A highly sensitive method for quantitative and structural identification of cobalamin and its application

Abstract

This invention belongs to the field of detection technology and relates to a highly sensitive method for quantitative analysis and structural identification of cobalamin, as well as its application. Using three common natural cobalamins with different structures found in the environment as standards, a full-scan, automatically triggered secondary mass spectrometry acquisition method is employed to obtain the cobalamin precursor ion and a series of high-abundance characteristic ion fragments. Based on the principle that fragments of the same cobalamin have the same fragment ion m / z and the fragment ion search function of the software, the structure of unknown cobalamin is identified. Then, a parallel reaction monitoring mass spectrometry quantitative method is used. A large number of interfering ions are filtered out by a quadrupole, and the cobalamin low-position ligand fragment ions with the highest abundance and high identifiability are used as quantitative ions to analyze the type and content of cobalamin in the sample. This invention, by combining two data acquisition methods of high-resolution mass spectrometry, can effectively eliminate interference from the endogenous matrix, improve the sensitivity of sample analysis, and enhance the specificity of quantification, thereby achieving highly sensitive detection of different types of cobalamin.

Description

Technical Field

[0001] This invention belongs to the field of detection technology and relates to a highly sensitive quantitative and structural identification method for cobalamin (liquid chromatography-high resolution mass spectrometry analysis and detection method for cobalamin) and its application. Background Technology

[0002] Cobalamin (e.g., vitamin B1) 12 Cobalamin is a class of organic compounds based on a complex porphyrin skeleton, containing cobalt ions. Its molecular weight is approximately 1,300-1,500 Daltons, making it the largest known non-polymeric natural biomolecule. The core structure of cobalamin is a conserved tetrapyrrole ring system. The nitrogen atom at the center of the tetrapyrrole ring is linked to a cobalt ion via four coordinate bonds to form a closed corrugated ring. The cobalt ion at the center of the corrugated 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 corrugated ring and the other end of the low-position ligand are connected by nucleotide ring side chains. Up to 18 natural cobalamin compounds have been discovered, and their structural diversity is mainly reflected in the low-position ligand, which is usually one of benzimidazole, phenol, or purine compounds. Figure 1 As a metalloenzyme coenzyme, 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 B1 12 As the most typical representative of natural cobalamin, it plays an important role in erythrocyte production, DNA synthesis, and nervous system function. For example, vitamin B... 12 Cobalamin deficiency not only leads to megaloblastic anemia and irreversible neurological damage, but may also accelerate the development of Alzheimer's disease, thus gaining wider attention in medicine and nutrition. However, only a small percentage of bacteria and archaea in nature (approximately 37%) can synthesize cobalamin de novo, and studies have shown that subtle structural differences can significantly affect the growth and metabolic capacity of cobalamin-dependent organisms. Therefore, establishing highly sensitive qualitative and quantitative analytical methods for cobalamin is not only crucial for a deeper understanding of its biological functions and role in the human body, but also of great significance for public health, food safety, and maintaining ecosystem function.

[0003] Traditional detection methods indirectly quantify cobalamin concentration by measuring the cell density of cobalamin-deficient microorganisms (e.g., *Lactobacillus*). This is based on the principle that the growth of *Lactobacillus* and the concentration of exogenously added cobalamin are linearly related within a certain range, thus enabling quantitative analysis of cobalamin. However, this microbial detection method cannot indicate structural differences in natural cobalamin (e.g., low-position ligands). High-performance liquid chromatography (HPLC) is another commonly used method for cobalamin analysis. A tandem UV-Vis absorption detector exhibits a strong absorption response to cobalamin around 361 nm and can distinguish cobalamin with different structures based on retention time. However, this method is susceptible to interference from impurity compounds in the sample and has very limited sensitivity, requiring a cobalamin concentration of at least 2 mg / L in the sample, which is far higher than the μg / L levels commonly found in environmental and biological samples.

[0004] The recently developed liquid chromatography-tandem mass spectrometry (LC-MS / MS) technique has overcome some of the shortcomings of traditional methods for analyzing cobalamin. However, most current mass spectrometry methods are only applicable to vitamin B1. 12 There are no mass spectrometry qualitative and quantitative analysis techniques applicable to other types of cobalamin, and the detection instruments are limited to triple quadrupole mass spectrometers. The main reasons may be as follows:

[0005] (1) Except for vitamin B 12 In addition, there are no other commercially available cobalamin standards; (2) cobalamin has a complex structure and a variety of high molecular weight derivatives, which makes its separation and identification difficult; (3) cobalamin has low ionization efficiency in ordinary mass spectrometers, 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 perform quantitative analysis on a limited number of target compounds with defined molecular weights. Therefore, mass spectrometry analysis of high molecular weight and structurally diverse cobalamin is very challenging. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a highly sensitive method for quantitative analysis and structural identification of cobalamin, and its application.

[0007] To achieve the above objectives, the technical problem that this invention needs to solve is:

[0008] A highly sensitive method for quantitative analysis and structural identification of cobalamin is proposed. Using three common natural cobalamins with different structures found in the environment as standards, a full scan-ddMS2 mass spectrometry acquisition method is employed to obtain cobalamin precursor ions and a series of high-abundance characteristic ion fragments. Based on the principle that identical 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, the structure of unknown cobalamin is identified. Then, a parallel reaction monitoring (PRM) mass spectrometry quantitative method is used. A large number of interfering ions are filtered out by a quadrupole, and the cobalamin low-position ligand fragment ions with the highest abundance and high identifiability are used as quantitative ions to analyze the type and content of cobalamin in the sample to be analyzed.

[0009] The standard mentioned is vitamin B. 12 5-Hydroxybenzimidazole type B 12 5-Methoxybenzimidazole type B 12 .

[0010] First, the cobalamin in the sample was qualitatively analyzed using a UHPLC-ESI-Q-Orbitrap Full scan-ddMS2 mass spectrometry method. In the Full scan, the precursor ion from the cobalamin standard was obtained; in the ddMS2, all precursor ions entered the collision cell for high-energy fragmentation, generating a series of characteristic cobalamin fragment ions. Based on the principle that fragments with the same cobalamin structure have the same m / z ratio (accurate to three decimal places) and the fragment ion search function of the Compound Discoverer software, the structural type of cobalamin contained in the sample was identified.

[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 was a Vanquish ultra-high performance liquid chromatograph; the chromatographic column was a Hypersil GOLD C1000. 18The column (2.1 mm × 100 mm, 2.6 μm, Thermo Fisher Scientific, USA) was used. The mobile phase consisted of 0.1% (v / v) formic acid aqueous solution (mobile phase A) and acetonitrile (mobile phase B). The flow rate was 0.3 mL / min. The column temperature was 30 °C. The gradient elution conditions were as follows: the initial 5% mobile phase B and 95% mobile phase A were increased to 15% mobile phase B within 2.8 min, and then linearly increased to 25% mobile phase B within 4.5 min. The 25% mobile phase B was further increased to 70%, and then the 70% mobile phase B was reduced 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 ionization (ESI) source; full scan-ddMS2 mode; positive ion scan 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. 6 100 ms; sheath gas and auxiliary gas pressures were 40 and 10 units, respectively; scan analysis time was 8 min; secondary scan range was m / z 90–1,400; ion fragmentation mode and energy: high-energy collision cell (ECD) and 30 eV; secondary scan resolution was 17,500; maximum C-trap capacity and maximum injection time for secondary scan were 1 × 10⁻⁶. 5 And 50ms.

[0016] The Full scan-ddMS2 mass spectrometry acquisition method allows for the acquisition of cobalamin precursor ions after a full scan, including vitamin B1. 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] Simultaneously, characteristic fragment ions of cobalamin can be obtained through automatically triggered secondary mass spectrometry scanning. Specifically, all standard fragment ions used to identify the structure of cobalamin are obtained by using Full scan-ddMS2 mass spectrometry, where all precursor ions are not selected by a quadrupole and are all subjected to high-energy fragmentation in the collision cell. All precursor ions are [M+2H]. 2+ After full scan-ddMS2 secondary scanning, all fragment ions used to identify the structure of the unknown cobalamin were obtained: Vitamin B1 12 →m / z 147.093, 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, through its ion fragment search function, identified m / z 147.093, 136.056, and 149.072 as representing vitamin B1, respectively. 12 5-Hydroxybenzimidazole type B 12 and 5-methoxybenzimidazole type B 12 The lower-position ligand ion [base+H] + m / z 359.099, 347.064, and 361.079 represent the ions [phosphoriboside + H] of the phosphoribosyl moieties containing the low-position ligands of the three cobalamin compounds, respectively. + m / z 997.448 corresponds to the [M-phosphoriboside+H] ion of three cobalamins containing a corrin ring but with the low-position ligand removed from the phosphate riboside structure. + ; m / z 456.729 and 912.438 correspond to m / z

[0020] The ion with m / z 997.448 minus C3H6N2O; m / z 1209.476 represents the ions of the three cobalamins without the lower ligand structure [M-base+H]. + m / z 1124.442 represents the ion obtained by subtracting C3H6N2O from m / z 1209.476.

[0021] After full scan-ddMS2 secondary scanning, the cobalamin structures corresponding to all characteristic fragment ions were predicted and identified using Compound Discoverer software: m / z 147.093, 136.056, and 149.072, representing vitamin B1, 147.093, 136.056, and 149.072, respectively. 12 5-Hydroxybenzimidazole type B 12 5-Methoxybenzimidazole type B 12 The lower-position ligand ion [base+H] + m / z 359.099, 347.064, and 361.079 represent the ions [phosphoriboside + H] of the phosphoribosyl moieties containing the low-position ligands of the three cobalamin compounds, respectively. + m / z 997.448 corresponds to the [M-phosphoriboside+H] ion of three cobalamins containing a corrin ring but with the low-position ligand removed from the phosphate riboside structure. + m / z 456.729 and 912.438 correspond to ions with C3H6N2O subtracted from m / z 997.448; m / z 1209.476 represents the three cobalamin ions without the lower ligand structure [M-base+H]. + m / z 1124.442 represents the ion obtained by subtracting C3H6N2O from m / z 1209.476.

[0022] Absolute quantitative analysis of cobalamin in samples was performed using a parallel reaction monitoring (PRM) secondary mass spectrometry acquisition method with UHPLC-ESI-Q-Orbitrap. Before the secondary scan of PRM, the cobalamin precursor ion obtained from Full scan-ddMS2 was entered into the inclusion list. High-resolution secondary mass spectra were then obtained after high-energy collisional fragmentation. Low-position ligand ions with high abundance and high identifiability were used as the quantitative ions for cobalamin. After obtaining the linear relationship between the standard concentration and the peak area of ​​the quantitative ion, absolute quantitative analysis of cobalamin in the sample was performed.

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

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

[0025] Liquid chromatography conditions:

[0026] The instrument was a Vanquish ultra-high performance liquid chromatograph; the chromatographic column was a Hypersil GOLD C1000. 18The column (2.1 mm × 100 mm, 2.6 μm, Thermo Fisher Scientific, USA) was used. The mobile phase consisted of 0.1% (v / v) formic acid aqueous solution (mobile phase A) and acetonitrile (mobile phase B). The flow rate was 0.3 mL / min. The column temperature was 30 °C. The gradient elution conditions were as follows: the initial 5% mobile phase B and 95% mobile phase A were increased to 15% mobile phase B within 2.8 min, and then linearly increased to 25% mobile phase B within 4.5 min. The 25% mobile phase B was further increased to 70%, and then the 70% mobile phase B was reduced 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 ionization (ESI) source; full scan-ddMS2 mode; positive ion scan 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. 6 100 ms; sheath gas and auxiliary gas pressures were 40 and 10 units, respectively; scan analysis time was 8 min; secondary scan range was m / z 90–1,400; ion fragmentation mode and energy: high-energy collision cell (ECD) and 30 eV; secondary scan resolution was 17,500; maximum C-trap capacity and maximum injection time for secondary scan were 1 × 10⁻⁶. 5 And 50ms.

[0029] The mass spectrometry quantification method of the PRM is to obtain the cobalamin precursor ion obtained by inputting it in the inclusion list, and obtain a high-resolution secondary mass spectrum after high-energy collision fragmentation. The low-position ligand with the highest abundance and high recognizability is used as the quantitative cobalamin ion.

[0030] The detection conditions used in the PRM mass spectrometry method for quantitative acquisition of cobalamin are described above. The liquid chromatography analysis conditions are consistent with those described above.

[0031] Mass spectrometry conditions are:

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

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

[0034] To determine the type of cobalamin in the sample, a sample pretreatment step is required. Take an appropriate amount of bacterial cells and perform a full scan-ddMS2 analysis using UHPLC-ESI-Q-Orbitrap. Compare the primary and secondary chromatograms of the sample and the standard to analyze the unknown cobalamin structure type in the sample.

[0035] To quantify the cobalamin concentration in the sample, a sample pretreatment step is required. The supernatant of the biological culture medium after pretreatment is subjected to PRM analysis by UHPLC-ESI-Q-Orbitrap. 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.

[0036] Advantages of this invention:

[0037] Due to the diverse types, low concentrations, difficult separation and purification, complex structures, and large molecular weight of cobalamin, there are currently very few mass spectrometry analysis techniques available for it. This 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. Using Full scan-ddMS2, the structural identification of cobalamin requires no prior method optimization, exhibiting good reproducibility and selectivity, making it suitable for the analysis of cobalamin in complex samples. Furthermore, the high-resolution quantitative scanning mode of PRM filters out a large number of interfering ions (especially in low-concentration matrix samples) using a quadrupole filter, improving both sample analysis sensitivity and quantitative specificity, enabling the detection of cobalamin at concentration levels in the μg / L range. Attached Figure Description

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

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

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

[0041] Figure 4 The mass spectrum of cobalamin identified in the sample provided in this embodiment of the invention.

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

[0043] Figure 6 This is a graph showing the quantitative analysis results of cobalamin in the sample to be tested provided in an embodiment of the present invention. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to specific implementation methods. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of application of the present invention.

[0045] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0046] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0047] Example 1: Structural Identification of Cobalamin

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

[0049] Weigh out an appropriate amount of vitamin B 12 Dissolve and dilute with deionized water to a concentration of 5 mg / L to obtain vitamin B. 12 Standard products ( Figure 1 ).

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

[0051] Methanogenous bacteria *Methanosarcina barkeri* strain Fusaro (NCBI ID: 269797) and *Sporomusa* sp. strain KB-1 (NCBI ID: 2184576) supplemented with 100 mM 5-methoxybenzimidazole were subjected to anaerobic culture. Fusaro used 0.3% methanol as a growth substrate, while KB-1 used 40 mM betaine. After culturing for 96-144 h, the bacterial cells were collected by centrifugation at 20,000 x g for 30 min at room temperature. The cells were then resuspended in 10 mL of deionized water, and cyanooxide was added for a water bath reaction (100 °C, pH 5.0, 30 min). Finally, 5-hydroxybenzimidazole-type B1 was obtained from the oxidation solutions of Fusaro and KB-1 cells. 12 and 5-methoxybenzimidazole B 12 The crude extracts of cobalamin were then processed using a syringe at C... 18 Elution was performed on an adsorption column (Waters Corporation), and the effluent was discarded; then, 10 mL of ultrapure water was used to rinse the remaining hydrophilic non-cobalamin impurities on the column; finally, 3 mL of methanol was used to elute the adsorbed C24 column. 18 Cobalamin was removed from the adsorption column, and the methanol eluent containing the cobalamin fraction was carefully collected. The obtained eluent was manually purified by HPLC as follows: elution steps: 18% mobile phase B (0.1% formic acid-methanol), 82% mobile phase A (0.1% formic acid-water). Mobile phase B was linearly increased to 20% over 34 minutes, then linearly increased to 90% over 2 minutes, held for 3 minutes, and then linearly decreased to the initial conditions over 2 minutes, held for 4 minutes. Detection was performed at 361 nm, and the chromatographically separated cobalamin fraction was collected from the mobile phase waste outlet. The collected fraction was diluted 5-fold with deionized water and reused using the above method. 18 Column concentration and purification. The cobalamin-methanol mixture was dried by rotary evaporation, dissolved in deionized water, and diluted to 5 mg / L to obtain 5-hydroxybenzimidazole type B. 12 and 5-methoxybenzimidazole type B 12 Standard products ( Figure 1 ).

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

[0053] An anaerobic enrichment culture system (DCB-1, USA) was established using approximately 2g of fine river sediment (latitude: 41°39′46″, longitude: 123°6′20″) as the initial inoculum, tetrachloroethylene as the electron acceptor, and acetic acid and hydrogen as the carbon source and electron donor, respectively. This resulted in an enriched culture of a mixed bacterial community dominated by *Dehalococcoides*. 100mL of this mixed bacterial community enrichment culture with unknown cobalamin composition was taken and filtered through a 0.22μm (47mm diameter) filtration membrane (Pall Life Sciences, USA) to collect the bacterial cells. The filter containing the bacterial cells was transferred to a 10mL centrifuge tube. Subsequently, it was resuspended in 5mL of deionized water for cyanooxidation (100℃, pH 5.0, 30min). Centrifugation at 20,000xg for 10 minutes yielded a cell-free supernatant, which was then transferred to a 2mL sample vial.

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

[0055] Five μL of each of the three standards and the processed supernatant sample were analyzed using a Vanquish ultra-high performance liquid chromatography-Thermo Q-Exactive quadrupole-electrostatic field orbital trap high-resolution tandem mass spectrometer in Full scan-ddMS2 mode. Chromatograms and mass spectrometers were obtained according to the set liquid chromatography and mass spectrometry conditions. The sizes of the precursor ions and characteristic fragment ions in the three cobalamin standards and the sample were compared. Based on the principle that fragments with the same cobalamin structure have the same m / z (accurate to three decimal places), the structure of the unknown cobalamin was determined. Compound Discoverer 3.0 software was used to search and predict cobalamin fragment ions to further verify the structure of the unknown cobalamin.

[0056] The specific conditions for high-resolution qualitative analysis of cobalamin in the standards and test samples are as follows:

[0057] Liquid chromatography conditions:

[0058] The instrument was a Vanquish ultra-high performance liquid chromatograph; the chromatographic column was a Hypersil GOLD C1000. 18The column (2.1 mm × 100 mm, 2.6 μm, Thermo Fisher Scientific, USA) was used. The mobile phase consisted of 0.1% (v / v) formic acid aqueous solution (mobile phase A) and acetonitrile (mobile phase B). The flow rate was 0.3 mL / min. The column temperature was 30 °C. The gradient elution conditions were as follows: the initial 5% mobile phase B and 95% mobile phase A were increased to 15% mobile phase B within 2.8 min, and then linearly increased to 25% mobile phase B within 4.5 min. The 25% mobile phase B was further increased to 70%, and then the 70% mobile phase B was reduced 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 ionization (ESI) source; full scan-ddMS2 mode; positive ion scan 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. 6 100 ms; sheath gas and auxiliary gas pressures were 40 and 10 units, respectively; scan analysis time was 8 min; secondary scan range was m / z 90–1,400; ion fragmentation mode and energy: high-energy collision cell (ECD) and 30 eV; secondary scan resolution was 17,500; maximum C-trap capacity and maximum injection time for secondary scan were 1 × 10⁻⁶. 5 And 50ms.

[0061] The precursor ion used to identify cobalamin is: Vitamin B1. 12 The parent ion of the standard is:

[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 / z 1343.538); 5-Methoxybenzimidazole type B 12 The parent ion is: [M+2H] 2+ (m / z679.278) and [M+H] + (m / z 1357.557)( Figure 2 ).

[0063] All characteristic fragment ions used to identify the structure of cobalamin are: Vitamin B1 12 →m / z 147.093, 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 the characteristic fragment ions used to identify the structure of cobalamin are: m / z 147.093, 136.056, 149.072, representing vitamin B1, 147.093, 136.056, 149.072, respectively. 12 5-Hydroxybenzimidazole type B 12 5-Methoxybenzimidazole type B 12 The lower-position ligand ion [base+H] + m / z 359.099, 347.064, and 361.079 represent the ions [phosphoriboside + H] of the phosphoribosyl moieties containing the low-position ligands of the three cobalamin compounds, respectively. + ;m / z

[0065] 997.448 corresponds to the [M-phosphoriboside+H] ion of three cobalamins containing a corrin ring but with the low-position ligand-containing phosphate riboside structural moiety removed. + m / z 456.729 and 912.438 correspond to the ion obtained by subtracting C3H6N2O from m / z 997.448; m / z 1209.476 represents the ions of the three cobalamins without the lower ligand structure [M-base+H]. + m / z 1124.442 represents the ion obtained by subtracting C3H6N2O from m / z 1209.476.

[0066] The results show that the sample tested achieved excellent detection results with a 5 μL injection. After a full scan of the primary mass spectrometer, a total of 1,286 precursor ions were obtained. Following a secondary scan of all precursor ions using full scan-ddMS2, the corresponding secondary mass spectra obtained after collision in the collision cell showed that the sample contained four cobalamin components. The primary and secondary mass spectra of three of the cobalamin components were completely consistent with those of the standard. Therefore, the three cobalamin components are vitamin B12.12 5-Hydroxybenzimidazole type B 12 and 5-methoxybenzimidazole type B 12 The precursor ion in 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 secondary mass spectrum showed that all fragment ions were identical to those of the three known standards, except for the low-position ligand (m / z 119.061) and the phosphoribosyl derivative containing the low-position ligand (m / z 331.072), which differed from the corresponding structures of the three standards. This indicates that, apart from the difference in the low-position ligand, the structure of this other cobalamin was completely identical to all fragment ions containing the corrin ring structure of the three standards. Importing the original mass spectrum into Compound Discoverer 3.0 software confirmed that this cobalamin was a benzimidazole type B with a non-methyl-substituted low-position ligand. 12 ( Figure 4 Ultimately, the mass spectrometry method for identifying the cobalamin structure described in Case Study 1 of this invention was proven to be feasible.

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

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

[0070] Take 10 μL each of the three different cobalamin standards (5 mg / L) from Example 1, mix them in a clean 2 mL centrifuge tube, and dilute with 940 μL of deionized water to prepare a 50 μg / L cobalamin mixed standard. Further serial dilution of this mixed standard with deionized water yielded cobalamin standard concentrations of 50, 25, 10, 5, 1, 0.5, and 0.1 μg / L. Transfer the standard to a 2 mL vial, and use an autosampler to dispense 10 μL for UHPLC-ESI-Q-Orbitrap analysis.

[0071] (2) Sample pretreatment for testing cobalamin content

[0072] Take 1 mL of the mixed bacterial culture enrichment broth from Example 1, centrifuge and transfer the supernatant to a new centrifuge tube; add 20 mM cyanide oxidant and 2 μL glacial acetic acid to the supernatant without bacteria, vortex mix for 30 seconds; place the sample in a boiling water bath and heat for 60 minutes; before the sample cools, centrifuge at 20,000 x g for 10 minutes, and transfer the treated and impurity-free supernatant sample to a sample vial.

[0073] (3) Preparation of quality control samples

[0074] To investigate the interference of the culture medium and sample pretreatment process of the mixed bacterial community enrichment broth in step (2) on the accuracy of cobalamin quantification in the test sample, this step uses blank culture medium as solvent to prepare vitamin B at a known concentration. 12 The quality control sample is prepared using the following steps: Take 10 μL of Vitamin B from Case 1. 12 The standard (5 mg / L) was placed in a clean 2 mL centrifuge tube, and 990 μL of blank culture medium without mixed enriched bacterial cells was added to obtain a 50 μg / L quality control sample. This was further diluted with blank culture medium to obtain 25 μg / L and 2.5 μg / L quality control samples. Three replicates were prepared for each quality control concentration, resulting in three consecutive analytical batches. The three sets of quality control concentration samples were pretreated according to the sample preparation method in step (2). The pretreated quality control samples were then transferred to sample vials for testing.

[0075] (4) Analytical steps of PRM for UHPLC-ESI-Q-Orbitrap

[0076] Take 10 μL each of the standards, processed supernatant, and quality control samples from steps (1), (2), and (3) above for analysis. Analyze the samples using a Vanquish ultra-high performance liquid chromatography-Thermo Q-Exactive quadrupole-electrostatic field orbital trap high-resolution tandem mass spectrometer in PRM scan mode. Obtain chromatograms and mass spectrometers according to the set liquid chromatography and mass spectrometry conditions. Select the corresponding low-position ligand ion [base+H] for each cobalamin standard. + As a quantitative ion, a standard curve with good linearity was obtained by plotting the concentration of cobalamin standard on the x-axis and the peak area on the y-axis, thereby calculating the cobalamin concentration in the test sample and the quality control sample.

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

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

[0079] Mass spectrometry conditions:

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

[0081] The characteristic fragment ions used for quantifying cobalamin are the corresponding low-position ligand ions [base+H] in each cobalamin structure. + They are: Vitamin B 12 Dimethylbenzimidazole (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] A standard curve with good linearity was obtained by plotting the concentration of cobalamin standard on the x-axis and the peak area on the y-axis. The results showed that the linear range for determining cobalamin concentration was 0.1-100 μg / L, and the LOQ was 0.1 μg / L. Therefore, vitamin B... 12 The working standard curve is y = 188,444x - 66,493, r 2 =0.9998; 5-hydroxybenzimidazole type B 12 The working standard curve is y = 156,950x - 68,893, r 2 =0.9995; 5-Methoxybenzimidazole type B 12 The working standard curve is y = 151,901x - 67,775, r 2 =0.9996( Figure 5 ).

[0085] The results showed that vitamin B was successfully detected in the processed supernatant sample. 12 The content is 32.65 μg / L, 5-hydroxybenzimidazole type B 12 The content was 25.63 μg / L, and 5-methoxybenzimidazole type B 12 The content was 6.14 μg / L ( Figure 6In addition, according to vitamin B... 12 The concentrations of each quality control sample were obtained from 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 media 12 Precision and accuracy of the measurement

[0087]

[0088] Given the linearity of the cobalamin standard curve and the accuracy of quality control samples, the absolute quantification method for cobalamin described in this invention exhibits good linearity, high accuracy, good reproducibility, sensitivity, and reliability, and can be applied to the absolute quantitative analysis of cobalamin in biological samples.

Claims

1. A highly sensitive method for quantitative and structural identification of cobalamin, characterized in that: Using three common natural cobalamins with different structures as standards, a full scan-ddMS2 mass spectrometry acquisition method was employed to obtain cobalamin precursor ions and a series of high-abundance characteristic ion fragments. Based on the principle that fragments of the same cobalamin have the same fragment ion m / z and the fragment ion search function of Compound Discoverer 3.0 software, the structure of unknown cobalamin was identified. Then, a parallel reaction monitoring (PRM) mass spectrometry quantitative method was used. A large number of interfering ions were filtered out by a quadrupole, and the cobalamin low-position ligand fragment ions with the highest abundance were used as quantitative ions to analyze the type and content of cobalamin in the sample to be measured.

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

3. The highly sensitive quantitative and structural identification method for cobalamin according to claim 1, characterized in that: The Full scan-ddMS2 mass spectrometry acquisition method allows for the acquisition of cobalamin precursor ions after a full scan, including vitamin B1. 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; Simultaneously, characteristic fragment ions of cobalamin can be obtained through automatically triggered secondary mass spectrometry scanning. Among them, all standard fragment ions used to identify the structure of cobalamin are: Vitamin B12... 12 →m / z 147.093, 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 highly sensitive quantitative and structural identification method for cobalamin according to claim 1 or 3, characterized in that: The Compound Discoverer software, through its ion fragment search function, identified m / z 147.093, 136.056, and 149.072 as representing vitamin B1, respectively. 12 5-Hydroxybenzimidazole type B 12 and 5-methoxybenzimidazole type B 12 low-position ligand ions [base+H] + m / z 359.099, 347.064, and 361.079 represent the ions [phosphoriboside + H] of the phosphoribosyl moieties containing the low-position ligands of the three cobalamin compounds, respectively. + m / z 997.448 corresponds to the [M-phosphoriboside+H] ion of three cobalamins containing a corrin ring but with the low-position ligand removed from the phosphate riboside structure. + m / z 456.729 and 912.438 correspond to the ion obtained by subtracting C3H6N2O from m / z 997.448; m / z 1209.476 represents the ions of the three cobalamins without the lower ligand structure [M-base+H]. + m / z 1124.442 represents the ion obtained by subtracting C3H6N2O from m / z 1209.

476.

5. The highly sensitive quantitative and structural identification method for cobalamin according to claim 1 or 3, characterized in that: The detection conditions used in the Full scan-ddMS2 mass spectrometry method for qualitative cobalamin acquisition; Liquid chromatography conditions: The instrument was a Vanquish ultra-high performance liquid chromatograph; the chromatographic column was a Hypersil GOLD C1000. 18 The column (2.1 mm × 100 mm, 2.6 μm) was used; the mobile phase consisted of mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (acetonitrile); the flow rate was 0.3 mL / min; the column temperature was 30 °C; the gradient elution conditions were as follows: the initial 5% mobile phase B and 95% mobile phase A were increased to 15% mobile phase B within 2.8 min, and then linearly increased to 25% mobile phase B within 4.5 min. The 25% mobile phase B was then increased to 70%, and then the 70% mobile phase B was reduced 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 ionization (ESI) source; full scan-ddMS2 mode; positive ion scan 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. 6 100 ms; sheath gas and auxiliary gas pressures were 40 and 10 units, respectively; scan analysis time was 8 min; secondary scan range was m / z 90–1,400; ion fragmentation mode and energy: high-energy collision cell ECD and 30 eV; secondary scan resolution was 17,500; maximum C-trap capacity and maximum injection time for secondary scan were 1 × 10⁻⁶. 5 And 50ms.

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

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

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

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