A non-targeted identification method for compounds based on bromine isotope labeling

Abstract

The present invention relates to the technical field of isotope labeling, and discloses a non-targeted identification method of compounds based on bromine isotope labeling, comprising the following steps: S1: bromine isotope probe labeling stage; S2: high-resolution chromatography-mass spectrometry detection stage; S3: data acquisition and analysis stage, S4: confirmation of labeled compounds; S5: structural analysis of labeled compounds. This scheme applies bromine isotope probe labeling to the non-targeted identification of compounds, and according to the new mechanism that bromine-labeled compounds are cracked in mass spectrometry to generate bromine-containing fragment ions corresponding to a pair of isotope parent ions, the specific two-dimensional signal formed by screening the fragment ions can quickly and accurately identify the compounds in the sample, improve the accuracy and efficiency of non-targeted identification of compounds, and improve the compound identification coverage, so that this scheme can be applied to the identification of compounds in samples containing complex matrices.

Description

Technical Field

[0001] The invention relates to the technical field of isotope labeling, and in particular to a non-targeted identification method of a compound based on bromine isotope labeling. Background Art

[0002] Chemical isotope labeling (CIL) combined with liquid chromatography-mass spectrometry (LC-MS) is currently the main method for non-targeted identification of a class of substances with the same characteristic groups from complex matrices. It is done by labeling the samples with "light" and "heavy" isotope probe compounds, merging the samples, and then using LC-MS analysis to identify the chromatographic peaks. The separately labeled compounds can produce a pair of chromatographic peaks with isotope mass difference and similar retention time, which can identify the labeled compounds. Therefore, LC-MS-based CIL technology is currently one of the most powerful strategies for screening a class of substances with the same functional groups and unknown compounds therein, and plays an important role in the fields of life sciences, medicine and health, environmental protection, industrial production, etc. For example, in the field of life sciences, screening of endogenous metabolite subgroups in the body such as aldehydes, carboxylates, lipids, carnitines, etc. is crucial to exploring cell metabolism, drug targets, disease mechanisms, etc.

[0003] The identification of compounds by non-targeted identification methods based on LC-MS and CIL technology is highly dependent on the retention time and accurate molecular weight of isotope chromatographic peak pairs. However, the commonly used isotope labels are 12 carbon / 13 carbon, 14 nitrogen / 15 Nitrogen, hydrogen / deuterium, etc., the "light" labeled and "heavy" labeled counterparts formed by their isotope labels are two independent compounds that undergo chromatographic elution separately, resulting in their chromatographic retention times and peak shapes always being slightly different, especially when the chromatographic retention fluctuates or the chromatographic separation is insufficient and the chromatographic peaks overlap. Therefore, relying solely on the retention time of conventional isotope chromatographic peak pairs can easily lead to misidentification of compounds and low recognition efficiency. In other words, the specificity of this isotopic feature is not enough to fully distinguish between labeled and unlabeled compounds, and this chromatographic-based isotopic feature is easily interfered with or mutated. Specifically, interfering ions are easily mistaken for signal ions, resulting in false positive results, and signal ions are not confident enough to confirm whether they are target compounds due to insufficient specificity of isotopic features, resulting in false negative results. This is also the main reason why the current non-targeted screening method based on LC-MS CIL technology has low recognition ability for labeled compounds.

[0004] Using specific data acquisition modes, such as precursor ion scanning or neutral loss scanning to monitor the common isotope fragments produced by the collision-induced dissociation (CID) of the labeled compound can improve the selectivity and sensitivity of the monitoring method, which is a method to improve the accuracy and coverage of compound identification. Similarly, the use of high-resolution mass spectrometry can eliminate some interferences and also improve identification accuracy. However, the recognition ability essentially depends on whether the specificity of the isotope characteristics is sufficient to fully distinguish between labeled and unlabeled compounds. Compared with conventional isotope labels, bromine (Br) elements can self-cleave to form 79 Br and 81 Br natural isotope. The isotopic characteristics of bromine-labeled molecules include the formation of a pair of parent ions with a mass number difference of 1.998Da and similar intensities, an isotopic chromatographic peak pair with a peak area ratio of approximately 1:1 and the same retention time. These characteristics make bromine isotope labeling also applicable to non-targeted identification methods based on LC-MS and CIL technology. However, the existing technology only has the characteristic of "bromine ions being able to produce isotopic chromatographic peak pairs with the same retention time and a peak area ratio of approximately 1:1 and a difference of 1.998Da", which is no different in essence from other conventional non-targeted screening methods based on LC-MS CIL technology, and the accuracy and coverage of its detection results are still not high.

[0005] In summary, when the existing non-targeted identification methods for unknown compounds are used to screen endogenous metabolite subgroups in the body, such as aldehydes, carboxylates, lipids, carnitines and other substances, the accuracy and coverage of the test results are not high, which affects the judgment of other test results based on compound identification. Therefore, the development of a non-targeted identification method for compounds with high efficiency, high accuracy, strong recognition ability and comprehensive coverage can not only effectively make up for the shortcomings of existing compound identification methods, but also has important significance for other tests based on compound identification (such as metabolomics research). Summary of the invention

[0006] The present invention has found through a large number of experiments that bromine isotopes can produce other more distinguishing isotopic characteristics, and provides a non-targeted identification method for compounds based on bromine isotope labeling to solve the technical problem of low recognition ability of existing compound identification methods.

[0007] To achieve the above object, the present invention adopts the following technical solution: a non-targeted identification method of compounds based on bromine isotope labeling, comprising the following steps:

[0008] S1: bromine isotope probe labeling stage, the bromine isotope probe is mixed with the sample to form a mixed solution, and the mixed solution reacts to obtain a sample containing a bromine labeled compound;

[0009] S2: High-resolution chromatography-mass spectrometry detection and data acquisition stage, the sample containing the bromine-labeled compound obtained in S1 is subjected to high-resolution chromatography-mass spectrometry detection to obtain chromatographic data and mass spectrometric data containing high-resolution secondary spectrum information;

[0010] S3: analysis phase, analyzing the mass spectrometry data containing high-resolution secondary spectrum information obtained in S2 to preliminarily identify the bromine-labeled compounds;

[0011] S4: Confirmation of the labeled compound: the bromine-labeled compound preliminarily identified in S3 is further confirmed to be a compound labeled with a bromine isotope probe through the chromatographic characteristics collected in S2;

[0012] S5: Structural analysis of labeled compounds, analyzing the structure of the compound through high-resolution secondary spectra and reference substances.

[0013] The principle of this scheme is:

[0014] Compounds containing bromine can form isotope parent ion pairs with a difference of 1.998Da and an abundance ratio of 100:98 in the mass spectrum. On the basis that the probe meets certain conditions, the isotope parent ion pair can be broken down in the mass spectrum to form isotope daughter ion pairs containing bromine with a difference of 1.998Da and an abundance ratio of 100:98. Each daughter ion in the bromine daughter ion pair corresponds to a pair of parent ions. Therefore, when a pair of isotope daughter ion pairs (difference of 1.998Da) is screened using data post-processing software (such as processing software MZmine2), a two-dimensional signal consisting of two pairs of parent ion pairs (both with a difference of 1.998Da) can be obtained. This highly restrictive isotope feature greatly improves the accuracy of screening. In addition, the bromine isotope daughter ion pairs are produced by ionization after the bromine-containing parent ion undergoes chromatographic separation. The two isotope daughter ion pairs with a difference of 1.998Da have exactly the same chromatographic characteristics, including the same retention time, a chromatographic peak area ratio of approximately 1:1, and the same chromatographic peak shape; in particular, the characteristics of the same chromatographic peak shape can effectively eliminate the interference of the matrix in the sample on the identification of the compound, which can further improve the accuracy of screening.

[0015] The advantages of this solution are:

[0016] 1. High recognition capability: Compared with the prior art, which has low recognition accuracy due to insufficient specificity and limited sensitivity of isotope characteristics, this solution significantly improves the accuracy and recognition capability of non-targeted identification of compounds through chromatographic data based on bromine isotope labeling and mass spectrometry data containing high-resolution secondary spectrum information. Specifically: This solution is based on the bromine-labeled compound in the mass spectrometry, which produces bromine-containing fragment ions corresponding to a pair of isotope parent ions. The specific two-dimensional signal formed by screening the fragment ions identifies the bromine-labeled compound, and on this basis, the chromatographic characteristics of the bromine isotope labeling are used to further confirm the bromine isotope-labeled compound. This dual isotopic feature can accurately distinguish between bromine-labeled and non-labeled compounds, significantly improving the accuracy and recognition capability of non-targeted identification of compounds.

[0017] 2. Wide coverage of identification results: Thanks to high recognition accuracy, high sensitivity and recognition stability, this scheme can detect and accurately confirm the target compound with high sensitivity and eliminate interfering signals, which greatly increases the number of compounds that can be identified and covers a wide range of results. The applicant found in the experiment that the number of aldehyde compounds that can be identified in urine samples using this scheme is 57, far exceeding the 6-16 aldehyde compounds that can only be identified by the prior art. Therefore, compared with previous technologies, the non-targeted recognition method of this scheme has greatly expanded the cognition of the existence and types of aldehyde compounds in urine, and is applied to life sciences or medical sciences, which is of great significance to their development.

[0018] Preferably, in S1, the bromine isotope probe is a bromine-containing compound comprising a bromine isotope, a reactive group and a breakable linker.

[0019] Beneficial effects:

[0020] 1. In this scheme, bromine is an element with stable isotopes. After the compound is labeled, the bromine-containing compound self-decomposes in the mass spectrometer to form 79 Br and 81 The parent ion of Br makes the labeled compound have bromine isotope characteristics. The reactive group is used to react with the target compound, so that the target compound in the sample is selectively labeled, so that the bromine isotope characteristics can be used to further selectively identify these labeled target compounds; a good reactive group should have the characteristics of rapid reaction with the target compound, complete reaction, mild reaction conditions, and single reaction product; and the easily breakable connecting bond facilitates the cleavage of the labeled compound (parent ion) in the mass spectrometer to produce a constant bromine-containing daughter ion pair and increase the relative abundance of the bromine-containing daughter ion pair in the secondary spectrum, thereby improving the selectivity of identification and the robustness and stability of compound identification.

[0021] 2. This scheme is based on the design of the probe chemical structure based on the mass spectrometry fragmentation behavior. By optimizing the probe chemical structure, in addition to improving the sensitivity, it also significantly improves the stability and robustness of compound identification (referring to the characteristics of the control system maintaining certain other performances under certain parameter fluctuations). The main points are: through the probe structure design, isotope daughter ion pairs with high relative abundance and mass spectrometry response are obtained, and the noise ions are distinguished by the absolute mass spectrometry response difference and the strict structural requirements (bromine-containing daughter ions with extremely high relative abundance need to be produced), which causes a huge difference between signal ions and noise ions; on the one hand, more stringent screening parameters are set to further improve the selectivity of identification (excluding noise ions), and on the other hand, this difference in relative and absolute abundance with noise ions can significantly improve the robustness and stability of compound identification (ensuring that signal ions can be identified). This stability allows this scheme to be used in biological samples with extremely large fluctuations in the content and type of interferences to stably (the results between parallel samples are very small) and accurately identify standard compounds with huge differences in chemical structure and content levels.

[0022] Preferably, in S1, the chemical structure of the bromine isotope probe is: reactive group-connecting bond-bromine isotope, and the bromine isotope part includes an easily ionized group.

[0023] Beneficial effects: This scheme facilitates the labeling of compounds and the fragmentation of compounds in mass spectrometry to produce constant high-abundance bromine-containing daughter ion pairs, thereby realizing rapid and selective identification of labeled compounds by screening bromine-containing daughter ion pairs. The presence of other isotopic elements or limiting factors in mass spectrometry fragments can further improve the selectivity and recognition efficiency of the method. Therefore, in the design of bromine-containing probes, the introduction of groups with high ionization efficiency (easily ionized groups) in the overall structure of the probe and the substructure that forms bromine-containing daughter ions can further improve the mass spectrometric response of the labeled compounds and daughter ions, thereby improving the compound recognition efficiency and accuracy of this scheme.

[0024] Preferably, the bromine isotope probe used in identifying aldehyde compounds in a sample is 5-bromothiophene-2-carboxylic acid hydrazide.

[0025] Beneficial effects: The applicant has found through long-term experiments that since the bromine-containing fragment ions produced by the compounds labeled with 5-bromothiophene-2-carboxylic acid hydrazide have higher relative abundance and mass spectrometric response and stronger labeling reaction efficiency, more aldehyde compounds in the sample can be identified by using 5-bromothiophene-2-carboxylic acid hydrazide as a bromine isotope probe. Prior to the use of 5-bromothiophene-2-carboxylic acid hydrazide, the applicant used bromine isotope probes including 5-bromofuran-2-carboxylic acid hydrazide, 5-bromopyridine-3-carboxylic acid hydrazide, 5-bromopyrimidine-2-carboxylic acid hydrazide and 5-bromo-2-hydroxybenzoic acid hydrazide to label a mixed standard of aldehyde compounds (a total of 16 aldehydes). The disadvantages of the mixed standard are as follows: 1) The low reaction efficiency leads to a greatly reduced recognition efficiency: the reaction efficiency of 5-bromofuran-2-carboxylic acid hydrazide with aldehydes is low, about 30-70%; the reaction efficiency of 5-bromopyridine-3-carboxylic acid hydrazide with different types of aldehydes is different, and the reaction efficiency with fatty aldehydes is the worst, about 25-65%; 2) when the collision energy is optimal, the relative abundance of the bromine-containing daughter ions generated by the cleavage is low or the cleavage is impossible: the relative abundance of the bromine-containing daughter ions generated by the cleavage of the derivative products of 5-bromopyrimidine-2-carboxylic acid hydrazide with aldehydes is low; 5-bromo-2-hydroxybenzoic acid hydrazide has no recognition capability because it cannot cleave to generate bromine-containing daughter ions. Therefore, the recognition ability of these bromine isotope probes is relatively low, and the number of aldehyde compounds identified is limited. Specifically, the number of aldehyde standards identified by 5-bromofuran-2-carboxylic acid hydrazide, 5-bromopyridine-3-carboxylic acid hydrazide, 5-bromopyrimidine-2-carboxylic acid hydrazide, and 5-bromo-2-hydroxybenzoic acid hydrazide are 10, 11, 14, and 0, respectively. However, when the applicant used 5-bromothiophene-2-carboxylic acid hydrazide to label the mixed standard of 16 aldehydes, all 16 aldehydes were identified, showing a very strong recognition ability.

[0026] Preferably, in S2, a full scan-daughter ion scan mode or an equivalent functional scan mode of a high-resolution liquid chromatography-mass spectrometer is used to collect mass spectral data containing high-resolution secondary spectral information.

[0027] Beneficial effects: This scheme can use high-resolution bromine-containing daughter ions (mass-to-charge ratio values ​​accurate to four decimal places) to identify compounds. This limiting factor can effectively exclude noise ions with similar mass-to-charge ratios and improve the accuracy of screening.

[0028] Preferably, in S3, the high-resolution mass spectrometry data containing secondary spectral signals collected in S2 are imported into mass spectrometry data post-processing software capable of performing mass spectrometry fragment screening for processing, and the mass spectrometry data post-processing software includes but is not limited to MZmine2.

[0029] Beneficial effects: This scheme uses the daughter ion or neutral loss screening mode of the software to screen the constant bromine-containing mass spectral fragments (including daughter ions or neutral loss molecules) generated by the fragmentation of bromine-labeled compounds to selectively identify bromine-labeled compounds. Since a pair of high-resolution isotope daughter ion pairs with a difference of 1.998Da are screened, this stringent restriction condition gives this scheme the advantage of more accurate identification of compounds in terms of mechanism. Combined with the probe structure optimization based on mass spectrometry fragmentation behavior in S1, daughter ions with high relative abundance and absolute mass spectrometry response are generated, so that this scheme can set a higher target daughter ion relative abundance screening threshold in the software (that is, only daughter ions above the set relative abundance threshold can be identified), reduce the interference of noise signals in the sample on the screening, and further enhance the advantage of more accurate identification of compounds in this scheme.

[0030] Preferably, in S3, the relative abundance of the detected target daughter ions is 80-100%, and the relative abundance screening threshold is 35-45%.

[0031] Beneficial effects: The relative abundance screening threshold of the target daughter ion in this scheme is a key parameter, which greatly affects the accuracy of identifying compounds. A value that is too small will cause noise ions to be misidentified, and a value that is too large will cause the target ions to be ignored; the applicant has found through long-term experiments that the relative abundance screening thresholds of the target daughter ions of 5-bromothiophene-2-carboxylic acid hydrazide, 5-bromofuran-2-carboxylic acid hydrazide, 5-bromopyridine-3-carboxylic acid hydrazide, and 5-bromopyrimidine-2-carboxylic acid hydrazide are 35%, 10%, 10%, and 5%, respectively. 5-bromo-2-hydroxybenzoic acid hydrazide is not suitable for this method because it cannot produce bromine-containing daughter ion pairs. The above scheme can obtain more accurate compound identification capabilities than the existing technical scheme.

[0032] Preferably, in S3, bromine-containing daughter ion pairs are screened by data post-processing software MZmine2, and the target compound can form a two-dimensional signal composed of four signal points based on bromine isotope characteristics, and the two-dimensional signal serves as a standard for identifying whether it is a bromine isotope probe-labeled compound.

[0033] Beneficial effects: The applicant has conducted in-depth research on the generation mechanism of two-dimensional signals based on bromine isotope characteristics. Since in a pair of bromine-containing daughter ions (difference of 1.998Da), any daughter ion corresponds to a pair of parent ion pairs (difference of 1.998Da), the positive compound can generate a pair of signal points representing the parent ion pair in the two daughter ion screening channels, forming a two-dimensional signal with a total of four signal points in the two channels. The formation of this two-dimensional signal mainly depends on the mass spectrometry response of the parent ion, the screening relative abundance threshold setting and the relative abundance of the daughter ions. Using the above scheme, this highly specific two-dimensional signal can more accurately determine the identified compound. The applicant compared the use of two-dimensional signals and the screening channel. 79The screening method with two signal points of Br precursor ion as identification criteria only 79 The method using the parent ion of Br as the identification standard will be affected by interference signals and produce false positive results. Taking the 5-bromothiophene-2-carboxylic acid hydrazide probe to identify aldehyde compounds in rat urine as an example, interference signals about twice the number of the target signal can be generated, while when the two-dimensional signal is used as the identification standard, the number of interference signals generated is only about 2% of the target signal.

[0034] Preferably, in S4, the identified bromine-labeled compound is further confirmed by the chromatographic data collected in S2, the chromatographic data including chromatographic peak shape, peak area ratio, and retention time; the judgment criteria of the chromatographic data are: the peak area ratio is 0.8-1.2, the chromatographic peak shape is that the difference in the horizontal and vertical coordinate values ​​corresponding to each monitoring point constituting the chromatographic peak in the chromatogram is ≤±20%, and the chromatographic retention time is that the numerical values ​​after the decimal point are all the same.

[0035] Beneficial effects: This scheme can further confirm the identified bromine-labeled compounds through chromatographic data. Specifically, the peak area ratio of the chromatographic peak corresponding to the isotopic ion pair should be between about 0.8-1.2; the retention time should be consistent at one decimal place; the difference in the horizontal and vertical coordinate values ​​corresponding to each monitoring point constituting the chromatographic peak is within ±20%. The peak shape of the chromatographic peak cannot be over-smoothed during integration. The peak shape of the chromatographic peak is kept in its original state by adjusting the chromatographic peak shape smoothing parameters to retain the characteristic curve of the chromatographic peak shape. The above scheme can further exclude false positive results and confirm the identified compounds.

[0036] Preferably, in S1, the bromine isotope probe further includes a second stable isotope, and the second stable isotope is 13 carbon, 15 Any of nitrogen and deuterium.

[0037] Beneficial effects: This scheme adds a second stable isotope in addition to the bromine isotope or other limiting factors, such as 13 carbon, 15 Nitrogen, deuteration, etc., the second stable isotope acts as a limiting factor for identification and helps to further improve the selectivity of the method, thereby more accurately identifying the compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a comparison chart of the isotopic characteristics produced by traditional isotope labeling and the isotopic characteristics produced by bromine isotope labeling in this scheme.

[0039] Figure 2 This is the flow chart of this scheme for determining whether it is a signal ion based on the bromine isotope characteristics.

[0040] Figure 3This is a flow chart of the non-targeted compound identification method based on the two-dimensional signal formed by the mass spectrometry characteristics of bromine isotopes.

[0041] Figure 4 This is a two-dimensional signal diagram generated by the bromine-labeled compound in Example 1 of this scheme using the data post-processing software MZmine2.

[0042] Figure 5 This is a comparison diagram of the chromatogram (A) and the secondary mass spectrum (B) of the rat urine sample labeled with the bromine-containing probe 5-bromothiophene-2-carboxylic acid hydrazide and the heptanal reference substance in Example 1 of this scheme. DETAILED DESCRIPTION

[0043] The following is further described in detail through specific implementation methods, but the implementation methods of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.

[0044] For a detailed comparison of the isotopic characteristics produced by traditional isotope labeling and the isotopic characteristics produced by bromine isotope labeling in this scheme, see Figure 1 . Compared with the prior art, which uses isotope groups to mark target compounds separately and then identifies a limited number of compounds, this scheme mainly uses the isotope characteristics of bromine isotopes, and selects probes with bromine elements to mark target compounds, which significantly improves the accuracy and number of target compound identification, and provides new ideas and methods for the non-targeted identification of compounds. This scheme specifically uses aldehyde compounds and mixed aldehyde compound standards in rat urine as target compounds, and uses specific embodiments to demonstrate a non-targeted identification method for compounds based on bromine isotope labeling. Using different reactive groups on the probe can achieve the separate labeling of different types of target compounds. Example 1 and Comparative Examples 1-4 respectively demonstrate the non-targeted identification of aldehyde compounds using different bromine isotope probes with hydrazides as reactive groups; the probe differences and main influencing factors for the non-targeted identification of compounds in samples in Example 1 and Comparative Examples 1 to 4 are shown in Table 1. Taking Example 1 as an example, the non-targeted identification method of compounds based on bromine isotope labeling in this scheme is explained.

[0045] Example 1

[0046] A non-targeted identification method based on bromine isotope labeling of compounds, such as Figure 2 , Figure 3 As shown, the specific steps include:

[0047] S1: bromine isotope probe labeling stage, the bromine isotope probe is mixed with the sample to form a mixed solution, and the mixed solution reacts to obtain a sample containing a bromine labeled compound;

[0048] The sample used was rat urine, and the sample pretreatment stage included: collecting urine samples from healthy Sprague-Dawley rats in a metabolic cage controlled by dry ice, centrifuging the urine immediately at 4°C, 14,000 rpm for 15 minutes after thawing, taking the supernatant, and filtering it through a 0.22 μm organic phase microporous filter membrane to obtain the filtrate for later use.

[0049] In this scheme, the bromine isotope probe is a compound including a bromine isotope, a reactive group, and a breakable linker, and the general chemical structure formula is: reactive group-linker-bromine isotope, and the bromine isotope part includes an easily ionized group. In addition, the bromine isotope probe also includes a second stable isotope, such as 13 carbon, 15 Nitrogen, deuterated, etc., according to the isotope type of the probe, there are two cases. The probe containing only bromine only needs to be labeled with the probe once; while the probe containing bromine and other isotopes at the same time needs to be labeled with the "light" and "heavy" probes containing bromine respectively, and then the samples are mixed in a ratio of 1:1. The bromine isotope probe used in this example to identify aldehyde compounds in rat urine is 5-bromothiophene-2-carbohydrazide (BTCH, CAS NO.: 98027-27-1).

[0050] Then, 60 μL of the filtrate was taken into a 1.5 mL EP tube, and 20 μL of 20 mg / mL 5-bromothiophene-2-carboxylic acid hydrazide solution, 1 μL of acetic acid, and 100 μL of acetonitrile (the volume ratio of the filtrate, bromine isotope probe, and acetic acid was 60:20:1) were added. After vortexing for 30 seconds, the mixture was reacted at 25°C for 2 hours. After the reaction was completed, the reaction solution was transferred to an injection bottle for mass spectrometry analysis.

[0051] S2: high resolution chromatography-mass spectrometry detection stage, the sample containing the bromine-labeled compound obtained in S1 is subjected to chromatography and mass spectrometry detection;

[0052] The ultra-high performance liquid chromatography-high resolution mass spectrometry analysis was performed using an UltiMate 3000 ultra-high performance liquid chromatography analysis system and a Q / Orbitrap high-resolution mass spectrometer (Thermo Fisher Scientific).

[0053] Among them, the liquid chromatography conditions are:

[0054] Column: Hypersil GOLD TMC18 chromatographic column (150×2.1 mm, 1.9 μm, Thermo Fisher Scientific). Mobile phase: Phase A is 0.1% formic acid aqueous solution, Phase B is methanol, and the flow rate is set to 0.3 mL / min. Gradient elution conditions: 0–1 min, 10–20% B; 1–4 min, 20–32% B; 4–6 min, 32–58% B; 6–16 min, 58–62% B; 14–20 min, 62–65% B; 20–27.5 min, 65–72% B; 27.5–28.5 min, 72–85% B; 28.5–30 min, 85% B; 30–30.5 min, 85–100%; 30.5–35 min, 100% B; 35–35.5 min, 100–10% B; 35.5–40 min, 10% B. The temperatures of the injector and column oven were 10°C and 40°C, respectively. The injection volume was 3 μL.

[0055] The mass spectrometry conditions were:

[0056] Atmospheric pressure electrospray ion source (ESI), positive ion acquisition mode: spray voltage 3.5 kV, capillary temperature 350 ° C, sheath gas flow rate 35, auxiliary gas flow rate 15, auxiliary gas heating temperature 350 ° C, purge gas: 2, S-Lens: 50, MS scanning range: m / z 200-800; the scanning resolution of the high-resolution mass spectrometer was set to 70000. The Full Scan-ddMS2 scanning mode was adopted, and the collision energy (CE) was 15%.

[0057] S3: Data collection and analysis phase, collect and analyze the data obtained in S2 including peak shape, peak area ratio, retention time, and obtain compound identification results.

[0058] The free and open source mass spectrometry data post-processing software MZmine2 was used to identify aldehyde compounds labeled with 5-bromothiophene-2-carboxylic acid hydrazide (BTCH).

[0059] Aldehyde compounds labeled with 5-bromothiophene-2-carboxylic acid hydrazide can generate two pairs of constant bromine-labeled fragments through collision-induced dissociation (CID) in LC-MS, which are bromine-containing product ions m / z 188.9004 / 190.8984.

[0060] First, the MS / MS data of rat urine samples were collected by high-resolution ultra-high performance liquid chromatography-quadrupole orbitrap mass spectrometer (UPLC-Q / Orbitrap-MS) in Full Scan-ddMS2 mode, and the data were imported into MZmine2 software. The diagnostic fragment filtering (DFF) module of MZmine2 software was used to screen the daughter ions m / z188.9005 and 190.8984 to identify aldehyde compounds. In the characteristic ion filtering mode, the m / z error range was within 5ppm, and the minimum diagnostic ion intensity of basepeak (MIB) screening threshold of target daughter ion relative abundance was 35%. Positive compounds can simultaneously generate two signal points with a difference of 1.998Da and the same retention time in the channels of m / z 188.9005 and 190.8984, which correspond to the precursor ion and isotope precursor ion of the positive compound, i.e., two-dimensional signal points. When a two-dimensional signal point appears, the signal point is identified as a candidate aldehyde compound, and the mass-to-charge ratio and retention time corresponding to the signal point are recorded. For example, in rat urine samples, two signal points with a difference of 1.998Da are generated in the m / z 188.9005 / 190.8984 channel. These two signal points correspond to the precursor ion m / z 317.0312 and the isotope precursor ion m / z 319.0288 of the compound, respectively, and the retention time is 23.03min. At this time, the signal point is identified as a candidate aldehyde compound (such as Figure 4 shown).

[0061] S4: Confirmation of labeled compounds

[0062] From the screening results of MZmine2, a series of ion pairs with a difference of 1.998Da and the same retention time were obtained. The extracted ion current chromatograms were compared. If the ion pair can simultaneously meet the conditions of having the same chromatographic peak shape and retention time and a peak area ratio of about 1:1, it is determined to be an aldehyde compound. The Human Metabolome Database (HMDB) database is used to further obtain potential compound information, such as the possible structure, molecular weight, molecular formula, CAS, and corresponding HMDB code of the aldehyde compound.

[0063] For example, the ion pair screened above: m / z 317.0312 / 319.0288, the ion pair has the same chromatographic peak shape, the retention time is 23.03min, and the peak area ratio is close to 1:1, which further confirms that the ion pair is a bromine-labeled aldehyde compound. Through the HMDB database screening, the molecular weight is 114.1045 and the molecular formula is C7H 14O, and the possible compound heptanal was obtained, whose CAS number is: 111-71-7, and HMDB code is HMDB0031475.

[0064] S5: Structural elucidation of labeled compounds

[0065] The above-mentioned chromatographic and mass spectrometric conditions were used to investigate the bromine isotope characteristics of the BTCH-labeled heptanal standard solution in LC-MS. This included three important information: chromatographic retention time, accurate molecular weight, and MS / MS spectrum. First, the theoretical mass-to-charge ratios of the precursor ion and isotope precursor ion of the bromine-labeled heptanal standard were 317.0318 and 319.0298, respectively. Figure 5 The extracted ion chromatographic peak pair of BTCH-labeled heptanal was shown. The chromatographic peak pair had a mass difference of 1.998Da, a peak area ratio of about 1:1, the same chromatographic peak shape, and a retention time of 23.12min. The retention time was close to that of the candidate aldehyde compounds screened in the urine samples mentioned above. Secondly, its precursor ions and isotope precursor ions can produce MS / MS spectra similar to product ions. The characteristic product ions include m / z 205.9279 / 207.9259, 188.9013 / 190.8992 and 162.9219 / 164.9199, which come from the cleavage of the NN covalent bond, amide bond, carbonyl and CC bond between thiophene, respectively. In addition, the cleavage of the NN bond also produces a neutral loss molecule of 204.9197 / 206.9161Da, and the structure of the corresponding aldehyde part is m / z 112.1127. These fragment ions are important bases for the structural analysis of aldehyde compounds. The ion pairs with a difference of 1.998Da screened in the above urine samples can also produce MS / MS spectra with similar product ions. The characteristic product ions are m / z205.9266 / 207.9248, 188.9000 / 190.8978 and 162.9204 / 164.9187, respectively, and the neutral loss is 204.9191 / 206.9171. The structure of the aldehyde part corresponding to the neutral loss is m / z 112.1121, which is highly consistent with the MS / MS spectrum of the BTCH-labeled heptanal standard. In addition, in the MS spectra of the precursor ions and isotope precursor ions of the BTCH-labeled heptanal standard, the precursor ion m / z 317.0316 and the isotope precursor ion m / z 319.0289 with a difference of 1.998Da can be observed simultaneously, and the ion pair has similar abundance. The aldehyde compounds screened in the above urine samples also meet this condition. In summary, the pair of ion pairs m / z 317.0312 / 319.0288 with a difference of 1.998Da and the same retention time screened by MZmine2 were determined to be heptanal (such as Figure 5 shown).

[0066] The recognition results of aldehyde compounds in rat urine in this example are shown in Table 2, and the differences in the recognition quantity of aldehyde compounds in rat urine in Comparative Examples 1-4 and other prior arts are shown in Table 3.

[0067] Table 1 Differences in probes for non-targeted identification of compounds in samples in Example 1 and Comparative Examples 1 to 4 and their main influencing factors

[0068]

[0069] Notes:

[0070] 1. Relative abundance: the percentage of the abundance of the ion to the base peak abundance.

[0071] 2. Reaction efficiency: the percentage of the ratio of the molar number of substrate labeled with the derivatization reagent to the molar number of unlabeled substrate.

[0072] 3. Target daughter ion relative abundance screening threshold: parameter Minimum diagnostic ion intensity of base peak (MIB) in mass spectrometry data post-processing software MZmine2

[0073] Experimental data show that Example 1 can produce bromine-containing daughter ions with high relative abundance (35%), high reaction efficiency (80-98%), high screening threshold MIB that can be set, and high absolute response, which fully shows that 5-bromothiophene-2-carboxylic acid hydrazide is the optimal probe for identifying aldehydes in rat urine, so 5-bromothiophene-2-carboxylic acid hydrazide is selected as a probe to identify aldehydes in rat urine. However, the probes in Comparative Examples 1-4 show different defects in different recognition results, making it impossible to better identify aldehyde compounds in rat urine: For example, the probe in Comparative Example 1 shows low reaction efficiency, low relative abundance, and low absolute response; the probe in Comparative Example 2 shows selectivity for labeling reactions of aldehydes of different structures, and the relative abundance of the daughter ions produced is different; the probe in Comparative Example 3 shows low relative abundance; the probe in Comparative Example 4 shows no daughter ions, so it cannot be screened.

[0074] Table 2 Example 1 Identification of aldehyde compounds in rat urine using bromine isotope labeling method using 5-bromothiophene-2-carboxylic acid hydrazide as probe

[0075]

[0076]

[0077]

[0078] The experimental results show that 57 aldehyde compounds were found in rat urine samples using bromine isotope labeling with 5-bromothiophene-2-carboxylic acid hydrazide as a probe. Among them, 8 aldehyde compounds have been reported in urine, and 20 aldehyde compounds were found in urine for the first time, such as succinic semialdehyde and phenylacetaldehyde. There are 29 unknown aldehyde compounds whose structures have not been resolved. This result shows that bromine isotope labeling has a strong ability to identify unknown compounds.

[0079] Experimental Example 1: Identification Effects of Different Probes on Aldehydes in Urine

[0080] Example 1, Comparative Examples 1-4 and other probes (see the probes used in Comparative Examples 5-12 in Table 3) are used to identify aldehyde compounds in urine samples; wherein, the samples in Comparative Examples 5-6 are rat urine samples, and the samples in Comparative Examples 7-12 are human urine samples; Comparative Examples 5-12 are identification judgments based on isotope-labeled chromatographic characteristics. The differences in the number of aldehyde compounds and the detection limit (sensitivity parameter) identified by different probes in Example 1 and Comparative Examples 1-12 for urine samples and 16 aldehyde mixed standards are shown in Table 3.

[0081] Table 3 Differences in the number of aldehyde compounds recognized by different probes in Example 1 and Comparative Examples 1-12

[0082]

[0083]

[0084] Experimental data show that when the probes in Comparative Examples 1-3 are used to identify a mixed sample of 16 aldehyde standards, only 10-14 types of aldehydes can be identified, and complete identification cannot be achieved; while the probes used in this solution can effectively identify all aldehydes in the mixed sample of 16 aldehyde standards, and the identification accuracy is significantly improved.

[0085] Compared with the probes in comparative documents 5-12 that can only identify 6-16 aldehydes in urine samples, Example 1 of this scheme uses 5-bromothiophene-2-carboxylic acid hydrazide as a probe and identifies and confirms based on the unique two-dimensional signal and chromatographic characteristics formed by bromine isotopes. A total of 57 aldehyde compounds (including 28 known structural aldehydes and 29 unknown structural aldehydes) were found in rat urine samples, which has higher recognition ability, compound recognition accuracy and recognition coverage. Therefore, applying the probes and non-targeted recognition methods in this scheme to compound recognition can more effectively improve the coverage of target compound recognition in samples, facilitate overall judgment based on more compounds identified in samples, avoid the judgment of the overall effect affected by incomplete recognition of compounds in samples by existing technologies, thereby reducing the objectivity and applicability of the test results. This scheme has higher recognition accuracy and a wider recognition range, which also enables this scheme to be applicable to the recognition of compounds in samples with more compound types or more complex matrices.

[0086] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A non-targeted identification method of compounds based on bromine isotope labeling, characterized in that: The compound is an aldehyde compound, comprising the following steps: S1: bromine isotope probe labeling stage, the bromine isotope probe is mixed with the sample to form a mixed solution, and the mixed solution reacts to obtain a sample containing a bromine labeled compound; The bromine isotope probe is a bromine-containing compound including a bromine isotope, a reactive group, and a breakable connecting bond; the chemical structure of the bromine isotope probe is: reactive group-connecting bond-bromine isotope, and the bromine isotope part includes an easily ionized group; The bromine isotope probe used to identify aldehyde compounds in the sample was 5-bromothiophene-2-carboxylic acid hydrazide; S2: High-resolution chromatography-mass spectrometry detection and data acquisition stage, the sample containing the bromine-labeled compound obtained in S1 is subjected to high-resolution chromatography-mass spectrometry detection to obtain chromatographic data and mass spectrometric data containing high-resolution secondary spectrum information; S3: analysis phase, analyzing the mass spectrometry data containing high-resolution secondary spectrum information obtained in S2 to preliminarily identify the bromine-labeled compounds; The mass spectrometry data containing high-resolution secondary spectrum information collected by S2 is imported into the mass spectrometry data post-processing software to screen for bromine-containing daughter ion pairs. The target compound can form a two-dimensional signal composed of four signal points based on the bromine isotope characteristics. The two-dimensional signal serves as a standard for identifying whether it is a bromine isotope probe-labeled compound; S4: Confirmation of the labeled compound: the bromine-labeled compound preliminarily identified in S3 is further confirmed by the chromatographic data collected in S2 to determine whether it is a compound labeled with a bromine isotope probe; S5: Structural analysis of labeled compounds, analyzing the structure of the compound through high-resolution secondary spectra and reference substances.

2. The method for non-targeted identification of compounds based on bromine isotope labeling according to claim 1, characterized in that: In S2, a full scan-daughter ion scan mode or an equivalent functional scan mode of a high-resolution liquid chromatography-mass spectrometer is used to collect mass spectral data containing high-resolution secondary spectral information.

3. The non-targeted identification method of compounds based on bromine isotope labeling according to claim 2, characterized in that: In S3, the relative abundance of the detected target product ions was 80-100%, and the relative abundance screening threshold was 35-45%.

4. The method for non-targeted identification of compounds based on bromine isotope labeling according to claim 3, characterized in that: In S4, the identified bromine-labeled compound is further confirmed by the chromatographic data collected in S2, the chromatographic data including chromatographic peak shape, peak area ratio, and retention time; the judgment criteria for the chromatographic data are: the peak area ratio is 0.8-1.2, the chromatographic peak shape is that the difference in the horizontal and vertical coordinate values ​​corresponding to each monitoring point constituting the chromatographic peak in the chromatogram is ≤±20%, and the chromatographic retention time is that the numerical values ​​after the decimal point are all the same.

5. The method for non-targeted identification of compounds based on bromine isotope labeling according to claim 4, characterized in that: In S1, the bromine isotope probe further includes a second stable isotope, and the second stable isotope is 13 carbon, 15 Any of nitrogen and deuterium.

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