Metabolin detection method based on liquid chromatography-mass spectrometry

Abstract

The invention discloses a liquid chromatography-mass spectrometry-based metabolite detection method, which comprises the following steps of: firstly, obtaining information such as retention time, parent ions and daughter ions of a metabolite standard substance by using ultra-high performance liquid chromatography-high resolution mass spectrometry; and then monitoring the intensity of metabolite ion pairs in a specific retention time window by using a multi-reaction monitoring mode (schemed MRM) of ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, optimizing mass spectrum parameters, and finally establishing a metabolite database with high coverage and high sensitivity. The relative abundance of various metabolites such as amino acid, nucleotide and sugar in animal and plant tissues and cells can be measured by utilizing the database. According to the invention, the high-resolution mass spectrum and the triple quadrupole mass spectrum are combined, so that the rapid establishment of the low-concentration metabolite high-throughput detection method is realized.

Description

Technical Field

[0001] The present invention relates to the field of biological metabolism technology, and in particular to a metabolite detection method based on liquid chromatography-mass spectrometry. Background Art

[0002] Metabolomics has important application potential in fields such as medicine and toxicology. By quantitatively analyzing the levels of metabolites in cells, tissues or body fluids, medical researchers can conduct disease diagnosis, prognosis assessment and drug development. For example, in some cancers, the levels of specific metabolites such as certain amino acids and organic acids may change significantly. These metabolites can be used as potential biomarkers for early diagnosis of the disease. Toxicology researchers can conduct work such as poison detection and identification, toxicity mechanism research and risk assessment / safety evaluation. For example, exposure to poisons usually leads to disorders in the body's metabolic pathways, such as abnormalities in important physiological processes such as energy metabolism, amino acid metabolism or lipid metabolism. Metabolomics can analyze these metabolic changes and reveal the mechanism of action of poisons.

[0003] Metabolomics can be divided into two research strategies: targeted and non-targeted. Targeted metabolomics is based on metabolite standards. After establishing a detection method for specific metabolites, the absolute / relative abundance of the target analyte in the sample is determined. Its advantages are strong specificity, high sensitivity, and good data reproducibility, but it can only detect known metabolites and has high a priori formal requirements (target metabolites need to be pre-selected); non-targeted metabolomics does not require standards, but instead obtains information on all substances in the sample indiscriminately, and then identifies metabolites by cleaning and collating the data and comparing it with the database. Its advantage is that it can obtain as many metabolite spectra as possible, comprehensively detect metabolites in the organism, and may discover new biomarkers, but the data it generates is huge, the analysis process is complex and time-consuming, and there are many false positive results.

[0004] Therefore, in order to balance specificity, sensitivity and coverage, it is urgent to develop a high-coverage targeted metabolite detection method. Summary of the Invention

[0005] The purpose of the present invention is to provide a metabolite detection method based on liquid chromatography-mass spectrometry to address the problems in the prior art of time-consuming parameter optimization and easy detector saturation during the establishment of targeted metabolomics methods.

[0006] The object of the present invention is achieved through the following technical solution: a metabolite detection method based on liquid chromatography-mass spectrometry, comprising the following steps:

[0007] (1) Dissolve the metabolite standard in 40% methanol-water solution;

[0008] (2) Using ultra-high performance liquid chromatography-high resolution mass spectrometry to collect chromatographic and mass spectrometric information of the standard sample;

[0009] (3) obtaining the liquid phase retention time, parent ion, and candidate product ion of each metabolite used in step (1);

[0010] (4) using ultra-high performance liquid chromatography-mass spectrometry to detect the response intensity of each ion pair in the metabolite standard under different mass spectrometry parameters to optimize the mass spectrometry parameters; wherein the ion pair refers to the combination of the parent ion obtained in step (3) and each candidate daughter ion;

[0011] (5) Extract metabolites from plant and animal cell tissues by quenching with organic solvents or grinding with liquid nitrogen;

[0012] (6) drying the metabolite-containing solution obtained in step (5) by a freeze dryer or a vacuum centrifugal concentrator to obtain a freeze-dried sample;

[0013] (7) The freeze-dried sample obtained in step (6) was reconstituted with 50% by volume acetonitrile aqueous solution, vortexed, and filtered through a 0.22 μm filter;

[0014] (8) Ammonium acetate (25 mmol / L), ammonia monohydrate (25 mmol / L) in water, and pure acetonitrile were used as mobile phases for ultra-high performance liquid chromatography-tandem mass spectrometry. Gradient elution was performed over 18 minutes to separate metabolites. Dynamic multiple reaction monitoring (DMRM) mode was used for quantitative analysis to achieve high-throughput quantification of multiple metabolites.

[0015] Furthermore, the high-resolution mass spectrometer is a quadrupole-time-of-flight mass spectrometer or an orbitrap mass spectrometer.

[0016] Furthermore, the chromatographic column of the ultra-high performance liquid chromatography is a BEH Amide chromatographic column, the particle diameter of the filler is 1.7 μm, the inner diameter is 2.1 mm, and the height is 100 mm.

[0017] Furthermore, in step (2), when using an ultra-high performance liquid chromatography-high-resolution mass spectrometer to collect chromatographic and mass spectrometric information of the standard sample, the primary mass spectrometric data is obtained by a full scan mode, and the secondary mass spectrometric data is obtained by a data-dependent acquisition mode or a data-independent scanning mode.

[0018] Furthermore, the step (3) specifically includes:

[0019] The characteristic ion information that may be generated in the primary mass spectrometry of each metabolite standard used in step (1) was searched in the HMDB database and the MassBank database. Then, the ion extraction function of the PeakView software was used to extract the ion signal corresponding to each metabolite, and its peak time was recorded as the liquid phase retention time of the metabolite; the characteristic ion with the highest intensity was recorded as the parent ion; and the top five ions in intensity among the secondary fragment ions corresponding to the parent ion were used as candidate daughter ions.

[0020] Furthermore, the mass spectrometer used in step (4) is a triple quadrupole mass spectrometer or a quadrupole-linear ion trap composite mass spectrometer.

[0021] Furthermore, the mass spectrometry parameters include declustering voltage and collision energy; wherein, the setting range of the declustering voltage is 60-100V; the collision energy is set to three groups: 10, 20, and 30eV in positive ion mode, and -10, -20, and -30eV in negative ion mode.

[0022] Furthermore, the operating temperature of the vacuum centrifugal concentrator is 25° C. and the rotation speed is 1000 r / min.

[0023] Furthermore, the mass spectrometry ion source temperature of the ultra-high performance liquid chromatography tandem mass spectrometry is: 650°C in positive ion mode, 550°C in negative ion mode; the ion source voltage is: 5500V in positive ion mode, 4500V in negative ion mode; the curtain gas is: 35 in positive ion mode, 30 in negative ion mode.

[0024] Furthermore, the conditions of the gradient elution are specifically as follows:

[0025] In the 0-1 min period, the volume ratio of ammonium acetate / ammonia monohydrate solution to pure acetonitrile solution was 5:95;

[0026] Within 1–14 min, the volume ratio of ammonium acetate / aqueous solution of ammonia monohydrate to pure acetonitrile solution increased from 5:95 to 35:65;

[0027] Within 14–16 min, the volume ratio of ammonium acetate / aqueous ammonia monohydrate solution and pure acetonitrile solution increased from 35:65 to 60:40;

[0028] Within 16-18 min, the volume ratio of ammonium acetate / aqueous ammonia monohydrate solution to pure acetonitrile solution was 60:40;

[0029] Within 18-18.1 min, the volume ratio of ammonium acetate / aqueous ammonia monohydrate solution and pure acetonitrile solution decreased from 60:40 to 5:95.

[0030] Furthermore, the dynamic multiple reaction monitoring mode specifically includes: setting seven key parameters: parent ion mass-to-charge ratio, product ion mass-to-charge ratio, liquid phase retention time, multiple reaction monitoring detection window, target scan time, declustering voltage and collision energy; dynamically allocating the detection window according to the compound retention time, and activating the corresponding multiple reaction monitoring channel only within the target expected retention time range.

[0031] The beneficial effects of the present invention are as follows: the present invention designs a convenient and rapid metabolite detection method establishment process, and establishes a high-coverage and highly sensitive metabolite database based on this process; the present invention simplifies the development process of metabolite standard detection methods, reduces the time consumption of parameter optimization, and solves the problem of easy detector saturation; the metabolite database established by the present invention has the functions of both non-targeted and targeted metabolomics; the metabolite detection method established by the present invention has high sensitivity, high recovery rate and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a flow chart of the metabolite detection method based on liquid chromatography-mass spectrometry of the present invention;

[0033] Figure 2 The total ion current of some standard products under acidic or alkaline mobile phase conditions;

[0034] Figure 3 It is the extracted ion current chromatogram of different product ions of a substance under acidic or alkaline mobile phase conditions;

[0035] Figure 4 is the recovery of the two internal standards;

[0036] Figure 5 is the matrix effect of cell samples on the two internal standards;

[0037] Figure 6 The intra-day and inter-day stability of this method;

[0038] Figure 7 The linear fit goodness of fit for the metabolites quantified by this method;

[0039] Figure 8 In order to use this method to analyze the stability and clustering degree of actual samples. DETAILED DESCRIPTION

[0040] Exemplary embodiments will be described in detail herein, with examples illustrated in the accompanying drawings. When the following description refers to the drawings, identical numbers in different figures represent identical or similar elements, unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims. It should be understood that the foregoing general description and the detailed description that follows are exemplary and illustrative only and do not limit the present application.

[0041] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. As used in this application and the appended claims, the singular forms "a," "an," "the," and "the" are intended to include the plural forms, unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0042] It should be understood that although the terms first, second, third, etc. may be used in this application to describe various information, these information should not be limited to these terms. These terms are only used to distinguish information of the same type from each other. For example, without departing from the scope of this application, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "at the time of..." or "when..." or "in response to determination." Moreover, the term "comprises," "comprising," or any other variant thereof is intended to cover non-exclusive inclusion, so that the process or method comprising a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process or method. In the absence of further restrictions, the elements defined by the statement "comprising a..." do not exclude the presence of other identical elements in the process, method, article, or device comprising the elements.

[0043] The present invention will be described in detail below with reference to the accompanying drawings. Unless there is any conflict, the features of the following embodiments and implementations may be combined with each other.

[0044] See also Figure 1 The metabolite detection method based on liquid chromatography-mass spectrometry of the present invention specifically comprises the following steps:

[0045] (1) Dissolve the metabolite standard in 40% methanol aqueous solution.

[0046] Preferably, the metabolite standards are derived from the mass spectrometry metabolite standard library provided by Merck. Of course, other commercially available metabolite standards may also be used.

[0047] Furthermore, the metabolite standards can be dissolved individually or mixed to prepare a mixed metabolite standard solution.

[0048] It should be noted that, in order to avoid mass spectrometry interference, the preparation of mixed standard solutions should avoid mixing compounds with the same molecular weight.

[0049] (2) Use ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) to collect chromatographic and mass spectrometric information of standard samples.

[0050] Preferably, the high-resolution mass spectrometer is a quadrupole-time-of-flight mass spectrometer (QTOF) or an orbitrap mass spectrometer (Orbitrap).

[0051] Furthermore, the chromatographic column for ultra-high performance liquid chromatography is an ACQUITY UPLC BEHAmide chromatographic column produced by Waters Corporation, with a filler particle diameter of 1.7 μm, an inner diameter of 2.1 mm, and a height of 100 mm.

[0052] Furthermore, when using an ultra-high performance liquid chromatography-high resolution mass spectrometer to collect chromatographic and mass spectrometric information of a standard sample, primary mass spectrometric data are obtained through a full scan mode (Full Scan); and secondary mass spectrometric data are obtained through a data-dependent acquisition mode (DDA) or a data-independent scanning mode (DIA).

[0053] (3) Obtaining the liquid phase retention time, parent ion, and candidate daughter ions of each metabolite used in step (1), wherein the combination of the parent ion and each candidate daughter ion constitutes an ion pair in mass spectrometry detection.

[0054] Specifically, the characteristic ion information that may be generated in the primary mass spectrum of each metabolite standard used in step (1) was queried in the HMDB database and the MassBank database, and then the "Extract Ions" function in the PeakView software was used to extract the primary mass spectrometry signal corresponding to each metabolite, and its peak time was recorded as the liquid phase retention time of the metabolite; the characteristic ion with the highest intensity was recorded as the parent ion (Q1), and the top five ions in intensity among the secondary fragment ions corresponding to the parent ion were recorded as candidate daughter ions (Q2).

[0055] (4) Using ultra-high performance liquid chromatography-mass spectrometry, the response intensity of each ion pair in the metabolite standard under different mass spectrometry parameters is detected to optimize the mass spectrometry parameters. The ion pair refers to the combination of the parent ion obtained in step (3) and each candidate daughter ion.

[0056] Furthermore, the mass spectrometer used in step (4) is a triple quadrupole mass spectrometer or a quadrupole-linear ion trap composite mass spectrometer, and the chromatographic column of ultra-high performance liquid chromatography is also an ACQUITY UPLC BEH Amide column.

[0057] Furthermore, the mass spectrometry parameters include declustering potential (DP) and collision energy (CE). DP is set in the range of 60-100 V, and CE is set in three groups: 10, 20, and 30 eV in positive ion mode, and -10, -20, and -30 eV in negative ion mode.

[0058] (5) Extract metabolites from animal and plant cell tissues by quenching with organic solvents or grinding with liquid nitrogen.

[0059] Specifically, animal cell samples were quenched and lysed using pre-cooled 80% volume percent methanol at -80°C, and the metabolite supernatant was collected after centrifugation. Plant tissue samples were minced and then ground in liquid nitrogen, followed by metabolite extraction using solvents such as ethanol and water.

[0060] (6) Drying the metabolite-containing solution obtained in step (5) by a freeze dryer or a vacuum centrifugal concentrator to obtain a freeze-dried sample.

[0061] Furthermore, the operating temperature of the vacuum centrifugal concentrator is 25° C. and the rotation speed is 1000 r / min.

[0062] (7) The freeze-dried sample obtained in step (6) was reconstituted with 50% by volume acetonitrile aqueous solution, vortexed and filtered through a 0.22 μm filter, and the filtrate was used for subsequent liquid chromatography tandem mass spectrometry analysis.

[0063] (8) 25 mmol / L ammonium acetate, 25 mmol / L ammonia monohydrate in water, and pure acetonitrile were used as mobile phases for ultra-high performance liquid chromatography-tandem mass spectrometry, and gradient elution was performed over 18 minutes to separate metabolites. Quantitative analysis was performed using the scheduled multiple reaction monitoring (MRM) mode to achieve high-throughput quantification of multiple metabolites.

[0064] Furthermore, the mass spectrometry ion source temperature of the ultra-high performance liquid chromatography tandem mass spectrometry is: 650°C in positive ion mode, 550°C in negative ion mode; the ion source voltage is: 5500V in positive ion mode, 4500V in negative ion mode; the curtain gas is: 35 in positive ion mode, 30 in negative ion mode.

[0065] Furthermore, the conditions for gradient elution are specifically as follows:

[0066] 0-1 min, 5:95 of ammonium acetate / ammonia monohydrate solution and pure acetonitrile solution;

[0067] 1-14 min, 5:95 to 35:65 of ammonium acetate / ammonia monohydrate solution and pure acetonitrile solution;

[0068] 14-16 min, 35:65 to 60:40 of ammonium acetate / ammonia monohydrate solution and pure acetonitrile solution;

[0069] 16-18 min, 60:40 of ammonium acetate / ammonia monohydrate solution and pure acetonitrile solution;

[0070] 18-18.1 min, 60:40 to 5:95 of ammonium acetate / ammonia monohydrate solution and pure acetonitrile solution.

[0071] Further, the dynamic multiple reaction monitoring mode specifically includes setting 7 key parameters: parent ion mass-to-charge ratio (Q1 Mass), daughter ion mass-to-charge ratio (Q2 Mass), liquid phase retention time (Time), multiple reaction monitoring detection window (MRM detection window), target scan time, DP, and CE. The sources of the 7 parameters are as follows: Q1 Mass is the parent ion obtained in step (3), Q2 Mass is the second strongest daughter ion detected in step (4), Time is the liquid phase retention time of the metabolite standard obtained in step (3), CE is the CE value required to obtain the intensity of the daughter ion, DP can be selected from 60-100 V, and Target Scan Time is set to 0.1 s. Unlike the traditional MRM method, the dynamic multiple reaction monitoring mode dynamically allocates the detection window according to the compound retention time, and only activates the corresponding MRM channel within the target expected retention time range, thereby improving the detection sensitivity and throughput.

[0072] Further, the conditions of the dynamic multiple reaction monitoring mode are specifically shown in Table 1 and Table 2.

[0073] Table 1: Multiple reaction mode condition information table (positive ion mode)

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] Table 2: Multiple reaction mode condition information table (negative ion mode)

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] The metabolite detection method based on liquid chromatography-mass spectrometry of the present invention will be described in detail below with reference to the examples, and the purpose and effect of the present invention will become more apparent.

[0113] Example 1

[0114] This example investigates the effect of mobile phase pH on the separation and signal intensity of metabolites during LC / MS / MS analysis, specifically including the following steps:

[0115] (1) Prepare 19 metabolite mixed standard samples, where the concentration of each metabolite is 4 mg / mL, and the solvent is 50% acetonitrile by volume. Use 50% acetonitrile to dissolve the metabolite mixed standard samples.

[0116] (2) Acidic mobile phase and alkaline mobile phase systems were used to detect standard samples respectively.

[0117] (3) In the acidic mobile phase system, mobile phase A was water containing 0.2% formic acid and 10 mM ammonium formate, and mobile phase B was acetonitrile.

[0118] (4) In the alkaline mobile phase system, mobile phase A is water containing 25 mM ammonia monohydrate and 25 mM ammonium acetate, and mobile phase B is acetonitrile.

[0119] Among them, the gradient elution conditions of the mobile phase system are specifically as follows:

[0120] In the 0-1 min period, the volume ratio of mobile phase A to mobile phase B was 5:95;

[0121] Within 1-14 min, the volume ratio of mobile phase A to mobile phase B increased from 5:95 to 35:65;

[0122] Within 14–16 min, the volume ratio of mobile phase A to mobile phase B increased from 35:65 to 60:40;

[0123] During 16-18 min, the volume ratio of mobile phase A to mobile phase B was 60:40;

[0124] Within 18–18.1 min, the volume ratio of mobile phase A to mobile phase B decreased from 60:40 to 5:95;

[0125] like Figure 2 As shown, Figure 2 (a) is the total ion current (TIC) of the metabolite mixture under acidic conditions. Figure 2 Figure (b) shows the total ion chromatogram of the metabolite standard mixture under alkaline conditions. The experimental results show that the TIC chromatogram shows more chromatographic peaks under alkaline conditions, especially between 10 and 15 minutes. Comparison of the extracted ion chromatograms (EICs) of each metabolite reveals that 15 metabolites elute under alkaline conditions, while only 8 elute under acidic conditions. Six of these metabolites exhibit higher peak intensities under alkaline conditions than under acidic conditions.

[0126] Furthermore, taking 3-acylurea propionic acid, a metabolite in the mixed standard sample, as an example, the chromatographic peaks of different ions produced under acidic / alkaline conditions were extracted, such as m / z 285.0817 ([2M-2H+Na] - )、m / z131.0457([MH] - ) and m / z 439.1171 ([3M-3H+2Na] - ).like Figure 3 As shown in the figure, by comparison, it can be found that the three extracted ions all elute under alkaline conditions and have the same retention time, which proves that they are indeed the chromatographic peaks of 3-ureide propionic acid; while under acidic conditions, only m / z 131.0457 elutes, and its retention time is inconsistent with the elute time under alkaline conditions. It can be judged that this peak is an impurity, and 3-ureide propionic acid does not form an ion that can be detected.

[0127] Example 2

[0128] This example investigates the recovery of metabolites measured using the method of the present invention after quenching, extraction, drying, and reconstitution of animal cell samples, specifically comprising the following steps:

[0129] (1) Using N-(4-aminobenzoyl)-L-glutamic acid (CAS: 4271-30-1, internal standard 1) and 4-fluoro-glutamic acid (CAS: 2708-77-2, internal standard 2) as internal standards and acetonitrile / water (50%, v / v) as solvent, mixed internal standard solutions with mass fractions of 1 ppm and 1000 ppm were prepared.

[0130] (2) Taking adherent animal cells cultured in a 6 cm culture dish as an example, the cells were rinsed twice with PBS pre-cooled at 4°C, and then 1.5 mL of 80% (v / v) methanol solution pre-cooled at -80°C was added, which was recorded as the post-spiked sample.

[0131] (3) Similar to step (2), 1.5 μL of internal standard solution with a mass fraction of 1000 ppm was added. The internal standard content in the mixture was 0.1 μg, which was recorded as the pre-spiked sample.

[0132] (4) The samples obtained in steps (2) and (3) were placed in a -80°C freezer for 20 minutes for rapid quenching and cell disruption, and then the cells were collected using a cell scraper.

[0133] (5) The sample obtained in step (4) was centrifuged at 14000 g for 5 minutes to obtain a supernatant.

[0134] (6) The metabolite-containing supernatant obtained in step (5) is dried using a vacuum centrifugal concentrator to obtain a freeze-dried pure sample and a pre-spiked sample.

[0135] (7) Add 100 μL of the 1 ppm mixed internal standard solution obtained in step (1) b to the lyophilized spiked sample obtained in step (6); add 100 μL of acetonitrile / water (50%, v / v) solution to the spiked sample before lyophilization, vortex thoroughly, and filter through a 0.22 μm organic filter membrane to obtain a sample.

[0136] (8) An aqueous solution of 25 mM ammonium acetate and 25 mM ammonia monohydrate (mobile phase A) and pure acetonitrile (mobile phase B) were used as mobile phases for liquid chromatography tandem mass spectrometry with a gradient elution of 18 minutes. The concentration of the internal standard in the sample obtained in step (7) was separated and quantified by multiple reaction monitoring mode (scheduled MRM).

[0137] Among them, the gradient elution conditions of the mobile phase system are specifically as follows:

[0138] In the 0-1 min period, the volume ratio of mobile phase A to mobile phase B was 5:95;

[0139] Within 1-14 min, the volume ratio of mobile phase A to mobile phase B increased from 5:95 to 35:65;

[0140] Within 14–16 min, the volume ratio of mobile phase A to mobile phase B increased from 35:65 to 60:40;

[0141] During 16-18 min, the volume ratio of mobile phase A to mobile phase B was 60:40;

[0142] Within 18-18.1 min, the volume ratio of mobile phase A to mobile phase B decreased from 60:40 to 5:95.

[0143] The calculation formula for internal standard recovery is:

[0144]

[0145] Among them, A 前加标样品 represents the peak area of ​​the internal standard in the pre-spiked sample, A 后加标样品 Represents the peak area of ​​the internal standard in the post-spiked sample.

[0146] like Figure 4 As shown, the recoveries of internal standard 1 and internal standard 2 measured in positive ion mode were 73.9% and 60.3%, respectively, and in negative ion mode were 67.7% and 65.2%, respectively. This result shows that the recovery rate of the method of the present invention is good.

[0147] Example 3

[0148] This example investigates the matrix effects of animal cell samples obtained after quenching, extraction, drying, and reconstitution, and metabolites measured using the method of the present invention, specifically comprising the following steps:

[0149] (1) Rinse the cultured animal cells twice with PBS pre-cooled at 4°C, add 80% (v / v) methanol solution pre-cooled at -80°C, freeze at -80°C for 20 minutes to achieve rapid cell quenching and lysis, and then collect the cells with a cell scraper (for adherent cells) or centrifugation (for suspended cells).

[0150] (2) Centrifuge the sample obtained in step (1) at 14,000 g for 5 minutes to obtain a supernatant.

[0151] (3) The metabolite-containing supernatant obtained in step (2) is dried using a vacuum centrifugal concentrator to obtain a freeze-dried sample.

[0152] (4) Using N-(4-aminobenzoyl)-L-glutamic acid (CAS: 4271-30-1, internal standard 1) and 4-fluoro-glutamic acid (CAS: 2708-77-2, internal standard 2) as internal standards and acetonitrile / water (50%, v / v) as solvent, a gradient internal standard solution with a mass fraction of 5-1000 ppb was prepared.

[0153] (5) The freeze-dried sample obtained in step (3) was redissolved in the internal standard acetonitrile / water solution obtained in step (4), vortexed thoroughly, and filtered through a 0.22 μm organic filter membrane to prepare a series of internal standard samples.

[0154] (6) An aqueous solution of 25 mM ammonium acetate and 25 mM ammonia monohydrate (mobile phase A) and pure acetonitrile (mobile phase B) were used as mobile phases for liquid chromatography tandem mass spectrometry with a gradient elution of 18 minutes. The concentrations of the internal standards in the samples obtained in steps (4) and (5) were separated and quantified by multiple reaction monitoring mode (scheduled MRM).

[0155] Among them, the gradient elution conditions of the mobile phase system are specifically as follows:

[0156] In the 0-1 min period, the volume ratio of mobile phase A to mobile phase B was 5:95;

[0157] Within 1-14 min, the volume ratio of mobile phase A to mobile phase B increased from 5:95 to 35:65;

[0158] Within 14–16 min, the volume ratio of mobile phase A to mobile phase B increased from 35:65 to 60:40;

[0159] During 16-18 min, the volume ratio of mobile phase A to mobile phase B was 60:40;

[0160] Within 18-18.1 min, the volume ratio of mobile phase A to mobile phase B decreased from 60:40 to 5:95.

[0161] The calculation formula for matrix effect is:

[0162]

[0163] Among them, A 加标样品 represents the peak area of ​​the internal standard in the spiked sample, A 纯内标 Represents the peak area of ​​the internal standard in the internal standard solution.

[0164] like Figure 5 As shown, the matrix effects experienced by both internal standards varied with internal standard concentration. In positive ion mode, the matrix effect experienced by internal standard 1 was 13%-51%, while that experienced by internal standard 2 was 74%-101%. In negative ion mode, the matrix effect experienced by internal standard 1 was 40%-148%, while that experienced by internal standard 2 was 67%-94%. This indicates that the matrix effects generated by the sample pretreatment and detection methods described are related to the properties of the analyte, with internal standard 2 experiencing less matrix interference than internal standard 1.

[0165] Example 4

[0166] This example examines the reproducibility and stability of metabolite samples on the same day and within three days of storage at -20°C, specifically including the following steps:

[0167] (1) Metabolite samples were tested three times a day within three days after pretreatment.

[0168] (2) An aqueous solution of 25 mM ammonium acetate and 25 mM ammonia monohydrate (mobile phase A) and pure acetonitrile (mobile phase B) were used as mobile phases for liquid chromatography-tandem mass spectrometry with an 18-min gradient elution. The concentrations of metabolites in the samples were separated and quantified using the multiple reaction monitoring (MRM) mode.

[0169] (3) Calculate the intra-day and inter-day changes in the peak area of ​​each metabolite, expressed as standard deviation.

[0170] Among them, the gradient elution conditions of the mobile phase system are specifically as follows:

[0171] In the 0-1 min period, the volume ratio of mobile phase A to mobile phase B was 5:95;

[0172] Within 1-14 min, the volume ratio of mobile phase A to mobile phase B increased from 5:95 to 35:65;

[0173] Within 14–16 min, the volume ratio of mobile phase A to mobile phase B increased from 35:65 to 60:40;

[0174] During 16–18 min, the volume ratio of mobile phase A to mobile phase B was 60:40;

[0175] Within 18-18.1 min, the volume ratio of mobile phase A to mobile phase B decreased from 60:40 to 5:95.

[0176] like Figure 6 As shown in the empirical cumulative distribution function diagram, the repeatability of three consecutive injections is excellent: in positive ion mode, the coefficient of variation of the peak area of ​​more than 90% of metabolites in three consecutive injections is less than 20%, and the coefficient of variation of more than 95% of metabolites is less than 30%; in negative ion mode, the coefficient of variation of the peak area of ​​more than 95% of metabolites in three consecutive injections is less than 20%, and the coefficient of variation of nearly 100% of metabolites is less than 30%. Figure 6 As shown in (a) in the figure. The stability of the three consecutive injections was good: in the positive ion mode, the coefficient of variation of the peak area of ​​more than 70% of the metabolites in three consecutive injections was less than 20%, and the coefficient of variation of more than 9% of the metabolites was less than 30%; in the negative ion mode, the coefficient of variation of the peak area of ​​more than 75% of the metabolites in three consecutive injections was less than 20%, and the coefficient of variation of more than 85% of the metabolites was less than 30%. Figure 6 As shown in (b) in .

[0177] Example 5

[0178] This example investigates the linearity of the method of the present invention in determining metabolites at different concentrations, aiming to verify the semi-quantitative accuracy of the method of the present invention. The method specifically includes the following steps:

[0179] (1) Metabolite samples from different sources were mixed to obtain quality control (QC) samples.

[0180] (2) The QC sample was diluted with acetonitrile / water (50%, v / v) solution to obtain a series of samples with concentrations of 0.1, 0.2, 0.4, 0.6, and 0.8 times that of the QC sample.

[0181] (3) An aqueous solution of 25 mM ammonium acetate and 25 mM ammonia monohydrate (mobile phase A) and pure acetonitrile (mobile phase B) were used as mobile phases for liquid chromatography-tandem mass spectrometry with an 18-min gradient elution. The concentrations of metabolites in the series of samples were separated and quantified using the multiple reaction monitoring mode (scheduled MRM).

[0182] (4) Perform linear fitting on the peak area of ​​each metabolite in QC samples of different concentrations to obtain the goodness of fit R 2 .

[0183] Among them, the gradient elution conditions of the mobile phase system are specifically as follows:

[0184] In the 0-1 min period, the volume ratio of mobile phase A to mobile phase B was 5:95;

[0185] Within 1-14 min, the volume ratio of mobile phase A to mobile phase B increased from 5:95 to 35:65;

[0186] Within 14–16 min, the volume ratio of mobile phase A to mobile phase B increased from 35:65 to 60:40;

[0187] During 16–18 min, the volume ratio of mobile phase A to mobile phase B was 60:40;

[0188] Within 18-18.1 min, the volume ratio of mobile phase A to mobile phase B decreased from 60:40 to 5:95.

[0189] like Figure 7 The empirical cumulative distribution function diagram shown shows that the quantitative linearity of this method is good: in the positive ion mode, more than 80% of the metabolites have a fit greater than 0.8, and about 70% of the metabolites have a linear fit greater than 0.9; in the positive ion mode, more than 80% of the metabolites have a fit greater than 0.8, and about 65% of the metabolites have a linear fit greater than 0.9.

[0190] Example 6

[0191] This example investigates the analytical capability of the method of the present invention in determining metabolites in actual samples and its stability during large-scale injection, specifically comprising the following steps:

[0192] (1) Human liver cancer cells HepG2 were used as test cells and cultured in 6 cm culture dishes until the confluence reached 80%.

[0193] (2) One control group and three experimental groups were set up, each with seven biological replicates.

[0194] (3) Rinse the cultured animal cells twice with PBS pre-cooled at 4°C, add 80% (v / v) methanol solution pre-cooled at -80°C, freeze at -80°C for 20 minutes to achieve rapid cell quenching and lysis, and then scrape the cells with a cell scraper.

[0195] (4) Centrifuge the sample obtained in step (3) at 14,000 g for 5 minutes to obtain a supernatant.

[0196] (5) The metabolite-containing supernatant obtained in step (4) is dried by a vacuum centrifugal concentrator to obtain a freeze-dried sample, wherein the operating temperature of the vacuum centrifugal concentrator is 4° C. and the rotation speed is 1000 r / min.

[0197] (6) 100 μL of 50% (v / v) acetonitrile aqueous solution was added to dissolve the freeze-dried sample and filtered to obtain 28 actual samples.

[0198] (7) Take equal amounts of samples from each experimental group and mix them to prepare quality control (QC) samples.

[0199] (8) An aqueous solution of 25 mM ammonium acetate and 25 mM ammonia monohydrate (mobile phase A) and pure acetonitrile (mobile phase B) were used as mobile phases for liquid chromatography-tandem mass spectrometry with an 18-min gradient elution. The concentrations of metabolites in the actual samples and QC samples were separated and quantified using the multiple reaction monitoring mode (scheduled MRM).

[0200] (9) Principal component analysis (PCA) was used to reduce the dimensionality of the obtained data and obtain two principal components.

[0201] Among them, the gradient elution conditions of the mobile phase system are specifically as follows:

[0202] In the 0-1 min period, the volume ratio of mobile phase A to mobile phase B was 5:95;

[0203] Within 1-14 min, the volume ratio of mobile phase A to mobile phase B increased from 5:95 to 35:65;

[0204] Within 14–16 min, the volume ratio of mobile phase A to mobile phase B increased from 35:65 to 60:40;

[0205] During 16–18 min, the volume ratio of mobile phase A to mobile phase B was 60:40;

[0206] Within 18-18.1 min, the volume ratio of mobile phase A to mobile phase B decreased from 60:40 to 5:95.

[0207] The sample analysis sequence adopts a distributed QC design: quality control samples are placed at the beginning and end of the sequence and inserted between each group of actual samples.

[0208] 129 and 163 metabolites were detected in positive and negative ion modes, respectively, totaling 226 after removing duplicates. Figure 7 The principal component analysis plot shows that the samples in each experimental group exhibit significant clustering characteristics, with good separation between groups. The QC samples are tightly clustered and evenly distributed across the data space, indicating excellent system stability. In summary, the method described in this paper demonstrates reliable quantitative repeatability in batch sample testing and is capable of meeting the needs of high-throughput analysis of complex metabolites.

[0209] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A metabolite detection method based on liquid chromatography-mass spectrometry, characterized in that: The following steps are involved: (1) Dissolve the metabolite standard in 40% methanol-water solution; (2) Using ultra-high performance liquid chromatography-high resolution mass spectrometry to collect chromatographic and mass spectrometric information of the standard sample; (3) obtaining the liquid phase retention time, parent ion, and candidate product ion of each metabolite used in step (1); (4) using ultra-high performance liquid chromatography-mass spectrometry to detect the response intensity of each ion pair in the metabolite standard under different mass spectrometry parameters to optimize the mass spectrometry parameters; wherein the ion pair refers to the combination of the parent ion obtained in step (3) and each candidate daughter ion; (5) Extract metabolites from plant and animal cell tissues by quenching with organic solvents or grinding with liquid nitrogen; (6) drying the metabolite-containing solution obtained in step (5) by a freeze dryer or a vacuum centrifugal concentrator to obtain a freeze-dried sample; (7) The freeze-dried sample obtained in step (6) was reconstituted with 50% by volume acetonitrile aqueous solution, vortexed, and filtered through a 0.22 μm filter; (8) Ammonium acetate (25 mmol / L), ammonia monohydrate (25 mmol / L) in water, and pure acetonitrile were used as mobile phases for ultra-high performance liquid chromatography-tandem mass spectrometry. Gradient elution was performed over 18 minutes to separate metabolites. Dynamic multiple reaction monitoring (DMRM) mode was used for quantitative analysis to achieve high-throughput quantification of multiple metabolites.

2. The method for metabolite detection based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The high-resolution mass spectrometer is a quadrupole-time-of-flight mass spectrometer or an orbital trap mass spectrometer.

3. The method for metabolite detection based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The chromatographic column of the ultra-high performance liquid chromatography is a BEH Amide chromatographic column, the particle diameter of the filler is 1.7 μm, the inner diameter is 2.1 mm, and the height is 100 mm.

4. The method for metabolite detection based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: In the step (2), when using an ultra-high performance liquid chromatography-high-resolution mass spectrometer to collect chromatographic and mass spectrometric information of the standard sample, the primary mass spectrometric data is obtained by a full scan mode, and the secondary mass spectrometric data is obtained by a data-dependent acquisition mode or a data-independent scanning mode.

5. The method for metabolite detection based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The step (3) specifically includes: The characteristic ion information that may be generated in the primary mass spectrum of each metabolite standard used in step (1) was searched in the HMDB database and the MassBank database. Then, the ion extraction function of the PeakView software was used to extract the ion signal corresponding to each metabolite, and its peak time was recorded as the liquid phase retention time of the metabolite; the primary ion with the highest intensity was recorded as the parent ion; and the top five ions in intensity among the secondary fragment ions corresponding to the parent ion were used as candidate daughter ions.

6. The method for metabolite detection based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The mass spectrometer used in step (4) is a triple quadrupole mass spectrometer or a quadrupole-linear ion trap composite mass spectrometer.

7. The method for metabolite detection based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The mass spectrometry parameters include declustering voltage and collision energy; wherein, the setting range of declustering voltage is 60-100V; the collision energy is set to three groups: 10, 20, and 30eV in positive ion mode, and -10, -20, and -30eV in negative ion mode.

8. The method for metabolite detection based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The operating temperature of the vacuum centrifugal concentrator is 25° C. and the rotation speed is 1000 r / min.

9. The method for metabolite detection based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The mass spectrometry ion source temperature of the ultra-high performance liquid chromatography tandem mass spectrometry is: 650°C in positive ion mode, 550°C in negative ion mode; the ion source voltage is: 5500V in positive ion mode, 4500V in negative ion mode; the curtain gas is: 35 in positive ion mode, 30 in negative ion mode.

10. The method for metabolite detection based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The conditions of the gradient elution are specifically: In the 0-1 min period, the volume ratio of ammonium acetate / ammonia monohydrate solution to pure acetonitrile solution was 5:95; Within 1–14 min, the volume ratio of ammonium acetate / aqueous solution of ammonia monohydrate to pure acetonitrile solution increased from 5:95 to 35:65; Within 14–16 min, the volume ratio of ammonium acetate / aqueous ammonia monohydrate solution and pure acetonitrile solution increased from 35:65 to 60:40; Within 16-18 min, the volume ratio of ammonium acetate / aqueous ammonia monohydrate solution to pure acetonitrile solution was 60:40; Within 18-18.1 min, the volume ratio of ammonium acetate / aqueous ammonia monohydrate solution and pure acetonitrile solution decreased from 60:40 to 5:

95.

11. The method for metabolite detection based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The dynamic multiple reaction monitoring mode specifically includes: setting seven key parameters: parent ion mass-to-charge ratio, product ion mass-to-charge ratio, liquid phase retention time, multiple reaction monitoring detection window, target scan time, declustering voltage and collision energy; dynamically allocating the detection window according to the compound retention time, and activating the corresponding multiple reaction monitoring channel only within the target expected retention time range.

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