Method for constructing multi-stage mass spectrum database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrum technology

Abstract

The invention relates to the technical field of chemical analysis, in particular to a method for constructing a multi-stage mass spectrum database of small molecular compounds based on a quadrupole-linear ion trap tandem mass spectrometry technology, which comprises the following steps: preparing standard stock solutions and standard working solutions of various small molecular compounds; optimizing single mass spectrum conditions; through different scanning modes of quadrupole rod-linear ion trap tandem mass spectrometry, mass spectrum information of the small molecule compound is obtained through TinyTrap software; acquiring a mass spectrum cracking rule, and confirming a fracture position; the method comprises the following steps of: determining retention time through TinyAcquisition software and TinyAnalysis software, and determining the retention time through the TinyAcquisition software and the TinyAnalysis software; related information of the small molecule compounds is summarized, and a multi-stage mass spectrum database is established. The method provided by the invention provides detailed information of each stage of mass spectrum of the small molecule compound, summarizes the mass spectrum splitting decomposition rules of various compounds, is beneficial to complex matrix detection, is beneficial to distinguishing and identifying isomeride, and provides a basis for subsequent practical application.

Description

Technical Field

[0001] The present invention relates to the field of chemical analysis technology, and in particular to a method for constructing a multi-level mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology. Background Art

[0002] Small molecule compounds such as mycotoxins, quinolones, sulfonamides, beta-receptor agonists, and tetracyclines are mainly found in the environment and food, which not only harm the environment but also threaten human health. Therefore, the residual detection of these small molecule compounds is of great significance. The existing detection of these small molecule compounds is mainly based on databases established by foreign instruments, which is costly for users to use. Summary of the invention

[0003] The present invention provides a method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology, so as to solve the above-mentioned technical problems existing in the prior art.

[0004] A method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology comprises the following steps: Step S1: preparing a standard stock solution and a standard working solution of a small molecule compound; Step S2: Optimizing single mass spectrometry conditions; Step S3: using different scanning modes of quadrupole-linear ion trap tandem mass spectrometry, the mass spectrum information of the parent ions and multistage ions of the small molecule compound is obtained in the TinyTrap software; Step S4: confirming the fragmentation position of the small molecule compound according to the mass spectrometry fragmentation rule; Step S5: Determine the retention time of small molecule compounds by TinyAcquisition and TinyAnalysis software; Step S6: Summarize the relevant information of small molecule compounds and establish a multi-level mass spectrometry database.

[0005] The present invention is the first to use quadrupole-linear ion trap tandem mass spectrometry technology and multi-stage fragmentation technology, that is, the parent ions of the target object are selected in the quadrupole, and then injected, captured and fragmented in the linear ion trap at the same time to enrich the secondary ions of the target object. The tertiary ions are obtained by capturing, separating and cracking the secondary ions in the ion trap.

[0006] The quadrupole-linear ion trap tandem mass spectrometer has the dual advantages of the quadrupole and the linear ion trap. Through the quadrupole screening function, the parent ion of the target is obtained, and the parent ion fragmentation and secondary ion capture, that is, secondary fragmentation, are performed in the linear ion trap. At the same time, multi-stage fragmentation can still be achieved in the linear ion trap. Multi-stage fragmentation is to perform fragmentation again on the basis of secondary fragmentation to obtain tertiary ions. Therefore, the multi-stage mass spectrometry database constructed for small molecule compounds based on the quadrupole-linear ion trap tandem mass spectrometry technology not only provides detailed information on the mass spectra at each level, but also summarizes the mass spectrometry fragmentation rules of various compounds, which is not only conducive to the detection of complex matrices, but also more conducive to the differentiation and identification of isomers, supplementing the current multi-stage database, and also providing a basis for subsequent practical applications. In addition, the quadrupole-linear ion trap tandem mass spectrometry technology already has domestically produced products and technologies in China, which is conducive to reducing the user's cost of use.

[0007] Furthermore, in step S1, the standard stock solution is diluted into a standard working solution as required, and injected into a quadrupole-linear ion trap tandem mass spectrometer at a flow rate of 4-6 μL / min using a direct injection method.

[0008] Furthermore, in step S1, the small molecule compound includes true toxin compounds, quinolone compounds, sulfonamide compounds, β-receptor agonist compounds, and tetracycline compounds.

[0009] Furthermore, in step S2, the single mass spectrometry conditions are optimized, an ESI source is used, and the ion spray needle position, scanning rate, ion guide RF AC voltage, multiplier voltage, ion source high voltage, high (low) vacuum gauge, sheath gas, auxiliary gas, backblowing gas, ion source temperature and atmospheric pressure interface temperature are optimized; preferably, the ion spray needle position is 1-1.5 mm, the ion source temperature is 150-180 ° C, and the atmospheric pressure interface temperature is 200-250 ° C.

[0010] Furthermore, in step S3, the scanning mode includes a daughter ion scanning mode and a multi-stage ion scanning mode; in the daughter ion scanning mode, the mass scanning range is input in the TinyTrap software according to the parent ion of the small molecule compound, the parent ion is input in the parent ion column, and the secondary ion is input in the daughter ion column. According to the response intensity, the ionization time, fragmentation q point, fragmentation time and fragmentation energy are optimized to obtain the parent ion, secondary ion and tertiary ion in turn.

[0011] Furthermore, the mass scan range was m / z 100-600.

[0012] Furthermore, in step S4, the mass spectrometry fragmentation rules include classifying the same type of compounds according to the fragmentation functional groups, and summarizing the same fragmentation rules of the same type of compounds.

[0013] In step S4, the mass spectrometry fragmentation rules of different compounds are described as follows according to their types: True toxins: (1) Aflatoxins (AFB1 / AFB2 / AFG1 / AFG2 / AFM1 / AFM2): Parent ion: in ESI + In this mode, [M+H] is formed. + The parent ion of AFB1 / AFB2 is secondary ion. After α-fragmentation and i-fragmentation, the alicyclic ketone in the structure of AFB1 / AFB2 undergoes neutral loss, losing one molecule of -CO. AFB1 / AFB2 forms secondary fragment ions of m / z 285 and m / z 287. After i-fragmentation, the alicyclic ketone in the structure of AFG1 / AFG2 undergoes distal rearrangement reaction to remove one molecule of H2O. AFG1 / AFG2 produces secondary fragment ions of m / z 311 and m / z 313. The furan ring of AFM1 / AFM2 is prone to neutral loss due to the low bond energy of CO bond. After removing one molecule of H2O, AFM1 / AFM2 forms secondary fragment ions of m / z 311 and m / z 313. Tertiary ion: The secondary daughter ion structure of AFB1 / AFB2 contains an ester bond, which is easy to lose one molecule of -CO. AFB1 / AFB2 forms m / z 257 and m / z The tertiary fragment ion is m / z 259; due to the migration of the π electron cloud, the C=C bond of AFG1 breaks and i breaks at the same time, and a molecule of -C2H4 is removed to form a tertiary fragment ion of m / z 243. The C=C bond of AFG2 breaks and a rearrangement reaction occurs to break the adjacent C=C bond, forming a tertiary fragment ion of m / z 285; AFM1 / AFM2 removes a molecule of -CO due to a distal rearrangement reaction after i break, generating tertiary fragment ions of m / z 282 and m / z 285. (2) ZEN / α-ZEL / β-ZEL / DON: parent ions: in ESI- mode, [M+H] - The parent ion; Secondary ions: In the cyclic lactone structure of ZEN / α-ZEL / β-ZEL, neutral loss is easy to occur to remove one molecule of CO2, forming secondary fragment ions of m / z 273, m / z 275 and m / z 275; DON is easy to undergo neutral loss of -CH2O to form a secondary fragment ion of m / z 265; Tertiary ions: The open-ring dihydroxybenzene side chain of ZEN is easy to undergo α-fragmentation, which breaks the -CC- single bond and forms a tertiary fragment ion of m / z 149; The open-ring dihydroxybenzene side chain of α-ZEL / β-ZEL is easy to undergo α-fragmentation to lose the terminal olefin, and neutral loss occurs to remove one molecule of H2O, forming tertiary fragment ions of m / z 231 and m / z 231. DON further loses a hydroxyl group to remove one molecule of H2O to form a double bond, and at the same time, ethylene oxide is cracked, neutral loss occurs to remove one molecule of -CH2O, and finally forms a tertiary fragment ion of m / z 217.

[0014] Quinolones: Quinolones are prone to form [M+H] + For molecular ions, the carboxyl CC bond is easily broken in tandem mass spectrometry, accompanied by the breakage of the carboxyl CO bond, followed by the breakage of the tertiary amine CN bond and CC bond mainly in the side chain.

[0015] Precursor ion: In ESI + In this mode, [M+H] is formed. + Parent ion; secondary ions: The carboxyl CC bond energy on the quinoline ring of NOR / FQ / ENR / PEF / SAP / SAR / CIP / LOM / DAF / OFX / DIF / ENX / ENR-d5 is low, and it is easy to undergo neutral loss to remove one molecule of CO2 to form secondary fragment ions of m / z 276, m / z 244, m / z 316, m / z 290, m / z 349, m / z 342, m / z 288, m / z 308, m / z 340, m / z 318, m / z 356, m / z 257, and m / z 321. Tertiary ions: N on the piperazine ring of NOR / FQ / ENR / PEF / SAP / SAR / CIP / LOM / DAF / OFX / DIF / ENX / ENR-d5 has a strong affinity for protons. NOR forms i-fragmentation and removes one molecule of HF, and FQ forms i-fragmentation and removes one molecule of CO. Then, the NOR / FQ benzene ring undergoes charge transfer and rearrangement reaction, and the charge is transferred to N to form tertiary fragment ions of m / z 256, m / z 202, m / z 245, m / z 233, m / z 292, m / z 299, m / z 245, m / z 265, m / z 255, m / z 261, m / z 299, m / z 229, and m / z 245.

[0016] Sulfonamides: Sulfonamides are easy to form [M+H] + Molecular ions are prone to SN bond breakage and CS bond breakage with -SO2-NH- group as the center in tandem mass spectrometry, accompanied by rearrangement reaction and molecular ion reaction with addition of water and methanol. + In this mode, [M+H] is formed. +Parent ion; Secondary ion: The mass spectrometry fragmentation of sulfonamides is centered on the -SO2-NH- group, and the SN bond or CS bond breaks. The loss of the R group is one of its typical fragmentation pathways. SIM / STZ / SBA / SCP / SDX / SMP / SDM / SMR / SCM / SMT / SMIZ / SSZ / SMZ / SDZ / SPD / SQX / SGN / SAN / SDX-d3 are all obtained according to the above rules: m / z 186, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 186, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156 156, m / z156, secondary fragment ions of m / z 156; the methoxy bond energy connected to the benzene ring of TMP is weak, and α-fragmentation can occur to lose two molecules of -CH3O to obtain a secondary fragment ion of m / z 230. Tertiary ions: SIM / STZ / SBA / SCP / SDX / SMP / SDM / SMR / SCM / SMT / SMIZ / SSZ / SMZ / SDZ / SPD / SQX / SGN / SAN / SDX-d3, under the attraction and transfer of charge center, lead to neutral loss of -SO2-NH-, lose 1 molecule of SO2 or SO, and obtain m / z 122, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 122, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108 The tertiary fragment ion is m / z 108; after the No. 3 N atom in the pyrimidine diamine structure of TMP obtains a proton, it easily attracts electrons on the adjacent C atom, causing rearrangement and breakage, losing one molecule of -CH2N to form a tertiary fragment ion of m / z 202.

[0017] β-Agonists: Precursor ion: In ESI - In this mode, [M+H] is formed. +Parent ion; Secondary ion: The benzene ring connected to the β-hydroxy side chain of β-receptor agonists is prone to neutral loss and remove one molecule of H2O to form secondary fragment ions conjugated with the benzene ring m / z 259, m / z 202, m / z 284, m / z 222, m / z 196, m / z 154, and m / z 209; the group on the benzene ring of PBT / TEB connected to the -NH- of phenylethanolamine undergoes α-fragmentation after obtaining a proton, forming secondary fragment ions of m / z 236 and m / z 152. Tertiary ions: After obtaining protons, the groups connected to -NH- of CLB / CMT / RCT / SLB / CLP / TBT / CMT-d7 and phenylethanolamine undergo α-fragmentation to form tertiary fragment ions m / z 203, m / z 160, m / z 164, m / z 166, m / z 154, m / z 118, and m / z 161. The hydroxyl group connected to C atom No. 2 of PBT is prone to neutral loss to remove one molecule of H2O, forming a double bond conjugated with the benzene ring. At the same time, the distal N atom No. 4 obtains a proton and attracts the electrons of the adjacent C atom, which is prone to i-fragmentation and loses one molecule of NH3, eventually forming a tertiary fragment ion of m / z 201. The phenylethanolamine side chain of TEB is further fragmented, and the double bond conjugated with the benzene ring undergoes α-fragmentation to form a tertiary fragment ion of m / z 152.

[0018] Tetracyclines: Precursor ion: In ESI + In this mode, [M+H] is formed. + The parent ion of TC / ETC / OTC / EOTC is secondary ion; the C atom of TC / ETC / OTC / EOTC is connected to both hydroxyl and methyl groups, and the CO bond energy is low, so it is easy to neutrally lose one molecule of H2O to form secondary fragment ions of m / z 427, m / z 427, m / z 443, and m / z 443; after the distal N of MTC / CTC / ECTC / DMC / DOC / MNC / TGC obtains a proton, it is easy to neutrally lose NH3, and finally form secondary fragment ions of m / z 426, m / z 462, m / z 462, m / z448, m / z 428, m / z 441, and m / z 569; tertiary ion: under the action of charge center transfer and distal rearrangement, the distal -NH2 of TC / ETC / OTC / EOTC obtains H +After that, it is easy to neutrally lose NH3 to form tertiary fragment ions of m / z 410, m / z 410, m / z 426, and m / z 426; the C atom No. 15 of MTC undergoes charge transfer, and the charge center will attract the electrons of the adjacent C atom to form a double bond. At the same time, due to the low bond energy of the CN bond, it is easy to neutrally lose one molecule of C2H7N to form a tertiary fragment ion of m / z 381; the C atom of CTC / ECTC / DMC / DOC / MNC is connected to a methyl group and a hydroxyl group at the same time, and it is easy to neutrally lose one molecule of H2O and form a double bond, and finally form tertiary fragment ions of m / z 444, m / z 444, m / z 430, m / z 410, and m / z410; the C atom No. 13 connected to the N atom No. 12 of TGC has three methyl groups, the electron cloud density increases, the reaction activity increases, and it is easy to be attracted by the N atom No. 12 to cause α-fragmentation, forming m / z The third fragment ion of 513.

[0019] Herbicide: Precursor ion: In ESI - In this mode, [M+H] is formed. + Parent ion; Secondary ion: The branched CN bond on the benzene ring of CHL / ATZ / SIA / IST / PRE is easy to break. Under the action of fragmentation energy, it is gradually fragmented into secondary fragment ions of m / z 174.0, m / z 123.8, m / z 164.9, and m / z 200.0; Tertiary ion: The branched groups on the benzene ring of ATZ / SIA / IST / PRE obtain tertiary fragment ions of m / z 145.9, m / z 95.7, m / z 119.7, and m / z 157.9 according to the energy level.

[0020] Furthermore, in step S5, a standard solution is prepared (the standard solution can be a single standard solution or a mixed standard solution), and quadrupole-linear ion trap liquid chromatography tandem mass spectrometry is performed on it. The sample is injected through TinyAcquisition software, and the extracted ion flow chromatogram of each compound is obtained by TinyAnalysis software to determine the retention time.

[0021] In order to obtain a more accurate chromatogram and an accurate retention time, further, in step S5, the chromatographic conditions are as follows: Mobile phase: including mobile phase A and mobile phase B, wherein mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol; chromatographic column: Elite C18 chromatographic column; flow rate is 0.1-0.5 mL / min; injection volume is 3-8 μL; column temperature: 20-30°C; The gradient elution program is as follows:

[0022] Furthermore, in step S6, the relevant information of the small molecule compound includes the English name, CAS number, molecular formula, molecular weight, scanning mode, parent ion, secondary ion, tertiary ion and its structural formula, and ion optical parameters, which are presented in a tabular form as a multi-level mass spectrometry database.

[0023] The present invention provides a method for constructing a multi-level mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology. The present invention is the first to construct a multi-level mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology and utilizes multi-level fragmentation technology. It not only provides detailed information on each level of mass spectra of small molecule compounds, but also summarizes the mass spectrometry fragmentation rules of various compounds, which is not only conducive to complex matrix detection, but also more conducive to the differentiation and identification of isomers, supplements the current multi-level database, and also provides a basis for subsequent practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 A schematic flow chart of a method for constructing a multi-stage mass spectrometry database for small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology provided in an embodiment of the present invention.

[0026] Figure 2 This is the multi-stage fragmentation mass spectrum of aflatoxin B1 obtained in an embodiment of the present invention.

[0027] Figure 3 This is the multi-stage fragmentation mass spectrum of aflatoxin G1 obtained in an embodiment of the present invention.

[0028] Figure 4 This is the multi-stage fragmentation mass spectrum of aflatoxin M1 obtained in an embodiment of the present invention.

[0029] Figure 5 This is the multi-stage fragmentation mass spectrum of zearalenone obtained in an embodiment of the present invention.

[0030] Figure 6 This is the multi-stage fragmentation mass spectrum of α-zearalenol obtained in an embodiment of the present invention.

[0031] Figure 7 This is the multi-stage fragmentation mass spectrum of β-zearalenol obtained in an embodiment of the present invention.

[0032] Figure 8This is the multi-stage fragmentation mass spectrum of vomitoxin obtained in an example of the present invention.

[0033] Fig. 9 This is the multi-stage fragmentation mass spectrum of norfloxacin obtained in an embodiment of the present invention.

[0034] Fig.10 This is the multi-stage fragmentation mass spectrum of enrofloxacin obtained in an embodiment of the present invention.

[0035] Fig.11 This is the multi-stage fragmentation mass spectrum of ofloxacin obtained in an embodiment of the present invention.

[0036] Fig.12 This is the multi-stage fragmentation mass spectrum of difloxacin obtained in an example of the present invention.

[0037] Fig.13 This is the multi-stage fragmentation mass spectrum of enoxacin obtained in an example of the present invention.

[0038] Fig.14 This is the multi-stage fragmentation mass spectrum of sulfadiazine obtained in an embodiment of the present invention.

[0039] Fig.15 This is the multi-stage fragmentation mass spectrum of trimethoprim obtained in an example of the present invention.

[0040] Fig.16 This is the multi-stage fragmentation mass spectrum of sulfanitrobenzene obtained in an embodiment of the present invention.

[0041] Fig.17 This is the multi-stage fragmentation mass spectrum of clenbuterol obtained in an embodiment of the present invention.

[0042] Fig.18 This is the multi-stage fragmentation mass spectrum of Penbutero obtained in an embodiment of the present invention.

[0043] Fig.19 This is a multi-stage fragmentation mass spectrum of Tubuterol obtained in an embodiment of the present invention.

[0044] Fig. 20 This is the multi-stage fragmentation mass spectrum of terbutaline obtained in the embodiment of the present invention.

[0045] Fig.21 This is the multi-stage fragmentation mass spectrum of cimaterol obtained in an embodiment of the present invention.

[0046] Fig. 22 This is the multi-stage fragmentation mass spectrum of tetracycline obtained in the embodiment of the present invention.

[0047] Fig.23 This is the multi-stage fragmentation mass spectrum of oxytetracycline obtained in an embodiment of the present invention.

[0048] Fig.24 This is the multi-stage fragmentation mass spectrum of chlortetracycline obtained in an example of the present invention.

[0049] Fig.25 This is the multi-stage fragmentation mass spectrum of doxycycline obtained in an embodiment of the present invention. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] The quadrupole-linear ion trap liquid chromatography-mass spectrometer used in the following examples is derived from the QLIT-6610 model.

[0052] Example

[0053] This embodiment provides a method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology, and its process flow chart is as follows: Figure 1 As shown (taking sulfonamide compounds as an example), the following steps are included: Step S1. Prepare standard stock solutions and standard working solutions of various small molecule compounds.

[0054] The standard stock solution was diluted to a standard working solution as needed, and a single standard of 1 μg / mL was injected into a quadrupole-linear ion trap tandem mass spectrometer at a flow rate of 5 μL / min using a syringe pump direct injection method.

[0055] Step S2. Because this instrument does not have mass spectrometry information for these small molecule compounds, it is necessary to optimize the single mass spectrometry parameters based on the mass-to-charge ratio of the parent ion of the known related compounds, and establish the primary and multi-stage mass spectrometry methods for small molecule compounds. The ion needle position is optimized to 1-1.5 mm, the ion source temperature to 150-180 °C, and the atmospheric pressure interface temperature to 200-250 °C, and the final mass spectrometry conditions are as follows: The mass spectrometry conditions were as follows: ESI source: scan rate 10000 Da / s, ion guide RF AC voltage 400 V, multiplier voltage -1150 V, ion source high voltage 4500 V, high vacuum gauge 1.5-1.8×10 -5 Torr, low vacuum gauge 0.9~1.3 Torr, sheath gas 3.2×10 5 Pa, auxiliary gas 1.6×10 5 Pa, back-blowing gas 3.4×10 3 Pa, ion source temperature 50 °C, atmospheric pressure interface temperature 280 °C.

[0056] Step S3. Using different scanning modes of quadrupole-linear ion trap tandem mass spectrometry, specific mass spectral information such as the mass-to-charge ratio of the secondary and tertiary ions of the small molecule compound with the highest corresponding intensity is obtained in the TinyTrap software according to the response intensity.

[0057] In the daughter ion scanning (QTPIS) mode, according to the parent ion of the compound, enter the mass scan range m / z 100-600 in the TinyTrap software, enter the parent ion in the "parent ion" column, and obtain the parent ion of the related compound when the fragmentation energy is 0 according to the mass-to-charge ratio obtained from the single mass spectrum, the ionization time, fragmentation q point, fragmentation time and other mass spectrum information of the parent ion of the known related compounds. Adjust the fragmentation energy and determine the mass spectral information such as the mass-to-charge ratio of the secondary ion according to the response intensity of the secondary ion; in the multi-stage daughter ion scanning (QTPISn) mode, in the "parent ion" column, enter the mass-to-charge ratio of the parent ion according to the single mass spectrum, and in the "daughter ion" column, enter the mass-to-charge ratio of the parent ion according to the single mass spectrum. Enter the mass-to-charge ratio of the secondary ion determined by the above method in the column. The optimization ranges of ionization time, fragmentation q-point, fragmentation time and fragmentation energy are 0.05-0.1, 0.25-0.35, 0.01-0.03 and 0-100 respectively. According to the response intensity of the secondary ion, obtain the specific mass spectral information such as ionization time, fragmentation q-point, fragmentation time and fragmentation energy. According to the fragmentation efficiency (90-95%), adjust the fragmentation energy, and determine the mass spectral information such as the mass-to-charge ratio of the tertiary ion based on the response intensity of the tertiary ion.

[0058] Taking sulfadiazine as an example, the known parent ion mass-to-charge ratio of sulfadiazine is 279. After positive ion scanning, the parent ion mass-to-charge ratio of sulfadiazine is obtained to be 279. After quadrupole-ion trap mass spectrometry product ion scanning (QTPIS), enter 279 in the "Precursor ion" column, enter 0.1 in the "Ionization time" column, enter 0-100 in the "Fragmentation energy" column, enter 0.25 in the "Fragmentation q-point" column, and enter "0.03" in the "Fragmentation time" column to obtain the secondary ion m / z 186. Similarly, select quadrupole-ion trap multistage product ion scanning (QTPISn), enter 279 186 in the "Precursor ion" column, enter 0.1 0.1 in the "Ionization time" column, enter 0-100 0-100 in the "Fragmentation energy" column, enter 0.25 0.25 in the "Fragmentation q-point" column, and enter "0.03 0.03" in the "Fragmentation time" column to obtain the tertiary ion m / z 122.

[0059] Step S4. Obtain the mass spectrometry fragmentation rules through literature research, and compare them with the mass spectrometry information of the relevant compounds obtained in step S3 to confirm the specific fragmentation position.

[0060] The same type of compounds are classified according to their cleavage functional groups, and the same cleavage rules of the same type of compounds are summarized.

[0061] True toxins: (1) Aflatoxins (AFB1 / AFB2 / AFG1 / AFG2 / AFM1 / AFM2): Parent ions: In ESI+ mode, all of them form parent ions of [M+H]+; Secondary ions: The alicyclic ketone in the structure of AFB1 / AFB2 undergoes neutral loss after α-fragmentation and i-fragmentation, losing one molecule of -CO, and AFB1 / AFB2 forms secondary fragment ions of m / z 285 and m / z 287; The alicyclic ketone in the structure of AFG1 / AFG2 undergoes distal rearrangement reaction after i-fragmentation to remove one molecule of H2O, and AFG1 / AFG2 produces secondary fragment ions of m / z 311 and m / z 313; The furan ring of AFM1 / AFM2 is prone to neutral loss due to the low bond energy of the C-O bond, and it loses one molecule of H2O, and AFM1 / AFM2 forms m / z 311 and m / z 313. Secondary fragment ion of m / z 313; Tertiary ion: AFB1 / AFB2 secondary daughter ion structure contains lactone bond, which is easy to lose a molecule of -CO, AFB1 / AFB2 forms tertiary fragment ions of m / z 257 and m / z 259; AFG1 / AFG2 due to π electron cloud migration, the C=C bond of AFG1 breaks and i breaks at the same time, after removing a molecule of -C2H4, a tertiary fragment ion of m / z 243 is formed, the C=C bond of AFG2 breaks and rearrangement reaction occurs at the same time to break the adjacent C=C bond, forming a tertiary fragment ion of m / z 285; AFM1 / AFM2 removes a molecule of -CO due to a distal rearrangement reaction after i break, generating tertiary fragment ions of m / z 282 and m / z 285. (2) ZEN / α-ZEL / β-ZEL / DON: Parent ion: In ESI-mode, all of them form parent ions of [M+H]-; Secondary ions: In the cyclic lactone structure of ZEN / α-ZEL / β-ZEL, neutral loss is prone to occur to remove one molecule of CO2, forming secondary fragment ions of m / z 273, m / z 275 and m / z 275; DON is prone to neutral loss of -CH2O to form secondary fragment ion of m / z 265; Tertiary ions: The open-ring dihydroxybenzene side chain of ZEN is prone to α-fragmentation, resulting in the cleavage of the -C-C- single bond, forming a tertiary fragment ion of m / z 149; the open-ring dihydroxybenzene side chain of α-ZEL / β-ZEL is prone to α-fragmentation to lose the terminal olefin, and at the same time, neutral loss occurs to remove one molecule of H2O, forming tertiary fragment ions of m / z 231 and m / z 231. DON further loses a hydroxyl group and removes one H2O molecule to form a double bond. At the same time, ethylene oxide is cracked, neutral loss occurs and one -CH2O molecule is removed, finally forming a tertiary fragment ion of m / z 217.

[0062] Quinolones: Quinolones are prone to form [M+H]+ molecular ions. In tandem mass spectrometry, the carboxyl CC bond is easily broken, accompanied by the breakage of the carboxyl CO bond, followed by the breakage of the tertiary amine CN bond and CC bond mainly in the side chain.

[0063] Parent ion: In ESI+ mode, parent ions of [M+H]+ are formed. Secondary ions: The C-C bond energy of the carboxyl group on the quinoline ring of NOR / FQ / ENR / PEF / SAP / SAR / CIP / LOM / DAF / OFX / DIF / ENX / ENR-d5 is low, and neutral loss easily occurs to remove one molecule of CO2, forming secondary fragment ions of m / z 276, m / z 244, m / z 316, m / z 290, m / z 349, m / z 342, m / z 288, m / z 308, m / z 340, m / z 318, m / z 356, m / z 257, and m / z 321. Tertiary ions: N on the piperazine ring of NOR / FQ / ENR / PEF / SAP / SAR / CIP / LOM / DAF / OFX / DIF / ENX / ENR-d5 has a strong affinity for protons. NOR forms i-fragmentation and removes one molecule of HF, and FQ forms i-fragmentation and removes one molecule of CO. Then, the NOR / FQ benzene ring undergoes charge transfer and rearrangement reaction, and the charge is transferred to N to form tertiary fragment ions of m / z 256, m / z 202, m / z 245, m / z 233, m / z 292, m / z 299, m / z 245, m / z 265, m / z 255, m / z 261, m / z 299, m / z 229, and m / z 245.

[0064] Sulfonamides: Sulfonamides are prone to form [M+H]+ molecular ions. In tandem mass spectrometry, they are prone to SN bond breaking and CS bond breaking centered on the -SO2-NH- group, accompanied by rearrangement reactions and molecular ion reactions with the addition of water or methanol. Parent ion: In ESI+ mode, parent ions of [M+H]+ are formed; Secondary ions: The mass spectrometry fragmentation of sulfonamides is centered on the -SO2-NH- group, and the SN bond or CS bond breaks. The loss of the R group is one of its typical fragmentation pathways. SIM / STZ / SBA / SCP / SDX / SMP / SDM / SMR / SCM / SMT / SMIZ / SSZ / SMZ / SDZ / SPD / SQX / SGN / SAN / SDX-d3 are all obtained according to the above rules: m / z 186, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 186, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156, m / z 156 Secondary fragment ions of m / z 156, m / z 156, m / z 156, and m / z 156; the methoxy bond energy connected to the benzene ring of TMP is weak, and α-fragmentation can occur to lose two molecules of -CH3O to obtain a secondary fragment ion of m / z 230. Tertiary ions: SIM / STZ / SBA / SCP / SDX / SMP / SDM / SMR / SCM / SMT / SMIZ / SSZ / SMZ / SDZ / SPD / SQX / SGN / SAN / SDX-d3, under the attraction and transfer of charge center, lead to neutral loss of -SO2-NH-, lose 1 molecule of SO2 or SO, and obtain m / z 122, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 122, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108, m / z 108 The tertiary fragment ion is m / z 108; after the No. 3 N atom in the pyrimidine diamine structure of TMP obtains a proton, it easily attracts the electrons on the adjacent C atom, causing rearrangement and breakage, losing one molecule of -CH2N to form a tertiary fragment ion of m / z 202.

[0065] β-Receptor agonists: Parent ion: In ESI- mode, parent ions of [M+H]+ are formed; Secondary ions: The benzene ring connected to the β-hydroxyl side chain of β-receptor agonists is prone to neutral loss and remove one molecule of H2O, forming secondary fragment ions conjugated with the benzene ring m / z 259, m / z 202, m / z 284, m / z 222, m / z 196, m / z 154, and m / z 209; the group on the benzene ring of PBT / TEB connected to the -NH- of phenylethanolamine undergoes α-fragmentation after obtaining a proton, forming secondary fragment ions of m / z 236 and m / z 152. Tertiary ions: After the groups connected to -NH- of CLB / CMT / RCT / SLB / CLP / TBT / CMT-d7 obtained protons, α-fragmentation occurred to form tertiary fragment ions m / z 203, m / z 160, m / z 164, m / z 166, m / z154, m / z 118, and m / z 161; the hydroxyl group connected to the C atom No. 2 of PBT was prone to neutral loss to remove one molecule of H2O, forming a double bond conjugated with the benzene ring. At the same time, the N atom No. 4 at the far end attracted the electrons of the adjacent C atom after obtaining a proton, and was prone to i-fragmentation, losing one molecule of NH3, and finally forming a tertiary fragment ion of m / z 201; the phenylethanolamine side chain of TEB was further fragmented, and the double bond conjugated with the benzene ring was α-fragmented to form a tertiary fragment ion of m / z 152.

[0066] Tetracyclines: Parent ion: In ESI+ mode, parent ions of [M+H]+ are formed; Secondary ions: The C atom of TC / ETC / OTC / EOTC is connected to both the hydroxyl group and the methyl group, and the C-O bond energy is low, so it is easy to undergo neutral loss to remove one molecule of H2O, forming secondary fragment ions of m / z 427, m / z 427, m / z 443, and m / z 443; After the distal N of MTC / CTC / ECTC / DMC / DOC / MNC / TGC obtains a proton, it is easy to undergo neutral loss of NH3, and finally form m / z 426, m / z 462, m / z 462, m / z 448, m / z 428, m / z 441, and m / z 443. The secondary fragment ion is 569; the tertiary ion: under the action of charge center transfer and distal rearrangement, the distal -NH2 of TC / ETC / OTC / EOTC easily undergoes neutral loss of NH3 after obtaining H+, forming tertiary fragment ions of m / z 410, m / z 410, m / z 426, and m / z 426; the C atom No. 15 of MTC undergoes charge transfer, and the charge center will attract the electrons of the adjacent C atom to form a double bond. At the same time, due to the low bond energy of the C-N bond, it is easy to undergo neutral loss to remove one molecule of C2H7N, forming a tertiary fragment ion of m / z 381; the C atom of CTC / ECTC / DMC / DOC / MNC is connected to both methyl and hydroxyl groups, and is easy to undergo neutral loss to remove one molecule of H2O and form a double bond, finally forming m / z 444, m / z 444, m / z 430, m / z 410, m / z The tertiary fragment ion is m / z 410; there are three methyl groups on the 13th C atom connected to the 12th N atom of TGC, the electron cloud density increases, the reaction activity increases, and it is easily attracted by the 12th N atom to cause α-fragmentation, forming a tertiary fragment ion of m / z 513.

[0067] Step S5. The retention time was determined to be 0.43-13.43 min by TinyAcquisition and TinyAnalysis software.

[0068] The standard solution was prepared with a concentration of 1 ppm and subjected to quadrupole-linear ion trap liquid chromatography tandem mass spectrometry detection. The sample was injected through TinyAcquisition software and the extracted ion current chromatogram of each compound was obtained by TinyAnalysis software to determine the retention time.

[0069] The chromatographic conditions were as follows: Mobile phase: 0.1% formic acid aqueous solution (A) and methanol (B); chromatographic column: Elite C18 column (4.6 mm×150mm., 5 μm); flow rate: 0.3 mL / min; injection volume: 5 μL; column temperature: 25 ℃

[0070] The gradient elution program is shown in Table 1 below: Table 1

[0071] S6. Summarize the relevant information of the compounds and establish a multi-level mass spectrometry database.

[0072] The relevant information of the compound, including its English name, CAS number, molecular formula, molecular weight, scanning mode, parent ion (secondary ion / tertiary ion) and its structural formula, ion optical parameters, etc., is summarized in a table as a multi-level mass spectrometry database, as shown in Table 2. The multi-level fragmentation mass spectra of each small molecule compound are shown in Figure 2-25 shown.

[0073] Table 2

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology, characterized in that: The steps include: Step S1: preparing a standard stock solution and a standard working solution of a small molecule compound; Step S2: Optimizing single mass spectrometry conditions and simultaneously obtaining mass spectrometry information of parent ions of small molecule compounds; Step S3: obtaining the mass spectrum information of multi-stage ions of small molecule compounds in TinyTrap software through different scanning modes of quadrupole-linear ion trap tandem mass spectrometry; Step S4: confirming the fragmentation position of the small molecule compound according to the mass spectrometry fragmentation rule and the mass spectrometry information of the parent ion and the multistage ion of the small molecule compound determined in steps S2 and S3; Step S5: Determine the retention time of small molecule compounds by TinyAcquisition and TinyAnalysis software; Step S6: Summarize the relevant information of small molecule compounds and establish a multi-level mass spectrometry database.

2. The method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology according to claim 1, characterized in that: In step S1, the standard stock solution is diluted to a standard working solution as needed, and injected into a quadrupole-linear ion trap tandem mass spectrometer at a flow rate of 4-6 μL / min using a direct injection method; the concentration of the standard working solution is 0.5-1.5 μg / mL.

3. The method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology according to claim 1, characterized in that: In the step S1, the small molecule compound includes true toxin compounds, quinolone compounds, sulfonamide compounds, β-receptor agonist compounds, and tetracycline compounds.

4. The method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology according to claim 1, characterized in that: In the step S2, the mass spectrometer adopts an ESI source, and the optimized single mass spectrometer conditions include ion needle position, scan rate, ion guide RF AC voltage, multiplier voltage, ion source high voltage, high vacuum gauge, low vacuum gauge, sheath gas, auxiliary gas, backblowing gas, ion source temperature and atmospheric pressure interface temperature; preferably, the ion needle position is 1-1.5 mm, the ion source temperature is 150-180 ° C, and the atmospheric pressure interface temperature is 200-250 ° C.

5. The method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology according to claim 1, characterized in that: In the step S3, the scanning mode includes a daughter ion scanning mode and a multi-stage ion scanning mode; in the daughter ion scanning mode and the multi-stage ion scanning mode, according to the mass-to-charge ratio of the parent ion of the small molecule compound, the mass scanning range is input in the TinyTrap software, the mass-to-charge ratio of the parent ion is input in the parent ion column, and according to the response intensity, the ionization time, fragmentation q point, fragmentation time and fragmentation energy are optimized to obtain the parent ion, secondary ion and tertiary ion in sequence.

6. The method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology according to claim 5, characterized in that: The mass scan range was m / z 100-600.

7. The method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology according to claim 1, characterized in that: In step S4, the mass spectrometry fragmentation rules include classifying the same type of compounds according to the fragmentation functional groups and summarizing the same fragmentation rules of the same type of compounds.

8. The method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology according to claim 1, characterized in that: In step S5, a standard solution is prepared and then subjected to quadrupole-linear ion trap liquid chromatography tandem mass spectrometry detection. Samples are injected using TinyAcquisition software and the extracted ion current chromatogram of each compound is obtained using TinyAnalysis software to determine the retention time.

9. The method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology according to claim 8, characterized in that: In step S5, the chromatographic conditions are as follows: Mobile phase: including mobile phase A and mobile phase B, wherein mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol; chromatographic column: Elite C18 chromatographic column; flow rate is 0.1-0.5 mL / min; injection volume is 3-8 μL; column temperature: 20-30°C; The gradient elution program is as follows: 。 10. The method for constructing a multi-stage mass spectrometry database of small molecule compounds based on quadrupole-linear ion trap tandem mass spectrometry technology according to claim 1, characterized in that: In step S6, the relevant information of the small molecule compound includes the English name, CAS number, molecular formula, molecular weight, scanning mode, parent ion, secondary ion, tertiary ion and its structural formula, and ion optical parameters, which are presented in a tabular form as a multi-level mass spectrometry database.

Images