A betaine lipid high-resolution mass spectrometry database, a construction method and application thereof, and a betaine lipid detection method

Abstract

The application provides a betaine lipid high-resolution mass spectrum database and a construction method and application thereof and a betaine lipid detection method, and relates to the technical field of analysis and detection.The application is based on the advantages of a super high performance liquid chromatography-mass spectrometer, analyzes the mass spectrum law of betaine lipids through a data-dependent acquisition mode, and constructs a primary and secondary mass spectrum database containing various DGTS type betaine lipids, DGTA type betaine lipids and DGCC type betaine lipids.Through the metabolite name, molecular formula, parent ion and characteristic fragment ion accurate mass information in the betaine lipid high-resolution mass spectrum database, the components in the sample can be quickly and accurately identified and annotated, meanwhile, the relative quantitative analysis is carried out according to the primary mass spectrum data of the corresponding metabolites, which has important significance for the research on the lipid metabolism pathway and functional activity in organisms, and the comprehensive deciphering of algal defense system and molecular regulation network.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, specifically to a high-resolution mass spectrometry database of betaine lipids, its construction method and application, and a method for detecting betaine lipids. Background Technology

[0002] Betaine lipids are structural and functional lipids widely found in various biological systems, including microalgae, bacteria, protozoa, fungi, and plants. They are phosphorus-free amphiphilic molecules composed of two fatty acid chains and a polar head molecule. Betaine lipids are structural analogs of phosphatidylcholine (PC). Currently, three types of betaine lipids are known: diacylglyceryl-N,N,N-trimethylhomo-serine (DGTS), diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA), and diacylglyceryl-carboxyhydroxymethylcholine (DGCC). DGTA and DGTS are isomers. DGTS is widely distributed across various biological systems, while DGTA and DGCC are found only in a few groups of algae. Betaine, along with phospholipids and glycolipids, constitutes the three major polar lipids in plants. Betaine plays an important role in maintaining the functional characteristics of cells during cellular life processes and under various environmental stresses. Furthermore, some betaine composed of special fatty acids also possess anti-inflammatory activity, making them potential anti-inflammatory agents in the pharmaceutical, nutritional, and functional food industries.

[0003] Currently, several analytical methods are available for the identification of betaine lipids in biological samples. After derivatization or acid hydrolysis, traditional analytical methods, including thin-layer chromatography, high-performance liquid chromatography (HPLC), gas chromatography (GC), and GC-MS, are used for the structural identification of betaine lipids in organisms. However, these methods are time-consuming and cannot effectively distinguish between isomers. GC-MS is a powerful tool for betaine structural identification, providing specific structural information for the identification of different betaine species. There are reports of analytical methods for the detection of betaine lipids in marine microalgae based on GC-MS, employing a data-independent acquisition (DIA) mode. However, this method is not suitable for high-throughput, rapid qualitative and quantitative analysis of large batches of samples or for complex matrices.

[0004] International databases currently contain limited information on betaine lipids. The LipidMAPS database, for example, has relatively little information on betaine lipids (9 for DGTS, 3 for DGTA, and 6 for DGCC). The LipidSearch database (Thermofisher) contains MS2 and MS3 data for approximately 1.7 million lipid molecules across 8 major classes and 300 subclasses, making it a widely referenced lipid library in lipidomics research; however, it lacks information on betaine lipid profiles. Therefore, there is an urgent need to develop a more efficient analytical method to achieve high-throughput and rapid screening of betaine lipids in biological samples. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a high-resolution mass spectrometry database of betaine lipids, its construction method and application, and a method for detecting betaine lipids. This invention constructs a high-resolution mass spectrometry database of betaine lipids, based on which high-throughput rapid screening of betaine lipids in biological samples can be achieved.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for constructing a high-resolution mass spectrometry database of betaine lipids, comprising the following steps:

[0008] The betaine-containing sample, antioxidant, and extractant are mixed and extracted to obtain an extract. The extract is then dried to obtain a total lipid extract. The betaine-containing sample includes algae, which include one or more of Micrococcus pluvialis, Brownia triangularis, and Pseudomicrococcus.

[0009] The total lipid extract was purified by Si SPE column purification using hexane-diethyl ether mixed solvent, methanol and chloroform-methanol-water mixed solvent as eluents in sequence. The eluent of methanol and chloroform-methanol-water mixed solvent was collected, dried and reconstituted to obtain betaine lipid solution.

[0010] The betaine lipid solution was detected by ultra-high resolution liquid chromatography-mass spectrometry to obtain betaine lipid mass spectrometry data. The secondary mass spectrometry data of the betaine lipid was analyzed by data-dependent acquisition mode to obtain the name and molecular formula of betaine lipid, and a high-resolution mass spectrometry database of betaine lipid was constructed.

[0011] The ultra-high resolution liquid chromatography-mass spectrometry (UHPLC-MS) detection includes UHPLC separation and mass spectrometry detection. The UHPLC separation conditions include: a mobile phase system comprising mobile phase A and mobile phase B; mobile phase A comprising ammonium formate, formic acid, acetonitrile, and water; and mobile phase B comprising formic acid, isopropanol, and acetonitrile. The elution method is gradient elution, with the following procedure: 0–4 min, the volume fraction of mobile phase A decreases from 60% to 55%; 4–7 min, the volume fraction of mobile phase A decreases from 55% to 50%; 7–14 min, the volume fraction of mobile phase A decreases from 50% to 45%; 14–19 min, the volume fraction of mobile phase A decreases from 45% to 0%; 19–22 min, the volume fraction of mobile phase A is 0%; 22–22.5 min, the volume fraction of mobile phase A increases from 0% to 60%; and 22.5–30 min, the volume fraction of mobile phase A is 60%.

[0012] Preferably, the betaine-containing sample further includes one or more fungi and plants;

[0013] The algae also include one or more of the following: Chlorella vulgaris, Microalgae yunnanensis, Leymus chinensis, Microdiatoms, Isochrysis galbana, Coccolithophora, Botrytis cinerea, Pavlova var. chinensis, Protodinium spp., Karenia mikimotoi, Haematococcus pluvialis, and kelp.

[0014] The mass ratio of the betaine-containing sample to the antioxidant was 1:0.003–0.05.

[0015] The extractant includes a chloroform-methanol mixed solvent, wherein the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 1 to 3:1;

[0016] The concentration of the antioxidant in the extractant is 50–100 μg / mL.

[0017] Preferably, the extraction includes sequentially performing oscillating extraction and ultrasonic extraction; the number of extractions is preferably 2 to 3 times; the time for a single oscillating extraction is 5 to 10 minutes; and the time for a single ultrasonic extraction is 20 to 30 minutes.

[0018] Preferably, the hexane-diethyl ether mixed solvent includes a first hexane-diethyl ether mixed solvent and a second hexane-diethyl ether mixed solvent, wherein the volume ratio of hexane to diethyl ether in the first hexane-diethyl ether mixed solvent is 3 to 5:1, and the volume ratio of hexane to diethyl ether in the second hexane-diethyl ether mixed solvent is 1 to 2:1.

[0019] The volume fraction of chloroform in the chloroform-methanol-water mixed solvent is 10-40%, and the volume fraction of water is 20-40%.

[0020] The solvent used for the resolution includes methanol.

[0021] Preferably, the ultra-high resolution liquid chromatography-mass spectrometry detection uses a C18 column; the sample chamber temperature is 4–8℃, the injection volume is 3–10 μL, and the mobile phase flow rate is 0.25–0.35 mL / min.

[0022] The concentration of ammonium formate in the mobile phase A is 5–15 mmol / L, the volume fraction of formic acid is 0.1–0.2%, and the volume fraction of acetonitrile is 60%.

[0023] The concentration of ammonium formate in the mobile phase B is 5–15 mmol / L, the volume fraction of formic acid is 0.1–0.2%, and the volume fraction of acetonitrile is 10%.

[0024] Preferably, the mass spectrometry detection conditions include: a scanning mass range of 200–1000 m / z, a first-order resolution of 70,000, and an automatic gain control target value of 5 × e⁵; a positive ion mode voltage of 3800 V, an ion transmission tube temperature of 350 °C, a desolvation gas nitrogen flow rate of 35 L / h, an auxiliary gas nitrogen flow rate of 15 L / h, a vaporization chamber temperature of 350 °C, a second-order resolution of 35,000, an automatic gain control target value of 2 × e⁵, and a collision energy of 35–40 eV.

[0025] Preferably, the method for constructing the betaine high-resolution mass spectrometry database includes:

[0026] Using Xcalibur TM The data processing system analyzes and summarizes the mass spectrometry fragmentation patterns of betaine to obtain primary and secondary mass spectrometry data of betaine.

[0027] Using mzVault 2.3 software, the name and molecular formula of betaine were entered into the database system;

[0028] The primary and secondary mass spectrometry data of betaine were imported into the library construction system, and the information was integrated to construct a high-resolution mass spectrometry database of betaine.

[0029] This invention also provides a high-resolution mass spectrometry database of betaine lipids obtained by the construction method described in the above technical solution.

[0030] The present invention also provides the application of the betaine high-resolution mass spectrometry database described in the above technical solution in the qualitative and / or quantitative detection of betaine.

[0031] This invention also provides a method for detecting betaine, comprising the following steps:

[0032] The betaine-containing sample to be tested, antioxidant and extractant are mixed and extracted to obtain an extract. The extract is then dried to obtain the total lipid extract of the sample to be tested.

[0033] The total lipid extract of the sample to be tested was purified by Si SPE column purification using hexane-diethyl ether mixed solvent, methanol and chloroform-methanol-water mixed solvent as eluents in sequence. The eluent of methanol and chloroform-methanol-water mixed solvent was collected, dried and reconstituted to obtain the betaine lipid solution to be tested.

[0034] The betaine lipid solution to be tested was detected by ultra-high resolution liquid chromatography-mass spectrometry to obtain the mass spectrometry data of betaine lipid;

[0035] Based on the mass spectrometry data of the betaine to be tested and the betaine high-resolution mass spectrometry database described in the above technical solution, the detection results of betaine in the sample containing betaine to be tested are obtained.

[0036] Leveraging the advantages of ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS), representative microalgae containing three types of betaine lipids—DGTS, DGTA, and DGCC—were selected. Mass spectrometric patterns of these betaine lipids were analyzed using a data-dependent acquisition mode, constructing primary and secondary high-resolution mass spectrometry databases for these three types of betaine lipids. These databases cover various DGTS, DGTA, and DGCC types of betaine lipids and include their molecular formulas, elemental composition, and primary (MS1) and secondary (MS2) mass spectrometry information. This allows for automatic retrieval of secondary mass spectra of unknown samples and qualitative detection of compounds by comparing corresponding characteristic fragment ions. Simultaneously, relative quantitative analysis of unknown samples can be performed based on the primary mass spectrum peak area combined with isotopic internal standards.

[0037] The betaine high-resolution mass spectrometry database constructed in this invention has broader coverage, stronger scalability, and higher throughput. In ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS), by searching the database for precise mass information such as metabolite names, molecular formulas, parent ions, and characteristic fragment ions, rapid and accurate identification, annotation, and high-throughput screening of betaine lipids in organisms can be achieved. Simultaneously, relative quantitative analysis based on the primary mass spectrometry data of the corresponding metabolites is of great significance for studying lipid metabolism pathways and functional activities in organisms, as well as for comprehensively deciphering algal defense systems and molecular regulatory networks. The betaine high-resolution mass spectrometry database constructed in this invention overcomes the insufficient identification accuracy caused by missing mass spectrometry information, different instrument types and conditions, and different data acquisition methods in existing methods. It is also of great significance for studying lipid metabolism pathways and functional activities in organisms, and for filling the gap in betaine analysis in lipidomics research.

[0038] Furthermore, this invention uses other algae (Chlorella vulgaris, Microalgae yunnanensis, Leymus chinensis, Microdiatoms, Isochrysis galbana, Pavlova spp., Coccolithophora, Botrytis cinerea, Prorocentrum spp., Karenia mikimotoi, Haematococcus pluvialis, kelp), fungi (e.g., Enoki mushroom, Termitomyces albuminosus, Pleurotus ostreatus), and plants (e.g., mosses) as betaine-containing samples to enrich the betaine high-resolution mass spectrometry database. The obtained betaine high-resolution mass spectrometry database covers 250 DGTS type betaines, 241 DGTA type betaines, and 139 DGCC type betaines.

[0039] The betaine high-resolution mass spectrometry database constructed in this invention enables high-throughput rapid screening of betaine in unknown samples. This is of great significance for the study of lipid metabolism pathways and functional activities in organisms, filling the gap in betaine analysis in lipidomics research, and comprehensively deciphering algal defense systems and molecular regulatory networks. Attached Figure Description

[0040] Figure 1 The primary and [M+H] compounds of betaine in *D. globulus* (DGTS) + Analysis of the second-order mass spectrum;

[0041] Figure 2 It is the primary and [M+H] betaine in *Dysmorpha triangularis* (DGTA). + Analysis of the second-order mass spectrum;

[0042] Figure 3 The primary and [M+H] compounds of betaine in *D. gypsum* (DGCC) + Analysis of the second-order mass spectrum;

[0043] Figure 4 Figure for partial data from the DGTS high-resolution mass spectrometry database of betaine;

[0044] Figure 5 Figure for a portion of the DGTA high-resolution mass spectrometry database of betaine;

[0045] Figure 6 Figure for partial data from the DGCC high-resolution mass spectrometry database of betaine;

[0046] Figure 7 A flowchart of the betaine screening process for CompoundDiscoverer software;

[0047] Figure 8 Parameter settings diagram for the Search mzVault module;

[0048] Figure 9 Image comparison of mass spectra of 14:0-22:6 DGTA and 16:1-18:1 DGTA;

[0049] Figure 10 A comparison chart of the relative contents of DGTA in the ranges of 14:0-22:6 and 16:1-18:1;

[0050] Figure 11 Image comparison of 16:0-18:1 DGTS and 16:0-16:1 DGCC mass spectrometry. Detailed Implementation

[0051] This invention provides a method for constructing a high-resolution mass spectrometry database of betaine lipids, comprising the following steps:

[0052] A betaine-containing sample, an antioxidant, and an extractant are mixed and extracted to obtain an extract. The extract is then dried to obtain a total lipid extract. The betaine-containing sample includes algae, specifically one or more of *Microcystis aeruginosa*, *Phaeodactylum tricornutum*, and *Pseudomicrocystis*.

[0053] The total lipid extract was purified by Si SPE column purification using hexane-diethyl ether mixed solvent, methanol and chloroform-methanol-water mixed solvent as eluents in sequence. The eluent of methanol and chloroform-methanol-water mixed solvent was collected, dried and reconstituted to obtain betaine lipid solution.

[0054] The betaine lipid solution was detected by ultra-high resolution liquid chromatography-mass spectrometry to obtain betaine lipid mass spectrometry data. The secondary mass spectrometry data of the betaine lipid was analyzed by data-dependent acquisition mode to obtain the name and molecular formula of betaine lipid, and a high-resolution mass spectrometry database of betaine lipid was constructed.

[0055] The ultra-high resolution liquid chromatography-mass spectrometry (UHPLC-MS / MS) detection includes UHPLC separation and mass spectrometry detection. The UHPLC separation conditions include: a mobile phase system comprising mobile phase A and mobile phase B; mobile phase A comprising ammonium formate, formic acid, acetonitrile, and water; and mobile phase B comprising formic acid, isopropanol, and acetonitrile. The elution method is gradient elution, with the following procedure: 0–4 min, the volume fraction of mobile phase A decreases from 60% to 55%; 4–7 min, the volume fraction of mobile phase A decreases from 55% to 50%; 7–14 min, the volume fraction of mobile phase A decreases from 50% to 45%; 14–19 min, the volume fraction of mobile phase A decreases from 45% to 0%; 19–22 min, the volume fraction of mobile phase A is 0%; 22–22.5 min, the volume fraction of mobile phase A increases from 0% to 60%; and 22.5–30 min, the volume fraction of mobile phase A is 60%.

[0056] The mass spectrometry detection employs a data-dependent acquisition mode.

[0057] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0058] A sample containing betaine lipids, an antioxidant, and an extractant are mixed and extracted to obtain an extract. The extract is then dried to obtain a total lipid extract.

[0059] In this invention, the betaine-containing sample includes algae, and preferably also includes one or more of fungi and plants. In this invention, the betaine-containing sample is preferably a lyophilized powder of betaine-containing sample. In this invention, the algae include one or more of *D. gloeocarpus*, *D. gloeoderma*, and *D. gloeocarpus*; the algae preferably also include one or more of *Chlorella vulgaris*, *Microalgae*, *D. gloeocarpus*, *D. gloeocarpus*, *Isochrysis galbana*, *P. gloeocarpus*, *P. gloeocarpus*, *Botrytis cinerea*, *Prorocentrum spp.*, *Karenella mikimotoi*, *Haematococcus pluvialis*, and kelp. In this invention, the fungi preferably include one or more of *Flammulina velutipes*, *Termitomyces albuminosus*, and *Pleurotus ostreatus*. In this invention, the plants preferably include mosses. This invention enriches the betaine high-resolution mass spectrometry database by using other algae, fungi and plants as betaine-containing samples. The obtained betaine high-resolution mass spectrometry database covers 250 DGTS type betaines, 241 DGTA type betaines and 139 DGCC type betaines.

[0060] In this invention, the antioxidant preferably includes 2,6-di-tert-butyl-p-cresol. In this invention, the mass ratio of algae to antioxidant is preferably 1:0.003 to 0.05, and in specific embodiments it can be 1:0.003, 1:0.005, 1:0.008, 1:0.01, 1:0.015, 1:0.02, 1:0.025, 1:0.03, 1:0.035, 1:0.04, 1:0.045, or 1:0.05; the mass of algae in this invention is based on the mass of the lyophilized algae powder. Considering that currently available commercial betaine standards are limited, only a few types of DGTS are available, while DGTA and DGCC standards are not yet available, it is impossible to construct a high-resolution mass spectrometry database of betaine based on standards. This invention utilizes betaine lipids from three types of algae, each exhibiting specific secondary mass spectrometry fragmentation patterns, including characteristic fragment ions of polar head, specific neutral loss fragment ions, and fatty acid chain fragment ions. This invention selects representative microalgae containing these three types of betaine lipids—DGTS, DGTA, and DGCC—for analysis of their lipid spectroscopic patterns. Combining literature reports and computer elemental simulations, the corresponding primary and secondary mass spectrometry information is calculated. Through the collection of relevant spectra from a large number of samples, a high-resolution mass spectrometry database for DGTS, DGTA, and DGCC is constructed. Furthermore, primary and secondary mass spectra of 250 DGTS, 241 DGTA, and 139 DGCC species were collected based on previously studied species.

[0061] In this invention, the extractant preferably includes a chloroform-methanol mixed solvent; the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is preferably 1 to 3:1, and in specific embodiments it can be 1:1, 1.5:1, 2:1, 2.5:1 or 3:1.

[0062] In this invention, the concentration of the antioxidant in the extractant is preferably 50-100 μg / mL, and in specific embodiments it can be 50 μg / mL, 55 μg / mL, 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL or 100 μg / mL.

[0063] In this invention, the mixing is preferably a mixture of algae and an antioxidant solution; the solvent in the antioxidant solution is preferably the extractant.

[0064] In this invention, the extraction preferably includes sequentially performing oscillating extraction and ultrasonic extraction; the number of extractions is preferably 2 to 3 times; and the extraction temperature is preferably room temperature. In this invention, the time for a single oscillating extraction is preferably 5 to 10 minutes, and in specific embodiments, it can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes; the oscillation speed for the oscillating extraction is preferably 1000 to 2000 r / min, and in specific embodiments, it can be 1200 r / min, 1500 r / min, 1800 r / min, or 2000 r / min. In this invention, the time for a single ultrasonic extraction is preferably 20 to 30 minutes, and in specific embodiments, it can be 20 minutes, 22 minutes, 25 minutes, 28 minutes, or 30 minutes; the frequency of the ultrasonic extraction is preferably 20 to 60 kHz, and in specific embodiments, it can be 20 kHz, 30 kHz, 40 kHz, 50 kHz, or 60 kHz.

[0065] After extraction, the present invention preferably further includes centrifuging the extraction system, with the supernatant being the extract. In this invention, the centrifugation speed is preferably 4000–10000 r / min, and in specific embodiments, it can be 4000 r / min, 5000 r / min, 6000 r / min, 7000 r / min, 8000 r / min, 9000 r / min, or 10000 r / min; the centrifugation time is preferably 10–20 min, and in specific embodiments, it can be 10 min, 12 min, 15 min, 18 min, or 20 min.

[0066] In this invention, the drying preferably includes nitrogen gas drying or rotary evaporation drying. This invention does not impose any particular limitations on the drying conditions, as long as the solvent is removed.

[0067] After obtaining the total lipid extract, the present invention sequentially uses a hexane-ethyl ether mixed solvent and a methanol and chloroform-methanol-water mixed solvent as eluents to purify the total lipid extract using a Si SPE column. The eluent of the methanol and chloroform-methanol-water mixed solvent is collected, dried, and then reconstituted to obtain a betaine lipid solution.

[0068] In this invention, the Si SPE column preferably comprises a CNWBOND Si SPE column (0.5-1g, 6mL). In this invention, the Si SPE column is preferably washed and equilibrated with n-hexane before use; the volume of n-hexane is preferably 6-12mL, and in specific embodiments it can be 6mL, 8mL, 10mL, or 12mL.

[0069] In this invention, the total lipid extract is preferably dissolved first, and the resulting total lipid extract solution is then purified using a SiSPE column. In this invention, the solvent used for dissolution preferably includes a chloroform-methanol mixed solvent, where the volume ratio of chloroform to methanol is preferably 1 to 3:1, and in specific embodiments, it can be 1:1, 1.5:1, 2:1, 2.5:1, or 3:1. In this invention, the mass ratio of the algae to the volume ratio of the solvent used for dissolution is preferably 1 mg: 3 to 30 μL, and in specific embodiments, it can be 1 mg: 3 μL, 1 mg: 5 μL, 1 mg: 10 μL, 1 mg: 15 μL, 1 mg: 20 μL, 1 mg: 25 μL, or 1 mg: 30 μL.

[0070] In this invention, the hexane-diethyl ether mixed solvent preferably includes a first hexane-diethyl ether mixed solvent and a second hexane-diethyl ether mixed solvent; the volume ratio of hexane to diethyl ether in the first hexane-diethyl ether mixed solvent is preferably 3 to 5:1, and in specific embodiments it can be 3:1, 3.5:1, 4:1, 4.5:1 or 5:1; the volume ratio of hexane to diethyl ether in the second hexane-diethyl ether mixed solvent is preferably 1 to 2:1, and in specific embodiments it can be 1:1, 1.2:1, 1.5:1, 1.8:1 or 2:1.

[0071] In this invention, the volume fraction of chloroform in the chloroform-methanol-water mixed solvent is 10-40%, and in specific embodiments it can be 10%, 15%, 20%, 25%, 30%, 35%, or 40%; the volume fraction of water in the chloroform-methanol-water mixed solvent is preferably 20-40%, and in specific embodiments it can be 20%, 25%, 30%, 35%, or 40%.

[0072] In this invention, the ratio of the mass of algae to the volume of each eluent during the purification process is preferably 1 mg: 0.1 to 1 mL. In specific embodiments, it can be 1 mg: 0.1 mL, 1 mg: 0.2 mL, 1 mg: 0.3 mL, 1 mg: 0.4 mL, 1 mg: 0.5 mL, 1 mg: 0.6 mL, 1 mg: 0.7 mL, 1 mg: 0.8 mL, 1 mg: 0.9 mL, or 1 mg: 1 mL.

[0073] In this invention, the drying preferably includes nitrogen gas drying or rotary evaporation drying. This invention does not impose any particular limitations on the drying conditions, as long as the solvent is removed.

[0074] In this invention, the solvent used for resolution preferably includes methanol, and the ratio of the mass of the algae to the volume of the solvent used for resolution is preferably 1 mg: 0.01 to 0.1 mL. In specific embodiments, it can be 1 mg: 0.01 mL, 1 mg: 0.02 mL, 1 mg: 0.03 mL, 1 mg: 0.04 mL, 1 mg: 0.05 mL, 1 mg: 0.06 mL, 1 mg: 0.07 mL, 1 mg: 0.08 mL, 1 mg: 0.09 mL, or 1 mg: 0.1 mL.

[0075] The present invention preferably further includes passing the obtained betaine lipid solution through a 0.22 μm filter membrane after reconstitution.

[0076] After obtaining the betaine lipid solution, the present invention performs ultra-high resolution liquid chromatography-mass spectrometry (UHPLC-MS / MS) on the betaine lipid solution to obtain mass spectrometry data of the betaine lipid. The secondary mass spectrometry data of the betaine lipid are analyzed by data-dependent acquisition mode to obtain the name and molecular formula of the betaine lipid, and a high-resolution mass spectrometry database of betaine lipid is constructed. The UHPLC-MS / MS detection includes UHPLC separation and mass spectrometry detection.

[0077] In this invention, the conditions for ultra-high performance liquid chromatography (UHPLC) separation include: the chromatographic column is preferably a C18 column, more preferably an ACQUITYUPLC BEH C18; the mobile phase system includes mobile phase A and mobile phase B, wherein mobile phase A includes ammonium formate, formic acid, acetonitrile, and water; and mobile phase B includes formic acid, isopropanol, and acetonitrile; the elution method is gradient elution, and the gradient elution program is shown in Table 1; the sample chamber temperature is preferably 4–8°C, and in specific embodiments, it can be 4°C, 5°C, 6°C, 7°C, or 8°C; the injection volume is preferably 3–10 μL, and in specific embodiments, it can be 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, or 10 μL; the mobile phase flow rate is preferably 0.25–0.35 mL / min, and in specific embodiments, it can be 0.25 mL / min, 0.3 mL / min, or 0.35 mL / min.

[0078] Table 1 Gradient elution program

[0079] Time / min Volume fraction of mobile phase A / % Mobile phase B volume fraction / % 0 60 40 4 55 45 7 50 50 14 45 55 19 0 100 22 0 100 22.5 60 40 30 60 40

[0080] In this invention, the concentration of ammonium formate in mobile phase A is preferably 5–15 mmol / L (mM), and in specific embodiments it can be 5 mM, 8 mM, 10 mM, 12 mM or 15 mM; the volume fraction of formic acid in mobile phase A is preferably 0.1–0.2%, and in specific embodiments it can be 0.1%, 0.12%, 0.15%, 0.18% or 0.2%; the volume fraction of acetonitrile in mobile phase A is preferably 60%.

[0081] In this invention, the concentration of ammonium formate in mobile phase B is preferably 5–15 mmol / L, and in specific embodiments it can be 5 mM, 8 mM, 10 mM, 12 mM or 15 mM; the volume fraction of formic acid in mobile phase B is preferably 0.1–0.2%, and in specific embodiments it can be 0.1%, 0.12%, 0.15%, 0.18% or 0.2%; the volume fraction of acetonitrile in mobile phase B is preferably 10%.

[0082] In this invention, the mass spectrometry detection employs a data-dependent acquisition mode. The preferred conditions for mass spectrometry detection in this invention include: the ion mode is positive ion mode; the primary mass spectrometry uses Full MS-ddMS2 mode for scanning, with a scanning mass range of 200–1000 m / z, a primary resolution of 70,000, and an automatic gain control target value of 5 × e⁵; the positive ion mode voltage is 3800 V, the ion transfer tube temperature is 350 °C, the desolvation nitrogen flow rate is 35 L / h, the auxiliary nitrogen flow rate is 15 L / h, and the vaporization chamber temperature is 350 °C; positive ion calibration is performed on the mass spectrometer before sample processing; the secondary mass spectrometry uses an automatic trigger mode; the secondary resolution is 35,000, the automatic gain control target value is 2 × e⁵, and the collision energy is 35–40 eV, which in specific embodiments can be 35 eV, 36 eV, 37 eV, 38 eV, 39 eV, or 40 eV.

[0083] While DIA (Data Independent Acquisition) produces a large number of secondary mass spectra, the quality is limited. Furthermore, as the instrument ages, the background of the chromatographic peaks increases, leading to more interference when using DIA to identify the structure of substances. This invention leverages the advantages of ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) and selects representative microalgae containing three types of betaine: *Dystropharia trifolia* (DGTS), *Dystropharia trifolia* (DGTA), and *Dystropharia pygmy* (DGCC). Using a data-dependent acquisition mode, the mass spectrometric patterns of these betaine are analyzed, constructing a primary and secondary high-resolution mass spectrometry database for the three types of betaine. This database covers 250 DGTS, 241 DGTA, and 139 DGCC species. The database also includes the molecular formula, elemental composition, and primary (MS1) and secondary (MS2) mass spectrometry information for betaine. This information can be used for automatic retrieval of secondary mass spectra of unknown samples. Qualitative detection of compounds can be performed by comparing corresponding characteristic fragment ions. Simultaneously, relative quantitative analysis of unknown samples can be conducted based on the peak area of ​​the primary mass spectrum combined with isotopic internal standards.

[0084] In this invention, the preferred method for constructing the betaine high-resolution mass spectrometry database includes:

[0085] Using Xcalibur TMThe data processing system analyzes and summarizes the mass spectrometry fragmentation patterns of betaine to obtain primary and secondary mass spectrometry data of betaine.

[0086] Using mzVault 2.3 software, the name and molecular formula of betaine were entered into the database system;

[0087] The primary and secondary mass spectrometry data of betaine were imported into the library construction system, and the information was integrated to construct a high-resolution mass spectrometry database of betaine.

[0088] This invention also provides a high-resolution mass spectrometry database of betaine lipids obtained by the construction method described in the above technical solution.

[0089] The present invention also provides the application of the betaine high-resolution mass spectrometry database described in the above technical solution in the qualitative and / or quantitative detection of betaine.

[0090] This invention also provides a method for detecting betaine, comprising the following steps:

[0091] The betaine-containing sample to be tested, antioxidant and extractant are mixed and extracted to obtain an extract. The extract is then dried to obtain the total lipid extract of the sample to be tested.

[0092] The total lipid extract of the sample to be tested was purified by Si SPE column purification using hexane-diethyl ether mixed solvent, methanol and chloroform-methanol-water mixed solvent as eluents in sequence. The eluent of methanol and chloroform-methanol-water mixed solvent was collected, dried and reconstituted to obtain the betaine lipid solution to be tested.

[0093] The betaine lipid solution to be tested was detected by ultra-high resolution liquid chromatography-mass spectrometry to obtain mass spectrometry fragmentation data of betaine lipid;

[0094] The detection results of betaine were obtained based on the mass spectrometry fragmentation data of the betaine to be tested and the betaine high-resolution mass spectrometry database described in the above technical solution.

[0095] This invention involves mixing the betaine-containing sample to be tested, an antioxidant, and an extractant for extraction, obtaining an extract, and drying the extract to obtain a total lipid extract of the sample to be tested. The total lipid extract is then purified using a SiSPE column with hexane-diethyl ether mixed solvent, methanol, and chloroform-methanol-water mixed solvent as eluents. The eluent from the methanol and chloroform-methanol-water mixed solvents is collected, dried, and reconstituted to obtain the betaine lipid solution to be tested. In this invention, the preparation method of the betaine lipid solution to be tested is preferably the same as the method used in the construction of the aforementioned betaine lipid high-resolution mass spectrometry database, and will not be repeated here. This invention does not specifically limit the betaine-containing sample to be tested, as long as it contains betaine. Specifically, biological samples are preferred, including one or more of algae, fungi, and plants. Algae are preferred to include one or more of the following: *Microcystis glomerata*, *Phaeodactylum tricornutum*, *Pseudomicrocystis*, *Chlorella vulgaris*, *Microcystis glomerata*, *Microcystis villosa*, *Microcystis glomerata*, *Isophyllus globosa*, *Coccolithophora*, *Botrytis cinerea*, *Pharbitis pulvinata*, *Prorocentrum spp.*, *Kalenella mikimotoi*, *Haematococcus pluvialis*, *Porphyra yezoensis*, and kelp. Fungi are preferred to include one or more of the following: *Termitomyces albuminosus*, *Flammulina velutipes*, and *Pleurotus ostreatus*. Plants are preferred to include mosses.

[0096] After obtaining the betaine lipid solution to be tested, the present invention performs ultra-high resolution liquid chromatography-mass spectrometry (UHPLC-MS / MS) on the solution to obtain mass spectrometric fragmentation data of the betaine lipids. In this invention, the conditions for the UHPLC-MS / MS detection are the same as those for the UHPLC-MS / MS detection during the construction of the aforementioned high-resolution betaine lipid mass spectrometry database, and will not be repeated here.

[0097] After obtaining the mass spectrometry fragmentation data of betaine, the present invention obtains the detection results of betaine in the sample containing betaine based on the mass spectrometry fragmentation data of the betaine to be tested and the betaine high-resolution mass spectrometry database described in the above technical solution.

[0098] In this invention, the detection results preferably include qualitative detection results and / or relative quantitative detection results; the principle for obtaining the detection results is "first-level quantitative + second-level qualitative".

[0099] In this invention, the method for obtaining the detection results preferably includes the following steps: loading the betaine high-resolution mass spectrometry database into the software CompoundDiscoverer 3.2, simultaneously importing the mass spectrometry fragmentation data of the betaine (i.e., the collected sample spectral data), and setting the corresponding workflow (see details). Figure 7 ), parameters (see details) Figure 8The system performs a database search and matching, where the mzVault module selects the betaine high-resolution mass spectrometry database. Based on the secondary characteristic fragment ions of the compounds in the betaine high-resolution mass spectrometry database, it rapidly screens and accurately identifies betaine in the betaine-containing sample to be tested, obtaining the qualitative detection results of betaine in the betaine-containing sample to be tested. Simultaneously, it extracts the peak area of ​​the primary mass spectrometry and compares it with the internal standard peak area of ​​betaine to perform relative quantitative analysis, obtaining the relative quantitative detection results of betaine in the betaine-containing sample to be tested.

[0100] In this invention, during the relative quantitative analysis process, when preparing the betaine lipid solution to be tested, the betaine lipid sample and the betaine lipid internal standard are added simultaneously. In this invention, the betaine lipid internal standard preferably includes a DGTS isotope internal standard (D9). In this invention, the mass ratio of the betaine lipid sample to the betaine lipid internal standard is preferably 10–50 mg:1 μg, and in specific embodiments it can be 10 mg:1 μg, 20 mg:1 μg, 30 mg:1 μg, 40 mg:1 μg, or 50 mg:1 μg.

[0101] This invention first utilizes an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometer (HPLC-Q-Orbitrap-HRMS) to obtain high-resolution mass spectrometry data of total lipid samples from three representative microalgae containing betaine lipids: *D. gloeocarpus* (DGTS), *D. gloeocarpus triangularis* (DGTA), and *D. gloeocarpus pseudomicrocarpus* (DGCC), through a data-dependent acquisition mode. This data includes primary and secondary ion fragmentation information. Then, secondary mass spectrometry is performed on molecules with even-numbered parent ions to determine their betaine composition and molecular composition based on the mass fragmentation mechanism. The identified betaine lipid mass numbers, elemental composition, adducts, and major secondary characteristic fragment ions are summarized and organized. For betaine fatty acid compositions reported in the literature but not detected in the above three algae, as well as some possible fatty acid compositions, the corresponding primary and secondary mass spectrometry information is calculated through elemental simulation. Based on the established mass spectrometry information, relevant samples were collected and their secondary mass spectra were analyzed using a data-dependent acquisition mode to obtain mass spectrometry data of betaine. Based on the principle of "primary quantitative analysis + secondary qualitative analysis", betaine in biological samples was rapidly screened and accurately identified according to the secondary characteristic fragment ions of compounds in the high-resolution mass spectrometry data. Simultaneously, the peak area of ​​the primary mass spectrometry was extracted and compared with the internal standard peak area of ​​betaine for relative quantitative analysis.

[0102] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of the betaine high-resolution mass spectrometry database, its construction method and application, and the detection method of betaine provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0103] The instruments used in this example were: a Q-Exactive quadrupole electrostatic track trap high-resolution mass spectrometer (ThermoFisher Scientific) equipped with an H-ESI II source; and an UltiMate 3000 high-performance liquid chromatography system with an autosampler. The instrument was an ultra-high performance liquid chromatography-quadrupole-electrostatic track trap high-resolution mass spectrometer.

[0104] Reagents used in the examples: chromatographically pure methanol, acetonitrile, isopropanol, ammonium formate, formic acid, and 2,6-di-tert-butyl-p-cresol (BHT, >99.9%) (Sigma-Aldrich, USA); chloroform, n-hexane, and diethyl ether (analytical grade, Sinopharm); pure water was obtained from a Milli-Q water purifier.

[0105] The standards used in the examples were: DGTS standard 1,2-Dipalmitoyl-sn-glycero-3-O-4-(N,N,N-trimethyl)homoserine (16:0-16:0DGTS) and DGTS isotope internal standard (1,2-di-palmitoyl-sn-glycero-3-O-4'-[N,N,N-trimethyl(D9)-homoserine), both purchased from Avanti Polar Lipids, USA. Both standards were dissolved in methanol, and the prepared solution concentration was 100 μg / mL. Since there are currently no commercially available standards for DGTA and DGCC, these two betaine esters were semi-quantitatively analyzed using DGTS standards.

[0106] Example 1

[0107] 1. Extraction and purification of betaine lipids

[0108] (1) Weigh 20 mg of microalgae freeze-dried powder, add 3 mL of chloroform-methanol mixed solvent (1:1, v / v) containing 50 μg / mL of 2,6-di-tert-butyl-p-cresol, shake for 5 min, extract by sonication for 20 min, then centrifuge at 4000 r / min for 15 min, and take the supernatant; repeat the extraction twice, combine the supernatants, and blow dry with nitrogen gas to obtain total lipid extract of microalgae; the microalgae freeze-dried powders are the algal powders of Micrococcus globosum (DGTS), Phaeodactylum tricornutum (DGTA) and Pseudomicrococcus globosum (DGCC) after freeze drying.

[0109] (2) The crude extract of total lipids was separated using a CNWBOND Si SPE column (1 g, 6 mL). Specifically, 6 mL of n-hexane was added to wash and equilibrate the column. The total lipid extract was dissolved in 200 μL of chloroform-methanol mixed solvent (2:1, v / v) and then loaded onto the CNWBOND Si SPE column along the column wall. Elution was performed sequentially with 6 mL of n-hexane-diethyl ether mixed solvent (4:1, v / v), 6 mL of n-hexane-diethyl ether mixed solvent (1:1, v / v), 6 mL of methanol, and 6 mL of chloroform-methanol-water mixed solvent (3:5:2, v / v / v). The eluents of methanol and chloroform-methanol-water mixed solvent were collected, concentrated and dried under nitrogen to remove the solvent, and then diluted to volume with 1 mL of methanol and filtered through a 0.22 μm filter membrane to obtain the sample solution to be tested.

[0110] 2. Instrument conditions

[0111] (1) Ultra-high performance liquid chromatography separation

[0112] The chromatographic column was an ACQUITYUPLC BEH C18 analytical column (id 2.1×100mm, particle size 1.7μm, pore size...). The flow rate was 0.25 mL / min. Mobile phase A consisted of acetonitrile-water (6:4, v / v) containing 10 mM ammonium formate and 0.1% formic acid, while mobile phase B consisted of isopropanol / acetonitrile (9:1, v / v) containing 10 mM ammonium formate and 0.1% formic acid. The mobile phase gradient is shown in Table 1. The sample chamber temperature was 4 °C, and the injection volume was 3 μL.

[0113] (2) Mass spectrometry conditions

[0114] Mass spectrometry was performed in positive ion mode using Full MS-ddMS2 mode. Mass range: m / z 400–1000, resolution 70000, automatic gain control target value 5×e5; positive ion mode 3800V, ion transfer tube temperature 350℃, solvent removal nitrogen flow rate: 35 L / h, auxiliary nitrogen flow rate: 15 L / h, vaporization chamber temperature 350℃; positive ion calibration was performed before sample processing; the second stage used automatic trigger mode, resolution 35000, automatic gain control target value 2×e5, collision energy 35–40 eV.

[0115] 3. Construction of a high-resolution mass spectrometry database for betaine lipids

[0116] (1) High-resolution mass spectra of betaine lipids in *D. globodactylis*, *D. globodactylis triangularis*, and *D. globodactylis pseudomicroscopica* (DGCC) were obtained using a data-dependent acquisition mode based on ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectra, as shown in the following figures. Figures 1-3 This includes primary and secondary mass spectrometry fragment ion information; via Xcalibur TM The data processing system processes the raw data files obtained from LC / MS, performing secondary mass spectra analysis on each even-numbered precursor ion, and conducting elemental simulations on both the precursor and daughter ions. Based on the mass spectrometric fragmentation patterns of the three types of betaine, it determines whether the substance is betaine and analyzes its molecular composition. The results show that DGTS and DGTA with the same fatty acid composition have essentially identical secondary mass spectra fragment ions, but the abundance ratios of the secondary mass spectra differ significantly at collision energies of 35–40 eV. In the secondary mass spectra fragments of DGTS, the characteristic fragment ion peak [C...]... 10 H 22 O5N] + As a base peak, its relative abundance is much greater than that of [M+H]. + Peak size of the parent ion peak; while in the secondary mass spectrometry fragments of DGTA, [M+H] + The parent ion peak, as the base peak, has a relative abundance much greater than that of the characteristic fragment ion peak [C]. 10 H 22 O5N] + The kurtosis of the peak can be used to distinguish between DGTS and DGTA.

[0117] Subsequently, the identified betaine mass numbers, molecular formulas, addition forms, and main secondary characteristic fragment ions were summarized and organized to obtain primary and secondary high-resolution mass spectrometry information databases for DGTS, DGTA, and DGCC. For some betaine fatty acid compositions that have been reported in the literature but not detected in these three algae, as well as some possible fatty acid compositions, the corresponding primary and secondary mass spectrometry information was calculated through elemental simulation to further improve the high-resolution mass spectrometry information database for betaine, as shown in Tables 2-4.

[0118] (2) Using mzVault 2.3 software, the basic information such as the name and molecular formula of betaine was entered into the library system. Following the methods for obtaining the mass spectrometry databases of DGTS, DGTA and DGCC, algae, fungi and plant samples containing the corresponding betaine were collected. The samples were Chlorella, Microalgae, Leymus chinensis, Microdiatoms, Isochrysis galbana, Prorocentrum mollissima, Karenia mikimotoi, Haematococcus pluvialis, Porphyra yezoensis, kelp, moss, Enoki mushroom, Termitomyces albuminosus and Pleurotus ostreatus. After the betaine mixture was analyzed under the conditions of extraction, purification and chromatographic separation, the raw files of the mass spectra of the corresponding betaine in the samples were imported into the mzVault 2.3 library system. The secondary mass spectra of each betaine compound were extracted, the background of the secondary mass spectra was subtracted and the mass number was corrected, and database entries were generated to construct a high-resolution mass spectrometry database of betaine.

[0119] (3) The betaine high-resolution mass spectrometry database information after the entry is completed is as follows: Figures 4-6 As shown, the database contains 250 DGTAs, 241 DGTSs, and 139 DGCCs. It includes the names, molecular formulas, and high-resolution MS1 ​​and MS2 information of betaine lipids, and can be saved in ".db" format for analysis using Compound Discoverer mass spectrometry software. Tables 2-4 show the main fragment information for the three betaine lipid profiles, including compound name, chemical formula, precursor ion, and fragment. Fragments include characteristic fragment ions and neutral loss of fatty acids ions.

[0120] Table 2. Fragmentation information from DGTS high-resolution mass spectrometry

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] Table 3. DGTA High-Resolution Mass Spectrometry Fragmentation Information

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] Table 4. High-resolution mass spectrometry fragmentation information for DGCC

[0136]

[0137]

[0138]

[0139]

[0140] 4. Construction of a high-throughput detection method for betaine lipids

[0141] The established high-resolution mass spectrometry database of betaine was loaded into the software CompoundDiscoverer 3.2, and the collected sample spectral data was imported. The corresponding workflow was set up. Figure 7 ),parameter( Figure 8 The system searches the database, with the mzVault module selecting the established high-resolution mass spectrometry database and matching the sample spectral data with the information in the high-resolution mass spectrometry database.

[0142] Example 2

[0143] High-throughput detection method and application of betaine based on high-resolution liquid chromatography-mass spectrometry

[0144] 1. Extraction and purification of betaine lipids

[0145] (1) Weigh 20 mg of freeze-dried *Phaeodactylum tricornutum* powder after logarithmic, plateau, and late plateau phases and add it to glass test tubes. Then add 10 μL of 100 μg / mL DGTS isotope internal standard (1,2-di-palmitoyl-sn-glycero-3-O-4'-[N,N,N-trimethyl(D9)-homoserine) and 3 mL of chloroform-methanol mixed solvent (1:1, v / v) containing 50 μg / mL 2,6-di-tert-butyl-p-cresol) to each test tube. Shake for 5 min, sonicate for 20 min, and centrifuge at 4000 r / min for 15 min. Collect the supernatant. Repeat the extraction twice, combine the supernatants, and dry with nitrogen to obtain the total lipid extract.

[0146] (2) The total lipid extract was separated using a CNWBOND Si SPE column (1 g, 6 mL). Specifically, 12 mL of n-hexane was added to wash and equilibrate the column. The total lipid extract was dissolved in 200 μL of a chloroform-methanol mixture (2:1, v / v) and then loaded onto the CNWBOND Si SPE column along the column wall. The column was eluted sequentially with 6 mL of a hexane-diethyl ether mixture (4:1, v / v), 6 mL of a hexane-diethyl ether mixture (1:1, v / v), 6 mL of methanol, and 6 mL of a chloroform-methanol-water mixture (3:5:2, v / v / v). The first two eluents were discarded, and the last two eluents were collected. The eluents were concentrated and dried under nitrogen to remove the solvent. The solution was then diluted to volume with 1 mL of methanol and filtered through a 0.22 μm filter to obtain the sample solution to be tested.

[0147] 2. Sample Analysis

[0148] (1) Ultra-high performance liquid chromatography separation

[0149] The chromatographic column was an ACQUITYUPLC BEH C18 analytical column (id 2.1×100mm, particle size 1.7μm, pore size...). The flow rate was 0.25 mL / min. Mobile phase A consisted of an acetonitrile-water mixture (6:4, v / v) containing 10 mM ammonium formate and 0.1% formic acid (v / v), and mobile phase B consisted of an isopropanol-acetonitrile mixture (9:1, v / v) containing 10 mM ammonium formate and 0.1% formic acid (v / v). The mobile phase gradient is shown in Table 1. The sample chamber temperature was 4 °C, and the injection volume was 3 μL.

[0150] (2) Mass spectrometry conditions

[0151] Mass spectrometry was performed in positive ion mode using Full MS-ddMS2 mode. Mass range: m / z 400–1000, resolution 70000, automatic gain control target value 5×e5; positive ion mode 3800V, ion transfer tube temperature 350℃, solvent removal nitrogen flow rate: 35 L / h, auxiliary nitrogen flow rate: 15 L / h, vaporization chamber temperature 350℃; positive ion calibration was performed before sample processing; the second stage used automatic trigger mode, resolution 35000, automatic gain control target value 2×e5, collision energy 35–40 eV.

[0152] (3) The algal betaine extract was injected into a chromatography-mass spectrometry instrument for elution and mass spectrometry detection, and the corresponding spectral data, including primary and secondary ion fragment information, were collected.

[0153] 3. Data Analysis

[0154] (1) The established high-resolution mass spectrometry database of betaine was loaded into the software CompoundDiscoverer 3.2, and the collected sample spectral data was imported. The corresponding analysis process and parameters were set for database searching, with the established high-resolution mass spectrometry database selected in the mzVault module. The results showed that after matching the sample spectral data with the high-resolution mass spectrometry database information, a total of 131 betaine molecules were identified, including 128 DGTAs and 3 DGTSs (Table 5).

[0155] Table 5. Betaine in *Phaeodactylum tricornutum* samples that matched the resolved mass spectrometry database.

[0156]

[0157]

[0158]

[0159]

[0160] (2) Taking the identified 14:0-22:6 DGTA and 16:1-18:1 DGTA as examples, the results are further interpreted. Figure 9 The image shows a mirror comparison of the two compounds and their matching against the high-resolution mass spectrometry database. The upper part of the image shows the secondary mass spectrometry information of the metabolites actually collected from the samples, and the lower part shows the characteristic fragment ions of the reference substances in the high-resolution mass spectrometry database. Through mirror comparison, it was found that the matching degree of the sample to be tested with the corresponding compound fragment ions in the high-resolution mass spectrometry database is 93.8% and 97.3%, respectively, which is a reliable result.

[0161] (3) Judging from the identification results, the number of betaine lipids identified by the method established in this invention is significantly greater than that reported in the literature for the same sample. Moreover, the detection and analysis process is convenient. It does not require secondary injection to obtain the secondary mass spectrum of the specific parent ion. It does not require manual mass spectrometry analysis of each sample. It can achieve high-throughput rapid screening of betaine lipids in multiple samples at the same time, which effectively improves the detection efficiency.

[0162] (4) While identifying betaine lipids, the peak area of ​​the primary mass spectrometry can be simultaneously extracted and combined with the peak area of ​​the isotope internal standard for relative quantitative analysis. Taking the identified 14:0-22:6 DGTA and 16:1-18:1 DGTA as examples, the contents of these two substances at different growth stages of *Phaeodactylum tricornutum* can be further compared. Figure 10 As shown, the contents of these two substances gradually accumulate with increasing cultivation time, indicating that the accumulation of these substances varies at different growth stages. Therefore, relative quantitative analysis can be used to compare and analyze the content of betaine under different conditions.

[0163] Example 3

[0164] High-throughput detection method and application of betaine based on high-resolution liquid chromatography-mass spectrometry

[0165] 1. Extraction and purification of betaine lipids

[0166] (1) Weigh 20 mg of the sample to be tested and add it to a glass test tube. Then add 10 μL of 100 μg / mL DGTS isotope internal standard (1,2-di-palmitoyl-sn-glycero-3-O-4'-[N,N,N-trimethyl(D9)-homoserine) and 3 mL of chloroform-methanol mixed solvent (1:1, v / v) containing 50 μg / mL 2,6-di-tert-butyl-p-cresol) to each test tube. Shake for 5 min, sonicate for 20 min, and centrifuge at 4000 r / min for 15 min. Collect the supernatant. Repeat the extraction twice, combine the supernatants, and dry with nitrogen gas to obtain the total lipid extract. The samples to be tested were freeze-dried moss, freeze-dried enoki mushroom, or freeze-dried laver.

[0167] (2) The total lipid extract was separated using a CNWBOND Si SPE column (1 g, 6 mL). Specifically, 12 mL of n-hexane was added to wash and equilibrate the column. The total lipid extract was dissolved in 200 μL of a chloroform-methanol mixture (2:1, v / v) and then loaded onto the CNWBOND Si SPE column along the column wall. The column was eluted sequentially with 6 mL of a hexane-diethyl ether mixture (4:1, v / v), 6 mL of a hexane-diethyl ether mixture (1:1, v / v), 6 mL of methanol, and 6 mL of a chloroform-methanol-water mixture (3:5:2, v / v / v). The first two eluents were discarded, and the last two eluents were collected, concentrated and dried under nitrogen to remove the solvent, and then diluted to 1 mL with methanol before being filtered through a 0.22 μm filter membrane.

[0168] 2. Sample Analysis

[0169] (1) Ultra-high performance liquid chromatography separation

[0170] The chromatographic column was an ACQUITYUPLC BEH C18 analytical column (id 2.1×100mm, particle size 1.7μm, pore size...). The flow rate was 0.25 mL / min. Mobile phase A consisted of acetonitrile / water (6:4, v / v) containing 10 mM ammonium formate and 0.1% formic acid, while mobile phase B consisted of isopropanol / acetonitrile (9:1, v / v) containing 10 mM ammonium formate and 0.1% formic acid. The mobile phase gradient is shown in Table 1. The sample chamber temperature was 4 °C, and the injection volume was 3 μL.

[0171] (2) Mass spectrometry conditions

[0172] Mass spectrometry was performed in positive ion mode using Full MS-ddMS2 mode. Mass range: m / z 400–1000, resolution 70000, automatic gain control target value 5×e5; positive ion mode 3800V, ion transfer tube temperature 350℃, solvent removal nitrogen flow rate: 35 L / h, auxiliary nitrogen flow rate: 15 L / h, vaporization chamber temperature 350℃; positive ion calibration was performed before sample processing; the second stage used automatic trigger mode, resolution 35000, automatic gain control target value 2×e5, collision energy 35–40 eV.

[0173] (3) The algal betaine extract was injected into a chromatography-mass spectrometry instrument for elution and mass spectrometry detection, and the corresponding spectral data, including primary and secondary ion fragment information, were collected.

[0174] 3. Data Analysis

[0175] (1) The established high-resolution mass spectrometry database of betaine was loaded into the software CompoundDiscoverer 3.2, and the spectral data of the collected samples were imported. The corresponding analysis procedures and parameters were set for database searching, with the established high-resolution mass spectrometry database selected in the mzVault module. The results showed that after matching the sample spectral data with the information in the high-resolution mass spectrometry database, a total of 44 betaine molecules were screened. Among them, 23 DGTS and 3 DGCC were detected in *Gnaphalium affine*, 17 DGTS were detected in moss, and 26 DGTS were detected in *Termitomyces albuminosus* (Table 6).

[0176] Table 6. Betaine (nmol / g dry powder) matched with high-resolution mass spectrometry databases for three different samples.

[0177]

[0178]

[0179] (2) Taking the identified 16:0-18:1DGTS and 16:1-16:1DGCC as examples, the results are further interpreted. Figure 11 This is a mirror image comparison of the two compounds above with high-resolution mass spectrometry databases. Figure 11 The upper half of the image shows the secondary mass spectrometry information of the metabolites actually collected from the sample, while the lower half shows the characteristic fragment ions of the reference substances in the high-resolution mass spectrometry database. Through mirror comparison, it was found that the matching degree between the sample to be tested and the corresponding compound fragment ions in the high-resolution mass spectrometry database was 91.7% and 97.3%, respectively, which is reliable.

[0180] (3) While identifying betaine lipids, relative quantitative analysis can be performed using the peak area of ​​primary mass spectrometry combined with the peak area of ​​the isotope internal standard. As shown in Table 6, the types of betaine lipids contained in samples from different species are different, and the content of the same betaine lipid varies greatly in samples from different species. Therefore, relative quantitative analysis can be used to compare the content of betaine lipids in different samples.

[0181] (4) Judging from the identification results, the method established in this invention identifies significantly more types of betaine than the number identified in the same sample reported in the literature. Moreover, the detection and analysis process is convenient and fast. It can obtain the secondary mass spectrum of a specific parent ion without the need for secondary injection. It does not require manual mass spectrometry analysis of each sample. It can achieve high-throughput rapid screening of betaine in multiple samples at the same time, which effectively improves the detection efficiency.

[0182] This invention leverages the advantages of ultra-high resolution liquid chromatography-mass spectrometry (HPLC-MS / MS) systems to establish a high-resolution mass spectrometry database containing 250 DGTS, 241 DGTA, and 139 DGCC species. This database includes betaine molecular formulas, elemental composition, and high-resolution MS1 ​​and MS2 information, enabling automatic retrieval of secondary mass spectra for unknown samples. Compound identification is achieved by comparing corresponding characteristic fragment ions. Simultaneously, relative quantitative analysis of samples can be performed based on primary mass spectrum peak areas combined with isotopic internal standards. The method provided by this invention offers broader coverage, greater scalability, and higher throughput, overcoming the limitations in identification accuracy caused by missing mass spectrometry information, different instrument types and conditions, and varying data acquisition methods in existing methods. Furthermore, it is of significant importance for research on lipid metabolism pathways and functional activities in organisms, and for filling the gap in betaine analysis in lipidomics research.

[0183] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a high-resolution mass spectrometry database of betaine, comprising the following steps: The sample containing betaine lipids, antioxidants, and extractants were mixed and extracted to obtain an extract. The extract was then dried to obtain a total lipid extract. The betaine-containing sample includes algae, which include one or more of Microcystis aeruginosa, Phaeodactylum tricornutum, and Pseudomicrocystis thunbergii. The total lipid extract was purified by Si SPE column purification using hexane-diethyl ether mixed solvent, methanol and chloroform-methanol-water mixed solvent as eluents in sequence. The eluent of methanol and chloroform-methanol-water mixed solvent was collected, dried and reconstituted to obtain betaine lipid solution. The betaine lipid solution was detected by ultra-high resolution liquid chromatography-mass spectrometry to obtain betaine lipid mass spectrometry data. The secondary mass spectrometry data of the betaine lipid was analyzed by data-dependent acquisition mode to obtain the name and molecular formula of betaine lipid, and a high-resolution mass spectrometry database of betaine lipid was constructed. The ultra-high resolution liquid chromatography-mass spectrometry (UHPLC-MS) detection includes UHPLC separation and mass spectrometry detection. The UHPLC separation conditions include: a mobile phase system comprising mobile phase A and mobile phase B; mobile phase A comprising ammonium formate, formic acid, acetonitrile, and water; and mobile phase B comprising formic acid, isopropanol, and acetonitrile. The elution method is gradient elution, with the following procedure: 0–4 min, the volume fraction of mobile phase A decreases from 60% to 55%; 4–7 min, the volume fraction of mobile phase A decreases from 55% to 50%; 7–14 min, the volume fraction of mobile phase A decreases from 50% to 45%; 14–19 min, the volume fraction of mobile phase A decreases from 45% to 0%; 19–22 min, the volume fraction of mobile phase A is 0%; 22–22.5 min, the volume fraction of mobile phase A increases from 0% to 60%; and 22.5–30 min, the volume fraction of mobile phase A is 60%.

2. The construction method according to claim 1, characterized in that, The betaine-containing sample also includes one or more fungi and plants; The algae also include one or more of the following: Chlorella vulgaris, Microalgae yunnanensis, Leymus chinensis, Microdiatoms, Isochrysis galbana, Coccolithophora, Botrytis cinerea, Pavlova var. chinensis, Protodinium spp., Karenia mikimotoi, Haematococcus pluvialis, and kelp. The mass ratio of the betaine-containing sample to the antioxidant was 1:0.003–0.

05. The extractant includes a chloroform-methanol mixed solvent, wherein the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 1 to 3:1; The concentration of the antioxidant in the extractant is 50–100 μg / mL.

3. The construction method according to claim 1 or 2, characterized in that, The extraction process includes sequential shaking extraction and ultrasonic extraction; the number of extractions is preferably 2 to 3 times; the time for a single shaking extraction is 5 to 10 minutes; and the time for a single ultrasonic extraction is 20 to 30 minutes.

4. The construction method according to claim 1, characterized in that, The n-hexane-diethyl ether mixed solvent includes a first n-hexane-diethyl ether mixed solvent and a second n-hexane-diethyl ether mixed solvent. The volume ratio of n-hexane to diethyl ether in the first n-hexane-diethyl ether mixed solvent is 3 to 5:1, and the volume ratio of n-hexane to diethyl ether in the second n-hexane-diethyl ether mixed solvent is 1 to 2:

1. The volume fraction of chloroform in the chloroform-methanol-water mixed solvent is 10-40%, and the volume fraction of water is 20-40%. The solvent used for the resolution includes methanol.

5. The construction method according to claim 1, characterized in that, The ultra-high resolution liquid chromatography-mass spectrometry (LC-MS) detection uses a C18 column; the sample chamber temperature is 4–8℃, the injection volume is 3–10 μL, and the mobile phase flow rate is 0.25–0.35 mL / min. The concentration of ammonium formate in the mobile phase A is 5–15 mmol / L, the volume fraction of formic acid is 0.1–0.2%, and the volume fraction of acetonitrile is 60%. The concentration of ammonium formate in the mobile phase B is 5–15 mmol / L, the volume fraction of formic acid is 0.1–0.2%, and the volume fraction of acetonitrile is 10%.

6. The construction method according to claim 1, characterized in that, The mass spectrometry detection conditions include: a scanning mass range of 200–1000 m / z, a first-order resolution of 70,000, and an automatic gain control target value of 5 × e⁵; a positive ion mode voltage of 3800 V, an ion transmission tube temperature of 350 °C, a desolvation nitrogen flow rate of 35 L / h, an auxiliary nitrogen flow rate of 15 L / h, a vaporization chamber temperature of 350 °C, a second-order resolution of 35,000, an automatic gain control target value of 2 × e⁵, and a collision energy of 35–40 eV.

7. The construction method according to claim 1, characterized in that, The methods for constructing the betaine high-resolution mass spectrometry database include: Using Xcalibur TM The data processing system analyzes and summarizes the mass spectrometry fragmentation patterns of betaine to obtain primary and secondary mass spectrometry data of betaine. Using mzVault 2.3 software, the name and molecular formula of betaine were entered into the database system; The primary and secondary mass spectrometry data of betaine were imported into the library construction system, and the information was integrated to construct a high-resolution mass spectrometry database of betaine.

8. The betaine high-resolution mass spectrometry database obtained by the construction method according to any one of claims 1 to 7.

9. The application of the betaine high-resolution mass spectrometry database of claim 8 in the qualitative and / or quantitative detection of betaine.

10. A method for detecting betaine, characterized in that, Includes the following steps: The betaine-containing sample to be tested, antioxidant and extractant are mixed and extracted to obtain an extract. The extract is then dried to obtain the total lipid extract of the sample to be tested. The total lipid extract of the sample to be tested was purified by Si SPE column purification using hexane-diethyl ether mixed solvent, methanol and chloroform-methanol-water mixed solvent as eluents in sequence. The eluent of methanol and chloroform-methanol-water mixed solvent was collected, dried and reconstituted to obtain the betaine lipid solution to be tested. The betaine lipid solution to be tested was detected by ultra-high resolution liquid chromatography-mass spectrometry to obtain mass spectrometry fragmentation data of betaine lipid; Based on the mass spectrometry fragmentation data of the betaine to be tested and the betaine high-resolution mass spectrometry database of claim 8, the detection results of betaine in the sample containing betaine to be tested are obtained.

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