RU2451936C1 - Method of detecting conjugated xenobiotics during doping tests in athletes - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention describes a method of detecting conjugated xenobiotics during doping tests in athletes, involving preparation of the analysed urine sample via enzyme hydrolysis in the presence of a buffer solution and an internal standard, separation of the hydrolysate, derivatisation thereof, followed by chromatographic/mass spectrometric analysis of the sample and recording the obtained results and determining presence of steroids, where when preparing the analysed sample, the urine sample is hydrolysed with a mixture of two enzymes: β-glucuronidase E.coli and arylsulfatase H.pomatia with volume ratio from 1:1 to 1:3, and the buffer used is a citrate buffer solution.

EFFECT: lower detection threshold of xenobiotics and wider range of detected compounds.

2 ex, 4 tbl, 6 dwg

Description

The invention relates to medicine, and more specifically to clinical chemistry, in particular to methods for determining the level of xenobiotics in the body, and can be used, for example, in doping control of athletes.

A known method for the determination of xenobiotics, such as steroids, by colorimetric analysis [1].

This method relates to methods of qualitative analysis and, accordingly, allows to determine only the presence of steroids, without determining their structural characteristics and, in fact, nature. In addition, according to the specified method, it is possible to analyze objects only with a very high content of steroids.

Also known are methods for determining xenobiotics by gas chromatography in combination with mass spectrometry [2-4].

A common drawback of such methods is the time-consuming and time-consuming stage of obtaining volatile derivatives, which significantly complicates the analysis, increases its duration and increases the cost of the process.

The closest in its technical essence and the achieved result to the claimed method is the GC-MS method for determining conjugated xenobiotics, including steroids in human urine by gas chromatography in combination with mass spectrometry (GC-MS) [5].

According to this method, a phosphate buffer solution, an internal standard, as well as an enzyme ( E. coli β-glucuronidase ) are added to a urine sample . The mixture is stirred and incubated, and after hydrolysis, a carbonate buffer solution and diethyl ether are added, then the organic layer is separated after centrifugation, evaporated to dryness, derivatized and analyzed by GC / MS. The main disadvantage of this method is the low degree of xenobiotic determination reliability and a high sensitivity threshold (for example, for epitestosterone up to 25-50 ng / ml), since this steroid is excreted in the urine in the form of both glucuronides and sulfates, and only glucuronides are analyzed.

The technical result, the achievement of which the creation of this invention is directed, is to increase the degree of reliability of the determination of xenobiotics by reducing the sensitivity threshold for determining and expanding the range of determined xenobiotics (KB).

The technical result is achieved by the fact that in the process of sample preparation, the hydrolysis of the analyzed urine sample is carried out with a mixture of two enzymes: E. coli β-glucuronidase and H. pomatia aryl sulfatase in a volume ratio of 1: 1 to 1: 3, and citrate buffer is used as a buffer solution solution.

It is known that during GC / MS analysis of CB, it is necessary to carry out several stages of sample preparation, since excretion of CB in urine occurs after phase II of metabolism, namely conjugation, during which esters of compounds are formed, and CB is excreted in the urine in the form of glucuronides and sulfates. Some CBs form the most long-lived metabolites in the sulfate fraction, therefore, they can be found in biological fluid for, for example, not 8 hours, but within 18 or 24 hours, which should increase the reliability of confirmation of doping by an athlete. Nevertheless, at present, concentrations of CB, including steroids, according to WADA standards, are calculated on the basis of the glucuronides of the corresponding compounds [6].

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

The temperature program can be set with the following parameters: rise from 188 to 232 ° C at a speed of 2 ° C / min, then from 232 to 300 ° C at 12 ° C / min, isotherm 5.33 min with a sample volume of 2 μl in the division mode flow (1:20), the temperature of the injector is 280 ° C. The carrier gas is helium (0.6 ml / min). The temperature of the ion source is 230 ° C, the temperature of the transition line is 290 ° C, the quadrupole is 150 ° C, the scanning speed is 1.69 cycles / s, the mass range in the full scan mode is 50-600 amu.

As reagents and materials, for example, N- methyl - N - (trimethylsilyl) trifluoroacetamide (MSTFA), β-glucuronidase from H. pomatia, aryl sulfatase from H. pomatia and sodium acetate (Sigma-Aldrich, USA) can be used; standard samples of terbutaline, formestane, carvediolol, morphine, timolol, androstenedione, epiandrosterone and stanozolol (Steraloids, Newport, USA); methanol, hydrochloric acid and acetic acid (Merck, Germany); potassium dihydrogen phosphate, sodium hydrogen phosphate and ammonium iodide (Riedel-de-Haën, Germany); potassium carbonate, potassium hydrogen carbonate and sodium sulfate (ChemMed, RF); diethyl ether (MedChemProm, RF); 1,4-dithio-DL-triethol (DTT), imidazole (Fluka, Switzerland); potassium citrate (Alfa Aesar, USA); internal standard - methyltestosterone (MT), D4-androsterone-glucuronide and D5-ethiocholanolone (LGC Standards, UK); deionized water (MilliQ, Germany).

Of course, equipment, materials and reagents of other manufacturers can be used, however, on the condition that in terms of quality characteristics they will ensure reproducibility of the research results in the scope of the claimed patent claims.

As auxiliary equipment the following can be used: automatic shaker of Glas-Col ^® company, USA for ZhEZh; a Hettich Rotina 46R centrifuge (Germany) to obtain a contrast interface between the organic and aqueous phases; evaporator of Barnstead Inc. (USA), combined with a Mistral-4 nitrogen generator from Schmidlin-DBS AG (Czech Republic) for evaporation of the organic extract. Methyl tert-butyl ether (MTBE) can be used as an extractant for LCE. Sodium bicarbonate and potassium carbonate can be used as pH adjusters.

The invention can be implemented as follows.

Urine samples are prepared, a buffer solution, an internal standard (methyltestosterone, for a final concentration of 500 ng / ml urine), the exact amount of the enzyme or enzyme mixture are added to them. The mixture is further stirred and incubated at elevated temperature for 1.0-1.5 hours. After hydrolysis is completed, carbonate buffer solution (pH 10.4) and diethyl ether are added to the mixture, shaken for 3-10 minutes, centrifuged, the organic is separated layer and evaporated to dryness and derivatized with a solution of MSTFA / NH ₄ I / DTT with heating for 20-40 minutes, the derivatizate was transferred to a vial and analyzed by GC / MS. The chromatographic and mass spectrometric characteristics of the reference substances, as well as all solutions and samples, 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 obtained the results are entered into software, for example, ChemStation from Agillent, USA.

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

Example 1

Prepare standard solutions of analytes (1 mg / ml), in particular terbutaline, formestane, carvediolol, morphine, timolol, androstenedione, epiandrosterone and stanozolol. Working solutions are prepared by dissolving an exact portion of xenobiotics - standards in methanol. Next, working solutions are obtained by diluting standard solutions to a content of 10 μg / ml. Standard solutions of each of the CBs (1 mg / ml) are prepared by dissolving the exact weight in the exact volume of methanol. To 3 ml of urine (from previously prepared samples) add 1 ml of buffer solution, 30 μl of the 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 20 minutes; the organic layer is separated, evaporated to dryness at 70 ° C for 40 minutes and derivatization is carried out - 50 μl of a solution of MSTFA / NH ₄ I / DTT (1000: 3: 2) when heated to 70 ° C for 30 minutes, the solution is transferred to a vial and analyzed further by GC / MS (parameters of the analytical procedure are given above).

With the permission of the ethics committee and the consent of the volunteers (5 people), they took doping (androstenedione and terbutaline - 1st; formestane and carvediolol - 2nd; morphine - 3rd; testosterone - 4th and stanozolol - 5th) . Sample preparation of urine samples of doped volunteers is carried out in the manner described above (add buffer solution, internal standard, exact amounts of enzyme or enzyme mixture, then mix the mixture and incubate at elevated temperature for 1.0-1.5 h) and then carry out GC / MS analysis. CB in urine samples is determined in the same order and with the same parameters as in standard solutions. The analysis results are presented in Table 1.

Table 1 Urine sample enzyme Buffer Found KB, concentration, ng / ml one E.coli + helix pomatia citrate Androstenedione, 120
Terbutaline, 430 2 E.coli + helix pomatia citrate Formestanan, 260
Carvediolol 120 3 E.coli + helix pomatia citrate Morphine, 1450 four E.coli + helix pomatia citrate Testosterone, 279 5 E.coli + helix pomatia citrate Stanozolol, 153

At the same time, these same urine samples are examined according to the method prescribed in the prototype (E. Coli enzyme, phosphate buffer). The analysis results are presented in Table 2.

table 2 Urine sample enzyme Buffer Found KB, concentration, ng / ml one E.coli phosphate Androstenedione not detected
Terbutaline, 180 2 E.coli phosphate Formestan, 100
Carvediolol 120 3 E.coli phosphate Morphine, 500 four E.coli phosphate Testosterone, 240 5 E.coli phosphate Stanozolol, 140

From the results presented in Table 1, it becomes obvious that the use of E. coli + helix pomatia enzymes for the hydrolysis of a mixture in citrate buffer allows us to expand the range of detectable CBs, as well as significantly lower the threshold for the determination of these compounds.

In particular, this can be seen from a comparison of the results obtained in Table 2 (the number of KB detected in the sample is ng / ml).

For comparison, analyzes of urine samples subjected to hydrolysis with various combinations of enzymes and buffers (see Table 3).

Table 3 one Enzyme E.coli Phosphate buffer By WADA 2 A mixture of E. coli + helix pomatia enzymes Citrate buffer According to the invention 3 Enzyme E.coli Citrate buffer For comparison four Enzyme helix pomatia Phosphate buffer For comparison 5 Enzyme helix pomatia Citrate buffer For comparison

Figure 1-5 shows the results of the analyzes for the investigated series of CBs, depending on the nature of the enzyme (s) and buffer.

From the presented illustrative materials, it follows that when using a mixture of enzymes (E. coli + helix pomatia) as a hydrolyzing medium in a citrate buffer, the lower detection limit of the studied KB compared with the known one (unit, or 100%) is reduced to at least 0.8 -0.35 (i.e. 1.2-1.65 times)

Example 2

A urine sample is prepared, a buffer solution, an internal standard (MT, for a final concentration of 500 ng / ml urine), the exact amount of the enzyme or enzyme mixture are added to it. The mixture is further mixed and incubated at elevated temperature for 1.0-1.5 hours and then the procedure is carried out as in Example 1, including the technique and conditions for GC / MS analysis, except that the urine sample is divided into five aliquots and each aliquot is hydrolyzed under the following conditions (table 4):

Table 4 Aliquot Enzyme E. coli (ml) Helix pomatia (ml) Note one 1,0 0.7 2 1,0 1,0 3 1,0 2.0 four 1,0 3.0 5 1,0 3,5

Figure 6 shows the results of the determination of carvediolol depending on the ratio of the volume of the mixture of enzymes used.

From Fig. 6 it follows that the optimal ratio of the volumes of enzymes used is 1: 2 - 1: 3. Going beyond the stated limits reduces the expected result of determining the ES.

As can be seen from the description and examples of the implementation of the method and comparative examples, the invention provides a decrease in the sensitivity threshold for determining ES, as well as expanding the range of defined compounds.

Information sources

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

2. Chem. farm. Journal, 1988, v. 22, p. 622.

3. RU 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.

Claims (1)

A method for determining conjugated xenobiotics during doping control of athletes, including preparing an analyzed urine sample by hydrolysis with an enzyme in the presence of a buffer solution and an internal standard, separating the hydrolyzate, derivatizing it with subsequent chromato / mass spectral analysis of the sample and recording the results and determining the presence of steroids, characterized in that in the preparation of the analyzed sample, the hydrolysis of the urine sample is carried out with a mixture of two enzymes: E. coli β-glucuronidase and arylsu H. pomatia lphatase with a volume ratio of 1: 1 to 1: 3, and a citrate buffer solution is used as a buffer.

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