RU2473079C1 - Method of detecting complex of xenobiotics in biological fluid during doping test and apparatus for realising said method - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: disclosed is a method of detecting a complex of xenobiotics in biological fluids during doping tests, wherein an analysed sample is divided into components by passing through a monolithic chromatographic column, wherein the flow rate is kept in the range of at least 500-1100 mcl/min; components are ionised with nitrogen in the presence of a methanol/water carrier in ratio of 20/80 and ionised components with mass difference of at least 10amu are successively extracted from the ion-orbital trap and fed for detection. The apparatus for detecting a complex of xenobiotics in biological fluid a sample input unit mounted in a temperature-controlled cabinet, an injector, a monolithic chromatographic column, an ion-orbital trap with a detector, a chemical ionisation unit with a built-in nitrogen generator placed between the output of the analysed substance from the chromatographic column and the input of the ion-orbital trap and a unit for controlling analysis progress, which is common for the system.EFFECT: enabling unambiguous detection and identification of chemical compounds and fragments thereof in arbitrary combinations with high reliability, accuracy and high rate of analysis.2 cl, 3 tbl, 1 dwg

Description

The invention relates to chromatographic determination of various chemical compounds and can be used in medicine, biology, ecology and doping control.

A known method of analyzing liquid preparations based on plant materials by chromatography, which involves the extraction of volatiles by stripping the initial sample with steam, the concentration of volatiles by extraction with a low boiling solvent followed by distillation of the solvent, and finally the determination of the components by gas or liquid chromatography [1].

The disadvantage of this technical solution is the low information content of the obtained data, in particular chromatographic spectra, which prevents the unambiguous identification of chemical compounds and their fragments in arbitrary combinations.

There is also a method for chromatographic identification of components of complex mixtures of organic compounds, including passing a substance through a system of series-connected columns filled with a sorbent of different polarity, sampling after each column, detecting on detectors of various types and identifying the substance to be determined by calculating the sensitivity coefficient and relative retention according to the data of two detectors [2].

The disadvantages of this method include the complexity of the analysis procedure, as well as the probability of obtaining false results in calculating the retention values of different columns.

There is also a method of identifying unknown substances by gas chromatography in combination with mass spectrometry. According to the indicated method, the chromatogram is recorded as a function of the retention time and the mass spectrum is recorded in a period of time corresponding to the exit of the substance from the column, and compared with the mass spectra of known substances in the database, then the retention index is determined and compared with those from the database and identify the substance by two parameters - the mass spectrum and the retention index [3].

The disadvantage of this method, despite the attractiveness of using the retention index, is the high probability of obtaining inaccurate analysis results, since the retention indices are initially tied to a specific column with certain parameters and often either are not reproduced, or on other, albeit similar equipment, are reproduced with distortion, which may lead to inaccurate interpretation of analysis results.

There is also a method of identifying unknown substances by liquid chromatography in combination with tandem mass spectrometry. According to the indicated method, the chromatogram is also recorded as a function of the retention time and the mass spectrum is recorded at a time corresponding to the exit of the substance from the column, and compared with the mass spectra of known substances in the database, the retention index is then determined and compared with those from the database data and identify the substance by two parameters - mass spectrum and retention index [4].

The disadvantage of this method is the high values of the boundary concentrations of the substances to be determined and the significant likelihood of inaccurate determination of substances close in structure due to identical fragmentation during collisional dissociation.

At the same time, the hardware used in the prototype of the process nevertheless does not allow to achieve the set technical result. The authors believe that the closest to the claimed device according to the technical characteristics and the achieved technical result of the device described in [5 and 6].

The specified device, for example [6], includes a sample entry unit mounted in a thermostat, an injector, a chromatographic column (CGC), an ion-orbital trap with a detector, connected to a common unit for automatic control of the analysis process for the system.

In this device, a sample of a test sample with a mobile liquid phase is separated into components by passing through a chromatographic column, the separated components are ionized, and ions are detected by separation through an orbital-ion trap (OIL) with the subsequent determination of the identity of each ion to a specific substance based on its analytical characteristics.

The technical result, the achievement of which the creation of this invention is directed, is to enable the unambiguous identification of chemical compounds in arbitrary combinations by increasing the accuracy of determining the characteristics of the investigated substances while increasing the threshold of sensitivity and efficiency of determination.

The technical result is achieved by the fact that the test substances are passed through a monolithic chromatographic column and the flow rate is maintained at least 500-1100 μl / min, the components are ionized in a nitrogen atmosphere in the presence of a methanol / water carrier, and the ratio of the carrier components in during the analysis, they change linearly from 20/80 to 90/10 for 5 minutes, and from the ion-orbital trap, ionized components with a mass difference of less than ^-4 least 10 amu

The authors propose an unexpected solution - to replace conventional CHC in a known device with a monolithic one. The latter allows to a certain extent to remove restrictions on the feed rate of the mobile phase (in our case, it is a mixture of methanol / water) in CHC with all the ensuing advantages:

- increase the feed rate of the test substance in the CHC to the necessary limits,

- accordingly, per unit time, increase the proton yield from the mobile phase, which allows ionizing a proportionally larger amount of the test substance,

- significantly reduce the duration of the analysis

- the ability to effectively use chemical ionization of the components of the investigated substance.

It is known that both the atomic mass and the molecular mass of substances are expressed not in whole but in fractional numbers, and, knowing the exact molecular weight of the compound, the elemental composition can be calculated (Molecular Fragment Calculator). On the other hand, if the measured mass of the protonated ion (m / z) is known, then the gross formula of the substance can be established from this mass. It should be emphasized that when determining mass up to the third sign (for example 345,253), the gross formula gives a list of three substances, one of which corresponds to stanozolol, and when determining mass up to the fourth sign (345,2533), the gross formula gives only one substance - stanozolol. It follows from the foregoing that the more accurately the mass of the protonated ion is determined during the analysis, the higher the accuracy of the determination of the desired substance.

It should be especially noted that, taking into account the foregoing, the claimed method for determining xenobiotics can be implemented both with the involvement of reference samples of the studied substances and without it.

As for the technological parameters of the implementation of the claimed technical solution, it should be noted that in tandem mass spectrometry (HPLC-MS / MS) the test sample is subjected to electrospray ionization in the registration mode of positive (or negative) ions. A characteristic feature of such an ionization process is a paradoxical phenomenon - the lower the flow rate of the eluent, the more productive the ionization process. At the same time, one should not lose sight of the fact that HPLC-MS / MS combines two processes: chromatography and mass spectrometry. And these processes are often contradictory in parameters, since the low flow rate of the eluent, although it contributes to more efficient ionization, negatively affects the selectivity.

At the same time, as mentioned above, the use of monolithic CHC allows (due to an increase in the speed of the mobile phase) per unit time to increase the proton yield from the mobile phase, which allows ionizing a proportionally larger amount of the test substance, and this leads to a more efficient use of chemical ionization of the components the investigated substance and, accordingly, to increase the sensitivity of the system. However, even in this case, an increase in the speed of the mobile phase above a certain level reduces the efficiency of the monolithic ChGK, and, accordingly, the sensitivity of the system decreases. Therefore, in the inventive method for determining xenobiotics, the parameters of the process are limited to such limits that optimally combine the implementation of chromatographic separation of the test sample and its spectral analysis.

Going beyond the declared parameters of the process leads to a deterioration in selectivity (supply of ionized components with a mass difference of 10 ^-3 to the detector), a decrease in sensitivity (a constant composition of the mobile phase, a decrease in the speed of the mobile phase or an increase beyond the declared limits), a sharp decrease in the sensitivity limit ( from 3 pg / ml to 30-80 pg / ml when replacing monolithic CHC with normal), etc.

Thus, in view of the foregoing, the device for implementing the proposed method includes a sample inlet thermostat mounted in the thermostat, an injector, CGC, an ion-orbital trap (OIL) with a detector, connected to a common unit for the automatic control of the analysis, and further includes a chemical unit ionization with a built-in nitrogen generator, located between the exit of the analyte from the chromatographic column and the entrance to the orbital-ion trap, and as a chromatographic column It comprises a monolithic chromatographic column.

Figure 1 presents a block diagram of the inventive device.

The device includes a thermostat 1, which contains a sample inlet 2, an injector 3 connected to a monolithic CHC 4, output 5 connected to a chemical ionization unit 6 with a nitrogen generator 7 attached to it. Chemical ionization unit 6 is connected in series with OIL 8 and detector 09. All the system is combined with an analysis progress control unit 10 with corresponding communications 11.

The device operates as follows.

The operating temperature of the thermostat 1 is set and, through the automatic control unit for the analysis 10, the necessary parameters of the chromatography-mass spectrometric determination of the analyzed sample are set. The analyzed sample is introduced into the sampling input unit 2, then through the injector 3 the sample enters the monolithic CHC 4, where it is divided into components, which, at the exit 5 from the column, enter the chemical ionization unit 6, into which nitrogen is supplied from the generator 7. At the same time chemical ionization 6 components of the sample are ionized (corona discharge, nitrogen atmosphere, the carrier is a mixture of methanol-water) and ions (positive) enter OIL 8, in which the ionized components of the sample are taken and fed into the detector 9, and the detection results I register in the automatic control unit of the analysis 10.

As an analytical system, for example, a Surveyor HPLC chromato-mass spectrometer combined with an OIL mass spectrometer (Bremen, Germany) and equipped with an ion source for chemical ionization with a nitrogen generator NM30LA (Peak Scientific, USA) can be used.

As a monolithic CGC, for example, PLPR-S 300 (Polymer Labs), Chromolith (Merck), Onyx Monolith C ₁₈ (Phenomenex, Germany) and other similar columns 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;

- vacuum concentrator from Barnstead Inc. (USA) for evaporation of the organic extract.

For the preparation of mobile phases and for the dissolution of comparison samples, for example, methanol for chromatography (Merck, Germany) and ultrapure water (ρ = 18.2 MΩ / cm obtained using a Direct Q, Millipore apparatus) can be used.

For liquid-liquid extraction can be used diethyl ether brand of analytical grade. (Medkhimprom, Russia).

Ionization is carried out by the action of a corona discharge in a nitrogen atmosphere at normal pressure.

Detection of detected substances is carried out in the registration mode of a full scan.

The invention can be implemented as follows.

Phosphate buffer and a solution of β-glucuronidase (Roche, Germany) are added to the studied sample of biological fluid and the sample is subjected to enzymatic hydrolysis, the hydrolyzate is cooled, a mixture of potassium carbonate and sodium hydrogen carbonate is added to a pH of 9-10, then the sample is extracted with diethyl ether and sodium sulfate. The organic extract is then centrifuged, the ether layer is evaporated to dryness, and the dry residue is dissolved in methanol. Thus prepared for analysis, the extract is then sent for chromatography-mass spectrometric determination, the parameters of which will be presented in examples of specific implementation of the claimed technical solution.

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

Example 1

Definition of xenobiotics.

The analysis is carried out on a Surveuor HPLC chromatography-mass spectrometric system combined with an OIL mass spectrometer (Bremen, Germany) and equipped with an ion source for chemical ionization with a nitrogen generator NM30LA (Peak Scientific, USA). Onyx Monolith C _{18 is} used as HCG.

Working solutions (the list of used substances is presented below in Table 1, obtained from LGC Promochem, Wesel, Germany) is prepared by dissolving xenobiotics in obviously not containing biological fluid (urine), and the content of each substance in the biological fluid depends on its class ( e.g. anabolics 10 ng / ml; beta-blockers and beta-agonists 50; diuretics 200; stimulants 100, etc.). To a urine sample (5 ml) with dissolved xenobiotic, 1 ml of phosphate buffer solution (pH 7.0) and 50 μl of β-glucuronidase solution are added and the sample is subjected to enzymatic hydrolysis (57 ° C, 3 hours), the hydrolyzate is cooled to room temperature, added it contains 100 mg of a mixture of potassium carbonate and sodium bicarbonate (2: 1) to a pH of 9-10, then the sample is extracted with 5 ml of diethyl ether with the addition of 500 mg of sodium sulfate. The organic extract is then centrifuged for 5 minutes at 2500 rpm, the ether layer is evaporated to dryness, the dry residue is dissolved in 50 μl of methanol. The extract thus prepared for analysis is then sent for chromatography-mass spectrometric determination with the following parameters: temperature of the focusing capillary - 280 ° C, current through the needle 5 μA, flow rate of the drying gas (nitrogen) 20 l / h; scanning speed 1 scan / sec; scanning range 100-500 amu; mass determination accuracy ≤2 million ^-1; Resolution 60,000 (at half height); sample volume 10 μl; mobile phase A: methanol-water 80:20, B: methanol-water 90:10; gradient elution 0-8 min: 50% B; 8-12 min: 100% B; flow rate 800 μl / min. The chromatographic and mass spectrometric characteristics of the sample are removed and recorded: retention time, molecular weight, and lower detection limit are determined. The lower detection limit is set as follows: prepare a xenobiotic solution of five different concentrations in each of 10 different biological fluid samples and analyze the 50 samples obtained. Selectivity is determined by comparing the results of the analysis of pure biological fluid (matrix) and with the addition of the corresponding xenobiotic. The results of the analysis are presented in Table 1.

Table 1 xenobiotic Gross formula Proton mass p. ion measured. Hold Time (min.) Mass Error (ppm) Lower detection limit
niya (ng) Xenobiotic Class one Boldenone C ₁₉ H ₂₆ O ₂ 07.27.2011 7.64 0.5 10 anabolic 2 Trenbolone C ₁₈ H ₂₂ O ₂ 271.1693 7.03 0.7 <0.2 anabolic 3 Anastrozole C ₁₇ H ₁₇ N ₅ 294.1719 5.33 1.2 <5 aromatase inhibitor four Dihydroeczemane C ₂₀ H ₂₆ O ₂ 299.2004 7.75 2.9 <5 aromatase inhibitor 5 Fenoterol C ₁₇ H ₂₁ NO ₄ 304.1549 2.90 1.5 <10 beta agonist 6 Atenolol C ₁₄ H ₂₂ N ₂ O ₅ 267.1709 2.22 2.4 fifty beta blocker 7 Bunolol C ₁₇ H ₂₅ NO ₃ 292.1913 6.85 1.6 <50 beta blocker 8 Cortisol C ₂₁ N ₃₀ O ₅ 363.2171 14.4 0.2 <0.1 corticotropin 9 Cortisone C ₂₁ H ₂₈ O ₅ 361.2014 15.3 0.3 <0.1 corticotropin 10 Amiloride C ₆ H ₈ N ₇ OCl 230.0552 1.60 2.2 125 diuretic eleven Ethylofrin C ₁₀ H ₁₅ NO ₂ 182.1176 6.21 3.9 <50 stimulant 12 Adrafinil C ₁₅ H ₁₅ NO ₃ S 312.0670 4.54 1/3 fifty stimulant

Example 2. Analysis of a biological fluid containing a mixture of xenobiotics.

The sample preparation procedure and analysis of the sample is carried out as in Example 1, except that the flow rate is maintained within 1000 μl / min and the biological fluid containing boldenone, trenbolone and dihydroexmestane is analyzed. The results of the analysis are presented in Table 2.

table 2 xenobiotic Gross formula Mass protonir. ion measured. Hold Time (min) Mass Error (ppm) Lower detection limit (ng) Theoretical molecular mass one Boldenone C ₁₉ H ₂₆ O ₂ 07.27.2013 7.64 0.5 10 07.27.2011 2 Trenbolone C ₁₈ S ₂₂ O ₂ 271.1691 7.03 0.7 <0.2 271.1698 3 Dihydroeczemane C ₂₀ H ₂₆ O ₂ 299.2007 7.75 2.9 <5 299.2011

Example 3 (comparative patent [4])

Acquisition of mass spectra, determination of characteristic ions, retention time and detection limits of cortisol and cortisone

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 analysis time with system stabilization the volume before the introduction of the next sample is 35 minutes Ionization at atmospheric pressure is carried out by electrospray in the mode of registration of negative 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; the gas flow rate (argon) in the impact chamber - 0.075 l / min; temperature in the ionization chamber 200 ° C; 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 3).

Table 3 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

Comparison of the results of the analysis according to the descriptions and Tables 1 and 3 clearly shows that the proposed method for detecting xenobiotics during doping control and the device for its implementation significantly exceed the known for the achieved technical result and provide the possibility of unambiguous identification of chemical compounds and their fragments in arbitrary combinations for at the same time increasing the accuracy and efficiency of determination.

Information sources

1. RU 2093822 C1, M.C. G01N 30/04, publ. 1997 year

2. RU 2069363 C1, M.C. G01N 30/02, publ. 1996 year

3. WO 2004/104571, M.C. G01N 30/00, publ. 2004 year

4. RU 2384846 C1, M.C. G01N 33/493, publ. 2010 year

5. Anal. Chem. 2000. V.72. V.6. P.1156 - prototype.

6. http://www.textronica.com/lcline/ltq_sens.html - prototype device

Claims (2)

1. A method for detecting a xenobiotic complex in biological fluids during doping control, in which a test sample is prepared, separated into components by passing through a mobile liquid phase chromatographic column, separated components are ionized, and ions are detected by separation through an orbital-ion trap, followed by determination of the identity of each ion to a specific substance on the basis of its analytical characteristics, characterized in that the separated components of the test substance are prop decompressed through a monolithic chromatographic column and the flow rate was maintained at least 500-1100 μl / min, the components were ionized in a nitrogen atmosphere in the presence of methanol / water support, and the ratio of the support components during the analysis changed linearly from 20/80 to 90/10 for 5 minutes, and from the ion-orbital trap, ionized components with a mass difference of at least 10 ^-4 amu are sequentially selected and fed for detection. 2. A device for detecting a xenobiotic complex in a biological fluid during doping control according to claim 1, including a sample inlet mounted in a thermostat, an injector, a chromatographic column, an ion-orbital trap with a detector, connected to a common unit for automatic analysis control, different the fact that it additionally includes a chemical ionization unit with a built-in nitrogen generator, located between the exit of the analyte from the chromatographic column and the entrance to the orbital a trap, and as a chromatographic column, the device includes a monolithic chromatographic column.