Method for high-sensitivity quantification and structural identification of cobalamin and application thereof

Abstract

The invention belongs to the technical field of detection, and relates to a method for high-sensitivity quantification and structural identification of cobalamin and application thereof. The method comprises the following steps: by taking three common natural cobalamin with different structures in the environment as standard substances, obtaining cobalamin parent ions and high-abundance series characteristic ion fragments by adopting a mass spectrum acquisition mode of full scanning-automatic triggering secondary mass spectrum scanning; according to the principle that the same cobalamin fragments have the same fragment ion m / z and the fragment ion searching function of software, the purpose of identifying the unknown cobalamin structure is achieved; then, a mass spectrum quantitative method for parallel reaction monitoring is adopted, a large number of interference ions are filtered out through a quadrupole, cobalamin low ligand fragment ions with the highest abundance and the identification degree serve as quantitative ions, and the type and content of cobalamin in a to-be-measured sample are analyzed. By combining two data acquisition modes of high-resolution mass spectrometry, interference of an endogenous matrix can be effectively eliminated, the sample analysis sensitivity is improved, and the quantitative specificity is enhanced, so that high-sensitivity detection of different types of cobalamin is realized.

Description

Technical Field

[0001] The invention belongs to the field of detection technology and relates to a highly sensitive quantitative and structural identification method of cobalamin (a liquid chromatography-high resolution mass spectrometry analysis and detection method of cobalamin) and applications thereof. Background Art

[0002] Cobalamin (eg, vitamin B 12 ) is a class of organic compounds based on a complex porphyrin skeleton and containing cobalt ions. Its molecular weight is about 1,300-1,500 Daltons, making it the largest known non-polymeric natural biomacromolecule. The core structure of cobalamin is a conservative tetrapyrrole ring system. The nitrogen atom at the center of the tetrapyrrole ring binds to the cobalt ion with four coordination bonds to form a closed corrin ring structure. The cobalt ion at the center of the corrin ring is covalently linked to one or two variable axial functional groups, namely the high-position ligand and the low-position ligand. The lower end of the corrin ring and the other end of the low-position ligand are connected by a nucleotide ring side chain. Up to 18 natural cobalamin species have been discovered so far, and their structural diversity is mainly reflected in the low-position ligand, which is usually one of the benzimidazole, phenolic or purine compounds ( Figure 1 ). As a metal coenzyme factor, cobalamin is widely involved in the biosynthesis of amino acids, DNA and fatty acids, as well as the biogeochemical cycles of carbon, halogens and heavy metals. Vitamin B 12 As the most typical representative of natural cobalamin, it plays an important role in red blood cell production, DNA synthesis and nervous system function. 12 Deficiency not only leads to megaloblastic anemia and irreversible nerve damage, but may also accelerate the development of Alzheimer's disease, which has attracted wider attention in medicine and nutrition. However, only a few bacteria and archaea (about 37%) in nature can synthesize cobalamin from scratch. Studies have shown that slight differences in its structure may significantly affect the growth and metabolic capacity of organisms that rely on cobalamin. Therefore, establishing a highly sensitive qualitative and quantitative analysis method for cobalamin will not only help to deeply understand its biological function and its role in the human body, but also has important significance for public health, food safety and maintaining ecosystem functions.

[0003] Traditional detection methods indirectly quantify the concentration of cobalamin by measuring the cell density of cobalamin-deficient microorganisms (e.g., Lactobacillus). The principle is that the growth of Lactobacillus is linearly related to the concentration of exogenously added cobalamin within a certain range, thereby achieving quantitative analysis of cobalamin. However, this microbial detection method cannot indicate the structural differences of natural cobalamin (e.g., low-lying ligands). High performance liquid chromatography is another commonly used method for cobalamin analysis. The tandem ultraviolet-visible absorption detector has a strong absorption response value for cobalamin near 361nm, and can distinguish some cobalamin with different structures based on retention time. However, this method is easily interfered by impurity compounds in the sample and has very limited sensitivity. It requires the cobalamin concentration in the sample to be at least 2 mg / L, which is much higher than the cobalamin concentration of μg / L commonly found in environmental and biological samples.

[0004] The development of liquid chromatography-tandem mass spectrometry in recent years has made up for some of the shortcomings of traditional cobalamin analysis methods. However, most current mass spectrometry analysis methods only focus on vitamin B 12 , there is no mass spectrometry qualitative and quantitative analysis technology that can be applied to other types of cobalamin, and the detection instrument type is limited to triple quadrupole mass spectrometer. The main reasons may be the following aspects:

[0005] (1) In addition to vitamin B 12 There are no other commercial cobalamin standards except for (1) 2) cobalamin has a complex structure and a variety of high molecular weight derivatives, which makes its separation and identification difficult; (3) the ionization efficiency of cobalamin in ordinary mass spectrometers is low, resulting in insufficient detection sensitivity, especially in low concentration samples, which limits its application; (4) conventional mass spectrometry quantitative methods based on MRM (multiple reaction monitoring) technology can only quantitatively analyze limited and molecular weight-defined target compounds. Therefore, mass spectrometry analysis of high molecular weight and structurally diverse cobalamin is very challenging. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a highly sensitive method for quantification and structural identification of cobalamin and its application.

[0007] In order to achieve the above purpose, the technical problem to be solved by the present invention is:

[0008] A highly sensitive quantitative and structural identification method for cobalamin is disclosed. Three natural cobalamin with different structures commonly found in the environment are used as standard substances. A full scan-automatically triggered secondary mass spectrometry scanning (Full scan-ddMS2) mass spectrometry acquisition method is adopted to obtain cobalamin parent ions and a series of highly abundant characteristic ion fragment data. The structure of unknown cobalamin is identified based on the principle that the same cobalamin fragments have the same fragment ion m / z (accurate to three decimal places) and the fragment ion search function of Compound Discoverer 3.0 software. Then, a mass spectrometry quantitative method of parallel reaction monitoring (PRM) is adopted to filter out a large number of interfering ions through a quadrupole, and the cobalamin low-position ligand fragment ions with the highest abundance and recognition are used as quantitative ions to analyze the type and content of cobalamin in the sample to be measured.

[0009] The standard substance is vitamin B 12 , 5-Hydroxybenzimidazole type B 12 , 5-methoxybenzimidazole type B 12 .

[0010] First, the mass spectrometry acquisition method of UHPLC-ESI-Q-Orbitrap Full scan-ddMS2 was used to qualitatively analyze the cobalamin in the sample. In the Full scan, the parent ions in the cobalamin standard can be obtained; in ddMS2, all parent ions enter the collision cell for high-energy fragmentation, thereby generating a series of cobalamin characteristic fragment ions. According to the principle that the same cobalamin fragment structure has the same fragment ion m / z (accurate to three decimal places) and the fragment ion search function of Compound Discoverer software, the purpose of identifying the cobalamin structure type contained in the sample to be tested is achieved.

[0011] The detection conditions used for full scan-ddMS2 mass spectrometry acquisition and identification of the cobalamin structure are as follows:

[0012] Liquid chromatography conditions:

[0013] The instrument is Vanquish ultra-high performance liquid chromatograph; the chromatographic column is Hypersil GOLD C 18Column (2.1 mm × 100 mm, 2.6 μm, Thermo Fisher, USA); mobile phases consisted of 0.1% (v / v) formic acid aqueous solution (mobile phase A) and acetonitrile (mobile phase B); flow rate was 0.3 mL / min; column temperature: 30°C; gradient elution conditions were: initial 5% mobile phase B and 95% mobile phase A increased to 15% mobile phase B within 2.8 min, linearly increased to 25% mobile phase B within 4.5 min, 25% mobile phase B continued to increase to 70%, then 70% mobile phase B decreased to 5% mobile phase B within 0.5 min, and maintained at 5% mobile phase B for 1.5 min;

[0014] Mass spectrometry conditions:

[0015] The instrument was a Thermo Q-Exactive quadrupole-electrostatic field orbital trap high-resolution tandem mass spectrometer; electrospray ion source (ESI); scanning mode was Full scan-ddMS2; positive ion scanning mode; spray voltage: 3.8 kV; source temperature: 350 °C; primary scan mass spectrometry resolution: 70,000; primary scan range: m / z 200-2,000; primary scan C-trap maximum capacity (AGC target) and maximum injection time: 3×10 6 and 100ms; sheath gas and auxiliary gas pressures were 40 and 10unit, respectively; scanning analysis time was 8min; secondary scanning range was m / z 90-1,400; ion fragmentation mode and energy: high energy collision cell (ECD) and 30eV; secondary scanning resolution was 17,500; secondary scanning C-trap maximum capacity and maximum injection time: 1×10 5 and 50ms.

[0016] The mass spectrometry acquisition method of Full scan-ddMS2 is to obtain the cobalamin parent ion after full scan, wherein vitamin B 12 The parent ion of the standard is: [M+2H] 2+ (m / z

[0017] 678.288) and [M+H] + (m / z 1355.577); 5-Hydroxybenzimidazole type B 12 The parent ion is: [M+2H] 2+ (m / z 672.270) and [M+H] + (m / z 1343.538); 5-methoxybenzimidazole type B 12 The parent ion is: [M+2H] 2+ (m / z 679.278) and [M+H] + (m / z 1357.557);

[0018] At the same time, the characteristic fragment ions of cobalamin can be obtained by automatically triggering the secondary mass spectrometry scanning, among which all the standard fragment ions used to identify the structure of cobalamin are (specifically, the mass spectrometry scanning method of Full scan-ddMS2 is adopted, and all parent ions are not selected by the quadrupole, but enter the collision cell for high-energy fragmentation. All parent ions [M+2H] 2+ After Full scan-ddMS2 secondary scanning, all fragment ions used to identify the unknown cobalamin structure were obtained): Vitamin B 12 →m / z147.093, 359.099, 456.729, 912.442, 997.448, 1124.442, 1209.476; 5-Hydroxybenzimidazole type B 12 →m / z 136.056, 347.064, 456.729, 912.442, 997.448, 1124.442, 1209.476; 5-methoxybenzimidazole type B 12 →m / z 149.072, 361.079, 456.729, 912.442, 997.448, 1124.442, 1209.476.

[0019] The Compound Discoverer software identified m / z 147.093, 136.056, and 149.072 as vitamin B12, respectively, through the ion fragment search function. 12 , 5-Hydroxybenzimidazole type B 12 and 5-methoxybenzimidazole type B 12 The low-lying ligand ion [base+H] + m / z 359.099, 347.064, 361.079 represent the ions of the phosphoribosyl glycoside structure containing the low-position ligand of the three cobalamin [phosphoriboside+H] + m / z 997.448 corresponds to the ion containing the corrin ring of the three cobalamin compounds but without the phosphate nucleoside structure containing the low-positioned ligand [M-phosphoriboside+H] + ; m / z 456.729 and 912.438 correspond to m / z

[0020] 997.448 represents the ion of base minus C3H6N2O; m / z 1209.476 represents the ions of the three cobalamin without the low-position ligand structure [M-base+H] + ; m / z 1124.442 represents the ion of C3H6N2O minus m / z 1209.476.

[0021] After Full scan-ddMS2 secondary scanning, the cobalamin structures corresponding to all characteristic fragment ions were predicted and identified according to the Compound Discoverer software: m / z 147.093, 136.056, and 149.072 represent vitamin B 12 , 5-Hydroxybenzimidazole type B 12 , 5-methoxybenzimidazole type B 12 The low-lying ligand ion [base+H] + m / z 359.099, 347.064, 361.079 represent the ions of the phosphoribosyl glycoside structure containing the low-position ligand of the three cobalamin [phosphoriboside+H] + m / z 997.448 corresponds to the ion containing the corrin ring of the three cobalamin compounds but without the phosphate nucleoside structure containing the low-positioned ligand [M-phosphoriboside+H] + m / z 456.729 and 912.438 correspond to ions based on m / z 997.448 minus C3H6N2O; m / z 1209.476 represents ions of the three cobalamin species without the low-position ligand structure [M-base+H] + ; m / z 1124.442 represents the ion of C3H6N2O minus m / z 1209.476.

[0022] The cobalamin in the sample was absolutely quantitatively analyzed using the secondary mass spectrometry acquisition method of PRM (parallel reaction monitoring) of UHPLC-ESI-Q-Orbitrap. Before the secondary scan of PRM, the cobalamin parent ion obtained in Full scan-ddMS2 was entered in the inclusion list, and a high-resolution secondary mass spectrum was obtained after high-energy collision fragmentation, with the low-position ligand ions with high abundance and recognition as the cobalamin quantitative ions. After obtaining the linear relationship between the standard concentration and the quantitative ion peak area, the cobalamin contained in the sample to be tested was absolutely quantitatively detected and analyzed.

[0023] The detection conditions used for PRM mass spectrometry acquisition and quantitative analysis of cobalamin are as follows: the liquid chromatography conditions are exactly the same as those in the Full scan-ddMS2 scanning mode.

[0024] The detection conditions used in the Full scan-ddMS2 mass spectrometry method for qualitative analysis of cobalamin are:

[0025] Liquid chromatography conditions:

[0026] The instrument is Vanquish ultra-high performance liquid chromatograph; the chromatographic column is Hypersil GOLD C 18Column (2.1 mm × 100 mm, 2.6 μm, Thermo Fisher, USA); mobile phases consisted of 0.1% (v / v) formic acid aqueous solution (mobile phase A) and acetonitrile (mobile phase B); flow rate was 0.3 mL / min; column temperature: 30°C; gradient elution conditions were: initial 5% mobile phase B and 95% mobile phase A increased to 15% mobile phase B within 2.8 min, linearly increased to 25% mobile phase B within 4.5 min, 25% mobile phase B continued to increase to 70%, then 70% mobile phase B decreased to 5% mobile phase B within 0.5 min, and maintained at 5% mobile phase B for 1.5 min;

[0027] Mass spectrometry conditions:

[0028] The instrument was a Thermo Q-Exactive quadrupole-electrostatic field orbital trap high-resolution tandem mass spectrometer; electrospray ion source (ESI); scanning mode was Full scan-ddMS2; positive ion scanning mode; spray voltage: 3.8 kV; source temperature: 350 °C; primary scan mass spectrometry resolution: 70,000; primary scan range: m / z 200-2,000; primary scan C-trap maximum capacity (AGC target) and maximum injection time: 3×10 6 and 100ms; sheath gas and auxiliary gas pressures were 40 and 10unit, respectively; scanning analysis time was 8min; secondary scanning range was m / z 90-1,400; ion fragmentation mode and energy: high energy collision cell (ECD) and 30eV; secondary scanning resolution was 17,500; secondary scanning C-trap maximum capacity and maximum injection time: 1×10 5 and 50ms.

[0029] The mass spectrometry quantification method of the PRM is to obtain the cobalamin parent ion obtained by inputting in the inclusion list, and obtain a high-resolution secondary mass spectrum after high-energy collision fragmentation, and use the most abundant and recognizable low-position ligand as the cobalamin quantitative ion.

[0030] The detection conditions used in the PRM mass spectrometry method for quantitative cobalamin collection include the following: wherein the liquid chromatography analysis conditions are consistent with the above description.

[0031] The mass spectrometry conditions were:

[0032] The instrument was a Thermo Q-Exactive quadrupole-electrostatic field orbital trap high-resolution tandem mass spectrometer; electrospray ion source (ESI); scanning mode was PRM; positive ion scanning mode; spray voltage: 3.8 kV; source temperature: 350 °C; sheath gas and auxiliary gas pressures were 40 and 10 units, respectively; scanning analysis time was 8 min; secondary scanning range was m / z 90-1,400; ion fragmentation mode and energy: high energy collision cell (ECD) and 35 eV; scanning resolution was 17,500; secondary scanning C-trap maximum capacity and maximum injection time: 2×10 5 and 100ms.

[0033] Before determining cobalamin in biological samples, a sample pretreatment step is required, and the supernatant after pretreatment is taken for full scan-ddMS2 and PRM scanning analysis by liquid chromatography-high resolution mass spectrometry.

[0034] The determination of the type of cobalamin in the sample to be tested requires a sample pretreatment step. An appropriate amount of the supernatant after pretreatment of the bacteria is taken for UHPLC-ESI-Q-Orbitrap Full scan-ddMS2 analysis. The primary and secondary chromatograms of the sample and the standard are compared to analyze the unknown cobalamin structure type in the sample to be tested.

[0035] The quantitative determination of the cobalamin concentration in the sample to be tested requires a sample pretreatment step. The supernatant of the biological culture fluid after pretreatment is subjected to UHPLC-ESI-Q-Orbitrap PRM analysis, and the peak area of ​​the characteristic low-position ligand ion fragments of cobalamin is substituted into the standard curve to determine the cobalamin content in the sample to be tested.

[0036] The advantages of the present invention are:

[0037] Due to the characteristics of cobalamin, such as many types, low content, difficult separation and purification, complex structure and large molecular weight, there are currently very few mass spectrometry analysis technologies for it. The present invention proposes for the first time a UHPLC-ESI-Q-Orbitrap cobalamin mass spectrometry analysis method based on Full scan-ddMS2 and PRM scanning acquisition technology. Through the scanning method of Full scan-ddMS2, the structural identification process of cobalamin does not require any preliminary method optimization, has good reproducibility and selectivity, and is suitable for the analysis of cobalamin in complex samples. In addition, the high-resolution quantitative scanning method of PRM filters out a large number of interfering ions (especially in low-concentration matrix samples) through the quadrupole, which improves the sensitivity of sample analysis while also enhancing the quantitative specificity, and can detect cobalamin at the μg / L concentration level. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1The structure of the cobalamin macromolecule provided in the embodiments of the present invention and the type of low-position ligands it carries.

[0039] Figure 2 The first-level full-scan mass spectra of three natural cobalamin standards with different structures provided in the embodiments of the present invention.

[0040] Figure 3 Secondary scanning mass spectra of three natural cobalamin standards with different structures provided in the embodiments of the present invention.

[0041] Figure 4 The mass spectrum of cobalamin identified in the sample to be tested provided in an embodiment of the present invention.

[0042] Figure 5 The standard curves of three natural cobalamin standards with different structures provided in the embodiments of the present invention.

[0043] Figure 6 This is a diagram showing the effect of quantitative analysis of cobalamin in a sample to be tested provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with specific implementation methods. The given embodiments are only for illustrating the present invention, but not for limiting the scope of application of the present invention.

[0045] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0046] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0047] Example 1: Structural Identification of Cobalamin

[0048] (1) Vitamin B 12 Preparation of standards

[0049] Weigh an appropriate amount of vitamin B 12 , dissolve in deionized water and dilute to 5 mg / L to obtain vitamin B 12 Standard( Figure 1 ).

[0050] (2) 5-Hydroxybenzimidazole type B 12 and 5-methoxybenzimidazole type B 12 Preparation of standards

[0051] The methanogen Methanosarcina barkeri strain Fusaro (NCBI ID: 269797) and the Sporomusa sp. strain KB-1 (NCBI ID: 2184576) with the addition of 100mM 5-methoxybenzimidazole were cultured anaerobically. The strain Fusaro used 0.3% methanol as a growth substrate, while the strain KB-1 used 40mM betaine as a substrate for growth. After 96h-144h of culture, the cells were collected by centrifugation at 20,000xg for 30 minutes at room temperature. Subsequently, the cells were suspended in 10mL of deionized water, and cyanide oxide was added for water bath heating reaction (100°C, pH 5.0, 30 minutes). Finally, 5-hydroxybenzimidazole type B can be obtained from the oxidation solutions of the Fusaro cells and KB-1 cells obtained above, respectively. 12 and 5-methoxybenzimidazole B 12 The crude extracts of cobalamin were injected into the syringe at C 18 The adsorption column (Waters) was used for elution, and the effluent was discarded; then 10 mL of ultrapure water was used to wash the hydrophilic non-cobalamin impurities remaining on the column; finally, 3 mL of methanol was used to elute the impurities adhering to the C 18 The cobalamin in the adsorption column was carefully collected, and the methanol eluate containing the cobalamin component was carefully collected. The obtained eluate was manually separated and purified by HPLC, and the specific operation was as follows: the elution step was 18% mobile phase B (0.1% formic acid-methanol), 82% mobile phase A (0.1% formic acid-water), and the mobile phase B was linearly increased to 20% within 34 minutes, and then the mobile phase B was linearly increased to 90% within 2 minutes, and maintained for 3 minutes, and then the mobile phase B was linearly reduced to the initial setting conditions within 2 minutes, and maintained for 4 minutes. The chromatographically separated cobalamin components were detected at a wavelength of 361nm, and the chromatographically separated cobalamin components were collected from the mobile phase waste liquid outlet. The collected components were diluted 5 times with deionized water and used again according to the above method. 18 Column concentration and purification. After rotary evaporation and drying, the cobalamin-methanol mixture was dissolved and diluted to 5 mg / L with deionized water to obtain 5-hydroxybenzimidazole type B 12 and 5-methoxybenzimidazole type B 12 Standards ( Figure 1 ).

[0052] (3) Pretreatment of samples containing unknown cobalamin components

[0053] An anaerobic enrichment culture system (the culture medium component is DCB-1, USA) was established with about 2 g of fine river sediment (latitude: 41°39′46″, longitude: 123°6′20″) as the initial inoculum source, tetrachloroethylene as the electron acceptor, acetic acid and hydrogen as the carbon source and electron donor, respectively, to obtain a mixed bacterial enrichment culture fluid dominated by Dehalococcoides. Take 100 mL of the mixed bacterial enrichment culture fluid with unknown cobalamin components, filter and collect the bacteria in the culture fluid with a 47 mm diameter 0.22 μm filter membrane (Pall Life Sciences, USA), and transfer the filter membrane containing the bacteria to a 10 mL centrifuge tube. Subsequently, it was suspended in 5 mL of deionized water for cyanide oxidation reaction (100°C, pH 5.0, 30 min). Centrifuge at 20,000xg for 10 minutes to obtain the supernatant to be tested without bacteria, and transfer it to a 2 mL injection bottle.

[0054] (4) UHPLC-ESI-Q-Orbitrap Full MS-ddMS analysis steps

[0055] Take 5 μL of each of the three standards and the treated supernatant sample for analysis, select the Full scan-ddMS2 scanning mode in the Vanquish ultra-high performance liquid chromatography-Thermo Q-Exactive quadrupole-electrostatic field orbital trap high-resolution tandem mass spectrometer, and obtain the chromatogram and mass spectrum according to the set liquid chromatography and mass spectrometry conditions. Compare the sizes of the parent ions and characteristic fragment ions in the three cobalamin standards and the samples to be tested, and judge the unknown cobalamin structure according to the principle that the same cobalamin fragment structure has the same fragment ion m / z (accurate to three decimal places); Compound Discoverer3.0 software is used to search and predict the cobalamin fragment ions to further verify the structure of the unknown cobalamin.

[0056] The specific conditions of the liquid chromatography-mass spectrometry analysis in the high-resolution qualitative method of cobalamin involved in the standards and samples to be tested are as follows:

[0057] Liquid chromatography conditions:

[0058] The instrument is Vanquish ultra-high performance liquid chromatograph; the chromatographic column is Hypersil GOLD C 18Column (2.1 mm × 100 mm, 2.6 μm, Thermo Fisher, USA); mobile phases consisted of 0.1% (v / v) formic acid aqueous solution (mobile phase A) and acetonitrile (mobile phase B); flow rate was 0.3 mL / min; column temperature: 30°C; gradient elution conditions were: initial 5% mobile phase B and 95% mobile phase A increased to 15% mobile phase B within 2.8 min, linearly increased to 25% mobile phase B within 4.5 min, 25% mobile phase B continued to increase to 70%, then 70% mobile phase B decreased to 5% mobile phase B within 0.5 min, and maintained at 5% mobile phase B for 1.5 min;

[0059] Mass spectrometry conditions:

[0060] The instrument was a Thermo Q-Exactive quadrupole-electrostatic field orbital trap high-resolution tandem mass spectrometer; electrospray ion source (ESI); scanning mode was Full scan-ddMS2; positive ion scanning mode; spray voltage: 3.8 kV; source temperature: 350 °C; primary scan mass spectrometry resolution: 70,000; primary scan range: m / z 200-2,000; primary scan C-trap maximum capacity (AGC target) and maximum injection time: 3×10 6 and 100ms; sheath gas and auxiliary gas pressures were 40 and 10unit, respectively; scanning analysis time was 8min; secondary scanning range was m / z 90-1,400; ion fragmentation mode and energy: high energy collision cell (ECD) and 30eV; secondary scanning resolution was 17,500; secondary scanning C-trap maximum capacity and maximum injection time: 1×10 5 and 50ms.

[0061] The parent ion used to identify cobalamin is: Vitamin B 12 The parent ions of the standards are:

[0062] [M+2H] 2+ (m / z 678.288) and [M+H] + (m / z 1355.577); 5-Hydroxybenzimidazole type B 12 The parent ion is: [M+2H] 2+ (m / z 672.270) and [M+H] + (m / z1343.538); 5-methoxybenzimidazole type B 12 The parent ion is: [M+2H] 2+ (m / z 679.278) and [M+H] + (m / z 1357.557)( Figure 2 ).

[0063] All characteristic fragment ions used to identify the structure of cobalamin are: Vitamin B 12 →m / z147.093, 359.099, 456.729, 912.438, 997.448, 1124.442, 1209.476; 5-Hydroxybenzimidazole type B 12 →m / z 136.056, 347.064, 456.729, 912.438, 997.448, 1124.442, 1209.476; 5-methoxybenzimidazole type B 12 →m / z149.072, 361.079, 456.729, 912.442, 997.448, 1124.442, 1209.476( Figure 3 ).

[0064] The fragment structures of cobalamin represented by all characteristic fragment ions used to identify the structure of cobalamin are as follows: m / z 147.093, 136.056, and 149.072 represent vitamin B 12 , 5-Hydroxybenzimidazole type B 12 , 5-methoxybenzimidazole type B 12 The low-lying ligand ion [base+H] + m / z 359.099, 347.064, 361.079 represent the ions of the phosphoribosyl glycoside structure containing the low-position ligand of the three cobalamin [phosphoriboside+H] + ; m / z

[0065] 997.448 corresponds to the ions of the three cobalamin compounds containing the corrin ring but without the phosphate riboside structure containing the low-positioned ligand [M-phosphoriboside+H] + m / z 456.729 and 912.438 correspond to ions based on m / z 997.448 minus C3H6N2O; m / z 1209.476 represents ions of the three cobalamin species without the low-positioned ligand structure [M-base+H] + ; m / z 1124.442 represents the ion of C3H6N2O minus m / z 1209.476.

[0066] The results show that when 5μL was injected, the sample to be tested achieved good test results. After the first-level mass spectrometry full scan, a total of 1,286 parent ions were obtained. After all parent ions were scanned by the Full scan-ddMS2 secondary scan, all parent ions in the sample were collided with the collision cell to obtain the corresponding secondary mass spectra, which showed that the sample contained four cobalamin components. The primary and secondary mass spectra of the three cobalamin components were completely consistent with those of the standard. Therefore, three of the cobalamin components are vitamin B12 , 5-Hydroxybenzimidazole type B 12 and 5-methoxybenzimidazole type B 12 The parent ion of the primary mass spectrum of another cobalamin is m / z 1327.543

[0067] [C 61 H 85 CoN 14 O 14 P] + and m / z 664.275[C 61 H 86 CoN 14 O 14 P] 2+ The fragment ions of the secondary mass spectrum are completely consistent with the corresponding structures of the three known standards, except for the low-position ligand (m / z 119.061) and the phospho-ribose derivative containing the low-position ligand (m / z 331.072). This shows that in addition to the difference in the low-position ligand, the structure of another cobalamin is completely consistent with all the fragment ions containing the corrin ring structure of the three standards. The original mass spectrum was imported into Compound Discoverer 3.0 software, confirming that this cobalamin is a benzimidazole-type B with a low-position ligand without methyl substitution. 12 ( Figure 4 ). Finally, it is confirmed that the mass spectrometry method for structural identification of cobalamin described in Case 1 of the present invention is feasible.

[0068] Example 2: Absolute quantitative analysis of cobalamin with different structures

[0069] (1) Preparation of mixed gradient dilution standard of cobalamin

[0070] Take 10 μL of each of the three different cobalamin standards (5 mg / L) in Example 1, mix the three in a 2 mL clean centrifuge tube, and add 940 μL of deionized water to dilute to make a 50 μg / L cobalamin mixed standard of equal concentration. The mixed standard is further diluted with deionized water to obtain cobalamin standards with concentrations of 50, 25, 10, 5, 1, 0.5, and 0.1 μg / L. The standard is transferred to a 2 mL injection bottle, and the autosampler takes 10 μL for UHPLC-ESI-Q-Orbitrap analysis.

[0071] (2) Pretreatment of samples to be tested for cobalamin content

[0072] Take 1 mL of the mixed bacterial enrichment culture medium in Implementation Case 1, transfer the supernatant by centrifugation to a new centrifuge tube; add 20 mM cyanide oxidant to the supernatant of the sterile body, add 2 μL of glacial acetic acid, and vortex mix for 30 seconds; place the sample in a boiling water bath and heat for 60 minutes; before cooling the sample, centrifuge at 20,000 x g for 10 minutes, and transfer the treated supernatant sample free of impurities to the injection bottle.

[0073] (3) Preparation of quality control samples

[0074] In order to investigate the interference of the culture medium and sample pretreatment process of the mixed bacterial enrichment culture in step (2) on the quantitative accuracy of cobalamin in the sample to be tested, this step uses blank culture medium as solvent to prepare vitamin B 12 The specific steps are as follows: Take 10 μL of vitamin B in Example 1 12 The standard (5 mg / L) was placed in a 2 mL clean centrifuge tube, and 990 μL of blank culture medium without mixed enriched bacteria was added to obtain a 50 μg / L quality control sample, which was further diluted with blank culture medium to obtain 25 and 2.5 μg / L quality control samples. Three replicate samples were prepared for each quality control concentration, and three analysis batches were prepared continuously. The three groups of quality control concentration samples were pre-treated according to the treatment method for the samples to be tested in step (2). The prepared quality control samples that have undergone pre-treatment were transferred to the injection bottle for testing.

[0075] (4) PRM analysis steps using UHPLC-ESI-Q-Orbitrap

[0076] Take 10 μL of the standard, the treated supernatant sample and the quality control sample in the above steps (1), (2) and (3) for analysis, select the PRM scanning mode in the Vanquish ultra-high performance liquid chromatography-Thermo Q-Exactive quadrupole-electrostatic field orbital trap high-resolution tandem mass spectrometer, and obtain the chromatogram and mass spectrum according to the set liquid chromatography and mass spectrometry conditions. Select the low-position ligand ion [base+H] corresponding to each cobalamin standard + As the quantitative ion. With the concentration of cobalamin standard as the horizontal axis and the peak area as the vertical axis, a standard curve with a good linear relationship was obtained, thereby calculating the cobalamin concentration in the test samples and quality control samples.

[0077] The specific liquid chromatography and mass spectrometry analysis conditions in the quantitative analysis method of cobalamin for the standards, treated supernatant samples to be tested and quality control samples involved are as follows:

[0078] The liquid chromatography conditions were exactly the same as in Example 1.

[0079] Mass spectrometry conditions:

[0080] The instrument was a Thermo Q-Exactive quadrupole-electrostatic field orbital trap high-resolution tandem mass spectrometer; electrospray ion source (ESI); scanning mode was PRM; positive ion scanning mode; spray voltage: 3.8 kV; source temperature: 350 °C; sheath gas and auxiliary gas pressures were 40 and 10 units, respectively; scanning analysis time was 8 min; secondary scanning range was m / z 90-1,400; ion fragmentation mode and energy: high energy collision cell (ECD) and 35 eV; scanning resolution was 17,500; secondary scanning C-trap maximum capacity and maximum injection time: 2×10 5 and 100ms.

[0081] The characteristic fragment ions used for quantification of cobalamin are the corresponding low-position ligand ions [base+H] in the structure of each cobalamin. + , respectively: Vitamin B 12 Bis(methylbenzimidazole) (m / z 147.093), 5-hydroxybenzimidazole type B 12 5-Hydroxybenzimidazole (m / z

[0082] 135.056), 5-methoxybenzimidazole B 12 5-methoxybenzimidazole (m / z

[0083] 149.072).

[0084] With the concentration of cobalamin standard as the horizontal axis and the peak area as the vertical axis, a standard curve with a good linear relationship was obtained. The results showed that the linear range of cobalamin concentration was 0.1-100 μg / L, and the LOQ was 0.1 μg / L. 12 The working curve is y = 188,444x-66,493, r 2 =0.9998; 5-Hydroxybenzimidazole type B 12 The working curve is y = 156,950x-68,893, r 2 =0.9995; 5-methoxybenzimidazole type B 12 The working curve is y = 151,901x-67,775, r 2 =0.9996( Figure 5 ).

[0085] The results showed that vitamin B12 was successfully detected in the treated supernatant samples. 12 The content is 32.65μg / L, 5-hydroxybenzimidazole type B 12 The content is 25.63μg / L, and 5-methoxybenzimidazole type B 12 The content is 6.14μg / L( Figure 6). In addition, according to vitamin B 12 The concentration of each quality control sample was obtained based on the standard curve, and the precision and accuracy were calculated. The results are shown in Table 1.

[0086] Table 1. Vitamin B in microbial culture medium 12 Precision and accuracy of measurements

[0087]

[0088] In view of the linearity of the cobalamin standard curve and the accuracy of the quality control samples, the absolute quantitative method of cobalamin described in the present invention has good linearity, high accuracy, good reproducibility, and is sensitive and reliable, and can be applied to the absolute quantitative analysis of cobalamin in biological samples.

Claims

1. A method for highly sensitive quantification and structural identification of cobalamin, characterized in that: Three natural cobalamin with different structures commonly found in the environment were used as standards. The full scan-automatic triggered secondary mass spectrometry scanning (Fullscan-ddMS2) mass spectrometry acquisition method was used to obtain the cobalamin parent ion and a series of high-abundance characteristic ion fragment data. The structure of unknown cobalamin was identified based on the principle that the same cobalamin fragments have the same fragment ion m / z and the fragment ion search function of Compound Discoverer 3.0 software. Then, the mass spectrometry quantitative method of parallel reaction monitoring (PRM) was used to filter out a large number of interfering ions through the quadrupole, and the cobalamin low-position ligand fragment ions with the highest abundance and recognition were used as quantitative ions to analyze the type and content of cobalamin in the sample to be measured.

2. The method for highly sensitive quantification and structural identification of cobalamin according to claim 1, characterized in that: The standard substance is vitamin B 12 , 5-Hydroxybenzimidazole type B 12 , 5-methoxybenzimidazole type B 12 .

3. The method for highly sensitive quantification and structural identification of cobalamin according to claim 1, characterized in that: The mass spectrometry acquisition method of Fullscan-ddMS2 is to obtain the cobalamin parent ion after full scanning, wherein vitamin B 12 The parent ion of the standard is: [M+2H] 2+ (m / z 678.288) and [M+H] + (m / z 1355.577); 5-Hydroxybenzimidazole type B 12 The parent ion is: [M+2H] 2+ (m / z 672.270) and [M+H] + (m / z 1343.538); 5-methoxybenzimidazole type B 12 The parent ion is: [M+2H] 2+ (m / z 679.278) and [M+H] + (m / z 1357.557); At the same time, the characteristic fragment ions of cobalamin can be obtained by automatically triggering the secondary mass spectrometry scan. Among them, all the standard fragment ions used to identify the structure of cobalamin are: Vitamin B 12 →m / z147.093, 359.099, 456.729, 912.442, 997.448, 1124.442, 1209.476; 5-Hydroxybenzimidazole type B 12 →m / z 136.056, 347.064, 456.729, 912.442, 997.448, 1124.442, 1209.476; 5-methoxybenzimidazole type B 12 →m / z 149.072, 361.079, 456.729, 912.442, 997.448, 1124.442, 1209.

476.

4. The method for highly sensitive quantification and structural identification of cobalamin according to claims 1 and 3, characterized in that: The Compound Discoverer software identified m / z 147.093, 136.056, and 149.072 as vitamin B12, respectively, through the ion fragment search function. 12 , 5-Hydroxybenzimidazole type B 12 and 5-methoxybenzimidazole type B 12 The low-lying ligand ion [base+H] + m / z 359.099, 347.064, 361.079 represent the ions of the phosphoribosyl glycoside structure containing the low-position ligand of the three cobalamin [phosphoriboside+H] + m / z 997.448 corresponds to the ion containing the corrin ring of the three cobalamin compounds but without the phosphate nucleoside structure containing the low-positioned ligand [M-phosphoriboside+H] + m / z 456.729 and 912.438 correspond to ions based on m / z 997.448 minus C3H6N2O; m / z 1209.476 represents ions of the three cobalamin species without the lower ligand structure [M–base+H] + ; m / z 1124.442 represents the ion of C3H6N2O minus m / z 1209.

476.

5. The method for highly sensitive quantification and structural identification of cobalamin according to claim 1 or 3, characterized in that: The detection conditions used in the Fullscan-ddMS2 mass spectrometry acquisition method for qualitative analysis of cobalamin. Liquid chromatography conditions: The instrument is Vanquish ultra-high performance liquid chromatograph; the chromatographic column is HypersilGOLD C 18 Column (2.1 mm × 100 mm, 2.6 μm, Thermo Fisher, USA); mobile phases consisted of 0.1% (v / v) formic acid aqueous solution (mobile phase A) and acetonitrile (mobile phase B); flow rate was 0.3 mL / min; column temperature: 30°C; gradient elution conditions were: initial 5% mobile phase B and 95% mobile phase A increased to 15% mobile phase B within 2.8 min, linearly increased to 25% mobile phase B within 4.5 min, 25% mobile phase B continued to increase to 70%, then 70% mobile phase B decreased to 5% mobile phase B within 0.5 min, and maintained at 5% mobile phase B for 1.5 min; Mass spectrometry conditions: The instrument was a Thermo Q-Exactive quadrupole-electrostatic field orbital trap high-resolution tandem mass spectrometer; electrospray ion source (ESI); scanning mode was Full scan-ddMS2; positive ion scanning mode; spray voltage: 3.8 kV; source temperature: 350 °C; primary scan mass spectrometry resolution: 70,000; primary scan range: m / z 200-2,000; primary scan C-trap maximum capacity (AGC target) and maximum injection time: 3×10 6 and 100ms; sheath gas and auxiliary gas pressures were 40 and 10unit, respectively; scanning analysis time was 8min; secondary scanning range was m / z 90-1,400; ion fragmentation mode and energy: high energy collision cell (ECD) and 30eV; secondary scanning resolution was 17,500; secondary scanning C-trap maximum capacity and maximum injection time: 1×10 5 and 50ms.

6. The method for highly sensitive quantification and structural identification of cobalamin according to claim 1 or 4, characterized in that: The mass spectrometry quantification method of the PRM is to input the cobalamin parent ion obtained in right 3 into the inclusion list, and obtain a high-resolution secondary mass spectrum after high-energy collision fragmentation, and use the most abundant and recognizable low-position ligand as the cobalamin quantitative ion.

7. The method for highly sensitive quantification and structural identification of cobalamin according to claim 1 or 6, characterized in that: The detection conditions used in the PRM mass spectrometry method for collecting quantitative cobalamin, wherein the liquid chromatography analysis conditions are completely consistent with those in claim 5. The mass spectrometry conditions were: The instrument was a Thermo Q-Exactive quadrupole-electrostatic field orbital trap high-resolution tandem mass spectrometer; electrospray ion source (ESI); scanning mode was PRM; positive ion scanning mode; spray voltage: 3.8 kV; source temperature: 350 °C; sheath gas and auxiliary gas pressures were 40 and 10 units, respectively; scanning analysis time was 8 min; secondary scanning range was m / z 90-1,400; ion fragmentation mode and energy: high energy collision cell (ECD) and 35 eV; scanning resolution was 17,500; secondary scanning C-trap maximum capacity and maximum injection time: 2×10 5 and 100ms.

8. The method for highly sensitive quantification and structural identification of cobalamin according to claim 1, 3 or 6, characterized in that: Before determining cobalamin in biological samples, a sample pretreatment step is required, and the supernatant after pretreatment is taken for Fullscan-ddMS2 and PRM scanning analysis by liquid chromatography-high resolution mass spectrometry.

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