RU2560066C1 - Method of sample preparation of bioorganic samples - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of sample preparation of bioorganic samples, including medical ones, for determining in them of isotopic ratio ¹⁴C/¹²C and ¹⁴C/¹³C using accelerative mass spectrometer (AMS). The method comprises oxidizing carbon contained in bioorganic sample to carbon dioxide. Oxidation is carried out in the liquid phase, at that the oxidizing agent is used as hydrogen peroxide, and the catalyst - as zeolite of type ZSM-5 with iron-containing active component. Carbon dioxide released as a result of oxidation is sent for analysis on the accelerative mass spectrometer AMS.

EFFECT: expanding the range of substances analysed for the isotopic composition of carbon.

2 cl, 4 ex

Description

The invention relates to methods for sample preparation of bioorganic, 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 isotope 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.

A promising application of this method is the study of metabolic products of drugs labeled with a radioactive isotope. The use of small doses of drugs will reduce not only the radiation level, but also reduce the negative impact of the drug itself. Moreover, the ability to analyze small samples will minimize biopsies of fat, muscle, and bone tissue. The ability to safely test new drugs 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, resulting in carbon dioxide.

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. The oxidation takes place overnight at 900 ° C. (EP 1257845, A2, B01D 59/44 G01N 1/28, 20.112002). 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 combustion with a liquid phase oxidation procedure. It is planned to use hydrogen peroxide as an oxidizing agent. The oxidation process will be catalyzed by zeolite containing iron, for example Fe-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, 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 into CO ₂ , with sulfur and nitrogen are transferred from the composition of organic substances to non-volatile inorganic compounds, mainly to sulfates and nitrates, dissolved in water.

The problem is solved by the method of sample preparation of bioorganic, including medical, samples for analysis on a UMS accelerating mass spectrometer, which includes the oxidation of carbon contained in the bioorganic sample to carbon dioxide in the liquid phase, hydrogen peroxide is used as an oxidizing agent, and zeolite is used as a catalyst type ZSM-5 with an active component containing iron, carbon dioxide released as a result of oxidation is sent for analysis on a UMS accelerating mass spectrometer.

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 carbon dioxide and evacuation, followed by thawing of CO ₂ and directing the purified gas to analysis for UMS accelerating mass spectrometer.

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 zeolite, for example, Fe-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 iron compounds, 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 quite strong oxidizing agents and can carry out the oxidation of 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 KA (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, the size of which 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 allows a much greater degree of mineralization to be achieved.

The invention is illustrated by the following examples.

Example 1

In a glass reactor, 10 ml of a solution containing 5 g / l of glucose, 20 g / l of iron-containing zeolite Fe-ZSM-5 prepared according to the known method is prepared (Sashkina K A (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 proceeds with stirring for 4 hours. The emitted carbon dioxide is sent directly to the analysis for UMC.

Example 2

In a glass reactor, 10 ml of a solution containing 5 g / l of urea, 20 g / l of iron-containing zeolite Fe-ZSM-5 prepared according to the known method (Sashkina K A (2013) Hierarchical zeolite FeZSM-5 as a heterogeneous Fenton-type catalyst is prepared 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, the released 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 urea, 20 g / l of iron-containing zeolite Fe-ZSM-5 prepared according to the known method (Sashkina K A (2013) Hierarchical zeolite FeZSM-5 as a heterogeneous Fenton-type catalyst is prepared 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, 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 carbon dioxide released 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 interferon, 20 g / l of iron-containing zeolite Fe-ZSM-5 with a hierarchical pore system prepared according to the known method (Sashkina K A (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, the carbon dioxide emitted is sent directly to the analysis for UMC or act according to the method described in example 3.

Claims (2)

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, zeolite of the ZSM-5 type with an iron-containing active component is used, the carbon dioxide released as a result of oxidation is sent for analysis on an accelerating mass spectrum UMS meter. 2. 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.