CN107389779B

CN107389779B - Rapid analysis method of chemical structure

Info

Publication number: CN107389779B
Application number: CN201710676249.8A
Authority: CN
Inventors: 张华俊; 吕云波
Original assignee: Suzhou Zhining Information Technology Co ltd
Current assignee: Guangzhou molecular information technology Co., Ltd
Priority date: 2016-11-01
Filing date: 2017-08-09
Publication date: 2020-05-12
Anticipated expiration: 2037-08-09
Also published as: CN107389779A

Abstract

The application provides a method for rapidly analyzing a chemical structure, which comprises the following steps: directly feeding a gas phase or liquid phase mixture into a gas phase or liquid phase mass spectrometer, then obtaining a primary mass spectrum of each component in the mixture by a soft ionization method, changing parameters to increase one or more disturbances to obtain a two-dimensional mass spectrum, and then analyzing the two-dimensional mass spectrum by a kinetic entropy minimum method to find each independent ion and a corresponding kinetic process; then, the chemical formula of each independent ion is calculated by utilizing the high-resolution mass-to-charge ratio data of the independent ions, or the structural formula of each independent ion is determined by comparing the chemical formula with the existing independent ion and kinetic database, so that the chemical formula or the chemical structure of each component in the mixture is determined. The method has the advantages of extremely low requirement on the original sample, no need of pretreatment on the sample, simple and convenient operation and accurate analysis result, and is favorable for quickly analyzing the structures of the unknown substance and the known substance in the mixture by using a gas phase/liquid phase mass spectrometer.

Description

Rapid analysis method of chemical structure

Technical Field

The application specifically relates to a method for rapidly analyzing chemical structures, which is used for rapidly finding chemical formulas or chemical structures of various unknown and known substance components in a mixture, and belongs to the field of chemical analysis.

Background

Finding the structure of unknown components in a mixture is an attractive field with a wide range of application values. For example, many unknown ingredients are unknown among various traditional Chinese medicine ingredients, and development of their medicinal value and commercial value is urgently needed. However, analyzing unknown components in a mixture is also very challenging. For example, nuclear magnetic resonance spectroscopy (NMRSpectroscopy) is a common technique in the field of chemical analysis, and the structure of an unknown substance can be obtained with high precision by analyzing the structure using the NMRSpectroscopy. However, analysis of material structure by nmr spectroscopy requires a relatively pure sample. The method usually needs dozens of kilograms of traditional Chinese medicines, and takes a lot of time, money and manpower after multiple times of separation and purification, and finally microgram-grade pure components are obtained, so that the chemical structure of the traditional Chinese medicines can be analyzed by utilizing a nuclear magnetic resonance spectroscopy. Therefore, the analysis of the components in the mixture by this method is time-consuming and labor-intensive, requires a very specialized person, and many trace components in the Chinese medicinal materials are lost or destroyed during the pretreatment process and thus cannot be found.

Mass Spectrometry (MS) is another important and common technique in the field of chemical analysis. Mass spectrometry is a chemical analysis means for analyzing various characteristics (e.g., organic matter component structure, isotope component, etc.) of a substance based on the mass-to-charge ratio (m/z) of an ionized substance by ionizing the substance, and is widely used in various fields. Mass spectrometry is a very common organic composition characterization technique. Mass spectrometers charge materials by means including soft ionization (ionization) and hard ionization (ionization). The soft ionization method is an ionization method having a relatively low energy, and therefore, during the ionization process, a small amount of fragments are generated, and molecular ions (molecular ions) are sometimes retained. Common soft Ionization methods include Chemical Ionization (CI), electrospray Ionization (ESI), Atmospheric Pressure Chemical Ionization (APCI), and the like. The hard ionization mode gives a larger energy to the sample, and high-speed electrons are adopted to impact the sample to cause chemical bond breakage or molecular rearrangement, so that a plurality of fragment ions are generated. Common hard Ionization methods include Electron bombardment (EI), and the like.

Since mass spectrometry is a detector, it is generally required to be combined with other chemical separation and analysis means to achieve more accurate separation and analysis. For example, mass spectrometry can be combined with gas chromatography or liquid chromatography (chromatography-mass spectrometry) to form a gas chromatography-mass spectrometer (abbreviated as gas mass spectrometry) or a liquid chromatography-mass spectrometer (abbreviated as liquid mass spectrometry). Chromatography separates various substances by using different adsorption forces of different substances on an adsorbent, so that a mixture is separated before entering a mass spectrum, and a mass spectrometer can analyze various components more accurately. Typically, a separate mass spectrometer is not used to analyze the mixture.

Compared with other analysis technologies, the method for analyzing the component structure of the known substance in the mixture by using the chromatography-mass spectrometry technology does not need to carry out excessive time-consuming and labor-consuming pretreatments such as purification on the known substance, can directly use the mixture for analysis, and is convenient and simple. It is very difficult to analyze the unknown substances in the mixture by using chromatographic techniques alone, because even if the unknown substances are separated by chromatography, the unknown substances cannot be identified without database alignment. In general, analysis of unknown structures requires the use of nuclear magnetic resonance or mass spectrometry. However, there are still many difficulties in analyzing the unknown substances in the mixture using mass spectrometry, such as:

1) typically, mass spectrometry is used to analyze unknown substances in a mixture, requiring the use of MSⁿThe technology is a tandem mass spectrometry technology, which brings a large amount of information, and mass spectrometry ion fragments obtained in different collision energy sections are mixed together, so that the mass spectrometry is interfered, and the difficulty of mass spectrometry is increased; in addition, as the number of secondary mass spectra increases, mass spectral information becomes more and more complex, and it becomes more and more difficult to analyze the structure.

2) The structural analysis of the gas phase composition is in fact very difficult compared to the liquid phase composition. For the analytical identification of unknown components in gas phase components, there is no effective technique today, the main reason for which is that gas phase unknown components are very difficult to enrich and purify. Furthermore, since the gas phase component is highly nonpolar and has a small molecular weight, and it is difficult to add charges to the liquid phase mass spectrum, it is difficult to detect the gas phase component with a liquid phase mass spectrometer, and thus it is very difficult to characterize the gas phase component.

3) Due to the complexity of mass spectrometry, the mass spectrometry of unknown substances is difficult to analyze, and usually requires a plurality of professional knowledge and experience, so that the analysis needs to be performed by skilled professionals, the cost is high, and the number of the professionals is small, thereby limiting the application of the mass spectrometry in the analysis of the components and the structures of the unknown substances.

4) The existing methods cannot automatically analyze with an instrument due to their high complexity and high requirements for personnel.

Disclosure of Invention

The main objective of the present application is to provide a method for rapidly analyzing chemical structure, which combines the method of minimum kinetic entropy with mass spectrometry to rapidly, accurately and automatically discover the chemical formula or chemical structure of various unknown and known components in gas/liquid phase mixture, so as to overcome the deficiencies of the prior art.

In order to achieve the above object, the present application adopts a technical solution comprising:

the embodiment of the application provides a method for rapidly analyzing a chemical structure, which comprises the following steps:

(1) feeding the gas phase or liquid phase mixture into a gas phase or liquid phase mass spectrometer;

(2) analyzing the mixture by a mass spectrometer in a soft ionization mode to obtain a primary mass spectrum of the mixture;

(3) selecting a primary mass spectrum peak of an unknown or known molecule from a plurality of ion peaks contained in the primary mass spectrum obtained in the step (2), selecting one or more parameters of the mass spectrometer during detection as disturbance conditions, setting a group of different values under the disturbance conditions, further analyzing under the different value conditions of the disturbance conditions to obtain a plurality of secondary mass spectra corresponding to the primary mass spectrum peak, and then integrating the plurality of secondary mass spectra obtained under the different disturbance conditions to obtain a two-dimensional mass spectrum data set;

(4) calculating the two-dimensional mass spectrum data set obtained in the step (3) through a kinetic entropy minimum algorithm to obtain each independent ion of the primary mass spectrum peak and a corresponding kinetic process;

(5) calculating the chemical formula of each independent ion obtained in the step (4) by using the detection result of a high-resolution mass spectrometer, or comparing each independent ion obtained in the step (4) and the corresponding kinetic process with a database of known independent ions and kinetic processes to determine the structural formula of each independent ion;

(6) and (5) deducing a chemical formula or a structural formula corresponding to the primary mass spectrum peak according to the chemical formula or the structural formula of each independent ion obtained in the step (5).

Further, the step (1) comprises the following steps: the gas phase or liquid phase mixture is directly fed into a gas phase or liquid phase mass spectrometer without any separation.

Further, in the step 1), the gas phase or liquid phase mass spectrometer has the function of obtaining more than two-level mass spectrum, and can provide various disturbances in the process of mass spectrum fragmentation at different levels.

Further, in step 1), the gas or liquid phase mass spectrometer includes, but is not limited to, a quadrupole time-of-flight mass spectrometer, a triple quadrupole mass spectrometer, an ion trap time-of-flight mass spectrometer, a tandem time-of-flight mass spectrometer, or an electrostatic field orbitrap mass spectrometer.

Further, soft ionization is adopted in the step 2) to charge various unknown and known components in the mixture

Further, the soft ionization method in step 2) includes, but is not limited to, a chemical ionization method, an electrospray ionization method, an atmospheric pressure chemical ionization method, or a matrix-assisted laser desorption ionization method.

Further, the perturbation condition adopted in step 3) includes any one or a combination of several of electric field intensity, magnetic field intensity, radiation intensity, vacuum degree, size and kind of collision molecule, and wavelength or intensity of excitation light source, but is not limited thereto.

Further, step 5) comprises: and calculating the chemical formula of each independent ion by using the high-resolution mass-to-charge ratio data of each independent ion.

Further, step 5) comprises: and analyzing different known components by a kinetic entropy minimum method to obtain independent ions and corresponding kinetic processes of the known or unknown components, and establishing a database based on the independent ions and the kinetic processes of the components.

Further, step 6) comprises: and (4) deducing the chemical formula of the corresponding unknown substance or known substance by using the chemical formula of each independent ion obtained in the step (5) and the molecular information obtained in the step (3).

Further, step 6) comprises: and (4) deducing the structural formula of the corresponding unknown substance or known substance by using the structural formula of each independent ion obtained in the step (5) and the molecular information obtained in the step (3).

Compared with the prior art, the method has the advantages that:

1) the method of the present application detects the chemical formula or structure of unknown and known components in a mixture, requiring only a few milligrams of sample, and very few samples.

2) By using the method, the mixture can be directly analyzed, and each component in the mixture sample can be analyzed without spending a large amount of manpower and material resources to carry out a large amount of pretreatment and separate and purify each component.

3) By using the method, each component can be smashed for multiple times, so that a smaller structure is obtained, and the molecular structure of the original substance is deduced after the molecular structure of the smaller structure is reduced.

4) With the method, the chemical formula or the chemical structure of each component in the mixture can be accurately analyzed by only repeating the steps 3) to 6) for the same sample.

5) When the method is used for analyzing gas phase/liquid phase substances, a computer algorithm can be added, high-resolution fragment peaks obtained by using a secondary mass spectrum are very quickly marked according to the positions of the high-resolution fragment peaks in a spectrogram and the isotope distribution of the high-resolution fragment peaks (because the molecular weight of components in a gas phase sample is small and the molecular weight of the fragments is smaller, the combination of elements such as C, H, O, N, S can be very conveniently obtained according to the high-resolution fragment peaks, and the structure of the gas phase/liquid phase substances can be quickly known). When the molecular structure of the fragment peak is obtained, the molecular formula of the parent peak (main unbroken peak) is easily reduced, so that the molecular structure information is obtained, and the chemical structure of the unknown or known substance in the mixture is found.

6) By utilizing the method, the chemical formula or structural formula of the unknown component in the gas phase/liquid phase mixture can be efficiently analyzed without the need of very experienced professionals, so that the cost is saved, and the time and the labor are saved.

Drawings

In order to more clearly explain the structural features and technical points of the present application, the present application will be described in detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a schematic diagram of a method for rapid analysis of chemical structures in one embodiment of the present application.

Fig. 2 is a two-dimensional mass spectrometry data set obtained by combining two-dimensional mass spectrometry maps obtained by dissociation of unknown molecules under different collision energies in example 2 of the present invention.

FIG. 3 is the kinetic process of the molecular fragmentation of the unknown substance in example 2 of the present invention.

FIGS. 4A-4E are mass spectra of individual ions A-E generated by fragmentation of the unknown molecule in example 2 of the present invention.

Fig. 5 is a two-dimensional mass spectrometry data set obtained by combining two-dimensional mass spectrometry maps obtained by dissociation of folate molecules under different collision gas pressures in example 3 of the present invention.

FIG. 6 is the kinetics of folic acid at different gas collision pressures.

Fig. 7 is a mass spectrum of individual ions A, B, C resulting from the breakdown of folic acid at different gas collision pressures.

Detailed Description

As described above, in view of the shortcomings of the prior art, the inventors of the present invention have conducted long-term research and extensive practice to provide a method for rapid analysis of chemical structure of the present application, the method can quickly, accurately and automatically discover and analyze the chemical formulas or chemical structures of various unknown and known components in a gas phase/liquid phase mixture by combining a kinetic entropy minimum method and a mass spectrometry analysis method, is particularly suitable for structural analysis of the unknown components in the gas phase mixture, thereby solving the problem of disturbing the analysis of the unknown gas phase components, rapidly analyzing the structural information of the unknown components based on a small amount of gas phase components, and for the analysis of the unknown components in the liquid phase mixture, the complexity of the data information of the multi-stage mass spectrum is greatly reduced, and the chemical formula or structural formula of the unknown components in the liquid phase mixture can be efficiently and accurately analyzed without professional personnel.

In summary, the present application provides a method of combining algorithms and mass spectrometry for the analysis of chemical formulas or chemical structures of unknown and known components in a mixture by first charging the various components in the mixture, preferably by soft ionization, while preserving the original molecular structure, and then using MS¹Obtaining molecular weight information of various components, changing a certain parameter to increase one or more disturbances to obtain a two-dimensional mass spectrum, analyzing the two-dimensional mass spectrum by a kinetic entropy minimum method to find independent ions and a kinetic process, and obtaining a molecular formula or a structural formula of the independent ions according to a certain rule, thereby deducing the molecular formula or the structural formula of the original molecule.

Further, a method for rapidly analyzing a chemical structure provided in an embodiment of the present application includes: and continuously and directly feeding a gas phase/liquid phase mixture sample into a gas phase/liquid phase mass spectrometer, and then obtaining a primary mass spectrum of each component in the mixture by using a mass spectrum soft ionization method. And then obtaining each secondary mass spectrum under different disturbance conditions, combining the secondary mass spectra into a two-dimensional mass spectrum, and applying a Kinetic entropy minimum method (Kinetic EM) to the two-dimensional mass spectrum for research to find independent ions and corresponding Kinetic processes in the two-dimensional mass spectrum. Then, the chemical formula of the independent ions in each secondary mass spectrum is calculated by utilizing the high-resolution mass-to-charge ratio (m/z) data of the independent particles; or comparing with the existing independent ion and dynamics database to determine the structural formula of the independent ion in each secondary mass spectrum; and then the chemical formula or chemical structure of each component in the mixture is determined.

The aforementioned Kinetic entropy minimization method (Kinetic EM) was developed based on an entropy minimization algorithm (entrypyration algorithm). Whereas The Entropy minimization algorithm (Entropy MinmizationAlgorithm) was developed based on Shannon Entropy (Shannon Entropy) (see C.E. Shannon, The Bell System technical Journal,27(1948) 379-. Marc Garland et al applied Shannon Entropy for the first time to chemical analysis (see Analytica Chimica acta 359(1998)303-310) and based thereon proposed a peak-lock Entropy Minimization method (BTEM, Band-Target Entropy Minimization, see Organometallics,21(2002) 1982-1990). The method for minimizing peak-locking entropy is mainly used for analyzing infrared mixed spectrum. In order to apply the entropy minimization algorithm to Mass spectrometry and to automatically analyze and remove the overlapped Spectrum, Zhang Hua also proposed tBTEM method (see J Am Soc Mass Spectrum,14(2003) 1295-. The multi-reconstruction entropy minimum method can be applied to mass spectrum analysis, a local optimization method is used for replacing a global optimization method, and pure spectrums are automatically searched.

To quickly and simply detect the structure of an unknown component in a mixture, zhanghuajun proposes a method for analyzing the structure of the unknown component by calculating each individual ion and the corresponding Kinetic process in a mass spectrometer by combining an entropy minimization algorithm, i.e., a Kinetic entropy minimization method (Kinetic EM, see CN 105842330A). The method for minimizing the kinetic entropy selects one or more parameters of a mass spectrometer as disturbance conditions, sets a group of different values under the disturbance conditions, detects a substance to be detected by the mass spectrometer under the different value conditions of the disturbance conditions to obtain a group of mass spectrum data (two-dimensional mass spectrum), simultaneously calculates various independent ions of the two-dimensional mass spectrum and the kinetic processes of the independent ions by applying the entropy minimization algorithm, and then analyzes the mass spectrum, thereby deducing the structural formula of the substance to be detected. The method avoids the core problem of analyzing unknown components by a common tandem mass spectrometry technology, namely the problem of mutual interference among ion information of different secondary mass spectrums, and can clearly and accurately give fragment information of each level mass spectrum to a user from complex secondary ions which are mutually interfered, so that the user can clearly know the crushing process of the unknown components, and further, the unknown components can be quickly and accurately determined.

The kinetic entropy minimum method utilizes an algorithm, and automatically discovers independent ions and corresponding kinetic processes in the crushing process without human guidance, so that the rapid analysis of the structure of an unknown substance in a mixture becomes possible. The method can be used for quickly and accurately carrying out qualitative analysis on the mixture according to the difference in the kinetic process, and is a simple method for analyzing the structure of an unknown substance no matter whether the component of the mixture is unknown or known, particularly for isomers, homologues and the like which are difficult to distinguish by a mass spectrometry technology.

The rapid analysis method for chemical structures is further developed on the basis of a kinetic entropy minimization method, and can rapidly, accurately and automatically analyze and identify the structures of unknown substances in a mixture based on a very small sample amount, particularly rapidly analyze the structures of all the unknown substances in a gas-phase mixture, thereby overcoming the long-standing problem in the field. Further, the method for rapidly analyzing a chemical structure provided by the embodiment of the present application specifically includes the following steps:

Further, an exemplary implementation process of the method for rapidly analyzing a chemical structure provided in the embodiments of the present application can be seen in fig. 1. Further, the step (1) comprises: the gas phase or liquid phase mixture is directly fed into a gas phase or liquid phase mass spectrometer without any separation.

The mode of directly advancing the gaseous phase/liquid phase mixture sample into gaseous phase/liquid phase mass spectrograph is not carried out any separation to this application, need not to separate the mixture, and the sample volume of needing is very little. Existing methods for analyzing unknown components in mixtures typically require a relatively large sample. For example, the use of NMR to analyze unknown components generally requires purer fractions and therefore requires numerous separations and purifications of the original mixture to obtain pure fractions, however, this method sometimes results in microgram-sized samples, and some unknown components are lost or destroyed even during the separation and purification process. Although the use of chromatographic techniques does not require excessive pretreatment of the mixture, such techniques can only analyze known components of the mixture, but cannot characterize unknown components. The method provided by the application can analyze the mixture by a direct sample injection mode, does not need to separate and purify the mixture to obtain a relatively pure component, only needs a few milligrams of sample, does not need to worry about that an unknown or known component is lost and damaged in the separation and purification process, and can simultaneously analyze the unknown and known components in the mixture. Therefore, compared with the existing analysis method, the method provided by the application is convenient and simple, saves time and labor, and greatly shortens the time for analyzing unknown and known components in the mixture.

Furthermore, the direct sample injection mode has another advantage. Usually, under different perturbations, obtaining a two-dimensional mass spectrum takes a relatively long time (several minutes), whereas a typical gas chromatography analysis of a mixture, with each pure component peak, only occurs in about 6-8 seconds or less; therefore, the conventional GC-MS analysis method uses the minimum kinetic entropy method to analyze, and the time window of peak emergence is not enough for a certain peak. However, the direct injection analysis method can be used for analytical analysis of the components in the sample by mass spectrometry continuously in a long time (several minutes to tens of minutes), so that the kinetic entropy minimization method can be applied easily.

When analyzing the chemical or structural formulae of unknown or known components in a mixture, the present application charges the components using a soft ionization method rather than a hard ionization method because the soft ionization method can preserve the molecular structure of the various components in the mixture. For example, when chemical ionization is used to ionize substances, then each substance is simply added CH₃ ⁺，C₂H₅ ⁺Etc., so that the original molecular structure can be preserved while ionizing it, thereby enabling Mass Spectrometry (MS) in the first order¹) The information of the original molecule is reserved, so that the method is favorable for finally splicing the independent ions back to the chemical formula or the chemical structure of the original molecule. Although the present application is equally applicable to hard ionization mass spectrometry for analyzing the structure of ions therein, hard ionization methods such as Electron Impact (EI) break up the components and only obtain ion peak-based MS of each component¹. Therefore, the generated independent ions can only be used for piecing together the structure of the fragment ion peaks, and the whole original molecular chemical formula or chemical structure cannot be obtained. In the EI ionization mode, if the voltage of EI is set very low, for example, 10V or less, molecular ion peaks can be obtained, and in this case, it can be considered as a soft ionization mode. Retains original molecular structure during ionization, and can be used in MS¹Much information is provided in the mass spectral peaks. For example, it can be selected from MS¹The molecular mass of the unknown or known component corresponding to a certain mass spectrum peak is known; secondly, the retained original molecular structure is broken down under disturbance to obtain the chemical formula and structure of the independent ion, and information of the chemical formula and structure of the original molecule is provided. Thus, the chemical formula or structure of the original molecule can be pieced back using the independent ion information and molecular mass generated by unknown or known components.

When MS is obtained¹Then, because the soft ionization mode is adopted, one MS is adopted¹The mass spectrum peak represents a component, and various components in the mixture are alongMS¹The m/z axis of (A) appears in the mass spectrum from small to large. Therefore, because the mass-to-charge ratios of the components are different, in the mass spectrum, the components in the mixture are separated, and the molecular formula or structural formula of a plurality of components in the mixture can be found from a sample by only selecting a certain ion and repeatedly applying the method for minimizing the kinetic entropy. However, this MS¹Mass spectral peaks provide only information on the mass of a molecule, but not directly on the structure of the molecule. Therefore, under the condition that one or more parameters are used as disturbance, different values of disturbance are set to further break up a certain MS¹The composition represented by the mass spectrum peak. Same MS¹Under different disturbance conditions, a plurality of secondary Mass Spectrograms (MS) can be obtained²) These MSs were combined²And (4) integrating the spectrograms to obtain two-dimensional mass spectrum data.

Because the ion mass and the ion composition of ions formed in the ion cracking process of the mass spectrum of a certain substance are fixed, the behavior of the ions is not interfered by other components, the detected amplitude change is caused only by the concentration change of the ions, and the ions can be regarded as independent ions. When it is to a certain MS¹When a plurality of secondary mass spectra are obtained by setting different values of disturbance on unknown or known components represented by mass spectrum peaks, the concentration of independent ions generated by the unknown or known components can change, but other characteristics are fixed, such as the m/z positions of the independent ions in the mass spectra and the proportion of parent ions and isotope ions. Because the behavior is independent, even if the peaks of the independent ions coincide, the independent ions can be distinguished in the two-dimensional mass spectrum. By using KineticEM, the kinetic change process of the independent ions can be obtained while each independent ion is distinguished.

After each independent ion is obtained by analysis, the chemical formula of each independent ion can be calculated by using the result of high-resolution mass spectrometry. Typically, mass-to-charge error ranges within 5ppm for high resolution mass spectrometry, so the possible chemical formulae for the isolated ion can be calculated from very accurate mass-to-charge values, or the range can be narrowed to a few possible chemical formulae. Furthermore, the chemical formula of the independent ion can be finally determined according to the information such as the proportion and the position between isotope peaks and other rules (nitrogen law). Therefore, the chemical formula of each independent ion can be calculated by combining a certain rule through high-resolution mass spectrometry. Since these rules can be expressed mathematically, the chemical formula of each individual ion can be automatically determined using a plurality of algorithms.

Even without a high resolution mass spectrometer, the structural formula of each individual ion can be found by comparison with existing individual ion and kinetic databases after obtaining the individual ion and corresponding kinetic data. By performing Kinetic EM analysis on different known components, independent ion and Kinetic data of the known components can be obtained, and an independent ion and Kinetic database based on the known components is established. Because known components are analyzed, the molecular formulas or structural formulas of the independent ions of the known components do not need to be known necessarily, but only the low-resolution spectrogram and the kinetic process of the independent ions of a certain component need to be known, and the data of the low-resolution spectrogram and the kinetic process are stored in a database. Therefore, when Kinetic EM is applied to a certain unknown substance or ion in low-resolution mass spectrum, as long as the spectrogram and the Kinetic process of the independent ion of the unknown substance or ion are known, the Kinetic EM can be compared with the existing independent ion and Kinetic database to obtain the structural formula corresponding to the mass spectrum data of the independent ion, so that the structural formula of the unknown substance or ion is obtained.

Finally, according to the chemical formula or structural formula of the obtained independent ion, and in MS¹The molecular weight of the molecular ion obtained in (1) can be estimated to determine the MS¹Chemical formula or structural formula of unknown or known component corresponding to mass spectrum peak.

In the present specification, the aforementioned "independent ions" are defined as: the mode of behavior of a component, group or fragment in a mixture is not disturbed by other components, groups or fragments. Its behavior pattern remains consistent, changing, only due to changes in its concentration causing changes in the detected amplitude (e.g., abundance), and its characteristics (e.g., pure spectrum) do not change.

Further, the mixture sample in the step 1) directly enters the mass spectrometer for analysis without separation in advance, and the direct sample injection manner includes, but is not limited to, direct insertion (direct injection), direct injection (direct injection), and the like.

Further, the mass spectrometer in the step 1) adopts a mass spectrometer which can obtain mass spectrum data with more than two levels and can provide various disturbances in mass spectrum fragmentation processes with different levels. Including Quadrupole time-of-flight mass spectrometer (Q-TOF-MS), Triple Quadrupole mass spectrometer (Triple Quadrupole MS), ion trap mass spectrometer (IT-MS), ion trap time-of-flight mass spectrometer (IT-TOF), tandem time-of-flight mass spectrometer (Triple TOF MS), electrostatic field Orbitrap mass spectrometer (LTQ-Orbitrap MS), and the like, without limitation thereto.

Further, the soft ionization method in step 2) includes, but is not limited to, Chemical Ionization (CI), electrospray ionization (ESI), Atmospheric Pressure Chemical Ionization (APCI), Matrix Assisted Laser Desorption Ionization (MALDI), and other common soft ionization methods. Further, the perturbation condition in the step 3) includes one or a combination of several of electric field intensity, magnetic field intensity, radiation intensity, vacuum degree, size and kind of collision molecule, wavelength or intensity of excitation light source, and is not limited thereto.

Wherein for MS²If the molecular weight of an independent ion in the ion is too large, the chemical formula of the independent ion cannot be determined by high-resolution mass spectrometry; or the database has no information of the independent ion, and MS for the independent ion can be adopted³Or MSⁿAnd (3) performing multiple applications of the Kinetic EM method by using the two-dimensional mass spectrum to obtain the chemical formula or structural formula of the independent ion.

Wherein, if it is desired to analyze as many components as possible in a certain mixture, it is only necessary to repeat the aforementioned steps 3) to 6). Namely, one MS is selected one by one¹Mass spectrum peak, two-dimensional mass spectrum obtained by changing certain parameter to increase one or more disturbances, information of independent ions obtained by the method of minimum kinetic entropy, and MS¹Chemical formula or structural formula corresponding to mass spectrum peak. And (3) repeating the steps 3) to 6) to obtain the chemical formulas or structural formulas of a plurality of components in the mixture.

Wherein the gas phase component has a relatively small molecular weight for the unknown component in the gas phase mixture, and therefore the mass to charge ratio of the fragment ions of the secondary mass spectrum is smaller. Since general molecules are composed of common elements such as C, H, O, N, S, Cl, and F, when each independent ion of a certain gas phase component is resolved, and the chemical formula of each independent ion is further obtained by high-resolution mass spectrometry, the corresponding structure corresponding to the chemical formula of the independent ion can be easily inferred. After obtaining the structure of these independent ions, the MS is performed according to this component¹The molecular weight information provided, the molecular formula and structure of the molecular ions are easily reduced. Thus, the molecular structure and other information of unknown or known components in the gas phase mixture can be obtained.

The present application provides only the basic concept of a method of analyzing a chemical formula or structural formula of a substance in a mixture, including but not limited to the steps described above. For example, if Electron Impact (EI) is used to charge the mixture, it can be charged in MS as well¹Individual fragment ion peaks were obtained. When wanting to know MS¹When the molecular formula or structural formula represented by one fragment peak is adopted, the fragment peak can be selected and broken under the condition that one or more parameters are used as disturbance conditions and the disturbance of different values is set, so that a plurality of MSs are obtained²Analyzing the formed two-dimensional mass spectrum data by using a kinetic entropy minimum method to obtain independent ions and a kinetic process, calculating the chemical formula of each independent ion by using a high-resolution mass spectrum, or obtaining the structural formula corresponding to each independent ion according to the comparison of an obtained independent ion and kinetic database and the existing independent ion and kinetic database, and finally calculating the MS according to the obtained chemical formula or structural formula of the independent ionⁿA molecular formula or a structural formula represented by one fragment peak. The method of the present application can therefore determine not only the chemical formula or structure of unknown and known components in a mixture, but also MSⁿThe corresponding chemical formula or chemical structure of a fragment peak in (a).

The technical solutions in the embodiments of the present application will be further described clearly and completely in the following description with reference to the drawings and the embodiments of the present application, and it is apparent that the described embodiments are some but not all of the embodiments of the present application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Unless otherwise defined, all terms used herein have the meanings commonly understood by those skilled in the art.

Example 1: gas phase composition structure rapid analysis based on gas phase mass spectrometer

On a GC-CI-Q-TOF-MS instrument from Agilent, the capillary chromatographic column used for separation in the GC instrument was detached from the GC and then connected to a short capillary tube of only 3 meters length with no separation efficiency, one end of which was connected to a 2ml sealed sample bottle (containing the liquid sample) and the capillary tube was kept above the sample level. The other end of the capillary tube is connected into the mass spectrometer. The sample vial and the capillary are disposed in a capillary chamber of a chromatograph. Setting a temperature rise program of the GC, for example, raising the temperature from 2 ℃ per minute and from 30 ℃ to 200 ℃; and mass spectrometry was started.

The liquid sample is slowly heated by the heating program of the GC, and simultaneously, the vaporized sample is directly absorbed into the mass spectrum by the negative pressure of the CI source of the mass spectrum. The composition of the inhaled gas varies with temperature. This can simplify MS since the number of components in the inspired gas is reduced (compared to the liquid sample) at each time period¹The complexity of mass spectrum can also reduce Ion Competition/inhibition (Ion Competition/Ion Suppression) and increase MS as much as possible¹Abundance of (c) in favor of the next step of MSⁿAn increase in the abundance of ions. Usually, under different perturbations, obtaining a two-dimensional mass spectrum takes a relatively long time (several minutes), whereas a typical gas chromatography analysis of a mixture, with each pure component peak, only occurs for about 6-8 seconds or less; therefore, the time window of peak appearance is not enough for a certain peak by using Kinetic EM analysis method in the conventional GC-MS analysis method. So a slow temperature ramp procedure allows enough time to observe MS¹And a certain MS is selected¹The peak of (a) is,and obtaining a two-dimensional mass spectrum under different disturbance conditions.

Selecting a certain MS¹Or MS over an m/z interval¹Peaks, which are broken up under different Collision-Induced Dissociation (CID) energies to obtain different MS²And time of flight (TOF) mass spectrometry, to obtain their high precision mass to charge ratio data.

By using Kinetic EM, a certain MS can be obtained¹Independent ion and kinetic processing of peaks and characterization of the MS¹The peaks can also analyze the components in the liquid sample one by one to obtain the structural information of the components.

Example 2: rapid analysis of composition structure based on liquid phase mass spectrometer

The experiment used a Saimer Fei LC-Quadrupole-Orbitrap-MS. An unknown substance is dissolved in a methanol solution to prepare a solution of 10ug/mL, the solution is placed in a bottle with a common solvent, and a sample is slowly injected into an ESI source by using a micro injection pump (syringe pump), wherein the injection rate is 0.1 mL/min. The mass spectrometric detection mode is ESI-.

MS containing species is obtained because the sample is slowly injected into the ESI source¹Mass spectrum of, the MS¹The mass spectrum of (A) can obtain the unknown molecular ion m/z as 440. For this MS¹Selecting different disturbance conditions to obtain a series of MS²To be analyzed, thereby. On this instrument, different CID energies (0 to 180eV) were selected to obtain a series of MSs²And (4) data. These MSs are combined²Can be combined to obtain a three-dimensional map, i.e. a two-dimensional mass spectral data set, as shown in fig. 2. Applying the EM algorithm on the two-dimensional mass spectral data set, the individual ions A, B, C, D and E, and their course of change, can be obtained, as shown in fig. 3.

As can be seen from the course of the kinetic changes, the fragmentation of this unknown was divided into five steps. The mass spectra of each individual ion shown in fig. 4A-4E may better illustrate the process of kinetic variation.

First, the independent ions a include molecular ions (molecular ions) m/z 440, and very small amounts of other ions (m/z 311, m/z 422, etc.). When the CID energy is gradually increased, the concentration of the independent ion A is gradually increased and then decreased after reaching the highest point. Next, the individual ions B are composed of a large number of ions located between m/z 250 and m/z 430, for example, 311, 422, 396, etc. It can be concluded that these ions are secondary ions formed by fragmentation of the individual ions a. The individual ions C are composed of a large number of ions located in the vicinity of m/z 175, and these ions are empirically tertiary ions formed by the continued fragmentation of secondary ions. By analogy, it can be obtained that the independent ion D is a fourth-order ion, and the independent ion E is a fifth-order ion.

Next, by careful analysis of the high resolution mass spectra, the molecular formula of the ions represented by the respective mass spectra data can be deduced. Because the error range of the mass-to-charge ratio is 5ppm by adopting the high-resolution mass spectrum, the possible chemical formula of the independent ion can be calculated according to the very accurate mass-to-charge ratio, or the range is narrowed to a plurality of possible chemical formulas.

First, from among the independent ions C, the ion peak m/z at the highest abundance was obtained as 175.0495, and the ion having this mass-to-charge ratio had only the possibility of one type of molecular species, i.e., C₇H₅N₅O^-Therefore, it is inferred that the parent molecule contains at least 7C atoms.

Then, the peak with higher abundance in independent ion B was obtained as m/z 311.0909, m/z 422.1230, m/z 396.1435, and m/z 293.0803. These ions give 3, 8, 3 possible molecular formulas, respectively. Three possible formulae where m/z is 311.0909 are: c₂H₁₅N₈O₁₀ ^-,C₁₆H₁₃N₃O₄ ^-And C₁₄H₁₁N₆O₃ ^-The former conclusion suggests that the parent molecule contains at least 7C atoms, and the latter conclusion suggests that the odd-numbered ion molecular weight and the even-numbered ion molecular weight according to the nitrogen law₁₄H₁₁N₆O₃ ^-. And eight possibilities of the peak at m/z 422.1230, excluding molecules with less than 14CAnd less than 5 molecules of N, it was concluded that only C was present in the 8 possibilities₁₉H₁₆N₇O₆ ^-And (4) correct.

Finally, the exact mass m/z of the molecular ion in the independent ion a is obtained as 440.1337, six molecular possibilities can be obtained when the mass-to-charge ratio error range is set to be within 5ppm, however, only C out of the six possibilities of the molecular ion is inferred from the previous inference, the number of C atoms obtained, and from the inference of nitrogen law₁₉H₁₈N₇O₆ ^-Is the only possibility for molecular ions. It can thus be concluded that the original formula is C₁₉H₁₉N₇O₆。

According to verification, the unknown substance is identified as folic acid molecule with the molecular formula of C₁₉H₁₉N₇O₆Consistent with the inferences of this embodiment. Thus, according to the methods of the present application, the molecular formula of the unknown can be successfully inferred.

Example 3: rapid analysis of composition structure based on liquid phase mass spectrometer

The liquid chromatography column used for the separation in the LC instrument was short-circuited (by pass) on a Shimadzu LC-IT-TOF instrument. The folic acid molecule in the previous embodiment is dissolved in methanol solution to prepare 10ug/mL solution, and the solution is placed in a bottle with solvent, and is directly pumped into an ESI source as a mobile phase without separation, wherein the injection rate is 0.2 mL/min. The mass spectrometric detection mode is ESI-.

MS according to folate molecules¹The spectrum gives the molecular ion m/z/═ 440, and on this instrument, the perturbation condition chosen is the perturbation of the collision gas (collision gas) pressure: we set the collision gas pressure from 0 to 100% with successive changes at 0.5% intervals, each lasting 500 MS, and finally obtain a set of 200 MS' s²Two-dimensional mass spectrum of the spectrum (fig. 5). Attached to, for this MS¹Other different perturbation conditions may also be selected, such as magnetic field strength, radiation intensity, size and type of collision molecule, wavelength or intensity of excitation light source, and the like, and similarly may be obtained, resulting in a series of MS²The peak of (a) is,thereby performing an analysis.

The two-dimensional data set was analyzed using an entropy minimization algorithm to obtain free standing ions A, B, C dissociated at different collision gas pressures, and their kinetic evolution (fig. 6). At the same time, a mass spectrum of the independent ion A, B, C can be obtained (fig. 7). It can be seen that under different collision gas pressures, folic acid dissociates into different independent ions, independent ion a is a molecular ion, m/z is 440, as the collision pressure increases, the molecular ion dissociates into independent ion B, and C, B contains two dissociated ions, m/z is 422 and m/z is 311, C includes m/z is 396, m/z is 378, m/z is 293, m/z is 175. As the collision pressure continues to increase, the concentration of free-standing ions B decreases and the concentration of C increases. When the collision gas pressure is very high, the C concentration also starts to decrease because neutrality is lost.

Since this example uses the IT-TOF, a high resolution mass spectrum can be obtained for each individual ion, similarly. According to the same logic of example 2, we can obtain the final chemical formula C of parent molecule ion according to the high resolution mass spectrum of each independent ion₁₉H₁₈N₇O₆ ^-。

Furthermore, the inventors have found that the chemical formula of other unknown substances can be determined accurately by analyzing the unknown substances according to the above schemes, and the accuracy of the determination can be verified by other characterization methods known in the art.

Finally, it should be noted that: the above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for rapid analysis of chemical structure, comprising:

(1) directly feeding the gas phase or liquid phase mixture into a gas phase or liquid phase mass spectrometer without any separation, wherein the gas phase or liquid phase mass spectrometer has the function of obtaining more than two-level mass spectra and can provide various disturbances in the process of mass spectrum fragmentation at different levels;

(6) deducing a chemical formula or a structural formula corresponding to the primary mass spectrum peak according to the chemical formula or the structural formula of each independent ion obtained in the step (5);

wherein the step (5) further comprises: and analyzing different known or unknown components by a kinetic entropy minimum method to obtain independent ions and corresponding kinetic processes of the components, and establishing a database based on the independent ions and the kinetic processes of the components.

2. The method for rapid analysis of chemical structure according to claim 1, characterized in that: in the step (1), the gas phase or liquid phase mass spectrometer comprises any one of a quadrupole time-of-flight mass spectrometer, a triple quadrupole mass spectrometer, an ion trap time-of-flight mass spectrometer, a tandem time-of-flight mass spectrometer or an electrostatic field orbitrap combined mass spectrometer.

3. The method for rapid analysis of chemical structure according to claim 1, characterized in that: in the step (2), various unknown and known components in the gas phase or liquid phase mixture are charged by adopting a soft ionization mode.

4. The method for rapid analysis of chemical structure according to claim 3, characterized in that: the soft ionization mode in the step (2) comprises any one of a chemical ionization method, an electrospray ionization method, an atmospheric pressure chemical ionization method, an electron bombardment ionization method and a matrix-assisted laser desorption ionization method.

5. The method for rapid analysis of chemical structure according to claim 1, characterized in that: the disturbance condition adopted in the step (3) comprises any one or combination of more of electric field intensity, magnetic field intensity, radiation intensity, vacuum degree, size and type of collision molecules, and wavelength or intensity of an excitation light source.

6. The method for rapid analysis of chemical structure according to claim 1, wherein step (5) comprises: and calculating the chemical formula of each independent ion by using the high-resolution mass-to-charge ratio data of each independent ion.

7. The method for rapid analysis of chemical structure according to claim 1, wherein step (6) comprises: and (4) deducing the chemical formula of the corresponding unknown substance or known substance by using the chemical formula of each independent ion obtained in the step (5) and the molecular information obtained in the step (3).

8. The method for rapid analysis of chemical structure according to claim 1, wherein step (6) comprises: and (4) deducing the structural formula of the corresponding unknown substance or known substance by using the structural formula of each independent ion obtained in the step (5) and the molecular information obtained in the step (3).