Mass spectrum system and method integrating online pretreatment and dynamic biological evaluation

Abstract

The invention belongs to the field of sample analysis, and provides a mass spectrum system and method integrating online pretreatment and dynamic biological evaluation. The system comprises a sample on-line pretreatment part which is used for obtaining a sample mother solution to be analyzed and separating and preparing concerned components in the sample mother solution; the dynamic biological evaluation part is used for performing multi-channel cell culture and real-time microscopic observation on concerned components in the sample; the dynamic biological evaluation part outputs at least two paths, and one path is connected with the multi-source chromatography-mass spectrometry analysis part so as to finish dynamic biological evaluation for analyzing related components at a cellular level; the other path is synchronously connected to a functional target plate site, and matrix-assisted laser desorption mass spectrometry and surface desorption mass spectrometry are carried out; the system control and data processing part is used for carrying out qualitative and / or quantitative analysis on the components of the metabolite of the dynamic biological evaluation part, and detecting sample injection mass spectrograms, sample difference metabolites and protein molecules in all treatment links of the sample.

Description

Technical Field

[0001] The present invention belongs to the field of sample analysis, and in particular relates to a mass spectrometry system and method integrating online pretreatment and dynamic biological evaluation. Background Art

[0002] The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] The separation and analysis of multiple components in complex samples and biological evaluation are of great significance for identifying the material basis composition of samples and studying the biological activity-toxicity effects. In the field of natural product research, especially in the field of medicinal plant research, the systematic screening, identification and activity evaluation of active substances can effectively accelerate the process of drug development and provide favorable technical support for the development of more effective drugs; in the field of food, the identification and biosafety evaluation of multiple components in food can provide important information for food industry research and development and safety assurance; in the field of environmental analysis, the screening and toxicity identification of pollutants in environmental samples, especially emerging pollutants and unknown pollutants, are crucial for environmental protection and pollution control.

[0004] However, in the separation and analysis stage, traditional solutions are mostly carried out in an offline step-by-step manner, which has the characteristics of complex and cumbersome operation process, offline operation in each stage, and high consumption of resources such as reagents; especially when facing the analysis of trace components in small volume samples, there are problems such as process environmental interference, potential contamination and component loss; in the biological evaluation stage, cell metabolite analysis and evaluation is an important way of biological evaluation, and in vitro cell culture is one of the most important technical means at present, but at this stage, biological evaluation cell culture is mainly based on ordinary static culture, which cannot effectively reflect the state of the cell microenvironment in the body. The reported toxicology, full chemical analysis and biological evaluation machines still use static culture in the evaluation part; in the analysis and identification stage, the commonly used online mass spectrometry system ion source is mainly based on a single setting, and switching requires cumbersome tuning and optimization of the system. When dealing with biological systems with coexistence in different mass ranges such as small molecule metabolites, peptides, nucleic acids or protein macromolecules, it still cannot fully meet the needs of actual sample analysis work. Summary of the invention

[0005] In order to solve the technical problems existing in the above-mentioned background technology, the present invention provides a mass spectrometry system and method integrating online pretreatment and dynamic biological evaluation, which can realize the sequential separation and analysis of mixed components in the sample and the dynamic biological evaluation.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: A first aspect of the present invention provides a mass spectrometry system integrating online pre-processing and dynamic biological evaluation.

[0007] A mass spectrometry system integrating online pretreatment and dynamic biological evaluation, comprising: a sample online pretreatment unit, a dynamic biological evaluation unit, a multi-source chromatography-mass spectrometry analysis unit, and a system control and data processing unit; The sample online pretreatment unit is used to perform time- and space-resolved sequential separation, enrichment, purification and basic preparation of multiple components according to the sample type, so as to obtain the sample mother solution to be analyzed and the separation and preparation of the components of interest therein; the sample online pretreatment unit outputs two paths through the first multi-way separation valve, which are respectively connected to the multi-source chromatography-mass spectrometry analysis unit and the dynamic biological evaluation unit; The dynamic biological evaluation unit is used to perform multi-channel cell culture and real-time microscopic observation of the components of interest in the sample; the dynamic biological evaluation unit outputs at least two paths through the second multi-way separation valve, one of which is connected to the multi-source chromatography-mass spectrometry analysis unit to complete the dynamic biological evaluation of the relevant components at the cellular level; the other is synchronously connected to multiple sites on the target plate to perform matrix-assisted laser desorption mass spectrometry analysis and surface desorption mass spectrometry analysis of proteins respectively; The system control and data processing unit is connected to the dynamic biological evaluation unit and the multi-source chromatography-mass spectrometry analysis unit, and is used to perform qualitative and / or quantitative analysis on the components of the metabolic fluid of the dynamic biological evaluation unit, and detect the injection mass spectra and sample differential metabolites and related differential proteins in all sample processing links.

[0008] As an embodiment, in the sample online pretreatment section, when the sample is a biological sample, the sample to be tested is extracted and enriched by magnetic solid phase extraction, then washed with a washing liquid, and then eluted with an eluent to extract the compound to be analyzed.

[0009] As an implementation mode, in the sample online pretreatment section, when the sample is a biological specimen and the sample amount reaches a set threshold, solid phase extraction and solvent extraction are used for extraction according to solid and liquid, respectively.

[0010] As an embodiment, in the sample online pretreatment section, when the sample is a natural product, high-speed countercurrent chromatography is used for separation preparation and online analysis after extraction.

[0011] As an embodiment, the dynamic biological evaluation unit includes a multi-channel injection pump module, a microscope module, a microfluidic chip / organ chip cell culture module and a micro solid phase extraction module; the multi-channel injection pump module mixes the cell culture fluid with the components of interest in the sample online and then introduces them into the microfluidic chip / organ chip cell culture module, the microscope module is used to observe the status of the cells in the chip, and the micro solid phase extraction module is used to perform online pre-treatment of cell metabolites, which, after treatment, enter the multi-source chromatography-mass spectrometry analysis unit for dynamic monitoring.

[0012] As an implementation mode, the dynamic biological evaluation unit further includes a CO2 control module and a temperature control module, and both the CO2 control module and the temperature control module are connected to the microfluidic chip / organ chip cell culture module.

[0013] As an embodiment, the multi-source chromatography-mass spectrometry analysis unit is composed of a liquid chromatograph and a high-resolution mass spectrometer integrated into one.

[0014] A second aspect of the present invention provides a method for operating a mass spectrometry system that integrates online preprocessing and dynamic biological evaluation.

[0015] A method for operating a mass spectrometry system integrating online preprocessing and dynamic biological evaluation, comprising: The sample online pretreatment unit performs time- and space-resolved sequential separation, enrichment, purification, and basic preparation of multiple components according to the sample type, obtains the sample mother solution to be analyzed and the separation and preparation of the components of interest therein, and divides them into two paths, one of which is transmitted to the dynamic biological evaluation unit, and the other is transmitted to the multi-source chromatography-mass spectrometry analysis unit; The dynamic biological evaluation unit performs multi-channel cell culture and real-time microscopic observation on the components of interest in the sample, and outputs at least two channels, one of which is connected to the multi-source chromatography-mass spectrometry analysis unit to complete the dynamic biological evaluation of the relevant components at the cellular level; the other channel is synchronously connected to multiple sites on the target plate to perform matrix-assisted laser desorption mass spectrometry analysis and surface desorption mass spectrometry analysis of proteins respectively; The system control and data processing unit receives data transmitted from the dynamic biological evaluation unit and the multi-source chromatography-mass spectrometry analysis unit, performs qualitative and / or quantitative analysis on the components of the metabolic fluid of the dynamic biological evaluation unit, and detects the injection mass spectra and sample differential metabolites and related differential proteins in all sample processing links.

[0016] The beneficial effects of the present invention are: The sample online pretreatment section of the present invention realizes the time and space resolved sequential separation, purification and preparation of multiple components in complex samples; the dynamic biological evaluation section completes the dynamic biological evaluation of analysis-related components at the cellular level; the multi-source chromatography-mass spectrometry analysis section is coupled to the sample online pretreatment section and the dynamic biological evaluation section, realizing the identification and quantitative analysis of each component of the sample and the detection of cell differential metabolites and proteins; finally, the system control and data processing section realizes the overall control of the system, data storage analysis and dynamic online evaluation.

[0017] Advantages of additional aspects of the present invention will be given in part in the following description, and in part will become obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] Figure 1 It is a schematic structural diagram of the online pretreatment and dynamic biological evaluation mass spectrometry analysis system of the present invention; Figure 2 This is a specific structural example of the present invention in which the output end of the sample online pretreatment unit is connected to the multi-source chromatography-mass spectrometry analysis unit. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0021] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0022] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0023] In the present invention, terms such as "upper", "lower", "left", "right", "front", "back", "vertical", "horizontal", "side", "bottom", etc. indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings. They are relational words determined only for the convenience of describing the structural relationships of the various parts or elements of the present invention, and do not specifically refer to any part or element in the present invention and should not be understood as limitations on the present invention.

[0024] In the present invention, terms such as "fixed connection", "connected", "connection", etc. should be understood in a broad sense, indicating that it can be fixedly connected, integrally connected or detachably connected; it can be directly connected or indirectly connected through an intermediate medium. Relevant scientific research or technical personnel in this field can determine the specific meanings of the above terms in the present invention according to specific circumstances, and they should not be understood as limiting the present invention.

[0025] The traditional mass spectrometry system is difficult to transfer samples between various links. There are problems such as cross-contamination, mutual interference and loss in the direct transfer of samples in multiple links. The present invention pre-classifies different samples and reserves a separate pretreatment port for subsequent system rewashing. The traditional mass spectrometry system has low sensitivity in biological evaluation and analysis of samples. The dynamic evaluation process requires real-time or timed detection. The concentration levels of differential proteins and metabolites are low, requiring convenient real-time collection and high-sensitivity analysis technology. The online micro-solid phase extraction purification of the present invention is mainly aimed at small molecules generated by the integrated evaluation part. The specific macromolecules to be measured are transferred to the online target plate for in-situ subsequent processing and analysis. In the analysis and identification link of traditional mass spectrometry systems, different types of molecules require different analysis techniques, and how to integrate multiple ion sources into a set of instrument system platforms is a difficult point. The present invention sets multiple ion sources on a platform through guide rails, and calls different source modules for analysis according to the characteristics of the analyzed samples and the requirements of experimental conditions.

[0026] like Figure 1 As shown, a mass spectrometry system integrating online pretreatment and dynamic biological evaluation according to an embodiment of the present invention comprises: a sample online pretreatment unit, a dynamic biological evaluation unit, a multi-source chromatography-mass spectrometry analysis unit, and a system control and data processing unit; The sample online pretreatment unit is used to perform time- and space-resolved sequential separation, enrichment, purification and basic preparation of multiple components according to the sample type, so as to obtain the sample mother solution to be analyzed and the separation and preparation of the components of interest therein; the sample online pretreatment unit outputs two paths through a first multi-way separation valve (such as a three-way separation valve), which are respectively connected to the multi-source chromatography-mass spectrometry analysis unit and the dynamic biological evaluation unit; The dynamic biological evaluation unit is used to perform multi-channel cell culture and real-time microscopic observation of the components of interest in the sample; the dynamic biological evaluation unit outputs at least two channels through a second multi-channel separation valve (such as a six-channel separation valve), one of which is connected to the multi-source chromatography-mass spectrometry analysis unit to complete the dynamic biological evaluation of the relevant components at the cellular level; the other channel is synchronously connected to multiple sites on the target plate to perform matrix-assisted laser desorption mass spectrometry analysis and surface desorption mass spectrometry analysis of proteins respectively; The system control and data processing unit is connected to the dynamic biological evaluation unit and the multi-source chromatography-mass spectrometry analysis unit, and is used to perform qualitative and / or quantitative analysis on the components of the metabolic fluid of the dynamic biological evaluation unit, and detect the injection mass spectra and sample differential metabolites and related differential proteins in all sample processing links.

[0027] In this embodiment, the sample online pretreatment unit is used to achieve the phase transformation of the target components in different types of samples, mainly to complete the extraction, purification, separation and basic purification preparation of the samples to be analyzed, which includes three modules, among which the pretreatment module-1 and the pretreatment module-2 are mainly used for extraction and separation, and the pretreatment module-3 is used for purification and preparation. The pretreatment multiple modules can be connected to the chromatography-mass spectrometer through an interface in sequence or separately to achieve real-time monitoring or detection. For example, the pretreatment part is mainly composed of automatic (magnetic) solid phase extraction, high-speed countercurrent chromatography, preparative chromatography, gel chromatography and quantitative concentrator.

[0028] Specifically, in the sample online pretreatment section, when the sample is a biological sample, the sample to be tested is extracted and enriched by magnetic solid phase extraction, then washed with a washing liquid, and then eluted with an eluent to extract the compound to be analyzed.

[0029] In the sample online pretreatment section, when the sample is a biological specimen and the sample amount reaches a set threshold, solid phase extraction and solvent extraction are used for extraction according to solid and liquid, respectively.

[0030] In the sample online pretreatment section, when the sample is a natural product, high-speed countercurrent chromatography is used for separation preparation and online analysis after extraction.

[0031] In some specific embodiments, the dynamic biological evaluation unit includes three modules, evaluation module-1 is a multi-channel microfluidic chip module, evaluation module-2 is an organ chip, and evaluation module-3 is a quality control module. Each module is independently controlled and is equipped with a CO2 (5%) control unit, a cell culture unit, an injection pump liquid delivery unit, a cell injection and metabolite extraction unit, and a chromatographic pretreatment, thereby realizing dynamic cell culture on the chip, real-time microscopic observation, and automated extraction and determination of metabolites.

[0032] For example, the dynamic biological evaluation unit includes a multi-channel injection pump module, a microscope module, a microfluidic chip / organ chip cell culture module and a micro solid phase extraction module; the multi-channel injection pump module mixes the cell culture fluid with the components of interest in the sample online and then introduces them into the microfluidic chip / organ chip cell culture module, the microscope module is used to observe the status of the cells in the chip, and the micro solid phase extraction module is used to perform online pre-treatment of cell metabolites, and after treatment, they enter the multi-source chromatography-mass spectrometry analysis unit for dynamic monitoring.

[0033] It should be noted here that the microfluidic chip / organ chip cell culture module integrates the microfluidic chip and the organ chip into a cell culture box.

[0034] In other embodiments, the dynamic biological evaluation unit further includes a CO2 control module and a temperature control module, and both the CO2 control module and the temperature control module are connected to the microfluidic chip / organ chip cell culture module.

[0035] In some optional embodiments, the multi-source chromatography-mass spectrometry analysis unit is composed of an integrated liquid chromatograph and a high-resolution mass spectrometer. For example, the high-resolution mass spectrometer is equipped with multiple ion sources such as electrospray, micro-liquid junction surface sampling ionization, desorption electrospray, atmospheric matrix-assisted laser desorption, and dielectric barrier discharge, and is integrated in a module, which can be quickly switched to achieve analysis of different substances. Among them, the mass spectrometry unit ion source component is a multi-source integrated type, which is convenient for switching and control. After ionization, the mass spectrometry inlet is connected to the differential ion migration component, which can synchronously complete the isomer gas phase ion separation. The chromatography-mass spectrometry part is used to complete the pre-treatment sample detection and the detection of the metabolic fluid of the biological evaluation part; the data processing and system control part is composed of a workstation and a display, which is used to complete the collection, sorting, analysis and system control of comprehensive data.

[0036] Countercurrent chromatography combined with chromatography-mass spectrometry and comprehensive evaluation as an example Figure 2 Description: After the extraction liquid is obtained through pretreatment, the mobile phase 2 sample is injected into the countercurrent chromatography 4 for efficient separation through the injection pump 1 and the injection valve 3, and UV 5 monitoring is carried out simultaneously. After passing through the splitter 6, it enters the mass spectrometer 7 and the collection unit 8 respectively; each separated component is identified online through the mass spectrometer 7, and the high-concern components are passed through the collection unit 8, directly or after the next step of processing; enter the biological evaluation part, and perform dynamic biological evaluation on the high-concern components in the sample, observe the survival status of the cells online, and detect the differential proteins and metabolites in the cells acted by the components and the control samples.

[0037] Due to the adoption of the above technical scheme, the present invention has the following advantages: the device is composed of four parts, including an online sample pretreatment unit, a dynamic biological evaluation unit, a multi-source chromatography-mass spectrometry analysis unit, and a system control and data processing unit. The online sample pretreatment unit realizes the time and space resolution sequential separation, purification and preparation of multiple components in complex samples; the dynamic biological evaluation unit completes the dynamic biological evaluation of related components at the cellular level; the multi-source chromatography-mass spectrometry analysis unit is coupled to the pretreatment unit and the dynamic biological evaluation unit to realize the identification and quantitative analysis of each component of the sample and the detection of cell differential metabolites and proteins; finally, the system control and data processing unit realizes the overall control of the system, data storage analysis and dynamic online evaluation.

[0038] The present invention provides an intensive device system for the analysis of compounds in complex systems and biodynamic evaluation of activity-toxicity screening, and has strong application and promotion value in complex sample analysis, especially in the screening of active substances in natural products.

[0039] The sample pretreatment module of the present invention adds a magnetic solid phase extraction module suitable for trace sample extraction and analysis, especially for trace clinical and biological samples. With the help of the high extraction efficiency of this module, specific target objects and targeted metabolites can be detected, thereby improving the applicability and extensiveness of the instrument system.

[0040] The biological evaluation of the present invention adopts microfluidics or organ chips for dynamic cell culture, highly simulates the dynamic microenvironment in vivo, and can integrate multiple functional units. The cell status can be observed microscopically in real time, and metabolites are dynamically extracted and measured. It has the advantages of low reagent consumption, fast speed, high throughput and sensitivity, etc.

[0041] The source area unit of the mass spectrometry module of the present invention integrates various types of ion sources and tandem differential ion migration modules, so it can complete the ionization of various types of molecules and the separation of ions; at the same time, it has an imaging ion source mobile system and a rapid in-situ analysis ion source, which reduces the dependence on various types of instruments and equipment and realizes one-stop separation analysis and in-situ analysis.

[0042] The data acquisition and data analysis processing system of the present invention integrates external device control and mobile software units on the basis of conventional acquisition software, and adds machine learning discriminant model algorithm software, which can realize rapid modeling and discriminant analysis of a variety of in-situ real-time mass spectrometry data.

[0043] In one or more embodiments, a method for operating a mass spectrometry system integrating online preprocessing and dynamic biological evaluation includes: The sample online pretreatment unit performs time- and space-resolved sequential separation, enrichment, purification, and basic preparation of multiple components according to the sample type, obtains the sample mother solution to be analyzed and the separation and preparation of the components of interest therein, and divides them into two paths, one of which is transmitted to the dynamic biological evaluation unit, and the other is transmitted to the multi-source chromatography-mass spectrometry analysis unit; The dynamic biological evaluation unit performs multi-channel cell culture and real-time microscopic observation on the components of interest in the sample, and outputs at least two channels, one of which is connected to the multi-source chromatography-mass spectrometry analysis unit to complete the dynamic biological evaluation of the relevant components at the cellular level; the other channel is synchronously connected to multiple sites on the target plate to perform matrix-assisted laser desorption mass spectrometry analysis and surface desorption mass spectrometry analysis of proteins respectively; The system control and data processing unit receives data transmitted from the dynamic biological evaluation unit and the multi-source chromatography-mass spectrometry analysis unit, performs qualitative and / or quantitative analysis on the components of the metabolic fluid of the dynamic biological evaluation unit, and detects the injection mass spectra and sample differential metabolites and related differential proteins in all sample processing links.

[0044] Among them, when the sample is a biological sample, the sample to be tested is extracted and enriched by magnetic solid phase extraction, and then washed with eluent, eluted with eluent, and the compound to be analyzed is extracted; when the sample is a biological sample and the sample amount reaches the set threshold, solid phase extraction and solvent extraction are used for solid and liquid respectively; when the sample is a natural product, high-speed countercurrent chromatography is used for separation, preparation and online analysis after extraction.

[0045] Specifically, the working process of the mass spectrometry system integrating online pretreatment and dynamic biological evaluation is as follows: Step 1: According to the type of sample and the basic physical and chemical properties of the relevant components, combined with the preset separation, analysis and / or biological evaluation specific goals, select the corresponding module in the pre-treatment part to extract, separate, basically purify and prepare the sample; Step 2: If the sample is a natural medicine, it is extracted with an organic solvent, separated and prepared by countercurrent chromatography, and a six-way valve is used to set different positions of the quantitative loop for injection or component collection; Step 3: To monitor the above pretreatment steps, each module has a separate three-way diverter valve / or six-way valve for diversion, which is connected to the chromatograph-mass spectrometer and the direct analysis ion source respectively to conduct a preliminary evaluation of the pretreatment effect; collect downstream processing; or return to the previous step; Step 4: Components that meet the requirements for biological evaluation enter multi-channel online dynamic biological evaluation, which is mainly based on microfluidic chips and / or organ chips, combined with different cells or combinations; the culture medium and corresponding components are respectively delivered into the chip flow path through the injection pump, and the culture medium, components and cells are mixed on the chip. The whole process can be observed and the survival status of the cells recorded under a microscope; the metabolic fluid of the cells exposed to the target components is further drawn by the injection needle and can be divided into several paths according to needs, for example, divided into three paths, one of which is subjected to microcolumn solid phase treatment and then subjected to chromatographic column separation and mass spectrometry detection; another path is directly deposited on conductive glass, and then subjected to atmospheric pressure matrix-assisted laser desorption mass spectrometry analysis; the third path can be selected to be subjected to online purification / enzymatic hydrolysis and then to nanoflow liquid phase-mass spectrometry analysis; Step 5: The data processing and system control part detects the mass spectra of all the samples in the separation, analysis and preparation processes, and performs qualitative and / or quantitative analysis on the components of the metabolite in the dynamic biological evaluation part. The data analysis system performs statistical analysis on the collected data to measure the differential metabolites and related differential proteins of the samples. At the same time, the real-time analysis data can be combined with machine learning for classification or discriminant model construction; Qualitative: Based on the primary spectrum data, secondary spectrum fragment ions and fine structure obtained by high-resolution mass spectrometry, combined with the properties of the research system, deduction is carried out, and existing databases are searched and compared; Quantification: For specific targets, standard linearity is established in advance, and the concentration level in the solution is calculated using the peak area and linear equation; Differential protein and metabolite detection: Based on the obtained spectra, differential metabolites and differential proteins are identified for each group of data using known statistical software; Real-time mass spectrometry is directly analyzed, from samples directly to mass spectrometry. After obtaining spectra through batch sample analysis, analytical discriminant models for different groups of samples are established, and the data are divided into training sets and test sets. Any of the following machine learning algorithms can be used to discriminate the model, such as (1) support vector machine with linear kernel (SVM-L), (2) SVM radical kernel (SVM-R), (3) SVM sigmoid kernel (SVM-S), (4) SVM polynomial kernel (SVM-P), (5) random forest (RF), (6) K-nearest neighbor (KNN), (7) linear discriminant analysis (LDA), (8) penalized discriminant analysis (PDA), (9) extreme gradient boosting (XGBoost), (10) adaptive boosting (AdaBoost), (11) naive Bayes (NB), (12) logistic regression (LR), (13) decision tree (DT) and (14) partial least squares discriminant analysis (PLS-DA). For unknown samples, the model can be brought in to achieve differentiation.

[0046] The mass spectrometry system integrated with online pretreatment and dynamic biological evaluation proposed by the present invention can realize online pretreatment, analysis and dynamic biological evaluation of various types of samples, and obtain the structure and biological activity information of the components or groups of interest. The system provides an intensive device system, which can reduce the consumption of resources such as reagents in complex sample analysis, especially the screening of active substances in natural products, functional ingredients of food, medicines and environmental analysis, and has high analysis efficiency and strong application and promotion value.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A mass spectrometry system integrating online preprocessing and dynamic biological evaluation, characterized in that: include: Sample online pretreatment department, dynamic biological evaluation department, multi-source chromatography-mass spectrometry analysis department and system control and data processing department; The sample online pretreatment unit is used to perform time- and space-resolved sequential separation, enrichment, purification and basic preparation of multiple components according to the sample type, so as to obtain the sample mother solution to be analyzed and the separation and preparation of the components of interest therein; the sample online pretreatment unit outputs two paths through the first multi-way separation valve, which are respectively connected to the multi-source chromatography-mass spectrometry analysis unit and the dynamic biological evaluation unit; The dynamic biological evaluation unit is used to perform multi-channel cell culture and real-time microscopic observation of the components of interest in the sample; the dynamic biological evaluation unit outputs at least two paths through the second multi-way separation valve, one of which is connected to the multi-source chromatography-mass spectrometry analysis unit to complete the dynamic biological evaluation of the relevant components at the cellular level; the other is synchronously connected to multiple sites on the target plate to perform matrix-assisted laser desorption mass spectrometry analysis and surface desorption mass spectrometry analysis of proteins respectively; The system control and data processing unit is connected to the dynamic biological evaluation unit and the multi-source chromatography-mass spectrometry analysis unit, and is used to perform qualitative and / or quantitative analysis on the components of the metabolic fluid of the dynamic biological evaluation unit, and detect the injection mass spectra and sample differential metabolites and related differential proteins in all sample processing links.

2. The mass spectrometry system integrating online preprocessing and dynamic biological evaluation as claimed in claim 1, characterized in that: In the sample online pretreatment section, when the sample is a biological specimen, the sample to be tested is extracted and enriched by micro solid phase extraction or magnetic solid phase extraction, then washed with a washing liquid, and then eluted with an eluent to extract the compound to be analyzed.

3. The mass spectrometry system integrating online preprocessing and dynamic biological evaluation as claimed in claim 1, characterized in that: In the sample online pretreatment section, when the sample is a biological specimen and the sample amount reaches a set threshold, solid phase extraction and solvent extraction are used for extraction according to solid and liquid, respectively.

4. The mass spectrometry system integrating online preprocessing and dynamic biological evaluation as claimed in claim 1, characterized in that: In the sample online pretreatment section, when the sample is a natural product, high-speed countercurrent chromatography is used for separation preparation and online analysis after extraction.

5. The mass spectrometry system integrating online preprocessing and dynamic biological evaluation as claimed in claim 1, characterized in that: The dynamic biological evaluation section includes a multi-channel injection pump module, a microscope module, a microfluidic chip / organ chip cell culture module and a micro solid phase extraction module; the multi-channel injection pump module mixes the cell culture fluid with the components of interest in the sample online and then introduces them into the microfluidic chip / organ chip cell culture module, the microscope module is used to observe the status of the cells in the chip, and the micro solid phase extraction module is used to perform online pre-treatment of cell metabolites, and after treatment, enter the multi-source chromatography-mass spectrometry analysis section for dynamic monitoring.

6. The mass spectrometry system integrating online preprocessing and dynamic biological evaluation as claimed in claim 5, characterized in that: The dynamic biological evaluation unit also includes a CO2 control module, which is connected to the microfluidic chip / organ chip cell culture module.

7. The mass spectrometry system integrating online preprocessing and dynamic biological evaluation as claimed in claim 5, characterized in that: The dynamic biological evaluation unit also includes a temperature control module, which is connected to the microfluidic chip / organ chip cell culture module.

8. The mass spectrometry system integrating online preprocessing and dynamic biological evaluation as claimed in claim 1, characterized in that: The multi-source chromatography-mass spectrometry analysis unit is composed of a liquid chromatograph and a high-resolution mass spectrometer integrated into one body.

9. A working method of a mass spectrometry system integrating online preprocessing and dynamic biological evaluation according to any one of claims 1 to 8, characterized in that: include: The sample online pretreatment unit performs time- and space-resolved sequential separation, enrichment, purification, and basic preparation of multiple components according to the sample type, obtains the sample mother solution to be analyzed and the separation and preparation of the components of interest therein, and divides them into two paths, one of which is transmitted to the dynamic biological evaluation unit, and the other is transmitted to the multi-source chromatography-mass spectrometry analysis unit; The dynamic biological evaluation unit performs multi-channel cell culture and real-time microscopic observation on the components of interest in the sample, and outputs at least two channels, one of which is connected to the multi-source chromatography-mass spectrometry analysis unit to complete the dynamic biological evaluation of the relevant components at the cellular level; the other channel is synchronously connected to multiple sites on the target plate to perform matrix-assisted laser desorption mass spectrometry analysis and surface desorption mass spectrometry analysis of proteins respectively; The system control and data processing unit receives data transmitted from the dynamic biological evaluation unit and the multi-source chromatography-mass spectrometry analysis unit, performs qualitative and / or quantitative analysis on the components of the metabolic fluid of the dynamic biological evaluation unit, and detects the injection mass spectra and sample differential metabolites and related differential proteins in all sample processing links.

10. The working method of the mass spectrometry system integrating online preprocessing and dynamic biological evaluation as claimed in claim 9, characterized in that: When the sample is a biological sample, the sample to be tested is extracted and enriched by magnetic solid phase extraction, and then washed with an eluent, and then eluted with an eluent to extract the compound to be analyzed; When the sample is a biological sample and the sample volume reaches the set threshold, solid phase extraction and solvent extraction are used for extraction according to solid and liquid respectively; When the sample is a natural product, high-speed countercurrent chromatography is used for separation, preparation and online analysis after extraction.

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