RU2390773C2 - Method of detecting xenobiotics in human urine during doping tests - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: in order to implement the method of detecting xenobiotics (corticosteroids, diuretics, narcotics, central nervous system (CNS) stimulators, anabolic steroids) in human urine, a solution of standard substances in an organic solvent is prepared, as well as a test sample through extraction, evaporation of the extract and dissolving the dry residue. Three samples are also prepared: first - by averaging urine samples (at least 10 urine samples of healthy people not taking medicinal drugs), second - by dissolving standard substances in the solution of the dry residue after evaporation of the averaged urine sample of healthy people not taking medicinal drugs and a third - by dissolving standard substances in the averaged urine sample of healthy people not taking medicinal drugs. Further, the prepared samples undergo chromatographic separation and mass spectrometric analysis, delay time values and the intensity ratio of characteristic ions of background substances in the prepared samples are recorded, and xenobiotics are detected by comparing the delay time values and the intensity ratio of characteristic ions in all solutions and samples, only considering those values which do not coincide with background substances.

EFFECT: lower threshold of detection sensitivity, high accuracy and reliability of the doping test.

4 tbl, 7 ex

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 xenobiotics, and can be used in doping control.

Known methods for the determination of organic substances, including xenobiotics (corticosteroids, diuretics, CNS stimulants, narcotic substances and anabolic steroids) by gas chromatography in combination with mass spectrometry, for example [1-3].

A common drawback of such methods is the time-consuming and time-consuming stage of obtaining volatile derivatives for corticosteroids, anabolic steroids, narcotic substances and diuretics, which significantly complicates the analysis and increases its duration.

The closest in technical essence and the achieved result to the claimed method is a method for determining xenobiotics in urine by high performance liquid chromatography in combination with mass spectrometry (HPLC-MS) [4].

According to the specified method, standard solutions of reference substances are prepared, chromatographic and mass spectrometric characteristics of the indicated solutions and the urine test sample are recorded and recorded in the scanning mode in the selected range, and the presence of xenobiotics is determined by comparing the recorded retention time and intensity ratios of the characteristic ions of reference substances and the sample .

The disadvantage of this method is the high threshold of sensitivity of determination, which consists in the fact that it does not allow to determine the studied substances at low concentrations, and the long duration of sample preparation, including enzymatic hydrolysis.

The technical result, the achievement of which the creation of this invention is directed, is to increase the information content and reliability of doping control by determining corticosteroids, diuretics, CNS stimulants, narcotic substances and a number of anabolic steroids in human urine in one sample and reducing the duration of sample preparation.

The technical result achieved is achieved by the fact that three samples are additionally prepared: the first by averaging urine samples (at least 10 urine samples of healthy people who did not take medications and obviously do not contain xenobiotics and their metabolic products), and the second by dissolving reference substances in the solution of the dry residue after evaporation of the average urine sample of healthy people who did not take drugs, and the third by dissolving the reference substances in the average urine sample of healthy people, n taking drugs, carry out chromatographic separation and mass spectral analysis of the prepared samples and record the retention time and the intensity ratio of the characteristic ions of the background substances in the prepared samples, and xenobiotics determine the comparison of the retention time and the ratio of the intensity of the characteristic ions in all solutions and samples, taking into account only those values that do not coincide with the background substances.

It is known that ethical standards in sports require the athletes to completely refuse to use any stimulants (xenobiotics) that artificially affect the body and central nervous system, and that is why the World Anti-Doping Agency (WADA) annually publishes lists of xenobiotics prohibited for use: corticosteroids, diuretics , narcotic substances, central nervous system (CNS) stimulants, anabolic steroids, beta-blockers, beta-2 agonists, polypeptide hormones (erythro oetin, growth hormone, insulin, corticotropin) and antiestrogens. Corticosteroids, CNS stimulants and beta-blockers are prohibited only for the period of competition. Diuretics, narcotic substances, anabolic steroids, beta-2 agonists, hormones and antiestrogens are constantly prohibited, i.e. in competitive and non-competitive cycles.

According to the approved WADA rules, the minimum required detection limit (MTPD) for corticosteroids, diuretics, CNS stimulants, narcotic substances, anabolic steroids and beta-blockers in human urine is 30, 250, 500, 200, 10 and 500 ng / ml, respectively, and for Clenbuterol and Stanozolol - 2 ng / ml [5]. The main necessary and sufficient parameters taken into account when determining xenobiotic compounds, due to the specificity of the HPLC-MS method, are the retention times and the intensity ratios of characteristic ions, however, at the same time, the reliability and accuracy of the determination of these parameters directly depends on the detection limits to which in turn, the parameters of the background substances in the analyzed urine sample have a significant effect.

Indeed, during electrospray ionization, a number of background compounds can form precursor ions identical with xenobiotics. In addition, such precursor ions in the collision chamber can fragment to form characteristic ions that are identical in molecular weight and, correspondingly, maximum intensity to those of the determined xenobiotics. In such cases, the interference of the peaks of the characteristic ion values distorts the desired parameters of the analysis results and leads to false conclusions about the detection limit and even the nature of the determined xenobiotic.

It is obvious that the more substances will be involved in the xenobiotics in the claimed method, the more universal it will be.

To determine xenobiotics as such can be selected, for example:

a) corticosteroids: cortisol, cortisone, flunisolide, flumethasone, fludrocortisone, fludrocortisone 21-acetate, budesonide, betamethasone, beclomethasone, methylprednisolone, deoxymethasone, triamcinolone, triamcinolone acetonide, desonolone dialozonolone dialozonolone dialozonolone dialefinolone dialefinolone dialefinolone diafenolone propionate;

b) diuretics: chlortiazide, hydrochlorothiazide, acetazolamide, chlortalidone, clopamide, trichloromethiazide, polythiazide, furosemide, bendroflumethiazide, benzthiazide, indapamide, bumetanide, probenecid, ethacrynic acid, amiloride azide, triamide;

c) CNS stimulants and narcotic substances: morphine, amphetamine, methylamphetamine, mephentermine, fenfluramine, norfenfluramine, strychnine, methadone, norephedrine, methylenedioxyethamphetamine (MDEA), methylenedioxymethamphetamine (MDMA), cocaine, ethylamphetamine, phenethyl methanefetamide, dimethylfetamide, dimethylfetamide, dimethylfetramonetamide, dimethylfetramidin codeine, niketamide, caffeine, butorphanol, norpsefedoephedrine, synephrine, adrafinyl, bupropion, ethamivan, pseudoephedrine, methylphenidate, modafinil and amitriptyline. 3'-OH-stanozolol, 16β-OH-stanozolol, 4α-OH-stanozolol, 4β-OH-stanozolol, heptaminol, carfedone, crotetamide andropropamide;

d) also such compounds as methylephedrine, ephedrine and foledrin, amfepramone and famphrofazone, testosterone, epitestosterone, gestrinone, clenbuterol, trenbolone, tetrahydrogestrinone, oxandrolone and oralturinabol, pemoline, mesocarb, selegilidin azidene azolefin azolefin azide, azolefin azide, azolefin azide, azolefin, azolefin, azolefin, azolefin, azolefin, azolefin, azole, azolefin, azolefin, azolefin, azolefin, azolefin, azolefin, azolefin, azolefin, azolefin, azolefin, azolefin, azolefin, azolefin, azolefinone amiphenazole, phentermine.

Standard analyte solutions can be prepared by dissolving the exact weights in the exact volume of a solvent, such as methanol. Working solutions can be obtained by dilution to an appropriate concentration.

Mixtures of methanol, ammonium acetate and formic acid, for example, can be used as eluent.

Diethyl ether can be used as an extractant for LCE.

Sodium bicarbonate and potassium carbonate can be used as pH adjusters.

As an HPLC-MS / MS system, 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) can be used). )

As auxiliary equipment can be used:

- automatic shaker from Glas-Col®, USA - for ZhEZh;

- a centrifuge brand Rotina 46R company Hettich (Germany) to obtain a contrasting interface between the organic and aqueous phases;

- an evaporator manufactured by Barnstead Inc. (USA) combined with a Mistral-4 nitrogen generator from Schmidlin-DBS AG (Czech Republic) to evaporate the organic extract;

- Eclipse XDB-C18 column, 150 × 2.1 mm, particle size 5 μm, pore size 100 Ǻ, Agilent (USA) for chromatographic separation.

As a mobile phase, the system can be used: a 0.05% solution of formic acid with a 20 mM solution of ammonium acetate (pH 3.0) (A) and methanol (B).

As internal standards (ISTDs), for example, fluoxymesterone, methyltestosterone and mefruzide can be used.

The invention can be implemented as follows.

Prepare solutions of reference substances in organic solvents, in the urine of healthy people who did not take drugs, in a solution of the dry residue after evaporation of the extract from the urine, and an average urine sample (by averaging at least 10 urine samples that do not contain xenobiotics and their metabolic products) .

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 fluoxymesterone, mefruzide and methyltestosterone are injected into the urine sample, mixed with solid buffer (sodium bicarbonate and potassium carbonate) and ammonium sulfate saline, the additives are dissolved by stirring, extracted with diethyl ether, the organic layer is separated quantitatively, evaporated, evaporated, evaporated. nitrogen, re-dissolve the dry residue in the mobile phase of the composition 40/60 methanol / 0.02% solution of formic acid with 20 mM solution of ammonium acetate and then one part of the sample is introduced into the HPLC-MS / MS system with ektroraspylitelnoy ionization mode registration of negative ions and a second portion - in the mode register positive ions with subsequent evaluation of the results obtained by comparing the registered parameters retention time and the ratio of the intensity of characteristic ions for all solutions and samples, taking into account only those values that do not coincide with the background substances.

It should be noted that the inventive method for determining xenobiotics can reduce the number of procedures in sample preparation and analysis by half compared with the prototype, which reduces the cost of doping control of one sample.

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 the studied xenobiotics

Prepare standard analyte solutions (1 mg / ml): cortisol, cortisone, flunisolide, flumethasone, fludrocortisone, fludrocortisone 21-acetate, budesonide, betamethasone, beclomethasone, methylprednisolone, deoxymethasone, triamcinolone, triamcinolone acetone-acetonefenone, acetone-acetone-acetone-acetone, acetamide, acetaminophen , prednisone, diphlorazone diacetate, clobetasol propionate, chlortiazide, hydrochlorothiazide, acetazolamide, chlortalidone, clopamide, trichloromethiazide, polythiazide, furosemide, bendroflumethiazide, benzthiazide, indapamide a, bumetanide, probenecid, ethacrylic acid, amiloride, triamtrene, spironolactone, acetazolamide, clopamide, morphine, amphetamine, methylamphetamine, mefentermin, fenfluramine, norfenfluramine, strychnine, methadamethaminetaminetaminetinamethinamidefinamidaminetinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinametinaminetin , petidine, hydrocodone, codeine, niketamide, caffeine, butorphanol, norpsefdoephedrine, synephrine, adrafinyl, bupropion, ethivivan, pseudoephedrine, methylphenidate, modafinil, amitripty ina, 3'-OH-stanozolol, 16β-OH-stanozolol, 4α-OH-stanozolol, 4β-OH-stanozolol, heptaminol, carfedone, crotetamide, cropropamide, methylephedrine, ephedrine, foledrin, amfepramone, famprofason, epesterone, testosterone, testosterone , Clenbuterol, Trenbolone, Tetrahydrogestrinone, Oxandrolone, Oralturninabol, Pemoline, Mesocarb, Selegiline, Torasemide, Xipamide, Dichlorophenamide, Metolazone, Prolintan, Cyclopentiazide, Amifenazole and Phentermine ** in Healthy Methanol, Phemerol the residue after evaporation of the averaged urine sample of healthy people who did not take drugs, and the averaged urine sample (by averaging at least 10 urine samples of healthy people who did not take drugs, and which did not contain xenobiotics and their metabolic products), then working solutions were diluted standard solutions to the appropriate concentration (μg / ml). Three types of working solutions prepared in this way are introduced into an HPLC-MS / MS system (a TSQ Quantum triple quadrupole chromatograph-mass spectrometer from Thermo Finnigan (USA) connected to a Surveyor high-performance liquid chromatograph equipped with an autosampler, high-pressure pump, and degasser Thermo 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 - 40% V; 8-9 min - 90% V; 12-18 min - 40% B, total analysis time, taking into account stabilization with the system before the introduction of the next sample is 18 minutes, Ionization at atmospheric pressure is carried out by electrospray in the registration mode of negative and positive ions.The voltage on the capillary is 3.8 kV; the temperature of the capillary is 245 ° C; the flow rate of the drying gas (nitrogen) is 0.45 l / min; gas flow (argon) in the collision chamber - 0.075 l / min; temperature in the ionization chamber 200 ° С; pressure on the atomizer - 2 atm. Detection of analytes 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), the delay time is 5 ms. Processing of the obtained data (mass spectra, characteristic ions, retention time and detection limits of the studied xenobiotics) is carried out using Xcalibur software version 1.3 of Thermo Finnigan, USA (see Table 1).

Table 1 №№ Detectable connection Retention Time (min) Like weight Precursor ion Characteristic, ions Lower detection limit, ng / ml one Amiloride 1.7 229 230 [M + H] + 171; 116 fifty 2 Acetazolamide 2.0 222 221 [M + H] - 221; 83 fifty 3 Acetazolamide 2.0 222 223 [M + H] + 164; 181 fifty four Benzthiazide 5.8 431 221 [M + H] - 308; 228 10 5 Bumetanide 8.6 364 221 [M + H] - 319; 207 twenty ... ... ... 110 Testosterone 9.6 288 289 [M + H] + 109; 97 0.5 111 Trenbolone 8.3 270 271 [M + H] + 253; 199 1,0 112 Epitestosterone 10.3 288 289 [M + H] + 109; 97 0.5 ** Note: the above xenobiotics were obtained from pharmaceutical and biomedical companies and laboratories of the Russian Federation, USA, Australia, Switzerland, Slovakia, Germany and others. The content of the main substance in the preparations in all cases exceeded 99%.

B. Sample preparation and test urine analysis

A urine sample (5 ml) is obtained from a volunteer who uses illegal drugs, 50 μl of an internal standard solution containing fluoxymesterone (50 ng / μl), mefruzide (10 ng / μl) and methyltestosterone (10 ng / μl) are added to it, alkalinized to pH 9.5-10.0 with a mixture of sodium bicarbonate and potassium carbonate (1: 2), add 5 g of ammonium sulfate and then extracted with 5 ml of diethyl ether for 10 min on an automatic extractor. It is centrifuged at 3000 rpm for 10 minutes, the organic extract is evaporated to dryness in a stream of nitrogen. The dry residue is dissolved in 100 μl of methanol, then 15 μl of the solution is introduced into the HPLC-MS / MS system with electrospray ionization at atmospheric pressure in the mode of registration of negative ions and 15 μl in the mode of registration of positive ions. The analysis is carried out with the following parameters. The flow rate of the mobile phase is 0.2 ml / min. The substances to be determined are separated by gradient elution under the conditions: 0 min - 40% B; 8-9 min - 90% B; 12-18 min - 40% V, the total analysis time, taking into account the stabilization of the system before entering the next sample, is 18 min. Ionization at atmospheric pressure is carried out by electrospray in the registration mode of negative and positive ions. The voltage on the capillary is 3.8 kV; capillary temperature - 245 ° С; flow rate of the drying gas (nitrogen) - 0.45 l / min; gas flow rate (argon) in the collision chamber - 0.075 l / min; temperature in the ionization chamber 200 ° C; spray pressure - 2 atm. Detection of analytes 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), the delay time is 5 ms.

The results are shown in Table 2.

Mass spectra, characteristic ions, retention time and detection limits of substances determined in the urine are compared with the recorded characteristics of reference substances and three additional samples (see Table 1).

table 2 No. Retention Time (min) Like weight Precursor ion Characteristic ions Lower detection limit, ng / ml Found connection one 1.9 165 166 [M + H] ⁺ 148 (10); 133 (20) 10 Ephedrine 2 1.8 299 300 [M + H] ⁺ 165 (40); 215 (20) 10 Codeine 3 2.8 178 179 [M + H] ⁺ 108 (20); 78 (30) 10 Niketamide

Thus, it was found that the urine sample contains: codeine, ephedrine and niketamide.

Examples 2-6

The analysis of urine samples was carried out as in Example 1, except that we examined urine samples taken from volunteers who used illegal drugs of various nature. The results of the analysis are presented in Table 3.

Control example

The preparation and analysis of a sample of the test urine of a volunteer who used illegal drugs was carried out from Example 1, however, additional samples were not prepared and, accordingly, when determining the presence of xenobiotics in the sample, the effect of background substances in the urine could not be taken into account. The results of the analysis are presented in Table 4.

Table 4 No. Retention Time (min) Like weight Precursor ion Characteristic ions Lower detection limit, ng / ml Found connection one 1.9 165 166 [M + H] ⁺ 148 (10); 133 (20) 25 Ephedrine 2 1.8 299 300 [M + H] ⁺ 165 (40); 215 (20) 25 Codeine 3 2,8 178 179 [M + H] ⁺ 108 (20); 78 (30) fifty Niketamide

As can be seen from the description of the given examples of the method and the control example, the inventive method provides a one-shot determination of a wide range of xenobiotics in human urine, which increases the information content and reliability of the doping control and the reduction in the duration of sample preparation.

Information sources

1. Chem. farm. Journal, 1988, T. 22, p. 622.

2. RU 2313086 C2, G01N 30/72, 2007.

3. J. Anal. Toxicol., 2005, v.29, No. 4, p. 217.

4. Rapid Communications in Mass Spectroscopy, 20 (22), pp. 3465-3476 - prototype.

5. The World Anti-Doping Code. The 2008 Prohibited List. International Standard for Laboratories. 2008.

Claims (1)

A method for determining xenobiotics in human urine during doping control, including the preparation of solutions of reference substances in an organic solvent, the preparation of an analyzed sample by extraction, evaporation of the extract and dissolution of the dry residue, chromatographic separation and mass spectral analysis of the standard solutions and the analyzed sample, as well as the registration of the obtained results with the subsequent determination of the presence of xenobiotics, characterized in that they additionally prepare three samples: the first - by averaging about urine nitrogen (at least 10 urine samples of healthy people who did not take medications and obviously did not contain xenobiotics and their metabolic products), the second - by dissolving the reference substances in a dry residue solution after evaporation of an average urine sample of healthy people who did not take medications and the third - by dissolving the reference substances in an averaged urine sample of healthy people who did not take medications, chromatographic separation and mass spectral analysis of copulating samples and record retention time indicators and intensity ratio of characteristic ions of background substances in the sample preparation, and xenobiotics is determined relative performance retention time and the intensity ratio of characteristic ions for all solutions and samples, taking into account only those values that do not coincide with the background substances.