9.0 with a solid buffer, liquid-liquid extraction is carried out with diethyl ether, the organic layer is evaporated to dryness in a stream of nitrogen and re-dissolved in the mobile phase. The reconstituted precipitate is then introduced into the HPLC-MS / MS system with electrospray ionization at atmospheric pressure in the mode of registration of negative ions. The chromatographic and mass spectrometric characteristics of the analytes in the sample are taken and recorded (at least three characteristic ions of each analyte are detected, the retention time, molecular weight, precursor ions, characteristic ions, and the lower detection limit are determined) and the results are entered into the software, for example Xcalibur version 1.3 from Thermo Finnigan, USA.

It should be noted that the inventive method for determining the intake of corticosteroids can reduce the duration of the doping control of a group of athletes and significantly reduce the cost of doping control.

For a better understanding, the invention can be illustrated, but not exhausted by the following examples of its specific implementation.

Example 1

A. Acquisition of mass spectra, determination of characteristic ions, retention time and detection limits of cortisol, cortisone and 6βOH-cortisol

Prepare standard analyte solutions (1 mg / ml): in methanol, then work solutions are obtained by diluting standard solutions to a content of 1.0 μg / ml. The working solutions thus prepared are introduced into an HPLC-MS / MS system (a TSQ Quantum Triple Quadrupole Analyzer (US) chromatograph-mass spectrometer connected to a Surveyor high-performance liquid chromatograph equipped with an autosampler, a high pressure pump and a Thermo degasser Finnigan (USA). The analysis is carried out at a flow rate of the mobile phase of 0.2 ml / min, The determined substances are separated by gradient elution under the conditions: 0 min 15% B; 10 min 60% B; 15 min 75% B, 25 min 85%, total time analysis taking into account the stabilization of the system Before entering the next sample is 35 minutes, Ionization at atmospheric pressure is carried out by electrospray in the registration mode of negative ions. The voltage on the capillary 3.8 kV; the temperature of the capillary 245 ° C; the flow rate of the drying gas (nitrogen) 0.45 l / min; the gas flow rate (argon) in the collision chamber 0.075 l / min; temperature in the ionization chamber 200 ° C; pressure on the atomizer 2 atm. Detection of the substances to be determined is carried out in the mode of registration of selective reactions (SRM). The peak width for the precursor ions and the corresponding characteristic ions on the first quadrupole (Q1) and the third quadrupole (Q3) is 0.5 amu (at half height), delay time 5 ms. Processing of the obtained data (mass spectra, characteristic ions, retention time and detection limits of the studied xenobiotics) is carried out using the Xcalibur software version 1.3 of Thermo Finnigan, USA (see Table 2).

table 2 No. Detectable connection Retention Time (min) Like weight Precursor ion Characteristic ions Lower detection limit ng / ml one cortisol 12.1 362 407 [M + UNSU] ^- 331, 297 2 2 cortisone 12,4 360 405 [M + UNSU] ^- 301, 329 2 3 6βOH-cortisol 2,4 378 423 [M + UNSU] ^- 347, 313 5

B. Sample preparation and test urine analysis

To a urine sample (5 ml), add 5 μl of an internal standard solution containing fluoxmyesterone (100 μg / ml), 0.1 g of solid buffer, 5.0 g of ammonium sulfate and 5 ml of diethyl ether, mix for 2 minutes, then centrifuged at 1000 rpm for 5 min, the organic layer was separated, evaporated to dryness in a stream of nitrogen at 40 ° C and the dry residue was redissolved in 50 μl of the mobile phase, 5 μl of the solution was taken and introduced into the HPLC-MS / MS system with electrospray ionization at atmospheric pressure in the mode of registration of positive ions. The analysis is carried out, as in part "A" and with the same process parameters.

Examples 2-4

The analysis of the studied urine samples is carried out, as in Example 1, part “B”, except that the samples obtained from different individuals are examined. The results of the analyzes are given in table.3.

Table 3 Cortisol / cortisone 6βOH-cortisol / cortisol Note Example 2 0.42 17.1 Norm Example 3 3,5 1,5 Corticotropin taken Example 4 0.75 14.9 Norm

From example 3 it is seen that the content of the analyte in the sample for cortisol, cortisone and 6βOH-cortisol is 645.3: 186.9 and 981.4 μg / ml, respectively, and the ratio of cortisol / cortisone reaches limits of 3.5, which significantly exceeds the weighted average value of 0.3-1.00 and the ratio of 6βOH-cortisol / cortisol is 1.5, which is significantly less than the weighted average of 10-30. According to the indicated results, they also judge the administration of corticotropins.

As can be seen from the description and examples of specific implementation, the inventive method for determining the intake of corticotropins can significantly reduce the duration of the doping control of a group of athletes and significantly reduce the cost of its implementation.

Information sources

1. http://www.utro.ru/articles/2006/11/13/600674.shtml

2. Rapid Comm. In Mass Spectroscopy. V.20, Issue 23, 2006 - prototype.

Claims (1)

A method for determining the intake of corticotropins in the doping control of athletes by analyzing a sample of biological fluid and detecting glucocorticosteroids, characterized in that urine is used as a biological fluid, while the athlete’s urine sample is subjected to high-performance liquid chromatography in combination with tandem mass spectrometry and the content of indirect biochemical markers is determined taking corticotropins - cortisol, cortisone and 6βOH-cortisol, and when the ratio in the cortisol / cortisone sample is higher f limits 0.3-1.00 and a ratio of 6βOH-cortisol / cortisol below the limit of 15.0 judge the intake of corticotropins.