RU2501011C2 - Calibration method of gas chromatography-mass spectrometry (gc-ms) system equipped with special software for determination of markers of microorganisms in tested sample of material of biological origin - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: qualitative and quantitative chromatography-mass spectrometric analysis of the tested biological material is performed for content of fatty acids and oxyacids; identification of a separate taxon of a microorganism is separated and performed as per its (taxon) profile and marker signs; for that purpose, specific ions of microorganism ions are determined by means of a mass fragmentography method with further determination of a full biocenosis of microorganisms (activators, taxons) in the investigated material of biological origin; applying an internal standard (template) method, components of the sample are identified as per fatty acids and oxyacids by comparing the obtained data to the standard database, and their quantitative content is determined; with that, before the measurement of a series of routine clinic samples of the tested material is carried out, a random sample of excrements is taken, thus accepting it as a reference mixture; based on the reference mixture, a chromatogram is built, and as per the obtained qualitative and quantitative characteristics of biocenosis of microorganisms of the sample of this reference mixture there determined are peaks of specific ions on time scale of retention for further calculation of composition of biocenosis of microorganisms in further series of clinic samples as per markers of those microorganisms; with that, qualitative and quantitative characteristics of biocenosis of microorganisms of the random excrement sample are taken as required and sufficient quantity characterising any tested material of biological origin, and identification of specific ion is performed based on detection of peaks of chromatogram of markers from standard (test) database.

EFFECT: improving accuracy and reliability; simplifying calibration.

13 cl, 12 dwg

Description

The invention can be used in medicine, ecology, biotechnology, veterinary medicine, crop production, food industry. It is used in the determination of microbial communities under the conditions of a prevailing amount of a substrate of biological origin, in particular for clinical routine medical analyzes of biological fluids and tissues (samples) of humans, animals and plants, as well as monitoring of therapeutic measures and correction of dysbiosis, food control and other substrates of biological origin .

The method relates to biochemical research methods using gas chromatography - mass spectrometry in mass fragmentography mode, i.e. using a method for measuring selective ions characterizing markers of microorganisms for molecular microbiological analysis.

The invention is known "A method for determining the content of ethyl alcohol and other metabolites in human blood", patent RU 2436088, publ. 12/10/2011, IPC G01N 33/48, G01N 30/02, in which the chromatograph is pre-calibrated with ethyl alcohol and other metabolites, as well as propylene glycol, using at least three calibration mixtures. The method allows to simultaneously determine the quantitative content in the human blood of ethyl alcohol, diethyl ether, acetaldehyde, acetone, methyl acetate, ethyl acetate, propyl alcohol, isobutyl alcohol, butyl alcohol, isoamyl alcohol and other metabolites by gas-liquid chromatography. However, the invention does not solve the calibration problem based on a calibration template (standard) obtained for complex microbiological communities in biological media using a limited number of components for calibration.

The invention is known "Method for the differentiation of plague and pseudotuberculosis microbes with simultaneous intraspecific differentiation of strains of the plague microbe", patent RU 2332464, publ. 08/27/2008, IPC C12Q 1/68, including the differentiation of microorganisms by comparing the amplicon size of the studied microorganism (strain) with a marker. However, the invention allows comparison of the size of the formed amplicon of the studied strains with markers only in a narrow field of application, where a mixture of amplicons of reference strains is used as a marker. In the invention, only standard markers from an existing database are used as a marker.

The invention is known "A method for identifying the causative agent of an infectious process in sterile biological environments of a macroorganism", patent RU 2146368, publ. 03.03.2000, IPC G01N 33/48, G01N 33/52, in which a quantitative chromatographic-mass spectrometric analysis of the test sample is carried out for the content of fatty acids, hydroxy acids and other chemical components and the subsequent identification of the pathogen by comparing the obtained data with a standard database. The method allows to identify the pathogen without preliminary seeding of the investigated clinical or other biological material. The sterile sample is subjected to chemical analysis by gas chromatography - mass spectrometry in mass fragmentography (GC-MS-MF) in order to detect chemical components - markers of potential pathogens contained in the biological substrate at a level of less than 1%. Recognition (identification) of microorganisms currently interacting with the immune system, of a macroorganism, is made by the presence of single markers specific to a given taxon (genus, species, group), as well as by their combination, quantitative ratio and material balance of individual sample chemicals. However, when identifying members of mixed infection and determining their concentration, a mathematical algorithm is used to analyze the superposition of part of the lipid profiles according to the composition of the lipid components of pure cultures of microorganisms, without taking into account the impact on the immune system of each individual microorganism, identification is carried out by superimposing contributions from different microorganisms and the background of the biological fluid . The basis of the calculations are only the already known calibration data and a standard database on the chemical composition of microorganisms. The invention uses only a standard data bank and does not use a calibration template (standard), made immediately before a series of measurements of samples of the studied material, which is used to calibrate the device. The disadvantage of this method is the need to verify the results of the integration of target chromatographic peaks of markers, which increases the analysis time and requires the participation of specially trained personnel. The latter is also associated with an element of subjectivity in the measurement results.

Closest to the proposed calibration method is the invention "A method for determining the generic (species) composition of the association of microorganisms", patent RU 2086642, publ. 08/10/1997, IPC C12N 1/00, C12N 1/20, C12Q 1/04, in which the chemical composition of the total biomass and the chemical composition of individual microorganisms is determined in advance and compiles a standard database. The method is based on the regularity and repeatability of the chemical composition of a certain type of microorganism and the additivity of the profiles of individual microorganisms in their total biomass. This method allows to increase the sensitivity of measurements and does not require the removal of individual strains and their growth, and the method can be applied regardless of the presence of a marker. However, the method uses initial information on the chemical composition of the total biomass determined by chromatography-mass spectrometry or chromatography with other types of detection and the chemical composition (profile) of individual microorganisms without first calibrating the device based on a calibration template (standard) made immediately before the series measuring samples of the test material that is used to calibrate this instrument. The disadvantage of this method is its inapplicability for the analysis of microbial communities, in conditions of a prevailing amount of a substrate of biological origin - for example, in biological fluids and tissues of humans, animals and plants, on food products and other similar materials.

The purpose of the proposed method is to determine the microecological status of the organism, infection and dysbiosis of the intestine and other organs of humans or animals, analysis of the composition of microbial communities, soil, silt, symbionts of plants, biotechnological products of the food industry, products and substrates of microbial processing of communal, agricultural and other wastes, and also microorganism contamination of food, pharmaceutical and other industries. The use of a calibration template (standard) for a long time for preliminary calibration of measuring instruments immediately before conducting a large series of measurements of samples of the studied biological material.

All known methods of microbiological analysis use a standard database with characteristics of markers of known microorganism taxa for fatty acids, since the composition of the fatty acids of most clinically significant microorganisms is well studied, its reproducibility is shown, and genus and species specificity are evaluated. (see Figure 1). Fatty acid profiles of many isolates found in microbial collections (e.g. Culture Collection, University ofGoteborg, Sweden) are also used for the same purposes.

Higher fatty acids, aldehydes and sterols in the composition of the cell wall with the possible assignment of them to a specific microorganism, allow specific fatty acids of a particular microorganism to be taken as a marker of this microorganism.

Each marker corresponds to a certain number of carbon atoms in the chain of the molecule, which, in turn, corresponds to the chromatographic retention time of the corresponding ion, therefore, it is possible to determine ultimately the microorganism of the studied community using selective ions using the reconstruction formula (see Figs. 2, 3, 4 the distribution of ions over time intervals in the GC-MS program for the analysis of microbial markers in samples of biological fluids and human tissues, indicating detectable markers and their corresponding micro -organisms). Chromatographic separation of the sample is traditionally carried out on a capillary column in the mode of selective ions, or mass fragmentography (MF), on a GC-MS system AT-5973 Agilent Technologies (Agilent Technologies Inc.) with periodic scanning of ions at time intervals. The time intervals and ions are selected in such a way as to selectively detect the markers of the identified types of microorganisms. In this case, traditionally use the available database of standard peak patterns on the timeline of the output of these ions. For automatic processing, the GC-MS is equipped with special software into which Method format programs are introduced to recognize chromatogram peaks from these standard peak patterns. Calibration of the device equipped with special software is carried out according to the same standard templates, however, their number is very large and there is no fixed principle of choosing from a large number of the required limited number of templates. This leads to the fact that during automatic processing these data require manual verification of peak measurements, since the peaks are often not completely separated in the chromatogram or they are comparable with the error of the device itself, since small peaks are in the vicinity of more intense ones. This, in turn, leads to false positive and false negative results. Therefore, it is necessary to calibrate the instrument before each new injection of the test sample to ensure reproducibility of the retention times of chromatographic peaks, markup of the program for MF time intervals and, therefore, to ensure the accuracy of automatic data processing. The time intervals are adjusted for the parameters of the chromatographic column and GC-MS of the user device. Standard templates are prepared according to the data of the standard database in advance from the whole set of characteristics of all microorganisms entered into the database on the basis of pure samples of these microorganisms.

If you know the necessary and sufficient number of microbial markers that characterize the microbial communities of a human, animal, plant, or any other product of biological origin that are significant for the evaluation of the studied material, then the task will be greatly simplified and the determination, as well as quantitative measurement of microorganisms in the community will be most accurate and reliable, since false positive and false negative results are excluded. The use of a fecal-based calibration template, when analyzing all the markers of the standard database, allows selecting groups of specific (selective) ions that characterize a particular marker of the microorganism that will be authentic to the task, for example, monitoring the treatment of a specific disease or determining the totality of the human microbial community, or plants, or test material with a prevailing biological component. Thus, the use of a calibration template (standard) based on an arbitrary sample of feces will make it possible to collectively determine the genus or type of microorganism present, or to exclude certain species from the list of putative microorganisms related to a specific material under study in accordance with the task of the study.

At the same time, from the whole set of characteristics of microorganisms included in the standard database, it is necessary to select such groups of markers of these microorganisms that are characterized by specific (selective) ions that will provide the necessary and sufficient number of identifiable (detectable) microorganisms for the microbial community under study in a specific sample of the original biological material, to provide the best possible definition of the community. Moreover, the combination of selected markers, and hence the parameters of determination by selective ions, will provide the most complete and reliable picture of the qualitative and quantitative characteristics of a particular microbial community, and will also allow for the most accurate monitoring of its changes in the process, for example, treatment of a specific disease.

At present. At present, traditional methods demonstrate the inability to assess the role of uncultivated microorganisms in the infectious and inflammatory process, primarily anaerobes, and also give frequent false-positive results and lead to the inability to adequately quantify (Persing, 1991; Fenollar et aL, 2006; Mikhailova et al. , 2008). The peaks corresponding to the LC of a particular marker (corresponding to a specific microorganism) have to be checked manually according to their correspondence to the following marker characteristics: absolute and relative retention time, confirmation of the presence of additional ions in them, and the ratio of ion areas. This significantly complicates and increases the cost of the study. As a result, traditional automated processing data requires manual verification of peak measurements. This is due to the fact that the chromatogram has incompletely separated peaks or these peaks are too small (i.e., commensurate with peaks from random impurities in the sample) and are adjacent to more intense peaks.

Thus, in determining the composition of the microbial community and in the quantitative measurement of microorganisms in it after reconstruction, it is necessary to ensure not only the collection of data for the bank of infectious pathogens, but also the possibility of creating a calibration template (standard) of microorganism markers, which minimizes manual editing of quantitative and qualitative characteristics of microorganisms defined for a particular sample. Moreover, the use of a calibration standard (template) obtained by the proposed method allows it (template) to be used for a long time without additional adjustment of parameters during reconstruction of the microbiological community of the sample.

Since the templates (standards), which are prepared on the basis of mixtures prepared in advance according to a certain rule, do not give a general picture of the microorganism community of the studied material (sample) taken from a specific organism or biological substance, the technology proposed by the Institute of Biomedical Problems of the Russian Academy of Sciences does not allow manual editing when processing measurement data.

A standard database of microorganism markers is created once and allows the introduction of additional data when new microorganisms are detected. This standard data bank corresponds to the standard complete set of templates corresponding to the complete set of peaks of specific (selective) ions of these markers. There are more than 200 of them, therefore, in the study of a specific biological material, the use of a full set is inappropriate and significantly increases the cost of the study.

The calibration template (standard) based on feces does not require repeated reference tests for subsequent series of measurements of the test material within at least 1 (one) month. The selection of the groups of selective ions detected by the GC-MS-MF study and the calibration of the time sequence of measurements of the selected ion groups are carried out in such a way as to measure the necessary markers according to the stated measurement task and to avoid the measurement of intense background substances of the prevailing biological fluid.

At the same time, the stages of preliminary seeding on nutrient media or the use of test biochemical materials are excluded.

In the present invention, a template is understood to mean a natural set of markers of microorganisms contained in the microorganisms themselves with specific characteristics of these markers expressed by a set of specific ions. Moreover, the template for all studied individuals and ecological niches and biotechnological products is the same.

The aim of the proposed method is to reduce the analysis time and reliability of measurements in a clinical routine analysis, monitoring of therapeutic measures and correction of dysbiosis. Achieving the goal is ensured by using one specially prepared calibration template (standard) before a series of measurements that contains the necessary and sufficient number of microbial markers characterizing the microbial communities of the human body, animal, plant, or any other product of biological origin.

The proposed method for calibrating GC-MS equipped with special software according to a calibration template based on an arbitrary sample of feces during chemodifferentiation of microorganisms, based on the quantitative determination of marker substances of microorganisms (fatty acids, aldehydes, alcohols, and sterols) allows not only monitoring the studied material in samples , but also to calculate the number of microorganisms of a particular taxon in the sample. In other words, to ensure the most adequate (authentic) definition of the microbial community inherent in a specific organism (the studied material), taking into account its change in time, i.e., to ensure the possibility of decomposition of the superposition of the entire pool of microbial markers, which makes it possible to assess the contribution from each of hundreds of microorganism species, present in the test material.

The use of a fecal-based calibration template was made possible due to the fact that feces are an ideal standard from the standpoint of analytical chemistry, because they contain the entire list of microorganisms - symbionts of humans or animals, and in a similar quantitative content to the observed composition of microbial markers in the blood, as well as microbiota of the intestinal wall or infected organs. (Luckey, 1987; Suau et al., 1999; Georgy A. Osipov, Natal'ya B. Boiko, Natal'ya F. Fedosova, Svetlana A. Kasikhina, Konstantin V. Lyadov. Comparative gas chromatography-mass spectrometry study of the composition of microbial chemical markers in feces. Microbial Ecology in Health and Disease, December 2009, Vol.21, No. 3-4, Pages 159-171.).

As a result of the proposed method for calibrating a system equipped with special software, the following technical result is achieved.

- expanding the functionality of the method, in particular:

- simultaneous measurement of the concentration of microbial markers authentic to the analyzed material, such as: blood, urine, biopsy samples and other biological fluids and tissues, as well as non-biological samples with prevailing biological material;

- the most accurate determination of the peak profiles of the chromatograms of the test sample, characterizing the state of the test material, which eliminates false positive and false negative results and expand the list of found microorganisms in the medium, i.e. to obtain more data on the presence of microorganisms in the sample, to increase the accuracy and reliability of identification of microorganisms belonging to the microbial community;

- the ability to control community microorganisms according to their most complete list, close to natural, in the material being studied, regardless of whether their presence in it is known or not

- exclude the full calibration of the scale of the device relative and absolute retention times during mass fragmentography before conducting a series of clinical studies of the material;

- reduce the analysis time in the conditions of clinical routine analysis, monitoring of therapeutic measures and correction of dysbiosis.

This technical result is achieved due to the fact that they carry out the proposed method for calibrating a gas chromatography system - mass spectrometry (GC-MS), equipped with special software, to determine microorganism markers in the studied sample of material of biological origin. The method consists in conducting a quantitative and quantitative chromato-mass spectrometric analysis of the biological material under study for the content of fatty acids and hydroxy acids, isolating and identifying an individual microorganism taxon by its (taxon) profile and marker characteristics, for which specific marker ions of microorganisms are determined by the mass method -fragmentography with subsequent identification of a complete community of microorganisms (pathogens, taxa) in the studied material of biological origin Using the method of internal standard (template), identify the components of the sample for fatty acids and hydroxy acids by comparing the data with a standard database, and determine their quantitative content, characterized in that before measuring a series of routine clinical samples of the studied material, an arbitrary sample of feces is collected, taking it as a reference mixture, a chromatogram is built on the basis of the reference mixture, and according to the obtained qualitative and quantitative characteristics of the microorganism community in the samples of this reference mixture, the peaks of specific ions are determined on the retention time scale for further calculation of the composition of the community of microorganisms in the next series of clinical samples using markers of these microorganisms, and the qualitative and quantitative characteristics of the community of microorganisms of an arbitrary sample of feces are taken as necessary and sufficient, characterizing any the studied material of biological origin, and the identification of a specific ion is carried out on the basis of detection of peak chromatograms of markers from a standard (test) database.

In particular, in the method, a device equipped with special software is calibrated against microbial markers of an arbitrary sample of feces at time intervals of their location on the time scale and peak areas.

For example, direct extraction of higher fatty acids by a chemical procedure from a sample of the reference mixture to be studied, on the basis of which time intervals for the GC-MS system are calibrated using standard test database chromatograms.

In the particular case, when implementing the method in the sample of the reference mixture and subsequent routine clinical samples, the LC and sterols are separated on a chromatograph in a high resolution capillary column, resulting in a graphic image of selective chromatograms of fatty acids and sterols using Method GC-MS software, which form a grid of marker peaks by retention time, and compare them with each other.

For example, a peak with an appropriate retention time for one or two specific ions is recorded on the chromatogram of the sample of the reference mixture, and the result is the measurement of the peak areas of the selected ions of a certain mass on a selective chromatogram (MF), identifying them as the marker of the corresponding microorganism in a routine clinical sample. In this case, the area of each peak of the chromatogram of a routine clinical sample is calculated from the chromatogram of the main ion of the marker of the sample of the reference mixture. In a particular case, the ion identification is additionally checked for authenticity by absolute and relative retention times, by the presence of confirming additional ions and the ratio of the ion areas of the identified markers only on the chromatogram of the sample of the reference mixture. Thus, the difference in the retention times of the markers relative to the values of the standard test retention times of the database chromatograms in the chromatogram of the sample of the reference mixture is taken no more than 0.1 min.

For example, in a routine clinical sample, the composition of the community of microorganisms is dynamically identified on a mass spectrometer, for which taxonomically significant fatty acids are identified on a chromatogram, identifying microorganisms by the chromatogram of fatty acids obtained on the sample of the reference mixture.

In this case, the concentration of the markers is calculated and assigned to specific microorganisms based on the calculation according to the calibration template by the formula:

N ₁ = Ai [MsV (q2 × M _sam × Ast)] / Ri ₁ ,

- where N _{1 is the} number of micro granite cells (taxon of the microorganism) per unit volume or weight of the sample of the reference mixture,

- K is a constant coefficient equal to = Mst / (q2 × M _sam × Ast) = Mst (mg) / (5.1 × 10 ^(-15) (g) × M _sam (mg) × Ast)

- Ai is the peak area of the marker,

- Mst - the number of the calibration template (standard) in the sample, in mg,

- M sam - the number of samples

- A st - peak area of the calibration template (standard)

- Ri ₁ - the percentage in% of the marker with index i in the profile of the LC of the determined microbe with number 1 (N ₁ ),

- q2 is a coefficient of 5.1 × 10 ^(-15) g, provided that 1 g of microbial biomass contains 5.9 × 10 ¹² cells of microorganisms,

- and for all subsequent routine clinical trials - according to the formula

- N ₂ = Ai × k / Ri ₂ , where

- Ai is the peak area of the i-th marker, according to which the calculations are carried out,

- Ri ₂ - content of the i-th marker (in%) in the composition of the FA of this microorganism.

For example, the chromatogram is taken as the template for the chromatograms of a subsequent series of routine clinical samples in the Method format of the GC-MS system software and the target peaks are taken in the required quantity for subsequent identification and calculation of the target peaks of the calibration template for sonva.

- In the particular case, the stability of the parameters entered into the special software of the GC-MS system is ensured by editing the characteristics of the calibration template entered in the program in the Method format, and all subsequent measurements of the characteristics of subsequent routine clinical samples and the processing of their chromatograms are carried out automatically using the program Enhanced Data Analysis.

Figure 1 - shows a table of markers for higher fatty acids, aldehydes and sterols in the composition of the cell wall with reference to the microorganisms defined for the calibration template for feces. The following notation of substances is accepted in the table: 17: 1 - 17 - the number of carbon atoms, the number after the colon - the number of double bonds; h is hydroxy acid; a, i - at the beginning means branching; sauce - cyclopropanoic acid, if the position of the hydroxyl is not indicated, then 3 - hydroxy acids.

Figure 2 - shows a table of groups of scanned ions corresponding to markers and, accordingly, defined microorganisms.

Figure 3 - shows a selective chromatogram of fatty acids (ion 87), which are markers and extracted from a pure culture of a microorganism taken from a standard database and adopted as a standard template.

Figure 4 - shows a selective chromatogram of hydroxy acids (ion 175), which are markers and extracted from a pure culture of a microorganism taken from a standard database and adopted as a standard template. In the chromatograms presented, the following designations of substances are accepted: 17: 1 - 17 - the number of carbon atoms, the number after the colon - the number of double bonds; h is hydroxy acid; a, i - at the beginning means branching; alc - at the end of the characters - alcohol, sous - cyclopropapic acid. For example, ha17 is 3-hydroxy-anteisoheptadecanoic acid, 2h24alc is 2-hydroxytetracosyl alcohol.

Figure 5 - shows the characteristics of some markers for measuring peak areas determined on a timeline. In this case, the ion retention time corresponds to the ion current signal, from which a baseline is selected for calculating the area.

Figure 6 shows the correct detection patterns for a series of calibrated measurements.

Figure 7 - shows a report of the results of the study. In the table, in order of columns, the sequence number of the marker of microorganisms, the abbreviation of the marker, the time of its peak exit, the main detection ion, and the area of the chromatographic peak in the selective chromatogram for this ion are shown from left to right.

On Fig - shows a table of the number of microorganisms, taking into account the possible origin of FAs from cells of different taxa (overlapping profiles of FAs and aldehydes).

Figure 9 - shows a table of the results of the study of microbial markers in the blood.

Figure 10 - shows a table of the results of the study of microbial markers in vaginal contents.

11 - shows a table of the results of the study of microbial markers in the biopsy of the intestinal wall.

On Fig - shows a table of the results of the study of microbial markers in the diagram of dysbiosis.

Scanning intervals are set for ion groups based on the retention times of each selective ion that are known. The peaks of the presented chromatograms are guided by the laws of their appearance on the chromatogram - i.e. absolute and relative retention times, with confirmation by additional ions and the ratio of the areas of ions. This characterizes the regularity of the location of the peaks on the timeline for fatty acids and aldehydes, which identify the marker. Chromatograms obtained on the basis of characteristics taken from a standard database based on test (pure cultures of microorganisms) biochemical preparations are taken as standard templates.

In the proposed method, Chromatograms obtained on the basis of characteristics taken from an arbitrary analysis of feces are taken as calibration templates.

On equipment, for example, an AT 5973 gas chromatography mass spectrometer (gas chromatograph with a mass selective mass production detector, manufactured by Agilent Technologies Inc., using gas chromatography combined with mass spectrometry (GC-MS), markers are detected in samples of the studied biological material.

For this, the GC-MS system is equipped with special software, for example, the Enhanced Data Analysis program and the Method format program.

Chromatograms obtained on the basis of characteristics obtained from an arbitrary sample of feces are taken as calibration templates for a series of samples performed on a specific task - either determining the community of microorganisms in the material, or its further monitoring.

By a series of samples is meant - all samples taken over a certain period of time from the N-th number of patients or from the N-th amount of test material, or all samples taken for a period of time determined by the doctor from one patient to monitor treatment.

The characteristics of the calibration template are retention times, including absolute and relative retention times, the presence or absence of additional ions and the ratio of the areas of ions that are used for the automatic identification of selective ions in the series of studies of samples of biological material, as well as the qualitative composition of the microbial community, contained in the calibration template.

The data of measurements of arbitrary samples of feces serve as a template for the accurate output of a regular GC-MS system integration program to the target peaks of mass fragmentograms to be integrated.

Using an arbitrary sample of feces, a calibration pattern of peaks is determined on a time scale that characterizes the necessary and sufficient list of microorganisms - symbionts of humans or animals, taking into account their quantitative content to the observed composition of microbial markers.

The holding times of the fecal-based calibration template along with other parameters are entered into the Method format program in accordance with the software resources of the GC-MS system.

The timeline is calibrated according to the time intervals of the release of ions also on the basis of a fecal-based calibration template.

Thus, they significantly reduce the time for automatic processing of the obtained data in a series of samples performed on a specific task due to the "enumeration" of only necessary and sufficient markers, and also eliminate false positive and false negative results due to the specified time intervals of the ions and peak areas of the chromatogram as a result calibration of the GC-MS system based on a fecal calibration template. In this case, further manual verification of the truth of the found characteristics in the sample is excluded. Calibration of the GC-MS system is carried out only once before the measurement of this series of samples. Within a month, this calibration template can be used. Therefore, the series of samples in which the community of microorganisms is determined can be increased accordingly. During this period of time, for example, in one month, in particular, it is possible to repeatedly take tests from the patient under monitoring.

All chromograms of a series of samples are processed automatically using the corresponding option of a standard data processing program. In Agilent Technologies instruments, this is the "Calculate" option in the "Quantitate" menu of the Enhanced Data Analysis program and in accordance with the manual for using the GC / MS system, including the standard report stage in the sheets of the EXCEL table.

The data obtained are entered into tables with formulas for the reconstruction of the microbial community, after which tables of the qualitative and quantitative composition of microorganisms of the community are obtained in comparison with the norm. During the reconstruction of the microbial community, the well-known method is used (see Beloborodova N.V., Osipov G.A. Homeostasis of small molecules of microbial origin and its role in the relationship of microorganisms with the host organism. // Vestnik RAMS, 1999, No. 7, p.25- 31.)

Thus, to calibrate the timeline and introduce refined calibration templates into the software for further automatic processing of the detected chromatogram peaks, quantitative calculation and identification of markers, and reconstruction of the microbiological community in the sample:

- from an arbitrary sample of feces, direct extraction of higher fatty acids (FA) and / or sterols is carried out using a chemical procedure;

- carry out the separation of LC and sterols on a chromatograph in a high resolution capillary column, resulting in a graphic image of selective chromatograms of fatty acids and sterols using Method GC-MS software, which form a network of marker peaks by retention time;

- fix the peak with the corresponding retention time for one or two specific ions,

- get the result of measuring the peak areas of the selected ions of a certain mass on a selective chromatogram (MF), identifying them as a marker of the corresponding microorganism

- the area of each peak is calculated by the chromatogram of the main marker ion

- additionally check the accuracy of ion identification by absolute and relative retention times, the presence of confirming additional ions and the ratio of the ion areas of the identified markers in this case

- take as a calibration standard the result of measurements of the chromatogram of the feces sample, taking the microbial community of the calibration standard (template) for the necessary and sufficient amount characterizing any studied material of biological origin, and the identification of a specific ion is carried out on the basis of detection (identification) of microbial markers from a standard database according to test standard patterns

- analyze the composition of the marker in dynamic mode on a mass spectrometer, for which they identify taxonomically significant fatty acids for chromatographic identification of microorganisms by fatty acids

- and calculate the concentration of markers and assign them to specific microorganisms of the calibration template according to the microbial community reconstruction formulas (see Technology approved by Roszdravnadzor "Assessment of the microecological status of a person by chromatography-mass spectrometry" (Resolution FS 2010/038 of February 24, 2010); and Osipov, G.A. Chromatography-mass spectrometric analysis of microorganisms and their communities in clinical samples for infections and dysbiosis./ Chemical analysis in medical diagnostics. - M .: Nauka, 2010. - P.293-368.)

- make a conclusion about the presence of identified microorganisms and determine their quantitative content in an arbitrary sample of feces, provided that the peak area of the marker is proportional to its concentration, and, consequently, the concentration of the corresponding microorganism.

- In the future, to determine the microbial community in a series of samples obtained from the studied material, which may be blood, urine, other biological fluids, the identification and quantitative calculation of markers is carried out on the basis of a calibration template obtained on the basis of an arbitrary sample of feces.

The final results of a series of clinical samples on the quantitative composition of the identified microorganisms are compared with the norm of its content in biological fluid or tissue (Jantzen, Bryn 1985; MeNabb et al., 1997; Weyant, 1999). And the qualitative composition of the community of microorganisms defined in a series of clinical samples is compared with the qualitative (nomenclature) composition of the community of microorganisms defined in an arbitrary sample of feces, which was used to obtain a calibration template.

Calibration of a gas chromatography - mass spectrometry (GC-MS) system equipped with special software and the further use of a calibration template based on fecal samples are used to determine the markers of microorganisms in the samples studied as follows.

1. A series of clinical analyzes of biological fluids or tissues for the content of microbial markers is being prepared in accordance with the Technology “Assessment of the Microecological Status of a Person by Chromatography-Mass Spectrometry” approved by the Federal Service for Healthcare (Resolution FS 2010/038 of February 24, 2010) - hereinafter simply “Technology”.

2. In parallel or in advance, prepare a calibration standard from an arbitrary sample of feces. To do this, using the well-known technology, simple fatty acids, hydroxy acids, fatty aldehydes and sterols necessary for complete calibration of the scale of relative and absolute retention times during mass fragmentography of a daily series of clinical tests are isolated from an arbitrary sample of feces. The calibration standard is suitable for calibration within a week.

3. Find markers of microorganisms for specific (selective) ions.

4. Determine retention times together with other parameters provided by the Technology in a series of clinical samples.

5. Enter the received data into the program of the Method format in accordance with the software resources of the GC-MS system. Since modern GC-MS systems, when used correctly, ensure parameter stability for months, the daily calibration process consists only in a small editing of the Method template.

6. Chromatograms are processed automatically using the appropriate option of the standard data processing program in Agilent Technologies instruments.

7. The following analyzes of clinical samples of this series are carried out automatically in the GC-MS system.

8. Issue a standard report in the sheets of the EXCEL table.

9. The analysis result is automatically printed in the form of a table containing a list of microorganisms, their number in comparison with the norm and accompanied by a graphical image of the result in the form of a histogram.

An express determination of a wide range of pathogens of the infectious process and control of dysbiosis in the biological environments of a macroorganism with external standardization by a close to the natural microbial community has not been carried out before.

To obtain the result in a series of clinical samples, the peak areas of the target ions are automatically determined using the method of determining the qualitative composition of the community of microorganisms optimized on an arbitrary sample of feces.

The proposed method is confirmed by the following examples, which do not cover all possible applications of the proposed method.

Example 1. A blood test for microecological status.

For analysis, whole blood in an amount of 40 μl is pipetted into a 1.5 ml vial with a screw cap with a teflon gasket, dried (with the cap removed) in an oven at 80 ° C with the addition of 40 μl of methanol to accelerate drying. 400 μl of 1M hydrochloric acid in methanol is poured onto the thickened sample, screwed tightly with a cap, and subjected to acid methanolysis at 80 ° C for 1 hour. To a cooled reaction medium was added 300 ng of a standard (tridecanoic acid deuteromethyl ester) dissolved in hexane. Then, extraction is carried out in two portions of 200 μl of hexane, shaking the mixture on a vortex and allowing it to settle for 5 minutes at room temperature. The combined extract was transferred to a clean vial, dried for 5-7 minutes at 80 DC and the dry residue was treated with 20 μl of N, O-bis (trimethylsilyl) trifluoroacetamide for 15 minutes at 80 ° C with the lid closed. 80 μl of hexane are added to the reaction mixture and, when analyzed using an autosampler, the mixture is transferred to a conical insert, which is placed in the same vial in which the silylation was carried out and screwed tightly on it with a lid. In this form, the sample is suitable for analysis within a week, if it is hermetically closed, and its evaporation does not occur. When manually entering the sample, a conical insert is not needed. For analysis, a mixture of esters in an amount of 2 μl is injected into the GC-MS injector of the system and analyzed using the Method program, for example, chromatographic separation of the sample is carried out on a capillary column with an HP-5ms methyl-silicone grafted phase Agilent Technologies with a length of 25 m and an inner diameter of 0.25 mm The carrier gas is helium. The analysis mode is programmed, the heating rate of the column thermostat is 7 ° C / min in the range of 135-320 ° C. Exposure at an initial temperature of 1.5 minutes Evaporator temperature 280 ° C, interface 250 ° C. The obtained chromatograms are processed automatically using the corresponding option of the standard data processing program. In Agilent Technologies instruments, this is the "Calculate" option in the "Quantitate" menu of Enhanced Data Analysis. The feces file taken at the beginning of the series of analyzes is used as a stencil to automatically search for target peaks in the working (raw) blood sample file.

The integrator report is removed in the corresponding software option of the GC-MS system. In Chemstation Agillent Instrumentation Software, the integrator report is shown in FIG. 7.

The data are automatically transferred to the group of formulas for calculating the number of microorganisms, taking into account the possible origin of FAs from cells of different taxa (overlapping profiles of FAs and aldehydes), which are shown in Fig. 8.

Next, the final report is automatically generated on the composition of microorganisms reconstructed by markers in the microecological status of the patient in comparison with the average norm (see Fig. 9).

The homeostasis of microbial markers in the blood and the adequacy of its profile to the composition of the intestinal microbiota of a healthy person, discovered as a result of systematic studies (4), provided a unique opportunity to monitor the state of the intestinal microbiota by a noninvasive express method - by blood analysis. Since the lipid components of dying microorganisms from other organs also enter the blood, it can be considered an express method for determining the microecological status of higher organisms.

Example 2. Analysis of a vaginal smear with nonspecific vaginitis.

The sample is prepared analogously to example 3 in the same way as blood - dried 40 μl of the sample (the entire sample, if the smear is on glass) and is subjected to acid methanolysis. To scan the peak areas of the target ions, use the feces file, first recorded in a series of analyzes as a matrix with which the exit times, search intervals and additional identification parameters are read. The integrator's report is removed in the appropriate software option of the GC-MS system and injected in the converted form into the EXCEL template for reconstruction of the microbial community of the vaginal smear. The result of the analysis is presented in a form similar to example 1. See Figure 10.

Example 3. Detection of dysbiosis by microbial markers in the biopsy of the intestinal wall.

A biopsy sample of the intestinal wall obtained during colonoscopy or intestinaloscopy in an amount of 4-8 mg is weighed immediately after receipt and stored until analysis in a frozen form at a temperature (-5 ° C) and below. For analysis, the sample is subjected to acid methanolysis as in example 3, without preliminary drying. To collect data and automatically integrate the peak areas of the target ions, a feces file is used, which was first taken in a series of analyzes as a matrix with which the exit times, search intervals and additional identification parameters are read. The integrator's report is removed in the appropriate software option of the GC-MS system and injected in the converted form into the EXCEL template for reconstruction of the microbial community of the vaginal smear. The result of the analysis is presented in a form analogous to example 1. See 11.

In addition, the same analysis data is presented in the form of dysbiosis, when the norm column data is subtracted from the patient’s measurement data column. In this case, the vertical grid line with the coordinate "0" - becomes the norm. Deviation in the positive direction - excessive growth of microorganisms, in the negative direction - deficiency of microbiota. See FIG. 12.

The proposed method provides high-precision determination of specific marker molecules, fatty acids, sterol aldehydes, which are part of the cell walls of microorganisms. Using a highly sensitive and selective method of gas chromatography - mass spectrometry (GC-MS), the proposed calibration method allows you to simultaneously determine and measure in the sample the presence and concentration of more than a hundred microbial markers. The analysis is carried out without additional repeated measurements of standard tests and additional growth of strains from the studied association using nutrient media. Identification and measurement is carried out directly in the analyzed biological material (sample), for example, blood, urine, biopsy specimens and other biological fluids and tissues, receiving immediately the measurement result, bypassing the preliminary seeding stage on nutrient media or using test biochemical materials. This provides the ability to monitor treatment in clinical practice.

Thus, the proposed method of calibration according to a calibration template (standard) obtained from feces ensures its use in medicine and consists in the qualitative and quantitative determination of molecular markers of microorganisms directly in biological fluid or tissue (sample) in order to determine the microecological status of the organism and its deviations from homeostasis (dysbiosis) as well as the identification or clarification of the etiology of the infectious and inflammatory process in any nosological forms of diseases in the clinical practice. When determining the microbial community in the test sample of biological material that characterizes, for example, food products, it is easy to determine the degree of suitability of the product for consumption by the composition and number of microorganisms in the sample.

Claims (13)

1. A method of calibrating a gas chromatography system - mass spectrometry (GC - MS), equipped with software, for determining microorganism markers in a test sample of a material of biological origin, which consists in conducting a qualitative and quantitative chromatographic-mass spectrometric analysis of the studied biological material on the content of fatty acids and hydroxy acids, isolate and identify an individual taxon of a microorganism by its (taxon) profile and marker characteristics, for which separate specific marker ions of microorganisms by mass fragmentography followed by the identification of a complete community of microorganisms (pathogens, taxa) in the test material of biological origin, using the internal standard method (template), identify the components of the sample for fatty acids and hydroxy acids by comparing the data with a standard database, and determine their quantitative content, characterized in that before measuring a series of routine clinical samples of the studied material, an arbitrary sample of feces is collected, taking it as a reference mixture, a chromatogram is built on the basis of the reference mixture, and, using the obtained qualitative and quantitative characteristics of the microorganism community, samples of this reference mixture are used to determine the peaks of specific ions on the retention time scale for further calculation of the composition of the microorganism community in the subsequent a series of clinical samples on the markers of these microorganisms, with qualitative and quantitative characteristics of the community of microorganisms arbitrary faeces samples taken as a necessary and sufficient quantity that characterizes the monitoring of any material of biological origin, and the identification of the specific ion is carried out on the basis of the detection peaks of the chromatogram from the standard markers (test) database. 2. The method according to claim 1, characterized in that they calibrate the device equipped with special software for microbial markers of an arbitrary sample of feces at time intervals for their location on the time scale and peak areas. 3. The method according to claim 1, characterized in that the direct extraction of higher fatty acids by means of a chemical procedure from the sample of the reference mixture to be studied, based on which time intervals are calibrated for the GC - MS system on standard test database chromatograms. 4. The method according to claim 1, characterized in that in the sample of the reference mixture and subsequent routine clinical samples, the LC and sterols are separated on a chromatograph in a high resolution capillary column, resulting in a graphic image of selective chromatograms of fatty acids and sterols using Method GC software - MS systems that form a grid of marker peaks by retention time, and compare them with each other. 5. The method according to claim 1, characterized in that a peak with an appropriate retention time for one or two specific ions is fixed on the chromatogram of the sample of the reference mixture, and the result is the measurement of the peak areas of the selected ions of a certain mass on a selective chromatogram (MF), identifying them as a marker the corresponding microorganism in a routine clinical trial. 6. The method according to claim 1, characterized in that the area of each peak of the chromatogram of a routine clinical sample is calculated from the chromatogram of the main ion of the marker of the sample of the reference mixture. 7. The method according to claim 1, characterized in that they additionally check the reliability of the identification of the ion by the absolute and relative retention times, the presence of confirming additional ions and the ratio of the ion areas of the identified markers only on the chromatogram of the sample of the reference mixture. 8. The method according to claim 1, characterized in that the discrepancy in the retention times of the markers relative to the values of the standard test retention times of the chromatograms of the database in the chromatogram of the sample of the reference mixture take no more than 0.1 minutes 9. The method according to claim 1, characterized in that in a routine clinical sample, the composition of the community of microorganisms is dynamically identified on a mass spectrometer, for which taxonomically significant fatty acids are identified on a chromatogram, identifying microorganisms by the chromatogram of fatty acids obtained on the sample reference mixture. 10. The method according to claim 1, characterized in that they calculate the concentration of markers and assign them to specific microorganisms based on the calculation according to the calibration template by the formula:
N ₁ = Ai [Mst / (q2 · M _sam · Ast)] / Ri ₁ ,
where N ₁ is the number of cells of microorganism (taxon of the microorganism) per unit volume or weight of the sample of the reference mixture;
K is a constant coefficient equal to k = Mst / (q2 · M _sam · Ast) = Mst (mg) / (5.1 · 10 ^(-15) (g) · M _sam (mg) · Ast);
Ai — marker peak area;
Mst is the number of calibration template (standard) in the sample, mg;
M _sam is the number of samples;
Ast is the peak area of the calibration template (standard);
Ri ₁ is the percentage in% of the marker with index i in the LC profile of the determined microbe with number 1 (N ₁ );
q2 - coefficient equal to 5.1 · 10 ^(-15) g, provided that 1 g of microbial biomass contains 5.9 · 10 ¹² cells of microorganisms,
and for all subsequent routine clinical trials - according to the formula
N ₂ = Ai · k / Ri ₂ ,
where Ai is the peak area of the i-th marker, according to which the calculations are carried out;
Ri ₂ - content of the i-th marker%, in the composition of the FA of this microorganism. 11. The method according to claim 1, characterized in that the chromatogram is taken as a template for the chromatograms of a subsequent series of routine clinical samples in the Method format of the software of the GC – MS system. 12. The method according to claim 1, characterized in that the peaks of specific ions are selected in the required amount for subsequent identification and calculation based on the peaks of the chromatogram of the markers of the calibration template. 13. The method according to claim 1, characterized in that the accuracy of the parameters entered into the special software of the GC-MS system is ensured by using the updated characteristics of the calibration template entered into the program in the Method format and all subsequent measurements of the characteristics of subsequent routine clinical samples and processing of their chromatograms is carried out automatically using the Enhanced Data Analysis program.

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