RU2617364C1 - Sampling method of bioorganic samples - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a bio sample preparation methods, including medical samples for the determination of isotopic ratios are ¹⁴C/¹²C and ¹⁴C/¹³C by accelerator mass spectrometer (AMS). The process is carried out using liquid phase oxidation system comprising a hydrogen peroxide decomposition catalyst as an oxidizer, and hydrogen peroxide. Exuding from the oxidation of carbon dioxide, if necessary, expose an additional cleaning and drying by successive operations: CO₂ adsorption preconcentration, CO₂ desorption with sorbent during heating, freezing of carbon dioxide and degassing followed by defrost of CO₂ and the direction of purified gas for analysis on accelerator mass spectrometer basis. If necessary, the purified carbon dioxide is subjected to catalytic graphitization, followed by pressing and obtain carbon tablets.

EFFECT: obtaining of solid or gaseous sample for analysis at the accelerator mass spectrometer.

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Description

The invention relates to methods for preparing bioorganic samples, including medical samples, for determining the isotopic ratio of ¹⁴ C / ¹² C and ¹⁴ C / ¹³ C in them using an accelerator mass spectrometer (UMS).

It is known that the UMS method allows the measurement of the isotopic ratio (the ratio of the concentration of a rare isotope to the total concentration of an element) from 10 ^-9 to 10 ^-15 in a sample weighing from micrograms to nanograms (US 5209919, A61K 51/04, G01N 33/60, 11.05 .1993). Due to its high sensitivity, the UMC method has found application in various fields of research, such as radiocarbon dating and earth sciences.

One of the applications of this method is the study of metabolic products of medicinal and biologically active substances labeled with a radioactive isotope. The use of small doses of the substance will reduce not only the radiation level, but also reduce the negative impact of the medicinal or biologically active drug itself. Moreover, the ability to analyze small samples will minimize biopsies of fat, muscle, and bone tissue. The ability to safely test new bioorganic substances in humans will reduce time and costs by avoiding animal testing and eliminating unsuitable drugs in the early stages of testing (Hellborg R (2003) Accelerator mass spectrometry - an overview. Vacuum, 70, 365-372).

For analysis on UMC, a bioorganic sample goes through several stages of sample preparation, the result is carbon dioxide, which can be directly used for isotope analysis, or, in most cases, a carbon tablet is obtained from it.

The most effective approach for producing carbon dioxide from concentrated samples is to burn a small amount of sample (US 8642953, H01J 49/34, 02/04/2014). However, this method is not applicable at low concentrations of the analyzed substances.

If the carbon concentration in the sample, which is an aqueous solution of organic matter, is small, then the stage of drying or evaporation of the sample is necessary. The dried sample is evacuated and sealed in a glass tube containing a calcined copper oxide wire. Oxidation takes place overnight at 900 ° C. (EP 1257845, A2, B01D 59/44 G01N 1/28, 11/20/2002). The disadvantages of this method of sample preparation include the possibility of losing volatile compounds at the drying stage, as well as the possibility of incomplete oxidation of sulfur and nitrogen compounds and the need for additional purification of the resulting gas from sulfur and nitrogen oxides at the next stage.

Thus, methods for conducting liquid-phase oxidation of samples are not known in the literature, in which carbon can be converted from a liquid bioorganic sample of any concentration directly into carbon dioxide, which is then sent to UMC analysis.

The described invention is an analogue of the traditional sample preparation procedure (US 8642953, H01J 49/34, 02/04/2014) in order to expand the capabilities of the analysis of liquid samples. It is proposed to replace the first two stages of sample preparation, namely, drying the sample and burning it, with the liquid-phase oxidation procedure. It is planned to use hydrogen peroxide as an oxidizing agent. The oxidation process will be catalyzed by any compound with catalytic activity in the decomposition of hydrogen peroxide, in particular, transition metals, for example platinum and its compounds, iron and its compounds, copper and its compounds (Cu (NO ₃ ) ₃ , CuSO ₄ , etc. ) or any combination thereof, in the form of solutions or deposited on a solid matrix, in particular on a zeolite, for example Cu-ZSM-5.

The invention solves the problem of expanding the spectrum of the analyzed substances on the isotopic composition of carbon using accelerator mass spectrometry.

EFFECT: obtaining a gaseous sample for analysis on an accelerating mass spectrometer or further converting it into a carbon tablet, in more detail, converting carbon from an aqueous solution of a bioorganic sample of any concentration directly to carbon dioxide, bypassing the drying and burning stages, while volatile compounds also turn in CO ₂ , while sulfur and nitrogen are transferred from the composition of organic substances to non-volatile inorganic compounds, mainly to sulfates and nitrates, dissolved in water e.

The problem is solved by the method of sample preparation of bioorganic, including biologically active and medical, samples for analysis on an accelerating mass spectrometer UMS, which includes the oxidation of carbon contained in a bioorganic sample to carbon dioxide in the liquid phase, hydrogen peroxide is used as an oxidizing agent, and as a catalyst using a compound having catalytic activity in the decomposition of hydrogen peroxide, such as: transition metals, for example, platinum and its compounds, iron and its compounds, copper and its compounds (Cu (NO ₃ ) ₃ , CUSO ₄ , etc.) or any combination thereof, in the form of solutions or deposited on a solid matrix, in particular on a zeolite, for example, Cu-ZSM-5, Pt- ZSM-5 emitted as a result of oxidation of carbon dioxide is sent for analysis on an accelerating mass spectrometer (UMC) or to the stage of graphitization of CO ₂ to obtain a carbon tablet and subsequent analysis on UMC.

If necessary, carbon dioxide released as a result of oxidation is subjected to an additional purification and drying procedure by successive operations: adsorption of CO ₂ on the sorbent, desorption of CO ₂ from the sorbent when heated, freezing of carbon dioxide and evacuation, followed by thawing of CO ₂ and directing the purified gas to analysis for accelerator mass spectrometer or for graphitization with subsequent analysis on UMC.

It is proposed to replace evaporation, drying and burning with a liquid phase oxidation procedure. It is planned to use hydrogen peroxide as an oxidizing agent. The oxidation process will be catalyzed by any compound that has catalytic activity in the decomposition of hydrogen peroxide, for example, copper-containing zeolite Cu-ZSM-5.

The advantages of the proposed oxidation method are the possibility of preparing a liquid bioorganic sample of any concentration and the absence of an evaporation and drying stage and, as a result, eliminating the possibility of loss of volatile compounds, as well as the complete oxidation of sulfur compounds to soluble and non-volatile sulfates, nitrogen to soluble and non-volatile nitrates.

It is known that transition metals, in particular compounds of copper, platinum, are catalysts for the decomposition of hydrogen peroxide. At certain pH values, various decomposition mechanisms can be implemented. At sufficiently low pH values (<10), the reaction proceeds through the formation of hydroxyl radicals. These radicals themselves are strong oxidizing agents and can oxidize organic molecules, but most of them, as a rule, are consumed with the formation of oxygen molecules. When using a heterogeneous catalyst, an increase in the contribution of the oxidation reaction is observed compared to homogeneous systems. This is explained by the adsorption of organic substances on the surface of the catalyst, on which the formation of hydroxyl radicals occurs. Organic molecules are close to the source of the formation of hydroxyl radicals, thereby increasing the likelihood of their interaction. Adsorption sites are released as the adsorbate is oxidized (Sashkina K A (2013) Hierarchical zeolite FeZSM-5 as a heterogeneous Fenton-type catalyst. Journal of Catalysis, 299, 44-52).

The use of zeolite-based catalysts is effective in the oxidation of small molecules such as phenol, ethanol, urea, thanks to the developed micropore system. However, the oxidation of large molecules occurs only due to the external surface of the catalyst due to steric difficulties, which explains the low degree of mineralization of molecules such as lignin, whose size is approximately 20 nm. There is a need to increase the outer surface of the catalyst. There are two approaches to this: reducing the size of the catalyst particles and using a certain template in the synthesis of zeolite to create an additional system of large pores. In the first case, the catalyst particles will coagulate to form larger particles. Thus, in both cases, the formation of catalyst particles with a hierarchical pore system will occur. The use of such catalysts in the oxidation reactions of large molecules makes it possible to achieve a significantly greater degree of mineralization.

The invention is illustrated by the following examples.

Example 1

In a glass reactor, 10 ml of a solution containing 5 g / l of urea, 20 g / l of copper-containing zeolite Cu-ZSM-5 prepared by the hydrothermal method according to the known method (Sashkina K A (2013) Hierarchical zeolite FeZSM-5 as a heterogeneous Fenton- is prepared type catalyst. Journal of Catalysis, 299, 44-52) followed by deposition of the active component by ion exchange according to a known procedure (Yashnik SA (2005) Catalytic properties and electronic structure of copper ions in Cu-ZSM-5, Catalysis Today, 110, 3-4, 310-325), and 2 M hydrogen peroxide. Air is pumped out of the reactor. The reaction takes place with stirring for 4 hours.

Next, the accumulated gas mixture is passed through a quartz capillary, which contains 1 g of sorbent - calcium oxide, at a temperature of 450 ° C. After passing the system was closed and pumped using a membrane pump, then the sorbent is heated to a temperature of 900 ° C and them moved secreted CO ₂ tube with a graphitization catalyst - iron powder in an amount of 8.6 mg, with the aid of freezing with liquid nitrogen. The capillaries are interconnected by means of SMC quick couplings and Luer-Lock taps.

The drawing shows a stand for drying, cleaning and concentration of carbon dioxide with further graphitization of CO ₂ . 1 - desiccant, 2 - pressure sensor, 3 - Fe catalyst, 4 - hydrogen supply, 5 - vacuum, 6 - sorbent, 7 - desiccant, 8 - the flow of gaseous oxidation products (СО ₂ , Н ₂ О, NO ₂ , etc. d.).

After the inlet, carbon dioxide is thawed, its pressure is determined, it is frozen again at the bottom of the quartz tube and hydrogen is injected to the ratio H ₂ : CO ₂ = 2.5-3. The amount of hydrogen introduced is monitored by the pressure sensor. Then the quartz tube is thawed and the total pressure in the system is measured, after which the bottom of the tube is placed in the furnace. Removing the water formed during graphitization is carried out with magnesium perchlorate. The kinetics of graphitization are monitored by pressure changes using Honeywell or MPX 4250 sensors. After graphitization is completed, a quartz cuvette with iron is removed and the catalyst is weighed after graphitization. Carbonized iron powder is pressed into aluminum caps for UMS analysis.

Example 2

In a glass reactor, 10 ml of a solution containing 5 g / l glucose, 5⋅10 ^-3 M CuSO ₄ and 2 M hydrogen peroxide. Air is pumped out of the reactor. The reaction proceeds with stirring for 4 hours. The emitted carbon dioxide is sent directly to the analysis for UMC.

Example 3

In a glass reactor, 10 ml of a solution containing 5 g / l of clarithromycin, 20 g / l of a zeolite containing a bimetallic active component, Fe-Cu-ZSM-5, is prepared, and FeZSM-5 was obtained hydrothermally by a known method (Sashkina K A (2013 ) Hierarchical zeolite FeZSM-5 as a heterogeneous Fenton-type catalyst. Journal of Catalysis, 299, 44-52), and copper is deposited by the ion exchange method according to the well-known procedure (Yashnik SA (2005) Catalytic properties and electronic structure of copper ions in Cu -ZSM-5, Catalysis Today, 110, 3-4, 310-325), and 2 M hydrogen peroxide. Air is pumped out of the reactor. The reaction takes place with stirring for 4 hours.

Next, the accumulated gas mixture is sent to the cleaning and drying procedure as follows. The gas mixture is passed through a quartz capillary, in which 1 g of sorbent - calcium oxide or magnesium oxide, is located at a temperature of 450 ° C (in the case of magnesium oxide - at a temperature of 200 ° C). After passing through the system, the system is closed and pumped out using a membrane pump, then the sorbent is heated to a temperature of 900 ° C, the released carbon dioxide is frozen and evacuated, followed by thawing of CO ₂ and dry and pure CO _{2 are} sent for analysis to UMS.

Example 4

In a glass reactor, 10 ml of a solution containing 5 g / l of sucrose, 20 g / l of silica gel with platinum impregnated with subsequent reduction with hydrogen, Pt-SiO ₂ and 2 M hydrogen peroxide is prepared. Air is pumped out of the reactor. The reaction takes place with stirring for 4 hours.

Next, the carbon dioxide emitted is sent directly to the analysis for UMC or act according to the method described in example 1.

Example 5

In a glass reactor, 10 ml of a solution containing 5 g / l of interferon, 20 g / l of platinum-containing zeolite Pt-ZSM-5 prepared by ion exchange according to a known method (Ismagilov ZR (2009) Deep desulphurization of diesel fuels on bifunctional monolithic nanostructured Pt-zeolite catalysts, Catalysis Today, 144, 3-4, 235-250), which has a hierarchical pore system obtained by a known method (Sashkina KA (2013) Hierarchical zeolite FeZSM-5 as a heterogeneous Fenton-type catalyst. Journal of Catalysis, 299, 44-52), and 2 M hydrogen peroxide. Air is pumped out of the reactor. The reaction takes place with stirring for 4 hours.

Next, act according to the method described in example 1.

Example 6

In a glass reactor, 10 ml of a solution containing 1 g / l glucose, 20 g / l platinum-containing beta zeolite Pt-BEA, prepared by ion exchange according to a known method (Ismagilov ZR (2009) Deep desulphurization of diesel fuels on bifunctional monolithic nanostructured Pt-zeolite catalysts, Catalysis Today, 144, 3-4, 235-250), and 2 M hydrogen peroxide. Air is pumped out of the reactor. The reaction takes place with stirring for 4 hours.

Next, act according to the method described in example 1.

Example 7

In a glass reactor, 10 ml of a solution containing 5 g / l of sucrose, 20 g / l of titanium dioxide coated with Cu-TiO ₂ and 2 M hydrogen peroxide impregnated with copper is prepared. Air is pumped out of the reactor. The reaction takes place with stirring for 4 hours.

Next, the carbon dioxide emitted is sent directly to the analysis for UMC or act according to the method described in example 1.

Claims (4)

1. The method of sample preparation of bioorganic, including medical, samples for analysis on an accelerating mass spectrometer UMS, including the oxidation of carbon contained in a bioorganic sample to carbon dioxide, characterized in that the bioorganic sample is oxidized in the liquid phase, hydrogen peroxide is used as an oxidizing agent, and as a catalyst, a compound having catalytic activity in the decomposition of hydrogen peroxide is used, the carbon dioxide released as a result of oxidation is sent for analysis on an accelerating mass spectrometer UMS or on the stage of graphitization of CO ₂ to obtain a carbon tablet and subsequent analysis on UMS. 2. The method according to p. 1, characterized in that as compounds having catalytic activity in the decomposition of hydrogen peroxide, transition metals, for example platinum and its compounds, copper and its compounds, or any combination thereof, are used, in the form of solutions or deposited on a solid matrix. 3. The method according to p. 1, characterized in that the carbon dioxide released as a result of oxidation is subjected, if necessary, to an additional purification and drying procedure by successive operations: adsorption of CO ₂ on the sorbent, desorption of CO ₂ from the sorbent when heated, freezing carbon dioxide and evacuating followed by thawing of CO ₂ and directing the purified gas for analysis on an accelerating mass spectrometer. 4. The method according to p. 1, characterized in that the graphitization of carbon dioxide released as a result of oxidation is carried out by sequential operations: adsorption of CO ₂ on the sorbent at low temperature, desorption of CO ₂ from the sorbent at high temperature, freezing carbon dioxide in the catalytic graphitization zone, catalytic graphitization of CO ₂ and pressing.

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