Method for steroid profiling in dope test of sportsmen

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely sports medicine, and may be used for steroid profiling in dope test of sportsmen. That is ensured by urine sample hydrolysis by a mixture of two enzymes: β-glucuronidase E.coli and arylsulphatase H.pomatia in volume ratio 1:1 to 1:3 in the presence of a citrated buffer solution and an internal standard. The hydrolysate is separated, derivatised that is followed by chromatic/mass-spectral analysis of the sample and recording of the derived results and stating the presence of endogenic steroids (ES). The ES value is determined in a sportsman for at least 10-15 times every 10-12 days. The analysis results are used to set minimum and maximum values of each of the derived ES. Said values are accepted to be a confidence range, and a steroid profile of the sportsman is drawn as a set of confidence ranges of all found ES.

EFFECT: invention provides steroid profiling, extending a variety of the stated ES and ensuring a higher degree of reliability of stating the ES.

2 cl, 12 dwg, 5 tbl, 5 ex

The invention relates to medicine, namely to clinical chemistry, in particular to methods for determining levels of endogenous steroids in the body, and can be used, for example, when the doping control athletes, and especially when preparing the athlete biological passport.

A known method for determining endogenous steroids (ES) colorimetric methods of analysis [1].

The way this relates to the methods of qualitative analysis and, accordingly allows to determine only the presence of ES, without determining their structural characteristics and nature. Moreover, according to the described method it is possible to analyze only objects with a very high content of ES.

There are also known methods for determining ES by gas chromatography coupled with mass spectrometry [2-4].

A common shortcoming of such methods is laborious and lengthy in time stage of obtaining volatile derivatives for ES, which significantly complicates the course of the analysis, increases the duration and cost of the process.

The closest in technical essence and the achieved result to the claimed method is GC-MS method for determining ES in human urine by gas chromatography coupled with mass spectrometry (GC-MS) [5].

At the specified method sample the eyes add phosphate buffer solution, an internal standard, and the enzyme β-glucuronidaseE.coli). The mixture is stirred and incubated and after hydrolysis add carbonate buffer solution and diethyl ether, then the organic layer was separated after centrifugation, evaporated to dryness, derivatized and analyze by method GC/MS.

The main disadvantage of this method is the low reliability of the determination of RN (in particular, when determining steroid profile) and high threshold (for example, to epitestosterone 10-12 ng/ml), because steroids are excreted in the urine in the form of glucuronidase and sulfates, and analysis are only glucuronidase.

The technical result, which directed the establishment of this invention is to increase the degree of reliability of determination of ES, in particular when determining steroid profile by lowering the threshold and expanding the range defined ES and, accordingly, determine the steroid profile.

The technical result is achieved by the fact that in the process of preparation of the hydrolysis of the analyzed sample of urine are a mixture of two enzymes: β-glucuronidaseE. coliand UkrainianH.pomatiain the ratio from 1:1 to 3:1 and as a buffer solution using citrate buffer solution, while the athlete determine Aut ES at least 10-15 times with intervals between tests not less than 10-12 days test results establish the minimum and maximum quantitative value for each of the identified ES, accept the specified values for the confidence interval and build longitudinal profile of the athlete as a set of confidence intervals all found ES.

It is known that GC/MS analysis of the ES it is necessary to spend several stages of sample preparation, because the excretion of ES in the urine occurs after phase II metabolism, namely conjugation, during which the formed esters of compounds and steroids are excreted in the urine in the form of glucuronidase and sulfates. Some steroids, such as Androsterone, etiocholanolone, testosterone and epitestosterone may appear in the urine in the form of sulfates with respect to the glucuronidase to 1:1. Other steroids, such as dehydroepiandrosterone, Androstenedione, estrone, estriol, can appear in the urine mainly in the form of sulfates. In turn sulfate are of interest for the purposes of doping control not only because of the endogenous ES, but also for the reason that some steroids constitute the most long-lived metabolites it is in the sulfate fraction and therefore they can be detected in biological fluids during, for example, not 6 hours, and within 18 or 24 hours, which should improve the reliability of the confirmation of doping athlete. However, men who in the present time, the concentration of the main endogenous ES, such as testosterone, epitestosterone, Androsterone, etiocholanolone, dehydroepiandrosterone and others, according to the standards WADA calculated from glucuronidase relevant ES [6]. Currently, there are about thirty ES man, but the authors based on their knowledge and experience, argue that to determine reliable steroid profile analytically, it is sufficient to explore the most typical 8-12 ES.

When implementing the claimed invention, the apparatus registration process can be used, for example, the chromatograph Agilent 6890, coupled with mass-selective detector Agilent 5973N with electron ionization (70 eV), column Restek Rxi - 1ms (length 12 m, internal diameter of 0.20 mm, the thickness of the stationary phase of 0.33 μm).

Temperature program can be set with the following parameters: rise from 188°C to 232°C at a rate of 2°C/min, then from 232°C to 300°C at 12°C/min, isotherm 5,33 min when the volume of injected sample 2 ál of the divide flow (1:20), injector temperature of 280°C. the carrier Gas - helium (0.6 ml/min). The temperature of the ion source is 230°C, the temperature transition line - 290°C, quadrupole - 150°C, scan rate was 1.69 cycles/sec, the mass range in the full scan mode - 50-600 Amu

As reagents and materials can be used, for example,N-methyl-N-(trimethylsilyl)-t is iterated (MSTFA), β-glucuronidase fromH.pomatia,arylsulfatase fromH.pomatiaand sodium acetate (Sigma-Aldrich, USA); standard samples of testosterone, epitestosterone, Androsterone, etiocholanolone, dihydrotestosterone, Androstenedione, estrone, estriol, 11β-hydroxyandrostenedione, 11β-hydroxyethykellulose and dehydroepiandrosterone (Steraloids, Newport, USA); methanol, hydrochloric acid and acetic acid (Merck, Germany); potassium dihydrophosphate, phosphate, sodium and ammonium iodide (Riedel-de-Haën, Germany); potassium carbonate, potassium bicarbonate and sodium sulfate (Hemmed, Russia); diethyl ether (Medienraum, Russia); 1,4-dithio-DL-trital (DTT), imidazole (Fluka, Switzerland); potassium citrate (Alfa Aesar, USA); internal standard - methyltestosterone, D4-androstene-glucuronide and D5-etiocholanolone (LGC Standards, UK); deionized water (MilliQ, Germany).

Of course, there may be used the equipment, materials and reagents from other manufacturers, provided, however, that the qualitative characteristics they will ensure the reproducibility of the results of research in the scope of the claimed patent claims.

As accessories can be used: automatic shaker firm Glas-Col^®, USA - for JJA; centrifuge brand Rotina 46R company Hettich (Germany) to obtain the contrast of the boundary surface between the organic and aqueous phases;- privately company Banstead Inc. (USA), combined with a nitrogen generator Mistral-4 firms Schmidlin-DBS AG (Czech Republic) for evaporation of the organic extract. As extractant for IGE can be used methyl tertiary butyl ether (MTBE). As pH regulators may be used sodium bicarbonate and potassium carbonate.

The invention can be implemented as follows.

Prepare urine samples, add them in buffer solution, internal standard (methyltestosterone, for a final concentration of 500 ng/ml of urine), the exact amount of enzyme or mixture of enzymes. The mixture is then stirred and incubated at an elevated temperature for 1.0 to 1.5 hours. After hydrolysis the mixture was added carbonate buffer solution (pH 10,4) and diethyl ether, shake for 3-10 min, centrifuged, separated the organic layer and evaporated it to dryness and derivatized solution of MSTFA/NH₄I/DTT by heating within 20-40 minutes, derivatized transferred into the vial and analyzed by method GC/MS. Remove and register chromatographic and mass spectrometric characteristics of the substances standards and all solutions and samples (detects at least three characteristic ions of each reference substance, determine the retention time, molecular mass precursor ions, characteristic ions, the lower limit of detection) and the resulting re is ulitity injected into the software, for example ChemStation company Agillent, USA.

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

Example 1

Prepare standard solutions of analytes (1 mg/ml), particularly testosterone, epitestosterone, Androsterone, etiocholanolone, dihydrotestosterone, Androstenedione, estrone, estriol, 11β-hydroxyandrostenedione, 11β-hydroxyethykellulose and dehydroepiandrosterone. Working solutions are prepared by dissolving accurately weighed ES - standards in methanol. Next get a working solution by diluting the standard solutions to the content of 10 μg/ml Standard solutions of each of the ESTER (1 mg/ml) prepared by dissolving accurately weighed in the exact volume of methanol. To 3 ml of urine (from previously prepared samples) add 1 ml of buffer solution, 30 μl of internal standard (MT, for a final concentration of 500 ng/ml of urine), the exact amount of the mixture of enzymes. The mixture is stirred and incubated at 55°C for 1 hour 10 minutes. After hydrolysis, add 1 ml of carbonate buffer solution (pH 10,4) and 5 ml of diethyl ether. Shake for five minutes, centrifuged at 3000 rpm for 5 min; the organic layer is separated, evaporated to dryness at 70°C for 40 minutes and spend the derivatization of 50 ál of a solution of MSTFA/NH₄I/DTT (1000:3:2) when heated to 70°C in t is within 30 minutes, the solution is transferred into the vial and analyzed further by method GC/MS (parameters of the analytical procedure described above).

The content of individual ES in urine is determined in the same manner and under the same parameters as in the standard solutions, with the exception that the hydrolysis of these samples are in the following order (see Table 1).

Table 1

Presented in Table 1 results becomes obvious that the use for hydrolysis of a mixture of enzymes E. coli + helix pomatia in citrate buffer allows to expand the range defined ES, as well as significantly reduce the detection threshold of these compounds. In particular, it can be seen from the comparison of the results obtained in Table 2 (the number in the sample ES ng/ml).

Table 2

Example 2 (comparative)

For comparison, carried out the analyses of the urine samples subjected to hydrolysis at various combinations of enzymes and buffers (see Table 3).

Table 3

The results for the studied series ES are shown in Fig.1-5.

From the presented illustrative materials implies that when used as a hydrolysing environment of a mixture of enzymes (E. coli+helix pomatia) in citrate buffer lower limit of detection was investigated ES compared with the known (unit, or 100%) is reduced at least to 0.8-0,35 (i.e. 1.2-1.65 times).

Example 3

Prepare the sample of urine, add in a buffer solution, internal standard (MT, for a final concentration of 500 ng/ml of urine), the exact amount of enzyme or mixture of enzymes. The mixture is then stirred and incubated at an elevated temperature during 1,0 -1,5 hour and further procedures which have carried out as in Example 1, including equipment and conditions for GC/MS analysis except that the urine sample is divided into five aliquot and each aliquot is subjected to hydrolysis under the following conditions (table 4).

Table 4

The results of determination of ES, in particular etiocholanolone shown in figure 6.

From figure 6 it follows that the optimal ratio of the amount of enzyme is 1:2 to 1:3. Output ratio for the stated limits reduces the expected result identify ES.

Example 4 (Definition of Stero the underwater profile)

Determine the longitudinal profile of the volunteer (biathlete, male, 23 years). Just taken 17 tests with an interval between analyses 10-15 days. It should be noted that the athlete for the entire observation period (220 days) did not take drugs and not used in a transfusion of blood. Confidence interval values of each specific ES set as the difference between the minimum and maximum values specified ES in time (in this case, during the time of observation - 220 days and according to the 17 analyses). Fig.7-10 presents the steroid profiles of athletes in the dehydroepiandrosterone, epitestosterone and testosterone and estriol, respectively. In other investigational ES steroid profiles are presented in Table 5. To determine steroid profile is selected by two minimum and maximum values of the total number of 17 values.

Table 5

Example 5 (control)

Determine the longitudinal profile of the volunteer (male, 25 years). Just taken 6 tests with an interval between analyses 10 days. It should be noted that volunteer for the entire observation period (60 days) did not take drugs and not used in a transfusion of blood. Confidence interval values of each specific ES set as in Example 4. On the 61st day of observation with the consent of the volunteer gave him to take the capsule 50 mg of testosterone and the decanoate (Andriol) and capsule DHEA 50 mg Control urine spend 8 hours after administration. Figure 11 and 12 presents the steroid profiles of the test for testosterone and DHEA. It should be noted that in this example, simultaneously determine the longitudinal profile of ES according to the method described in the prototype (hydrolysis of E. coli in phosphate buffer), while the lower limit value profile for testosterone was 32 ng/ml, and DHEA was discovered only after taking the drug.

As can be seen from the description and examples of the method and the comparative and reference examples, the invention provides a reduction in the threshold detection sensitivity of ES, the expansion of the range defined compounds while increasing the reliability of determination of steroid profile.

Sources of information

1. EN 2190853 C2, G01N 33/74, 2002

2. Chem. Pharm. journal, 1988, t, s.

3. EN 2313086 C2, G01N 30/72, 2007.

4. J. Anal. toxicol., 2005, v. 29, No. 4, p.217.

5. WADA Technical Document - TD2004EAAS prototype.

6. Wada Prohibited List, Version 5.0, 2010.

The method of determining the steroid profile in doping control of athletes, including hydrolysis of the sample of urine by an enzyme in the presence of buffer solution and the internal standard, the separation of the hydrolysate, the derivatization him, followed by gas chromatography-mass spectral analysis of the sample and recording the obtained results and definitions n the availability of endogenous steroids (ES), characterized in that in the preparation of the sample hydrolysis of the urine sample is performed with the mixture of two enzymes: β-glucuronidase E.coli and Ukrainian H.pomatia in a volume ratio from 1:1 to 1:3, and the buffer using citrate buffer solution, and the athlete determine ES at least 10-15 times with intervals between tests not less than 10-12 days, based on the test results establish the minimum and maximum quantitative value for each of the identified ES, accept the specified values for the confidence interval and build longitudinal profile of the athlete as a set of confidence intervals all found ES.