A stable isotope-labeled aniline derivative reagent-based carboxylic acid compound targeting-non-targeting synergistic analysis method based on double library system

Abstract

The application belongs to the field of environmental analysis chemistry and mass spectrometry, and particularly relates to a carboxylic acid compound targeted-non-targeted collaborative analysis method based on stable isotope labeled aniline derivative reagent and double library system. The method uses aniline and d5-aniline as derivative reagents to perform amide derivative treatment on carboxylic acid compounds, and realizes high-sensitivity detection in combination with liquid chromatography-mass spectrometry. Further, a self-built double library system containing a derivative spectrum library and a non-derivative spectrum library is constructed, and in combination with targeted quantification and non-targeted screening, the collaborative identification and quantitative analysis of known and unknown carboxylic acid compounds are realized. The actual PM2.5 sample is analyzed by using the method, 52 kinds of carboxylic acid compounds are detected, 13 of which are known carboxylic acids with standard samples, and 39 of which are carboxylic acids without standard samples but verified and confirmed by the double library collaborative verification, which verifies the effectiveness of the application.

Description

Technical Field

[0001] This invention belongs to the field of environmental analytical chemistry and mass spectrometry, specifically relating to a targeted-non-targeted synergistic analysis method for carboxylic acid compounds based on a stable isotope-labeled aniline derivative reagent and a dual-library system. Background Technology

[0002] Carboxylic acids are important polar oxygen-containing organic components in atmospheric particulate matter (PM2.5), playing a crucial role in the formation of secondary organic aerosols, particulate nucleation, and hygroscopic regulation. However, due to the wide variety of carboxylic acids, their strong polarity, and significant differences in volatility, traditional detection methods still have significant shortcomings in terms of sensitivity, coverage, and quantitative accuracy.

[0003] Currently, the analysis of carboxylic acid compounds mainly relies on liquid chromatography-mass spectrometry (LC-MS), but this method is mostly concentrated on a few target compounds with available standards, making it difficult to cover the complex components in actual samples. While non-targeted analysis can improve detection coverage, its quantitative capability is weak, and the reliability of the results still needs further improvement. Most existing methods lack effective internal standard correction methods, making it difficult to achieve high-precision quantitative analysis of carboxylic acid compounds in complex matrices.

[0004] Therefore, developing a carboxylic acid analysis method that combines high sensitivity, high accuracy, and high coverage is of great significance for the systematic study of carboxylic acid compounds in complex environmental samples. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a targeted-non-targeted synergistic analysis method for carboxylic acid compounds based on a stable isotope-labeled aniline derivative reagent and a dual-library system.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A targeted-untargeted synergistic analysis method for carboxylic acid compounds based on a stable isotope-labeled aniline derivatizing reagent and a dual-library system includes the following steps:

[0008] S1. Construct a dual-library system including derived spectral libraries and non-derived spectral libraries;

[0009] The derivatized spectral library is constructed by collecting chromatographic-mass spectrometry information after derivatizing carboxylic acid compounds with standards using aniline derivatizing reagent; the non-derivative spectral library is constructed by collecting data from underivative samples in IDA mode, screening them using a public mass spectrometry library, and verifying them by isotope derivatization using aniline derivatizing reagent.

[0010] S2. The samples to be tested are divided into a first sample to be tested and a second sample to be tested. After the first sample to be tested is derivatized with aniline, liquid chromatography-mass spectrometry is performed in MRM mode, and the results are compared with the derivatized spectral library to perform targeted quantitative analysis of carboxylic acid compounds with standards. The second sample to be tested is not derivatized, and liquid chromatography-high resolution mass spectrometry is performed in IDA mode, and the results are compared with the non-derivatized spectral library to perform non-targeted screening and confirmation of carboxylic acid compounds without standards.

[0011] Preferably, the aniline derivatizing agent in step S1 includes aniline and / or d5-aniline, wherein aniline reacts with a carboxylic acid compound to generate a benzamide derivative, and d5-aniline reacts with a carboxylic acid compound to generate a d5-benzamide derivative.

[0012] Preferably, the construction of the derivatized spectral library in step S1 includes:

[0013] (1) A carboxylic acid compound containing a standard is reacted with an aniline derivatizing agent in the presence of a condensing agent to obtain a derivatized product;

[0014] (2) The retention time, parent ion, characteristic daughter ion, declustering voltage and collision energy parameters of the derivatives were collected by liquid chromatography-tandem mass spectrometry, and a derivative mass spectrometry library for targeted identification and quantitative analysis was established.

[0015] Preferably, when constructing the derivatization library, the aniline derivatizing reagent is aniline; the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole.

[0016] Preferably, the construction of the non-derived mass spectrometry library in step S1 includes:

[0017] (a) Liquid chromatography-high resolution mass spectrometry was used to acquire primary and secondary mass spectrometry information of the underrivatized sample using IDA mode;

[0018] (b) Match the primary and secondary mass spectrometry information with a public spectral library to obtain candidate carboxylic acid compounds;

[0019] (c) Isotope derivatization verification of the candidate carboxylic acid compounds using aniline derivatizing reagents;

[0020] (d) Incorporate candidate carboxylic acid compounds verified by isotope derivatization into the non-derivative mass spectrometry library.

[0021] Preferably, the first-level scanning range of the IDA mode (information-dependent acquisition mode) in step (a) is 50-1000 m / z, and the second-level scanning range is 50-1000 m / z.

[0022] Preferably, the public spectral library mentioned in step (b) includes one or more of NIST, MassBank, and HMDB; the matching includes one or more of precise mass number matching, retention time matching, and secondary mass spectrometry fragment matching.

[0023] Preferably, the isotope derivation verification in step (c) includes:

[0024] ① The candidate carboxylic acid compounds were derivatized using aniline and d5-aniline, respectively;

[0025] ② The aniline-carboxylic acid derivatives and d5-aniline-carboxylic acid derivatives obtained by detection;

[0026] ③ When the aniline-carboxylic acid derivative and the d5-aniline-carboxylic acid derivative have corresponding mass shift characteristics, and the retention time and peak area response relationship between the two meet the preset requirements, the candidate carboxylic acid compound is confirmed to have passed the isotope derivatization verification.

[0027] Preferably, the preset requirements are: retention time deviation ≤ 0.5 min and peak area ratio 0.5 to 2.0.

[0028] Preferably, the aniline derivatization treatment in step S2 is carried out in the presence of a condensing agent, the derivatization temperature is 40–50°C, and the derivatization time is 10–20 min; the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole. More preferably, the derivatization temperature is 45°C, and the derivatization time is 10 min.

[0029] Preferably, when performing liquid chromatography-mass spectrometry detection in MRM mode in step S2, the chromatographic conditions include: mobile phase A is 0.1% formic acid / water; mobile phase B is acetonitrile; the mobile phase elution gradient is 0–20 min; the mass spectrometry conditions include: ion source temperature 550℃; ion spraying voltage 5500 V; Gas 1, Gas 2 and Curtain gas are 30, 15 and 20 psi respectively; sampling is in MRM positive ion mode.

[0030] Preferably, the sample to be tested in step S2 is an extract of atmospheric particulate matter (PM2.5); the extract of the PM2.5 sample is prepared by the following steps:

[0031] The filter membrane containing PM2.5 was cut into pieces and added to an organic solvent for ultrasonic extraction; the resulting extract was filtered, concentrated, and reconstituted to obtain the sample to be tested.

[0032] Preferably, the determination of the results of the collaborative analysis includes:

[0033] Carboxylic acid compounds that match the aforementioned derivatization library are considered to be completely identified.

[0034] Carboxylic acid compounds that match the non-derived spectral library and are verified through isotope derivatization are considered to be essentially identified.

[0035] The present invention has the following advantages over the prior art:

[0036] 1. This invention uses aniline / d5-aniline as a derivatizing reagent to react with carboxylic acid compounds under mild conditions via an amidation reaction, generating benzamide derivatives. This derivatization reaction significantly improves the response intensity and stability of carboxylic acid compounds in liquid chromatography-mass spectrometry (LC-MS), increasing the detection sensitivity by 1-2 orders of magnitude compared to direct detection. Simultaneously, by using d5-aniline as a stable isotopic internal standard and employing isotope dilution mass spectrometry for quantitative analysis, errors caused by sample pretreatment, matrix effects, and instrument fluctuations can be effectively corrected, significantly improving the quantitative accuracy and repeatability of carboxylic acid compounds in samples from complex environments.

[0037] 2. This invention establishes a derivatization library for carboxylic acid compounds with available standards, recording multidimensional mass spectrometry parameters such as retention time, mother ion-daughter ion channels, declustering voltage, and collision energy to achieve high-confidence targeted quantitative analysis. Simultaneously, for carboxylic acid compounds without standards, a strategy combining IDA high-resolution mass spectrometry acquisition of underrivatized samples, public library matching, and isotope derivatization verification is employed to construct a non-derived library, enabling non-targeted screening. The parallel construction and synergistic application of these two libraries ensures both accurate quantification of known compounds and reliable identification of unknown compounds, overcoming the technical contradictions of traditional methods that result in either "accurate quantification but narrow coverage" or "broad coverage but inaccurate quantification."

[0038] 3. The synergistic method of this invention was used to analyze actual PM2.5 samples, detecting a total of 52 carboxylic acid compounds. Among them, 13 were known carboxylic acids with standards, and 39 were carboxylic acids without standards but confirmed by dual-library synergistic verification, significantly expanding the coverage of carboxylic acid detection. This method is simple to operate, has high analytical throughput, and good reliability, meeting the high-throughput analysis needs of carboxylic acid compounds in complex environmental samples. It can provide reliable technical support for the source apportionment of polar oxygen-containing organic compounds in atmospheric particulate matter and the study of the formation mechanism of secondary organic aerosols. Attached Figure Description

[0039] Figure 1 This is a schematic diagram illustrating the principle of the derivatization reaction between aniline / d5-aniline and carboxylic acid;

[0040] Figure 2 This is a schematic flowchart of the targeted-non-targeted synergistic analysis method for carboxylic acid compounds based on a stable isotope-labeled aniline-derived reagent and a dual-library system, according to the present invention. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0042] The ultra-high performance liquid chromatography of this invention uses an LC-30AD (Shimadzu Corporation, Japan) system and a 6500 Q-TRAP mass spectrometer (AB SCIEX, USA) equipped with an ion-driven turbine ESI source; the chromatographic column for ultra-high performance liquid chromatography is a Waters Atlantis T3 3μm (100mm × 2.1mm).

[0043] Example 1: Pretreatment of PM2.5 samples and preparation of derivatization reagent stock solution

[0044] Atmospheric particulate matter (PM2.5) samples were collected on quartz fiber filter membranes. One-quarter of the filter membrane was taken, cut into small pieces, and placed in a 15 mL centrifuge tube. 10 mL of 80% acetonitrile aqueous solution was added, and the mixture was extracted using ultrasonication in an ice bath for 15 min. This process was repeated three times. The three extracts were combined, filtered through a 0.22 μm polytetrafluoroethylene (PTFE) filter membrane, and the filtrate was collected. The filtrate was then concentrated to dryness and reconstituted with acetonitrile to a final volume of 1 mL to obtain the PM2.5 sample extract, which was then used for subsequent derivatization analysis or non-derivatized non-targeted analysis.

[0045] The aniline (C6H5NH2) and d5-aniline (C6D5NH2) used in this invention are both commercially available reagents and can be used directly. A 0.1 mmol / mL solution of aniline and a d5-aniline solution were prepared with acetonitrile as stock solutions for derivatization.

[0046] Example 2: Construction of Derivatized Spectral Libraries

[0047] Carboxylic acid compounds with standards were selected and derivatized with aniline derivatizing reagents. The retention time (RT), parent ion (Q1), characteristic daughter ion (Q3), declustering voltage (DP), and collision energy parameter (CE) of the obtained carboxylic acid derivatives were collected by liquid chromatography-tandem mass spectrometry. Based on RT, Q1, Q3, DP, and CE, a derivatization spectral library was established.

[0048] In one specific embodiment, the carboxylic acid compound containing the standard includes dihydroxybenzoic acid, pyromellitic acid, malic acid, o-toluic acid, m-toluic acid, p-toluic acid, maleic acid, fumaric acid, lauric acid, succinic acid, citric acid, lactic acid, and propionic acid, and carboxylic acid standard solutions with a concentration of 0.1 mmol / mL are prepared respectively.

[0049] Take 10 μL of carboxylic acid standard solution, add 10 μL of aniline solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution (EDCI, 5 eq), and 1-hydroxybenzotriazole (HOBt, 4 eq), and react at 45 °C with shaking for 10 min. After the reaction is complete, dilute to 1 mL with acetonitrile, and perform LC-MRM analysis on the derivatized product.

[0050] LC conditions: Column: Waters Atlantis T3 (100 mm × 2.1 mm, 3 μm); Mobile phase A: 0.1% formic acid / water, Mobile phase B: acetonitrile; Gradient elution program: 0–15 min, 2% B; 1–8 min, 2% → 50% B; 8–10 min, 50% → 90% B; 10–12 min, 90% B; 12–13 min, 90% → 2% B; 13–15 min, 2% B; Flow rate: 0.3 mL / min; Column temperature: 40°C; Injection volume: 5 μL.

[0051] MS conditions: Electrospray positive ion mode was used, and the scanning method was multiple reaction monitoring (MRM); ion source temperature was 550°C; electrospray voltage was 5500 V; curtain gas was 20 psi; nebulizer gas was 30 psi; and auxiliary gas was 15 psi.

[0052] Since the chromatographic retention time and mass spectrometry fragmentation pattern of aniline and d5-aniline derivatives are exactly the same, this embodiment only uses aniline as an example to establish a derivatization library. The obtained parameters such as retention time, parent ion, daughter ion and collision energy are also applicable to d5-aniline labeled internal standards.

[0053] The MRM parameters of 13 carboxylic acid compounds with standards are shown in Table 1 below. Their characteristic fragment ion is m / z 94 (aniline core). This fragment appears stably and can be used to confirm targeted quantification.

[0054] Table 1

[0055] Serial Number carboxylic acid <![CDATA[Q1(m / z)]]> <![CDATA[Q3(m / z)]]> RT(min) DP(V) CE(eV) 1 Dihydroxybenzoic acid 230.1 94 15.9 120 30 2 Tristyric acid 286.1 94 17 130 38 3 malic acid 210.1 94 10.8 110 28 4 o-Toluene 212.1 94 19.2 120 35 5 m-Toluene 212.1 94 7.4 120 35 6 p-Toluene 212.1 94 13 120 35 7 maleic acid 192.1 94 13.7 125 32 8 fumaric acid 192.1 94 6.55 125 32 9 Lauric acid 276.3 94 0.74 115 40 10 Succinic acid 194.1 94 8.12 125 34 11 Citric acid 268.1 94 15.9 135 45 12 lactic acid 166.1 94 17 105 25 13 propionic acid 150.1 94 10.8 105 30

[0056] Example 3: Construction of non-derived spectral libraries

[0057] (a) IDA data acquisition

[0058] The PM2.5 sample extract prepared in Example 1 or its quality control sample (QC sample, prepared by mixing 84 PM2.5 samples) was taken and directly subjected to LC-high resolution mass spectrometry analysis in information-dependent acquisition (IDA) mode without derivatization to obtain the primary and secondary mass spectrometry information of the candidate compounds.

[0059] The IDA acquisition parameters were as follows: primary scan range m / z 50–1000, scan frequency 0.15 s / spectrum, declustering voltage 80 V; intensity threshold for triggering secondary fragmentation >100 cps, a maximum of 15 precursor ions triggered per cycle, dynamic exclusion time 10 s; secondary collision energy 30 V ± 15 eV. Each sample was acquired 6 times.

[0060] (b) Public spectral library matching

[0061] The acquired raw data (.wiff format) was converted to .mzML format and imported into MS-DIAL software for peak extraction, feature extraction, peak identification, and library matching to obtain candidate carboxylic acid compounds. Public spectral libraries included NIST 20, MassBank, and HMDB. Matching metrics included one or more of the following: precise mass number, retention time, secondary mass spectrometry fragmentation information, and neutral loss features. The primary mass deviation was set to ≤0.01 Da, and the secondary mass deviation to ≤0.005 Da.

[0062] (c) Verification of isotope derivatization

[0063] For the candidate carboxylic acid compounds obtained from the initial screening, further isotopic derivatization verification was performed using aniline and d5-aniline. Pure (if available) or enriched actual samples of the candidate carboxylic acid compounds were derivatized with aniline and d5-aniline under the same conditions. The light label (aniline-carboxylic acid derivative) and the heavy label (d5-aniline-carboxylic acid derivative) were detected by MRM. If the retention times of the light and heavy derivatives were consistent (deviation ≤ 0.5 min) and the peak area ratio was within the range of 0.5–2.0, the candidate compound was confirmed and included in the non-derivative spectral library.

[0064] Using the above method, 39 candidate carboxylic acid compounds without standards were screened from actual PM2.5 samples. After isotope derivatization verification, all of them were confirmed to be included in the non-derivative spectral library, and MRM detection parameters were established for them (see Table 2 in Example 7).

[0065] Example 4: Dual-library collaborative detection of test samples

[0066] Based on the foregoing embodiments, this invention provides a targeted-non-targeted synergistic analysis method for carboxylic acid compounds based on a stable isotope-labeled aniline derivatizing reagent and a dual-library system. The specific process is as follows:

[0067] The PM2.5 sample extract prepared in Example 1 was divided into two portions (the first sample to be tested and the second sample to be tested):

[0068] First sample to be tested (targeted quantification): Take 50 μL of sample extract, add 10 μL of aniline derivatizing reagent (0.1 mmol / mL), 10 μL of EDCI (0.1 mmol / mL), and 10 μL of HOBt (0.1 mmol / mL), react at 45℃ for 10 min, then add 10 μL of internal standard mixture labeled with d5-aniline derivatizing reagent (0.1 mmol / mL), and detect according to the LC-MRM conditions of Example 2. Compare the detected chromatographic peaks with the derivatization library (Table 1), and confirm the target analyte based on parameters such as retention time and MRM channels. Absolute quantification is performed using the isotope dilution method: calculate the peak area ratio of aniline derivative to d5-aniline internal standard, and substitute it into the standard curve to calculate the concentration of carboxylic acid in the sample.

[0069] Second sample to be tested (non-targeted screening): Take 50 μL of sample extract, without derivatization, and directly perform LC-high resolution mass spectrometry analysis according to the IDA conditions in Example 3. The collected data are compared with the non-derivative spectral library (Table 2) for confirmation of non-targeted screening of carboxylic acid compounds without standards.

[0070] The results of the above collaborative analysis are determined according to the following hierarchy:

[0071] (1) Carboxylic acid compounds that match the derivatization spectral library and have standards and standard curves are determined to be "completely determined" and their absolute concentrations are reported;

[0072] (2) Carboxylic acid compounds that match the non-derived spectral library and are verified by d0 / d5 isotope derivatization are determined to be "basically confirmed" and their relative concentrations are reported.

[0073] Example 5: Analysis Results of Actual PM2.5 Samples

[0074] The method of this invention was used to analyze PM2.5 samples from a certain location. The samples were divided into two parts, and derivatized MRM analysis and non-derivatized IDA analysis were performed respectively according to Example 4.

[0075] The results showed that a total of 52 carboxylic acid compounds were detected (see Table 2). Among them, 13 carboxylic acids with commercially available standards were completely identified by derivatization spectral library and absolute quantification was performed using the d5-aniline internal standard method, with concentrations ranging from 0.1 to 1000 ng / mL. 3 The remaining 39 are carboxylic acids without commercially available standards. They were confirmed through initial screening using a non-derivative spectral library and verification by aniline / d5-aniline isotope derivatization. Although absolute quantification is not possible, the identification results are reliable and can be reported as "basically certain".

[0076] Method repeatability test: The same actual sample was analyzed 6 times under the same conditions. The relative standard deviation of the retention time of each compound was <2%, and the relative standard deviation of the peak area ratio of the light / heavy labeled derivatives was <15%, indicating that the method has good repeatability.

[0077] Table 2

[0078] Serial Number name Q1(m / z) Q3(m / z) RT(min) source 1 Dihydroxybenzoic acid 230.1 94 15.9 Completely certain 2 Tristyric acid 286.1 94 17 Completely certain 3 malic acid 210.1 94 10.8 Completely certain 4 o-Toluene 212.1 94 19.2 Completely certain 5 m-Toluene 212.1 94 7.4 Completely certain 6 p-Toluene 212.1 94 13 Completely certain 7 maleic acid 192.1 94 13.7 Completely certain 8 fumaric acid 192.1 94 6.55 Completely certain 9 Lauric acid 276.3 94 0.74 Completely certain 10 Succinic acid 194.1 94 8.12 Completely certain 11 Citric acid 268.1 94 15.9 Completely certain 12 lactic acid 166.1 94 17 Completely certain 13 propionic acid 150.1 94 10.8 Completely certain 14 Formic acid 122.025 94 4.1 Basically determined 15 Acetic acid 136.05 94 5.88 Basically determined 16 glyoxylic acid 150.035 94 7.28 Basically determined 17 glycine 151.067 94 7.44 Basically determined 18 Isobutyric acid 164.105 94 1.59 Basically determined 19 oxalic acid 241.035 94 13.78 Basically determined 20 lactic acid 166.078 94 5.97 Basically determined 21 3,3-Dimethicone 176.116 94 10.25 Basically determined 22 Isovalerate 178.132 94 10.77 Basically determined 23 malonic acid 255.062 94 10.67 Basically determined 24 hexanoic acid 192.158 94 13.07 Basically determined 25 Oxaloacetic acid 283.072 94 19.57 Basically determined 26 p-Aminobenzoic acid 213.136 94 11.75 Basically determined 27 salicylic acid 214.121 94 8.74 Basically determined 28 Hexadienoic acid 293.109 94 7.42 Basically determined 29 bitter 220.211 94 15.6 Basically determined 30 α-Ketoglutarate 297.098 94 11 Basically determined 31 glutamic acid 298.129 94 11.04 Basically determined 32 3-Phenylacetic acid 226.174 94 4.81 Basically determined 33 o-hydroxyphenylacetic acid 228.147 94 6.79 Basically determined 34 whey acid 232.096 94 12.75 Basically determined 35 nonanoic acid 234.238 94 16.53 Basically determined 36 p-hydroxycinnamic acid 240.158 94 6.04 Basically determined 37 m-Nitrobenzoic acid 243.119 94 4.81 Basically determined 38 vanillic acid 244.147 94 4.79 Basically determined 39 gallic acid 246.12 94 13.67 Basically determined 40 Decanoic acid 248.265 94 4.77 Basically determined 41 Quinoline-2-carboxylic acid 249.168 94 1.58 Basically determined 42 Shikimic acid 250.151 94 1.57 Basically determined 43 Dihydroxymandelic acid 260.146 94 17.9 Basically determined 44 Undecanoic acid 262.291 94 18.15 Basically determined 45 azelaic acid 339.221 94 13.7 Basically determined 46 dodecanoic acid 276.318 94 18.9 Basically determined 47 Tridecanoic acid 290.344 94 19.3 Basically determined 48 Tetradecanoic acid 304.371 94 13.16 Basically determined 49 Palmitic acid 332.424 94 19.77 Basically determined 50 heptadecanoic acid 346.451 94 17.57 Basically determined 51 stearic acid 360.477 94 18.1 Basically determined 52 Octadecano 360.477 94 18.56 Basically determined

[0079] Note: All carboxylic acid compounds in this table were detected in actual PM2.5 samples. Among them, 13 carboxylic acid compounds with commercially available standards have been labeled with MRM parameters in Example 2 and are judged as "fully certain"; the remaining 39 are aldehydes without standards but confirmed by dual-library co-validation and are judged as "basically certain".

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A targeted-non-targeted synergistic analysis method for carboxylic acid compounds based on a stable isotope-labeled aniline-derived reagent and a dual-library system, characterized in that, Includes the following steps: S1. Construct a dual-library system including derived spectral libraries and non-derived spectral libraries; The derivatization library is constructed by collecting chromatographic-mass spectrometry information after derivatizing carboxylic acid compounds with standards using aniline derivatizing reagents; The non-derivative spectral library was constructed by collecting underivative samples in IDA mode, screening them in a public mass spectrometry library, and verifying their isotope derivatization using aniline derivatization reagent. S2. The samples to be tested are divided into a first sample to be tested and a second sample to be tested. After the first sample to be tested is derivatized with aniline, liquid chromatography-mass spectrometry is performed in MRM mode, and the results are compared with the derivatized spectral library to perform targeted quantitative analysis of carboxylic acid compounds with standards. The second sample to be tested is not derivatized, and liquid chromatography-high resolution mass spectrometry is performed in IDA mode, and the results are compared with the non-derivatized spectral library to perform non-targeted screening and confirmation of carboxylic acid compounds without standards.

2. The collaborative analysis method according to claim 1, characterized in that, The aniline derivatizing reagent in step S1 includes aniline and / or d5-aniline, wherein aniline reacts with a carboxylic acid compound to generate a benzamide derivative, and d5-aniline reacts with a carboxylic acid compound to generate a d5-benzamide derivative.

3. The collaborative analysis method according to claim 1, characterized in that, The construction of the derivatized spectral library in step S1 includes: (1) A carboxylic acid compound containing a standard is reacted with an aniline derivatizing agent in the presence of a condensing agent to obtain a derivatized product; (2) The retention time, parent ion, characteristic daughter ion, declustering voltage and collision energy parameters of the derivatives were collected by liquid chromatography-tandem mass spectrometry, and a derivative mass spectrometry library for targeted identification and quantitative analysis was established.

4. The collaborative analysis method according to claim 3, characterized in that, When constructing the derivatization library, the aniline derivatizing reagent is aniline; the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole.

5. The collaborative analysis method according to claim 1, characterized in that, The construction of the non-derived mass spectrometry library in step S1 includes: (a) Liquid chromatography-high resolution mass spectrometry was used to acquire primary and secondary mass spectrometry information of the underrivatized sample using IDA mode; (b) Match the primary and secondary mass spectrometry information with a public spectral library to obtain candidate carboxylic acid compounds; (c) Isotope derivatization verification of the candidate carboxylic acid compounds using aniline derivatizing reagents; (d) Incorporate candidate carboxylic acid compounds verified by isotope derivatization into the non-derivative mass spectrometry library.

6. The collaborative analysis method according to claim 5, characterized in that, The isotope-derived verification described in step (c) includes: ① The candidate carboxylic acid compounds were derivatized using aniline and d5-aniline, respectively; ② The aniline-carboxylic acid derivatives and d5-aniline-carboxylic acid derivatives obtained by detection; ③ When the aniline-carboxylic acid derivative and the d5-aniline-carboxylic acid derivative have corresponding mass shift characteristics, and the retention time and peak area response relationship between the two meet the preset requirements, the candidate carboxylic acid compound is confirmed to have passed the isotope derivatization verification.

7. The collaborative analysis method according to claim 6, characterized in that, The preset requirements are: retention time deviation ≤ 0.5 min and peak area ratio 0.5 to 2.

0.

8. The collaborative analysis method according to any one of claims 1 to 7, characterized in that, The aniline derivatization treatment in step S2 is carried out in the presence of a condensing agent, the derivatization temperature is 40-50°C, and the derivatization time is 10-20 min; the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole.

9. The collaborative analysis method according to any one of claims 1 to 7, characterized in that, The sample to be tested in step S2 is an extract of atmospheric particulate matter (PM2.5); the PM2.5 sample extract is prepared through the following steps: The filter membrane containing PM2.5 was cut into pieces and added to an organic solvent for ultrasonic extraction; the resulting extract was filtered, concentrated, and reconstituted to obtain the sample to be tested.

10. The collaborative analysis method according to any one of claims 1 to 7, characterized in that, The determination of the results of the collaborative analysis includes: Carboxylic acid compounds that match the aforementioned derivatization library are considered to be completely identified. Carboxylic acid compounds that match the non-derived spectral library and are verified through isotope derivatization are considered to be essentially identified.

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