CN115184530B - Multistage online energy resolution mass spectrometry and application thereof in precise structural identification - Google Patents

Abstract

The application belongs to the technical field of analytical chemistry, and provides a multistage online energy-resolved mass spectrometry (online ER) for accurately identifying chemical component structures ² -MS), wherein the first dimension employs an on-line energy-resolved mass spectrometry to construct a fragmentation curve of related ions based on a multi-reaction monitoring (MRM) mode, and the correlation of Optimal Collision Energy (OCE) and substructure connection is established by gaussian curve fitting, and the second dimension employs a post-collision induced dissociation energy-resolved mass spectrometry (post-CID ER-MS) based on a three-level Mass Spectrometry (MS) ³ ) Mode or tertiary Multiple Reaction Monitoring (MRM) ³ ) The mode constructs a cleavage curve of the relevant ion, and identifies the substructure of the chemical component. In comparison with the structural identification of traditional chemical components, the online ER ² The MS increases the analysis dimension of mass spectrum, identifies the substructure and provides connection information of the substructure, so that the accurate identification of the structure of the unknown compound is realized on an LC-MS analysis platform, and the method has high innovation and wide application prospect.

Description

A multi-stage online energy-resolved mass spectrometry method and its application in precise structural identification

Technical field

The invention belongs to the technical field of analytical chemistry, and specifically relates to a multi-stage online energy resolution mass spectrometry method and its application in accurate identification of structures. More specifically, the present invention relates to a method for accurately identifying the chemical structure of unknown compounds through multi-stage online energy-resolved mass spectrometry using a mass spectrometry system with two collision cells.

Background technique

Liquid chromatography-mass spectrometry (LC-MS/MS) combines the advantages of strong separation ability of liquid chromatography and high sensitivity of mass spectrometry, and has become the most important tool for analyzing the chemical composition of complex systems such as traditional Chinese medicine, food, and biological samples. Tandem high-performance ion trap-time-of-flight mass spectrometers (IT-TOF-MS), tandem quadrupole-time-of-flight mass spectrometers (Q-TOF-MS), and tandem quadrupole orbitrap mass spectrometers (Q Exactive-Orbitrap MS) are used. Resolution mass spectrometry can not only obtain the precise molecular weight of a compound, but also infer the fragmentation mode of the compound through multi-level mass spectrometry data, providing more information for the identification of compounds at the substructure level. However, since the structural differences between isomers are only different substructures and/or different connection methods, they often present very similar multi-level mass spectra. As a result, the structural identification of unknown chemical components based on each mass spectrometry system can only give one result. The group of isomers greatly restricts the in-depth revelation of the chemical composition of each sample. Energy-resolved mass spectrometry (ER-MS) is a mass spectrometry method that uses different methods to allow ions to absorb energy and decompose, and then study the ion decomposition method and how the product abundance changes with energy. It has been widely used. on the distinction between isomers. Generally speaking, ER-MS mainly establishes the fragmentation curve of the parent ion or product ion (i.e., the correlation curve between relative ion abundance and collision energy) by injecting chemical reference substances, and uses Sigmoid curves and Gaussian curves to fit the parent ions respectively. The fragmentation curves of the product ions and the product ions further realize the distinction of isomers through the difference in the half response collision energy (CE ₅₀ ) of the parent ion or the optimal collision energy (OCE) of the product ions. In order to overcome the dependence of ER-MS on reference substance monomers, the applicant constructed an online energy-resolved mass spectrometry (online ER-MS) in the early stage, which added a new mass spectrometric dimension to LC-MS analysis and applied it widely. Rapid and effective differentiation of isomers in traditional Chinese medicine and biological samples. However, online ER-MS combined with LC-MS still cannot accurately identify the chemical structure of the compound substructure, making it difficult to accurately identify the structure of unknown chemical components. Therefore, there is a need to establish a new method that can simultaneously obtain substructural information (in addition to elemental composition) and connection patterns.

In order to solve the above-mentioned defects in the existing technology, the applicant proposed energy-resolved mass spectrometry after collision-induced dissociation on the basis of using high-resolution multi-level mass spectrometry to determine the precise mass numbers of parent ions and product ions, and accurately calculating the elemental composition. The concept of (post collision-induced dissociation energy-resolved mass spectrometry, post-CID ER-MS) proposes a second-dimensional online energy-resolved mass spectrometer, which allows the product ions to continue fragmenting in the second collision cell to construct the grandchild ion The relationship curve between relative abundance and excitation energy (i.e., daughter fragmentation curve) studies the fragmentation behavior from product ions to grandchild ions, thereby revealing the chemical structure of the product ions. Combining online energy-resolved mass spectrometry with energy-resolved mass spectrometry after collision-induced dissociation, a multi-level energy-resolved mass spectrometry is formed, which can not only reveal the connection information between fragments, but also obtain the chemical structure of each fragment ion, thereby accurately identifying chemicals. structure.

Contents of the invention

In order to solve the above technical problems, the present invention provides a method for establishing a multi-stage online energy-resolved mass spectrometry using a mass spectrometry system with multiple collision cells to achieve accurate identification of the chemical structure of unknown compounds, so that the structure annotation has higher credibility.

Specifically, the present invention is implemented through the following technical solutions:

In a first aspect, the present invention provides a multi-stage online energy-resolved mass spectrometry (online ER ² -MS), which adopts a mass spectrometry system with dual collision cells. The multi-stage online energy-resolved mass spectrometry method With two dimensions of analysis, the mass spectrometry method consists of the following steps:

First, in the first dimension, online energy-resolved mass spectrometry is used to construct the fragmentation curve of relevant ions based on multiple reaction monitoring (MRM) mode. Through Gaussian curve fitting, the correlation between the optimal collision energy (OCE) and the substructure connection mode is established;

Then the second dimension uses post-collision-induced dissociation energy-resolved mass spectrometry (post-CID ER-MS) to construct fragmentation curves of relevant ions based on three-level mass spectrometry (MS ³ ) mode or three-level multiple reaction monitoring (MRM ³ ) mode. Identify the substructure of chemical components by comparing with reference substances or establishing the correlation between excitation energy and structure.

As an optional method, among the above mass spectrometry methods, ^1st online ER-MS is an online energy-resolved mass spectrometry method. The MRM mode in the HPLC-Qtrap-MS system is used to detect the compound to be tested and construct the related ion abundance. The relationship curve with collision energy, apply quantum chemistry to calculate the bond length and bond dissociation energy of the candidate structure, build a quantitative structure-collision energy relationship model, and reveal information on the connection mode of substructures.

As an optional method, among the above mass spectrometry methods, ^2nd online ER-MS is collision-induced dissociation post-energy-resolved mass spectrometry (post-CID ER-MS), using the MS ³ mode in the HPLC-Qtrap-MS system. The compound to be tested is detected, and a relationship curve between relative ion abundance and excitation energy is constructed to characterize the substructure information of the compound to be tested.

As an optional method, in the above mass spectrometry method, the mobile phase, chromatographic column model, flow rate, column temperature, elution program and injection volume used in the mass spectrometry method are adjusted according to the compound to be measured.

As an optional method, in the above mass spectrometry method, the ion source parameters are set as follows: ion source temperature: 400-550°C, spray voltage in negative ion mode is set to -4500V; collision gas (CAD) is High; curtain gas (Curtain Gas) ) range is 25-35psi; GS1 and GS2 are 15-55psi; among them, the curtain gas, GS1, GS2 and ion source temperatures are adjusted according to the flow rate.

As an optional method, in the above mass spectrometry method, the ^1st online ER-MS adopts multiple reaction monitoring (MRM) scanning mode, the injection voltage (EP) is -10V; the collision chamber injection voltage (CXP) is -16V . Construct a series of pseudo-ion pairs (PITs), corresponding to a set of progressive CE values, –30-–80eV, Step is set to 1eV, with the response value as the ordinate, and the progressive collision energy as the abscissa. Further use GraphPad Prism 8.0 Perform Gaussian curve fitting to obtain the optimal collision energy (OCE) of the target ion; obtain the half response collision energy (CE ₅₀ ) of the target ion through Sigmoid curve fitting.

As an optional method, in the above mass spectrometry method, the ^2nd online ER-MS adopts the third-level mass spectrometry (MS ³ ) scanning mode, the scanning speed is 10000 Da/s; the linear ion trap fixed fill time (Fixed LIT fill time ) is 10ms; the excitation time (Excitation Time) is 25ms; 16 MS ³ experiments are set, corresponding to a set of progressive AF2 values, 0.005-0.175V, Step is set to 0.01V, with the response value as the ordinate, progressive formula The excitation energy is taken as the abscissa, and Gaussian curve fitting is further performed through GraphPad Prism 8.0 to obtain the optimal excitation energy of the target ion; through Sigmoid curve fitting, the half response excitation energy of the target ion is obtained.

In a second aspect, the present invention provides the application of the mass spectrometry method described in the first aspect above in accurately identifying the chemical structure of unknown compounds in complex systems.

As an alternative, in the above applications, the unknown compound is preferably a bile acid compound.

As an alternative, in the above applications, the complex system includes biological reagents, drugs, food and clinical samples.

Compared with the existing technology, the present invention has the following beneficial effects:

The multi-stage online energy-resolved mass spectrometry provided by the present invention can be applied to the accurate identification of bile acid component structures in different complex systems. For different complex systems, the mobile phase, chromatographic column model, flow rate, column temperature, and elution program Chromatographic parameters such as injection volume and injection volume are adjusted according to the sample to be tested. The curtain gas, GS1, GS2 and ion source temperature plasma source parameters are adjusted according to the sample injection flow rate. The MS ¹ and MS ² scan ranges, CE and CES values can be adjusted according to the molecular weight range of the compound types in the complex system. Based on the characteristics of the double collision cell of the triple quadrupole-linear ion trap composite mass spectrometer, ^1st onlineER-MS is based on the multiple reaction monitoring (MRM) mode to construct the fragmentation curve (i.e. relative ion abundance or relative ion) of related ions (mainly product ions). The relationship curve between peak area and collision energy), through Gaussian curve fitting, establishes the correlation between the optimal collision energy (OCE) and the connection mode of the substructure. ^2nd online ER-MS is based on the three-level mass spectrometry (MS ³ ) mode or the three-level multiple reaction monitoring (MRM ³ ) mode to construct the fragmentation curve (i.e. relative ion abundance or relative peak) of related ions (mainly grandchild ions or product ions) The relationship curve between area and excitation energy), by comparing with the reference substance or establishing the correlation between excitation energy and structure, the substructure of the chemical component can be identified. ER ² -MS can improve the accuracy of structural identification of bile acid components, for example, and achieve the distinction of isomers.

Description of drawings

The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

In the attached picture:

Figure 1 Extracted ion chromatogram of m/z 471.3>97 in UMS in LC-sMRM mode (A), high-resolution MS ² spectrum of compound 1-10 (B), ion pair of free cholic acid sulfonate compound 3-10 The fragmentation curve of m/z 471.3>97 (C) and the distribution diagram of the relationship between OCE _{m/z 471.3>97} and the substitution position of -SO ₃ group (D).

Figure 2 Fragmentation curve of free cholic acid sulfonate compound 3-10 ion pair m/z 471.3>391.3>391.3 and the corresponding free cholic acid compound isomer reference substance ion pair m/z 391.3>391.3> Fragmentation curve of 391.3.

Figure 3 OCE _{m/z 471.3＞97} distribution diagram of compounds 1 and 2 (A), the fragmentation curve of the free cholic acid compound isomer reference substance ion pair m/z 391.3＞391.3＞391.3 and compounds 1 and 2 Fragmentation curve (B) for ion pair m/z 471.3>391.3>391.3.

Figure 4 Fragmentation curve of glycine cholic acid sulfonate compound isomer reference substance ion pair m/z 528.3>448.3>74 and the corresponding glycine cholic acid compound isomer reference substance ion pair Fragmentation curve of m/z 448.3>448.3>74.

Figure 5 Extracted ion chromatogram of m/z 624.3＞448.3 in LC-sMRM mode (A), high-resolution MS ² spectrum of compound 11-14 (B), ion pair m/z 624.3＞448.3＞74 of compound 11-14 cleavage curve (C).

Figure 6 The fragmentation curve of the ion pair m/z 578.3＞498.3＞80 of the taurine cholic acid sulfonate compound isomer reference substance and the corresponding taurine cholic acid compound isomer reference substance ion pair Fragmentation curve of m/z 498.3>498.3>80.

Figure 7 Extracted ion chromatogram of m/z 674.3＞498.3 in LC-sMRM mode (A), high-resolution MS ² spectrum of compound 15-19 (B), ion pair m/z 674.3＞498.3＞80 of compound 15-19 Fragmentation curves (C) of compounds 15, 16, 17 and 19 (D) for ion pairs m/z 674.3>498.3.

Detailed ways

The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the scope of the present invention.

If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field shall be followed, or the product instructions shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased through regular channels.

The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

Example 1 Preparation of experimental materials and samples

1.1 Experimental materials

Reference substances: Glycinoursodeoxycholic acid-3-sulfonate (GUDCA-3-S, batch number: IR-15693), glycinchenodeoxycholic acid-3-sulfonate (GCDCA-3-S, batch number: : IR-15685), glycodeoxycholic acid-3-sulfonate (GDCA-3-S, batch number: IR-15689), glycinoursodeoxycholic acid (GUDCA, batch number: ZS-20013), glycosides Aminochenodeoxycholic acid (GCDCA, batch number: ZS-20040), glycinodeoxycholic acid (GHDCA, batch number: ZS-20023), glycinodeoxycholic acid (GDCA, batch number: ZS-20152), taurine Ursodeoxycholic acid-3-sulfonate (TUDCA-3-S, batch number: IR-15744), taurochenodeoxycholic acid-3-sulfonate (TCDCA-3-S, batch number: IR-15736 ), taurodeoxycholic acid-3-sulfonate (TDCA-3-S, batch number: IR-15740), tauroursodeoxycholic acid (TUDCA, batch number: ZS-20014), taurochenodeoxy Cholic acid (TCDCA, batch number: ZS-20033) and taurodeoxycholic acid (TDCA, batch number: ZS-20026), murine deoxycholic acid (MDCA, batch number: ZS-20309), ursodeoxycholic acid (UDCA) , batch number: ZS-20012), chenodeoxycholic acid (CDCA, batch number: ZS-20028), hyodeoxycholic acid (HDCA, batch number: ZS-20019), deoxycholic acid (DCA, batch number: ZS-20145) , Chenodeoxycholic acid-3-sulfonate (CDCA-3-S, batch number: IR-15657), deoxycholic acid-3-sulfonate (DCA-3-S, batch number: IR-15669) are all Purchased from Shanghai Zhenzhun Biotechnology Co., Ltd.; the purity of the above reference substances was greater than 98% when tested by high performance liquid chromatography (HPLC); mass spectrometry grade acetonitrile (ACN) and formic acid (FA) were purchased from Thermo Fisher Company in the United States, and were deionized The water was Milli-Q ultrapure water (18.2 MΩ·cm) made in the laboratory.

LC-20AD ^XR analytical high-performance liquid chromatograph (including binary high-pressure gradient pump, vacuum degasser, autosampler, column oven and controller, Shimadzu Corporation, Japan); ACQUITY UPLC HSS T3 chromatographic column (100mm×2.1mm, 1.8μm, Waters Company, USA); Qtrap 6500 mass spectrometer (Sciex Company, USA); Milli-Q ultrapure water purification system (Millipore Company, USA); ME204 electronic analytical balance (Mettler Toledo Company, Switzerland) , accuracy is 0.1mg).

1.2 Reference substance sample preparation

Precision weighing reference substances GUDCA-3-S, GCDCA-3-S, GDCA-3-S, GUDCA, GCDCA, GHDCA, GDCA, TUDCA-3-S, TCDCA-3-S, TDCA-3-S, TUDCA , TCDCA, TDCA, UDCA, CDCA, HDCA, DCA, MDCA, CDCA-3-S, and DCA-3-S in appropriate amounts, respectively, were dissolved in DMSO to prepare a reference substance stock solution with a mass concentration of 1 mg·mL ^-1 . Take an appropriate amount of each reference substance stock solution, dilute it with 50% acetonitrile aqueous solution to a mixed reference substance solution of 1 μg·mL ^-1 , and set aside.

1.3 Preparation of model sample (universal metabolome standard, UMS)

In order to construct a mixed sample containing an absolute portion of natural cholic acid components, we widely introduced samples containing cholic acid components, such as dry powders of bear bile powder, bezoar, snake gall, chicken gall, duck gall and rat bile, and were accurately weighed respectively. Take 20 mg and mix thoroughly, weigh another 20 mg from the mixed sample powder, add 10 times of 70% methanol aqueous solution for ultrasonic extraction for 30 min, centrifuge at 12000 r·min ^-1 for 10 min at 4°C, take the supernatant, concentrate and dry under nitrogen. Redissolve in 50 μL of 50% acetonitrile aqueous solution, vortex for 1 min, and centrifuge again at 12000 r·min ^-1 for 10 min at 4°C. Take the supernatant to obtain the model sample (UMS) stock solution.

1.4 Experimental methods

A multi-stage online energy-resolved mass spectrometry method was established to detect the free cholic acid sulfonate isomers, glycine cholic acid sulfonate and glucuronate isomers, and taurine in vivo metabolites of bile traditional Chinese medicines. The identification of cholic acid sulfonate and glucuronate isomers is taken as an example to confirm the effectiveness and progress of the method of the present invention.

1.4.1 Establishment of first-dimensional online energy-resolved mass spectrometry ( ^1st online ER-MS)

The mixed reference solution was introduced into the LC-Qtrap 6500 mass spectrometer. Chromatographic column: Acquity UPLC HSS T3 column (2.1mm×100mm, 1.8μm, Waters Company, USA); mobile phase: 0.1% (v/v) formic acid water (A) and acetonitrile (B); elution program: 0– 5min, 25–35%B; 5–15min, 35%–41%B; 15–20min, 41%–42%B; 20–25min, 42%–100%B; 25–25.1min, 100%–25 %B; 25.1–30min, 25%B; flow rate: 0.25mL/min; column oven: 40°C; injection volume: 2μL. The ion source parameter settings are as follows: negative ion mode acquisition, the atomizer, heater, curtain gas or collision gas are all N ₂ ; atomizer gas (GS1): 50psi; heater gas (GS2): 50psi; curtain gas: 30psi; Ion spray needle voltage: –5500V; Heater gas temperature: 450°C; Collision-activated dissociation (CAD) gas: High. Use MRM mode to optimize parameters for all target ion transitions. Taking the ion transition m/z 528.3>97 as an example, the series of pseudo-ion transitions (PITs) are set to 528.301>97, 528.302>97, 528.303>97...528.350>97, and the corresponding CE values are respectively -70, -71, -72···-120eV, and DP are all -120V. Define the maximum response value in the series of pseudo-ion pairs as 1, calculate the relative response values of all pseudo-ion pairs, import the matrix of relative response values and collision energies into GraphPadPrism, and establish a fitting curve of CE and relative response values, which is 1 ^st Fragmentation curves of online ER-MS. Similar to the concept of half effective concentration (EC ₅₀ ), the CE corresponding to when the relative abundance of the ion pair is 0.5 is defined as the half response collision energy (CE ₅₀ ); the CE corresponding to the vertex of the parabola is defined as the optimal collision energy (OCE).

1.4.2 Establishment of second-dimensional online energy-resolved mass spectrometry ( ^2nd online ER-MS)

The mixed reference solution was introduced into the LC-Qtrap 6500 mass spectrometer. Chromatographic column: Acquity UPLC HSS T3 column (2.1mm×100mm, 1.8μm, Waters Company, USA); mobile phase: 0.1% (v/v) formic acid water (A) and acetonitrile (B); elution program: 0– 5min, 25–35%B; 5–15min, 35%–41%B; 15–20min, 41%–42%B; 20–25min, 42%–100%B; 25–25.1min, 100%–25 %B; 25.1–30min, 25%B; flow rate: 0.25mL/min; column oven: 40°C; injection volume: 2μL. The ion source parameter settings are as follows: negative ion mode acquisition, the atomizer, heater, curtain gas or collision gas are all N ₂ ; atomizer gas (GS1): 50psi; heater gas (GS2): 50psi; curtain gas: 30psi; Ion spray needle voltage: –5500V; Heater gas temperature: 450°C; Collision-activated dissociation (CAD) gas: High. Use MS ³ mode to optimize parameters for all target ion transitions. Taking the ion pair m/z 528.3>448.3>74 as an example, set up 16 MS ³ experiments, AF2 corresponds to 0.005, 0.015, 0.025, 0.035, 0.045, 0.055, 0.065, 0.075, 0.085, 0.095, 0.105, 0.125, 0.135 respectively. , 0.145, 0.155V, CE is -45eV, DP is -120V. Define the maximum response value in the ion pair in the series of MS ³ experiments as 1, calculate the relative response value of the ion pair in all MS ³ events, import the matrix of relative response value and AF2 into GraphPad Prism, and establish a fitting of AF2 and relative response value The curve is the fragmentation curve of ^2nd online ER-MS. Define AF2 corresponding to the half response excitation energy when the relative abundance of the ion pair is 0.5; define AF2 corresponding to the vertex of the parabola as the optimal excitation energy.

Experimental results of Example 2

2.1 Differentiation of free cholic acid sulfonate isomers of metabolites in vivo of bile traditional Chinese medicine based on multi-stage online energy-resolved mass spectrometry

Based on the pMRM ion transition m/z 471.3>97 in Qtrap, the dihydroxy free cholic acid sulfonate compounds in the model sample were detected. A total of 10 peaks appeared. Compounds 9 and 10 were compared with the standard. They are accurately identified as CDCA-3-S and DCA-3-S respectively. According to the literature [Lin M, Chen X, Wang Z, Wang D, Zhang JL. Global profiling and identification of bile acids by multi-dimensional data mining to reveal a way of Eliminate abnormal bile acids. Anal Chim Acta. 2020 Oct 2; 1132:74-82.] Compounds 3-8 are designated as UDCA-7-S, UDCA-3-S, CDCA-7-S, and HDCA-6-S respectively. , DCA-12-S and HDCA-3-S, comparing the high-resolution mass spectrometry data collected in QTOF-MS, the quasi-molecular ions of these free cholic acid sulfonate compounds are all m/z 471.3([M–H ] ^– , C ₂₄ H ₃₉ O ₇ S ^– ), mainly neutral losses of SO ₃ (80Da) and H ₂ O (18Da) occur, and fragment ions m/z 391.3 ([MH-SO ₃ ] ^- are produced in MS ² , C ₂₄ H ₃₉ O ₄ ^– ), 373.3 ([M–H–SO ₃ –H ₂ O] ^– , C ₂₂ H ₃₉ O ₃ ^– ) and 97 ([HSO ₄ ] ^– ). Among them, 97([HSO ₄ ] ^– ) is the characteristic ion of bile acid sulfonate compounds. Therefore, the ion pair m is constructed for the eight free bile acid sulfonate compound isomers of the identified compounds 3-10. /z 471.3>97 cleavage curve (Figure 1A), calculated by software, UDCA-3-S, UDCA-7-S, HDCA-3-S, HDCA-6-S, CDCA-3-S, CDCA-7 The OCE _{m/z 471.3>97} of -S, DCA-3-S and DCA-12-S are -107.1, -100.9, -106.1, -103.8, -106.6, -102.9, -105.6 and -99.0eV respectively, the same as There are certain differences in OCE between isomers. As shown in Figure 1B, the size of OCE _{m/z 471.3>97} and the substitution position of the -SO ₃ group show a certain pattern: 3-OH>6-OH>7-OH>12-OH. Therefore, ^1st ER-MS can construct a relationship curve between characteristic ion abundance and collision energy based on multiple reaction monitoring (MRM) mode, obtain the optimal collision energy required for the fragmentation of characteristic ions, and achieve the distinction of isomers. And the connection position information of the -SO ₃ group can be determined based on the distribution of OCE _{m/z 471.3>97} .

Free cholic acid sulfonate compounds such as UDCA-3-S, HDCA-3-S, CDCA-3-S and DCA-3-S are usually sulfated in vivo by the corresponding free cholic acid. The metabolites produced, such compounds usually undergo hydrolysis in aqueous acidic media to shed the sulfo group, producing the corresponding free bile acids UDCA, HDCA, CDCA and DCA respectively. Since the precursor ions mainly undergo gas-phase decomposition ion reactions in the collision cell of Qtrap-MS in the CID manner, the fragment ions produced should theoretically be consistent with the quasi-molecular ions of the hydrolysis products produced after the chemical hydrolysis of the compound. Therefore, the fragmentation curves of the ion pair m/z 471.3>391.3>391.3 were constructed for the identified dihydroxy free cholic acid sulfonate compounds 3-10, and the ion pairs were constructed for the corresponding dihydroxy free cholic acid compound reference substances. For the fragmentation curve of m/z 391.3>391.3>391.3, the corresponding half response excitation energy was obtained. As shown in Figure 2, there are obvious differences in the fragmentation curves of the sulfonate compound ions of UDCA, HDCA, CDCA and DCA at m/z 471.3>391.3>391.3, but they are significantly different from the corresponding free bile acids UDCA, HDCA and CDCA. It is almost consistent with the fragmentation curve corresponding to the ion pair m/z of DCA 391.3>391.3>391.3. Therefore, ^2nd online ER-MS can characterize the parent core skeleton information of the isomer molecules of free cholic acid sulfonate compounds by constructing a relationship curve between relative ion abundance and excitation energy.

After analyzing the quasi-molecular ions and fragment ions in the high-resolution data of compounds 1 (t _R =7.76min) and 2 (t _R =8.47min), it was found that compounds 1 and 2 are both dihydroxy free cholic acid sulfonate compounds. Fragmentation curves of ion pair m/z 471.3>97 were constructed for compounds 1 and 2 through ^1st online ER-MS. The best OCEs were -103.4 and -106.3eV respectively, which were distributed in -SO ₃ substituted in 6-OH and The OCE _{m/z 471.3>97} range at the 3-OH position (Figure 3A), therefore, compounds 1 and 2 belong to the dihydroxy free cholic acid sulfonic acid substituted by -SO ₃ at the 6-OH and 3-OH positions, respectively. Salt compounds. Based on the ^2nd online ER-MS, the fragmentation curves of the ion pair m/z 471.3>391.3>391.3 were constructed for compounds 1 and 2 respectively, and the ion pair m/z were constructed for the free cholic acid standards UDCA, CDCA, HDCA, DCA and MDCA. The results show that the cleavage curves of compounds 1 and 2 are consistent with those of MDCA (Figure 3B). Therefore, compounds 1 and 2 belong to the sulfonate metabolites of MDCA. Combined with the information obtained by ER ² -MS, compounds 1 and 2 could be accurately assigned as MDCA-6-S and MDCA-3-S, respectively.

2.2 Identification of glycine cholic acid sulfonate or glucuronate isomers of the in vivo metabolites of bile traditional Chinese medicine based on multi-stage online energy-resolved mass spectrometry

Free bile acid combines with glycine through an amide bond to form glycine cholic acid compounds. The reference substances glycine cholic acid compound sulfonates GUDCA-3-S, GCDCA-3-S, and GDCA-3-S are The molecular ion peaks ([MH] ^- ) given in the MS ¹ spectrum are all m/z 528.3. After neutral loss of SO ₃ (80Da), the fragment ion m/z 448.3 appears in the MS ² spectrum, and characteristic fragments are produced. Ions m/z 97 and 74 were assigned to sulfate [HSO ₄ ] ^- and glycine ([NHCH ₂ CO ₂ H] ^- ) residues, respectively. Since m/z 74 is the characteristic ion of glycinic cholic acid compounds, the ion pairs m/z 528.3>448.3> were constructed for the reference substances GUDCA-3-S, GCDCA-3-S, and GDCA-3-S respectively. Fragmentation curves of 74 were constructed for reference substances GUDCA, GCDCA and GDCA respectively with ion pair m/z 448.3>448.3>74. According to software calculations, the optimal excitation energies of GUDCA-3-S, GCDCA-3-S and GDCA-3-S ions for m/z 528.3>448.3>74 are 0.09667, 0.09122 and 0.1017V respectively, and their corresponding Gan The optimal excitation energies of the ion pairs m/z 448.3>448.3>74 of the ammonia-type cholic acid compounds GUDCA, GCDCA and GDCA are 0.09781, 0.08998 and 0.1021V respectively. It can be seen from Figure 4 that there are certain differences in the cleavage curves between isomers of glycine cholic acid sulfonate compounds, but they are consistent with the cleavage curves of their corresponding glycine cholic acid compounds. Therefore, based on ^2nd online ER-MS, the relationship curve between relative ion abundance and excitation energy can be constructed to characterize the parent core skeleton information of the glycine-type cholic acid sulfonate isomer molecule.

Glucuronidation is an important II binding reaction in organisms. Glycine cholic acid compounds are catalyzed by uridine diphosphate glucuronosyltransferases (UGTs) to form more hydrophilic glycine cholic acid glucuronide. Acid compounds. Based on the pMRM ion transition m/z 624.3>448.3 in Qtrap, the dihydroxyglycine-type cholic acid glucuronate compounds in the model sample were detected. A total of 4 peaks appeared, which were compound 11 (t _R =6.85min) , 12 (t _R =7.60min), 13 (t _R =10.35min) and 14 (t _R =10.52min) (Figure 5A). The high-resolution data of all four show quasi-molecular ion peaks ([MH] ^- ) It is m/z 624.3, and the predicted molecular formula is C ₃₂ H ₅₁ NO ₁₁ (Figure 5B). By constructing the fragmentation curves of the ion pairs m/z 624.3>448.3>74.0 for these four compounds, it was found that the fragmentation curves of compounds 11 and 13 were consistent with the fragmentation curves of GUDCA. Therefore, compounds 11 and 13 belong to the glucuronidation derivatives of GUDCA. things. By comparison with the literature [Lin M, Chen :74-82.], compounds 11 and 13 were designated as GUDCA-7-G and GUDCA-3-G respectively. Compounds 12 and 14 are consistent with the cleavage curves of GDCA and GHDCA, respectively. Therefore, compounds 12 and 14 belong to the glucuronidated derivatives of GDCA and GHDCA, respectively.

2.3 Identification of taurine-type cholic acid sulfonate or glucuronate isomers of the in vivo metabolites of bile traditional Chinese medicine based on multi-stage online energy-resolved mass spectrometry

Free bile acid combines with taurine through an amide bond to form taurine cholic acid compounds. The reference standards are taurine cholic acid compound sulfonates TUDCA-3-S, TCDCA-3-S, and TDCA-3- The molecular ion peaks ([MH] ^- ) given by the MS ¹ spectrum of S are all m/z 578.3. After neutral loss of SO ₃ (80Da), the fragment ion m/z 498.3 appears in the MS ² spectrum, and the Characteristic fragment ions m/z 97 ([HSO ₄ ] ^- ) and 80 ([SO ₃ ] ^- ). Since m/z 80 is a characteristic ion of taurine-type cholic acid compounds, ion pairs m/z 578.3>498.3>80 were constructed for TUDCA-3-S, TCDCA-3-S, and TDCA-3-S respectively. Fragmentation curves were constructed for TUDCA, TCDCA and TDCA with m/z 498.3>498.3>80. According to software calculations, the optimal excitation energies of ions of TUDCA-3-S, TCDCA-3-S, and TDCA-3-S for m/z 578.3>498.3>80 are 0.1377, 0.1432, and 0.1316V respectively, and their corresponding N The optimal excitation energies of the ion pairs m/z 498.3>498.3>80 for the sulfonate cholic acid compounds TUDCA, TCDCA, and TDCA are 0.1381, 0.1408, and 0.1321V respectively. It can be seen from Figure 6 that there are certain differences in the cleavage curves between isomers of taurine cholic acid sulfonate compounds, but they are consistent with the cleavage curves of their corresponding taurine cholic acid compounds. Therefore, the relationship between relative ion abundance and excitation energy is constructed based on ^2nd online ER-MS, which can characterize the parent core skeleton information of taurine cholic acid sulfonate isomer molecules.

Taurine cholic acid compounds are catalyzed by uridine diphosphate glucuronosyltransferase (UGT) to form more hydrophilic taurine cholic acid glucuronide compounds. Based on the pMRM ion transition m/z 674.3>498.3 in Qtrap, the dihydroxytaurine-type cholic acid glucuronate compounds in the model sample were detected, and a total of 5 peaks appeared, which were compound 15 (t _R = 4.30min) , 16 (t _R =4.72min), 17 (t _R =6.97min), 18 (t _R =7.31min) and 19 (t _R =8.10min) (Figure 7A). The high-resolution data all showed quasi-molecular ion peaks. ([MH] ^- ) is m/z 674.3, and the predicted molecular formula is C ₃₂ H ₅₃ NO ₁₂ S (Figure 7B). By constructing the fragmentation curves of the ion pairs m/z 674.3>498.3>80 for these four compounds (Figure 7C), it was found that the fragmentation curves of compounds 15 and 16 were consistent with the fragmentation curves of TUDCA. Therefore, compounds 15 and 16 belonged to TUDCA. Glucuronidated derivatives. Compounds 17 and 19 are consistent with the cleavage curves of TDCA and both belong to glucuronidated derivatives of TDCA, while compound 18 is consistent with the cleavage curve of TCDCA. Therefore, compound 18 belongs to the glucuronidated derivatives of TCDCA. By comparison with the literature [Lin M, Chen :74-82.], compounds 15 and 16 can be designated as TUDCA-7-G and TUDCA-3-G respectively; compounds 17 and 19 can be accurately designated as TDCA-12-G and TDCA-3-G.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the invention. In this way, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims (8)

1. A multi-stage online energy-resolved mass spectrometry method, characterized in that: the mass spectrometry method adopts a mass spectrometry system with dual collision cells, the multi-stage online energy-resolved mass spectrometry method has two analysis dimensions, and the mass spectrometry method includes the following step: First, in the first dimension, online energy-resolved mass spectrometry is used to construct the fragmentation curve of related ions based on multiple reaction monitoring mode. Through Gaussian curve fitting, the correlation between the optimal collision energy and the substructure connection mode is established; Then in the second dimension, energy-resolved mass spectrometry after collision-induced dissociation is used to construct the fragmentation curve of related ions based on the three-level mass spectrometry mode or the three-level multiple reaction monitoring mode. By comparing with the reference substance, the correlation between the excitation energy and the structure is established, and the identification substructure of chemical components, The first dimension online ER-MS is an online energy-resolved mass spectrometry method. It uses the MRM mode in the HPLC-Qtrap-MS system to detect the compounds to be tested, constructs the relationship curve between the relative ion abundance and the collision energy, and uses quantum chemistry to calculate the candidate structure. Bond length and bond dissociation energy, construct a quantitative structure-collision energy relationship model, and reveal information about substructure connection methods, The second dimension online ER-MS is energy-resolved mass spectrometry after collision-induced dissociation. It uses the MS ³ mode in the HPLC-Qtrap-MS system to detect the compounds to be tested, and constructs a relationship curve between the relative ion abundance and excitation energy to characterize the compounds to be tested. Substructural information of test compounds. 2. The mass spectrometry method according to claim 1, characterized in that: the mobile phase, chromatographic column model, flow rate, column temperature, elution program and injection volume used in the mass spectrometry method are adjusted according to the compound to be measured. 3. The mass spectrometry method according to claim 1, characterized in that: the parameters of the ion source are set as follows: ion source temperature: 400-550°C, the spray voltage in the negative ion mode is set to -4500 V; the collision gas is High; the curtain gas The range is 25-35 psi; GS1 and GS2 are 15-55 psi; among them, the curtain gas, GS1, GS2 and ion source temperatures are adjusted according to the flow rate. 4. The mass spectrometry method according to claim 1, characterized in that: the first-dimensional online ER-MS adopts a multi-reaction monitoring scanning mode, the injection voltage is -10 V; the collision chamber injection voltage is -16 V, and a series of Pseudo ion pair, corresponding to a set of progressive CE values, –30 - –80 eV, Step is set to 1 eV, with the response value as the ordinate, the progressive collision energy as the abscissa, and further perform a Gaussian curve through GraphPad Prism 8.0 Through fitting, the optimal collision energy of the target ion is obtained; through Sigmoid curve fitting, the half response collision energy of the target ion is obtained. 5. The mass spectrometry method according to claim 1, characterized in that: the second-dimensional online ER-MS adopts the third-level mass spectrometry scanning mode, and the scanning speed is 10000 Da/s; the fixed filling time of the linear ion trap is 10 ms; and the excitation The time is 25 ms; 16 MS ³ experiments are set up, corresponding to a set of progressive AF2 values, 0.005-0.175 V, Step is set to 0.01 V, with the response value as the ordinate, and the progressive excitation energy as the abscissa. Further pass GraphPad Prism 8.0 performs Gaussian curve fitting to obtain the optimal excitation energy of the target ion; through Sigmoid curve fitting, the half response excitation energy of the target ion is obtained. 6. Application of the mass spectrometry method according to any one of claims 1 to 5 in the accurate identification of the chemical structure of unknown compounds in complex systems. 7. The application according to claim 6, characterized in that: the unknown compound is a bile acid compound. 8. The application according to claim 6, characterized in that the complex system includes biological reagents, medicines, food and clinical samples.