WO2023193323A1 - 一种快速、全面、稳定的未知毒物的鉴定方法 - Google Patents

一种快速、全面、稳定的未知毒物的鉴定方法 Download PDF

Info

Publication number
WO2023193323A1
WO2023193323A1 PCT/CN2022/091819 CN2022091819W WO2023193323A1 WO 2023193323 A1 WO2023193323 A1 WO 2023193323A1 CN 2022091819 W CN2022091819 W CN 2022091819W WO 2023193323 A1 WO2023193323 A1 WO 2023193323A1
Authority
WO
WIPO (PCT)
Prior art keywords
reaction
mass
phase
mass spectrometry
tested
Prior art date
Application number
PCT/CN2022/091819
Other languages
English (en)
French (fr)
Inventor
楼燕
杨希
Original Assignee
浙江大学医学院附属第一医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 浙江大学医学院附属第一医院 filed Critical 浙江大学医学院附属第一医院
Publication of WO2023193323A1 publication Critical patent/WO2023193323A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/90—Programming languages; Computing architectures; Database systems; Data warehousing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material

Definitions

  • This application relates to the technical field of poison analysis, and in particular to a rapid, comprehensive and stable identification method of unknown poisons.
  • the purpose of this application is to provide a rapid, comprehensive, and stable identification method of unknown poisons, so as to achieve rapid, comprehensive, accurate, and stable identification of the types of unknown poisons.
  • the specific technical solutions are as follows:
  • the first aspect of this application provides a rapid, comprehensive and stable identification method for unknown poisons, including:
  • the chromatography and mass spectrometry data include at least one of retention time, precise molecular mass, isotope distribution, precursor ion information, product ion fragment information, precursor ion spectrum, product ion spectrum, structural formula, name and category;
  • the poisoning drug database includes a prototype compound database and/or a metabolite database; the prototype compound database includes chromatography and mass spectrometry data of compounds; the metabolite database includes compounds obtained by deriving metabolic reaction models. Chromatographic and mass spectrometric data of metabolites.
  • the second aspect of this application provides the use of identifying unknown poisons with mass numbers in the range of 50-1200 within 20 minutes according to the identification method provided by the first aspect of this application.
  • This application provides a rapid, comprehensive and stable identification method for unknown poisons, based on a prototype compound database including nearly 9,000 compounds and their chromatography and mass spectrometry data, and/or including nearly 9,000 compounds deduced by metabolic reaction models.
  • the poisoning drug database of the metabolite database of the obtained metabolites and their chromatographic and mass spectrometry data is used to detect the compounds in the test sample and blank sample through the detection method of ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry of the present application.
  • the chromatography and mass spectrometry data of the difference substance are compared to obtain the chromatography and mass spectrometry data of the difference substance.
  • the type of unknown poison in the sample to be tested can be determined. Covering small molecule compounds with mass numbers in the range of 50-1200, the detection range has strong coverage, and can simultaneously detect thousands of compounds in one injection, and can determine the type of unknown poisons within 20 minutes, achieving rapid, effective and comprehensive , accurately and stably identify the types of unknown poisons; the method has the advantages of simplicity, high sensitivity, high resolution, fast analysis speed, and high accuracy. When used in clinical practice, it can narrow the scope of unknown poisons and reduce screening time in combination with clinical symptoms. , can quickly and accurately screen out exogenous substances in patients, provide timely theoretical basis for the selection of clinical treatment plans, and accurately assist patient rescue.
  • Figure 1 is a flow chart of an unknown poison identification method provided by this application.
  • Figure 2 is a chromatogram of the sample to be tested and the blank sample in Example 1, where the top is the sample to be tested and the bottom is the blank sample.
  • Figure 3 is a chromatogram of the difference product in Example 1.
  • Figure 4 is a chromatogram of flufenpyrazone in the sample to be tested in Example 1.
  • Figure 5 is the mass spectrum of flufenpyrazone in the sample to be tested in Example 1 under low channel collision energy and high channel collision energy.
  • Figure 6 is a chromatogram of the sample to be tested and the blank sample in Example 2, where the upper part is the sample to be tested and the lower part is the blank sample.
  • Figure 7 is a chromatogram of the difference product in Example 2.
  • Figure 8 is a chromatogram of scopolamine hydrobromide in the sample to be tested in Example 2.
  • Figure 9 is a mass spectrum of scopolamine hydrobromide in the sample to be tested in Example 2 under high channel collision energy.
  • Figure 10 is a mass spectrum of scopolamine hydrobromide in the sample to be tested in Example 2 under low channel collision energy.
  • Figure 11 is a chromatogram of phenobarbital in the sample to be tested in Example 2.
  • Figure 12 is a mass spectrum of phenobarbital in the sample to be tested in Example 2 under high channel collision energy.
  • Figure 13 is a mass spectrum of phenobarbital in the sample to be tested in Example 2 under low channel collision energy.
  • Figure 14 is a chromatogram of phenobarbital + O + H2 + SO3, the metabolite of phenobarbital in Example 2.
  • Figure 15 is a mass spectrum of phenobarbital + O + H2 + SO3, the metabolite of phenobarbital in Example 2, under low channel collision energy.
  • Figure 16 is a mass spectrum of the metabolite phenobarbital + O + H2 + SO3 of phenobarbital in Example 2 under high channel collision energy.
  • Figure 17 is the precursor ion chromatogram of phenobarbital with a precursor ion mass-to-charge ratio m/z 233.093 in the sample to be tested in Example 2.
  • Figure 18 is the precursor ion chromatogram of scopolamine hydrobromide with a precursor ion mass-to-charge ratio m/z 384.081 in the sample to be tested in Example 2.
  • Figure 19 is a chromatogram of scopolamine hydrobromide + 2 ⁇ (-H2) + C2H2O, the metabolite of scopolamine hydrobromide in Example 3.
  • Figure 20 is a mass spectrum of scopolamine hydrobromide +2 ⁇ (-H2)+C2H2O, a metabolite of scopolamine hydrobromide, at low channel collision energy in Example 3.
  • Figure 21 is a mass spectrum of scopolamine hydrobromide +2 ⁇ (-H2)+C2H2O, a metabolite of scopolamine hydrobromide in Example 3, under high channel collision energy.
  • the first aspect of this application provides a rapid, comprehensive and stable identification method for unknown poisons, including:
  • the chromatography and mass spectrometry data include at least one of retention time, precise molecular mass, isotope distribution, precursor ion information, product ion fragment information, precursor ion spectrum, product ion spectrum, structural formula, name and category;
  • the poisoning drug database includes a prototype compound database and/or a metabolite database; the prototype compound database includes chromatography and mass spectrometry data of compounds; the metabolite database includes compounds obtained by deriving metabolic reaction models. Chromatographic and mass spectrometric data of metabolites.
  • the preparation of the sample solution to be tested includes: taking the whole blood sample to be tested from the poisoned patient, centrifuging, taking the supernatant, and obtaining the plasma sample; taking the plasma sample, adding methanol, vortexing for 2-5 minutes, and centrifuging, Take the upper layer solution to obtain a sample solution to be tested; the volume ratio of the plasma sample to the methanol is 1:(2-4).
  • the preparation of the sample solution to be tested includes: taking the whole blood sample to be tested from the poisoned patient, centrifuging it at 3500-4500 rpm for 8-12 min, taking the supernatant to obtain the plasma sample; taking the plasma sample, adding methanol and vortexing for 2 -5 min, centrifuge at 12500-14000 rpm for 8-12 min, take the upper layer solution, and obtain the sample solution to be tested; the volume ratio of the plasma sample to the methanol is 1:(2-4).
  • the whole blood sample to be tested from a poisoned patient centrifuge it at 4000 rpm for 10 minutes, transfer the supernatant to a clean centrifuge tube, and obtain a plasma sample; accurately transfer 100 ⁇ L of the plasma sample, place it in a 1.5 mL centrifuge tube, and add 300 ⁇ L of methanol, vortex for 3 minutes, centrifuge at 13200 rpm for 10 minutes, take the upper layer solution to obtain the sample solution to be tested, and take 100 ⁇ L of the sample solution to be tested into a sample bottle with a lined tube for sample injection and detection.
  • the injection volume is 2 ⁇ L.
  • a blank sample is set as a blank control.
  • a healthy person's whole blood sample can be prepared by taking a healthy person's whole blood sample and using the same method as the preparation of the sample solution to be tested.
  • Blank sample solution use the same detection method as the sample solution to be tested to detect the blank sample solution.
  • the preparation method of the sample solution to be tested in this application uses plasma plus methanol precipitation, which can be used for sample detection, and the sample usage is small. For example, in this application, 100 ⁇ L of plasma can be used for detection, and the type of unknown poison can be identified. .
  • the preparation method of the sample solution to be tested in this application is simple, fast and convenient, and is conducive to timely detection and identification of unknown poisonous substances.
  • the binary comparison analysis is to turn on dynamic background subtraction, exclude the compounds contained in the blank sample from thousands of compounds in the sample to be tested, remove the real-time dynamic background, and obtain the chromatogram of the difference.
  • Mass spectrometry data at the same time, combined with the clinical symptoms of poisoned patients, the range of unknown poisons is narrowed, thereby reducing the screening time. This method is fast and can improve the efficiency of identification of unknown poisons in the samples to be tested.
  • the compound composition in the patient's plasma sample is complex.
  • Using the blank plasma of a healthy person as a blank control for real-time dynamic background subtraction can reduce the interference of endogenous substances in the patient's plasma sample and narrow the scope of unknown poisons.
  • the sample to be tested selects different matching screening methods based on the poisoning time between the sample collection time and the poisoning time of the poisoned patient, including:
  • the obtained chromatography and mass spectrometry data of the differential substances were matched and screened with the prototype compound database to initially determine the identity of the unknown poisons in the samples to be tested. Type; then perform matching screening with the metabolite database to further determine the type of unknown poison in the sample to be tested;
  • the obtained chromatography and mass spectrometry data of the differential substances are matched and screened with the metabolite database to initially determine the types of unknown poisons in the samples to be tested. ; Then perform matching screening with the prototype compound database to further determine the type of unknown poison in the sample to be tested.
  • the above matching screening method can be used to further improve the efficiency of matching screening, making the identification of unknown poisons more rapid, comprehensive and accurate.
  • the rapid, comprehensive and stable identification method of unknown poisons provided by this application can be classified and analyzed according to the collection time of samples after taking medicine for different patients. That is, for samples to be tested within 24 hours after taking medicine, first based on accurate molecular mass, retention time, The prototype compound is screened from multiple dimensions such as isotope distribution and product ion fragments, and is supplemented by metabolite identification for multi-faceted confirmation; for samples to be tested more than 24 hours after taking the drug, since the prototype compound may have been metabolized at this time, the compounds in the sample to be tested The prototype content is low. At this time, the type of unknown poison can be inferred by identifying the metabolites in the sample to be tested.
  • the process is carried out based on the precise molecular mass, retention time, primary mass spectrometry, and secondary mass spectrometry information of the differential substance and the established poisoning drug database containing the precise molecular mass, retention time, primary mass spectrum, and secondary mass spectrometry information of the compound.
  • Similarity comparison is used to match and confirm the structure of the compound fragments, thereby completing the screening and confirmation of the target compound, and determining the type of unknown poison in the sample to be tested; the similarity comparison is a commonly used technical means in this field, and those skilled in the art
  • the comparison method can be selected according to actual needs.
  • the identification method further includes: constructing the poisoning drug database.
  • the poisoning drug database can be continuously expanded to increase the data of compounds or metabolites in the database, so that the poisoning drug database can be updated sustainably and further improve the comprehensiveness and accuracy of identification of unknown poisons. sex.
  • the compounds include 6416 traditional Chinese medicine ingredients, 2302 poison ingredients and pesticide ingredients, 23 amino acid ingredients, 46 sedative drug ingredients, psychotropic drug ingredients and non-steroidal Anti-inflammatory drug ingredients.
  • the prototype compound database includes an importable commercial database and a self-built database; in this application, the prototype compound database of this application is constructed by importing a commercial database and a self-built database.
  • the commercial database mentioned in this application is a database that can be purchased and used on the market, such as the traditional Chinese medicine ingredient database containing 6416 compounds and their chromatography and mass spectrometry data, the poison ingredient and pesticide ingredient database containing 2302 compounds and their chromatography and mass spectrometry data, An amino acid database containing 23 compounds and their chromatography and mass spectrometry data; the self-built database may include chromatography and mass spectrometry data of compounds obtained by ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry using standard products.
  • the database can include the chromatography and mass spectrometry data of the compounds disclosed in published articles, and can also use customized functions, such as editing the structural formula of compounds, to expand the self-built database.
  • the prototype compound database can be used to infer possible poison types based on the symptoms of clinical patients, and can be selectively imported into commercial databases and/or self-built databases, making this application's method of identifying unknown poisons in samples to be tested more targeted. This method further improves the speed and accuracy of identifying unknown poisons in samples to be tested.
  • the sedative drug ingredients, psychotropic drug ingredients and non-steroidal anti-inflammatory drug ingredients include the following compounds: olanzapine, amisulpride, aripiprazole, amitriptyline, aspirin, moxa Sicitalopram, ibuprofen, piroxicam, phenylbutazone, imipramine, doxepin, desipramine, acetaminophen, flurbiprofen, fluvoxamine, perphenazine, fluperzine Fluoxetine, fluphenazine, quetiapine, chlorpromazine, clozapine, rofecoxib, clomipramine, risperidone, loxoprofen, mianserin, Clobemide, maprotiline, meloxicam, nedometone, naproxen, nimesulide, paliperidone, paroxetine, nortriptyline, trazodone,
  • the self-built database includes a database of chromatography and mass spectrometry data of compounds obtained by ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry detection using standard products; exemplarily, Table 1 is used The standard products of 46 compounds were detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometer, and the chromatographic and mass spectrometric data of 46 compounds were obtained;
  • the chromatographic conditions of the ultra-high performance liquid chromatography include:
  • Chromatographic column octadecylsilane bonded silica gel chromatographic column; mobile phase: phase A is 0.8-1.2mol/L ammonium acetate aqueous solution, phase B is 0.8-1.2mol/L ammonium acetate methanol solution; use volume fraction 1 -98% phase A, 2-99% phase B, gradient elution; column temperature: 43-47°C; flow rate: 0.4-0.5mL/min; injection volume: 1-5 ⁇ L;
  • the mass spectrometry conditions of the quadrupole time-of-flight mass spectrometer include:
  • mass spectrometry parameters include:
  • the chromatographic conditions of the ultra-high performance liquid chromatography include:
  • Phase A is 1mol/L ammonium acetate aqueous solution (volume ratio of ammonium acetate and water is 1:99, solution pH is 5.0)
  • phase B is 1mol/L ammonium acetate methanol solution (volume ratio of ammonium acetate and methanol is 1:99, solution pH is 5.0); gradient elution includes: 0-0.25min, 2%B; 0.25-12.25min, 2-99%B ;12.25-13.00min, 99%B; 13.00-13.01min, 99-2%B; 13.01-17.00min, 2%B;
  • the mass spectrometry conditions of the quadrupole time-of-flight mass spectrometer include:
  • mass spectrometry parameters include:
  • the collision energies were 15V, 30V, and 45V respectively, and the mass scan was the precursor ion mass-to-charge ratio of the standard.
  • Leucine-enkephalin (5ng/mL) is used as a calibration solution throughout the entire analysis to determine whether there is any deviation in the mass axis of the instrument and to control the accuracy of the instrument.
  • the metabolite database includes chromatography and mass spectrometry data of metabolites obtained by deriving compounds through metabolic reaction models; wherein, the metabolic reaction model can be constructed by setting metabolic reaction pathways; the metabolite database , based on the correlation between the prototype compound-group-metabolic reaction and the correlation between metabolic reaction-metabolite, the metabolites produced by the metabolic reaction of the prototype compound and their chromatography and mass spectrometry data are deduced, including the precise molecular mass and isotopes of the metabolites. Distribution, parent ion information, product ion fragment information, structural formula, name and category; multi-dimensional identification of metabolite types in the sample to be tested based on accurate molecular mass, isotope distribution, product ion fragments and other information.
  • the metabolic reaction includes phase I reaction and/or phase II reaction; because the metabolism of the drug in the body may be directly excreted from the body after the phase I reaction, or it may be directly excreted from the body after the phase II reaction, or it may be It is a phase I reaction combined with a phase II reaction and then excreted from the body.
  • the metabolic characteristics of phase I reaction and phase II reaction in the human body are comprehensively considered: metabolites are deduced through phase I reaction and/or phase II reaction, which can be further supported.
  • the identified types of unknown poisons in the sample to be tested do exist, reducing the possibility of false positives.
  • the set metabolic reaction pathway is realized through a preset program, including:
  • the phase I reaction includes at least one of oxidation reaction, reduction reaction and hydrolysis reaction;
  • the oxidation reaction includes C-hydroxylation, N-hydroxylation, N-dealkylation, O-dealkylation, At least one of oxidative deamination, oxidation of N, S, P, desulfidation, dehalogenation, alcohol oxidation and aldehyde oxidation;
  • the reduction reaction includes at least one of aldehyde reduction, azo reduction and nitro reduction.
  • the hydrolysis reaction includes at least one of ester hydrolysis, amide hydrolysis and epoxide hydrolysis;
  • the Phase II reaction includes at least one of glucosylation, glucuronic acid conjugation, sulfation, methylation, acetylation, amino acid conjugation, glutathione conjugation, fatty acid conjugation, and condensation reaction; the Phase II reaction
  • the reaction may also include customized metabolic pathways or modification types, which are not specifically limited by this application, as long as the purpose of this application can be achieved.
  • the results of the phase I reaction correspond to the changes in the database data parameters, and then map to the structural modifications, such as mass number.
  • An increase of 16 Da may be due to an oxidation reaction, and an increase in mass number of 2 Da may be due to a reduction reaction;
  • the phase II reaction is also the same as above.
  • the mass number change value is different, and different binding groups can be inferred; for example, glucuronic acid binding, the mass number increases by 176Da.
  • the prototype compound undergoes a phase I reaction to obtain a phase I metabolite, and the structural changes between the phase I metabolite and the prototype compound are reflected in the mass change value; wherein the mass number increases by 16 Da, and the phase I metabolite
  • the reaction is an oxidation reaction; the mass number increases by 2 Da, and the phase I reaction is a reduction reaction;
  • the prototype compound or the phase I metabolite undergoes the phase II reaction to obtain the phase II metabolite.
  • the structural changes between the phase II metabolite and the prototype compound are reflected in the mass change value; among them, the mass number increases by 162 Da, and the phase II reaction is Glycosylation; the mass number increases by 176 Da, the phase II reaction is glucuronic acid conjugation; the mass number increases by 80 Da, the phase II reaction is sulfation; the mass number increases by 42 Da, the phase II reaction is acetylation; the mass number increases by 14 Da, the phase II reaction It is methylation; the mass number increases by 57 Da, and the phase II reaction is glycine binding; the mass number increases by 289 Da, and the phase II reaction is glutathione binding.
  • the type of the unknown toxic prototype compound in the sample to be tested can be quickly and accurately obtained; according to the obtained differences Comparing chromatography and mass spectrometry data with the metabolite database, through matching screening, the types of metabolites in the sample to be tested can be quickly and accurately obtained, and then the types of unknown toxic prototype compounds in the sample to be tested can be determined.
  • This application uses a prototype compound database combined with a metabolite database to identify unknown poisons in the samples to be tested, which can effectively reduce false positive and false negative results, making the identification results more accurate;
  • the database is based on the constructed prototype compound database including nearly 9000 compounds and their chromatography and mass spectrometry data, and/or including the metabolites and their chromatography and mass spectrometry data derived from nearly 9000 compounds through metabolic reaction models.
  • the poisoning drug database of the metabolite database enables a more comprehensive determination of the types of unknown poisons in the samples to be tested.
  • the screening conditions include: the precise molecular mass deviation range is ⁇ 10mDa, the retention time deviation range is ⁇ 0.5min; the response intensity of the differential substance is ⁇ 100 , the total number of product ion fragments of the differential substance is ⁇ 2.
  • the chromatography and mass spectrometry data of the differential substance are matched and screened with the poisoning drug database to determine the type of unknown poison in the sample to be tested;
  • the screening conditions for the differential substance include: the differential substance The response intensity is ⁇ 100, and the total number of product ion fragments of the differential substance is ⁇ 2;
  • the screening conditions include: the precise molecular mass of the differential substance and the precise molecular mass of the compound or metabolite in the poisoning drug database The deviation range is ⁇ 10mDa, and the deviation range between the retention time of the difference substance and the retention time of the compound or metabolite in the poisoning drug database is ⁇ 0.5min; among them, when the compound or metabolite in the poisoning drug database does not have a retention time
  • the screening conditions include: the deviation range between the precise molecular mass of the differential substance and the precise molecular mass of the compounds or metabolites in the poisoning drug database is ⁇ 10 mDa.
  • the response intensity of the differential substance can be increased as needed to narrow the possible range of unknown poisons.
  • This application has no special limitations on it, as long as the purpose of this application can be achieved.
  • the The response intensity of the differential substance can be set to be greater than or equal to 1000, 2000, 3000, and 5000 in sequence; the deviation range of the precise molecular mass and the deviation range of the retention time can be narrowed as needed to narrow the range of possible types of unknown poisons.
  • the screening conditions include: the precise molecular mass deviation range is ⁇ 5mDa, the retention time deviation range is ⁇ 0.1min; the response intensity of the differential substance is ⁇ 5000 ; Wherein, when there is no retention time data for compounds or metabolites in the poisoning drug database, the screening conditions include: the precise molecular mass of the difference substance and the precise molecular mass of the compound or metabolite in the poisoning drug database.
  • the deviation range is ⁇ 5mDa.
  • sample solution to be tested and the blank sample solution are detected using an ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometer to obtain the chromatographic and mass spectrometric data of the compounds in the sample to be tested and the blank sample;
  • the chromatographic conditions of the ultra-high performance liquid chromatography include:
  • Chromatographic column octadecylsilane bonded silica gel chromatographic column; mobile phase: phase A is 0.8-1.2mol/L ammonium acetate aqueous solution, phase B is 0.8-1.2mol/L ammonium acetate methanol solution; use volume fraction 1 -98% phase A, 2-99% phase B, gradient elution; column temperature: 43-47°C; flow rate: 0.4-0.5mL/min; injection volume: 1-5 ⁇ L;
  • the mass spectrometry conditions of the quadrupole time-of-flight mass spectrometer include:
  • MS E continuum mode electrospray ion source, positive and negative ion detection modes, mass spectrometry parameters include:
  • the chromatographic conditions of the ultra-high performance liquid chromatography include:
  • phase A is 1mol/L ammonium acetate aqueous solution (volume ratio of ammonium acetate and water is 1:99, solution pH 5.0)
  • phase B is 1mol/L ammonium acetate methanol solution (volume ratio of ammonium acetate and methanol is 1:99, solution pH 5.0); gradient elution includes: 0-0.25min, 2%B; 0.25-12.25min, 2-99%B ;12.25-13.00min, 99%B; 13.00-13.01min, 99-2%B; 13.01-17.00min, 2%B;
  • the mass spectrometry conditions of the quadrupole time-of-flight mass spectrometer include:
  • MS E continuum mode electrospray ion source, positive and negative ion detection modes, mass spectrometry parameters include:
  • the low-channel collision energy is 6eV; the high-channel collision energy is 20-30eV; the mass scanning range is 50-1200m/z.
  • leucine-enkephalin (5ng/mL) is used as a calibration solution throughout the entire analysis to determine whether there is a deviation in the mass axis of the instrument and to control the accuracy of the instrument.
  • This application uses an ultra-high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometer.
  • the detection method is determined and no additional method exploration is required.
  • the experimental operation is fast, the output of sample collection results is stable, and the characteristic information of the compound such as accurate molecular weight and retention is obtained.
  • Chromatography and mass spectrometry data such as time, isotope distribution, and product ion fragments are highly specific, and can detect thousands of compounds simultaneously in one injection, and can determine the type of unknown poisons within 20 minutes, achieving comprehensive, fast, stable, and Accurately identify the types of unknown poisons in samples to be tested.
  • the specific experimental instrument can also be an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer.
  • This application has no special limitations on this, as long as it can achieve the purpose of this application.
  • This application provides a fast, comprehensive and stable identification method for unknown poisons, based on the poisoning drug database of this application, and based on the matching screening of the chromatography and mass spectrometry data of the obtained differential substances and the constructed poisoning drug database, to achieve rapid and Comprehensively, stably and accurately identify the types of unknown poisons; when the poisoning drug database is used in clinical practice, it can be constructed only once, retaining the database, realizing reuse of the database, greatly improving the efficiency of identification of unknown poisons, and improving clinical screening. Speed and accuracy in detecting unknown poison types.
  • the second aspect of this application provides the use of identifying unknown poisons with mass numbers in the range of 50-1200 within 20 minutes according to the identification method provided by the first aspect of this application.
  • This application provides a rapid, comprehensive and stable identification method for unknown poisons, based on a prototype compound database including nearly 9,000 compounds and their chromatography and mass spectrometry data, and a metabolic database including nearly 9,000 compounds derived from metabolic reaction models. Metabolite database of substances and their chromatography and mass spectrometry data.
  • the flow chart of the identification method is shown in Figure 1. Through the detection method of ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry of the present application, the sample to be tested is detected.
  • This application provides a rapid, comprehensive and stable identification method of unknown poisons, which has served 20 clinical cases and accurately identified the types of exogenous poisons in poisoned patients. See Table 2, which provides a theory for the selection of clinical treatment plans in a timely manner. Based on this, precise assistance in patient rescue was achieved.
  • Preparation of the sample solution to be tested Take the whole blood sample to be tested from the patient with unknown pesticide poisoning (6 hours after taking the drug), centrifuge at 4000 rpm for 10 minutes, transfer the supernatant to a clean centrifuge tube, and obtain the plasma sample; accurately pipette 100 ⁇ L of the plasma sample , put in a 1.5mL centrifuge tube, add 300 ⁇ L of methanol, vortex for 3 minutes, centrifuge at 13200 rpm for 10 minutes, take the upper layer solution to obtain the sample solution to be tested, and take 100 ⁇ L of the sample solution to be tested into the injection bottle with a lined tube for sample injection For detection, the injection volume was 2 ⁇ L.
  • Preparation of blank sample solution Take a healthy human whole blood sample and prepare a blank sample solution using the same method as the preparation of the sample solution to be tested.
  • the chromatographic conditions of the ultra-high performance liquid chromatography include:
  • phase A is 1mol/L ammonium acetate aqueous solution (volume ratio of ammonium acetate and water is 1:99, solution pH 5.0)
  • phase B is 1mol/L ammonium acetate methanol solution (volume ratio of ammonium acetate and methanol is 1:99, solution pH 5.0); gradient elution includes: 0-0.25min, 2%B; 0.25-12.25min, 2-99%B ;12.25-13.00min, 99%B; 13.00-13.01min, 99-2%B; 13.01-17.00min, 2%B;
  • the mass spectrometry conditions of the quadrupole time-of-flight mass spectrometer include:
  • MS E continuum mode electrospray ion source, positive and negative ion detection modes, mass spectrometry parameters include:
  • the low-channel collision energy is 6eV; the high-channel collision energy is 20-30eV; the mass scanning range is 50-1200m/z.
  • Leucine-enkephalin (5ng/mL) is used as a calibration solution throughout the analysis to determine whether there is any deviation in the mass axis of the instrument.
  • the traditional Chinese medicine ingredient database containing 6416 compounds and their chromatography and mass spectrometry data By importing the traditional Chinese medicine ingredient database containing 6416 compounds and their chromatography and mass spectrometry data, the poison ingredients and pesticide ingredients database containing 2302 compounds and their chromatography and mass spectrometry data, the amino acid database containing 23 compounds and their chromatography and mass spectrometry data, and Database of sedative drug ingredients, psychotropic drugs and non-steroidal anti-inflammatory drugs including chromatographic and mass spectrometric data of 46 compounds detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry using standards , construct a prototype compound database.
  • the binary comparison analysis is the component of the sample to be tested and the blank reference component, and compare the data. , remove the real-time dynamic background, and obtain the chromatography and mass spectrometry data of the differential substance; wherein, the chromatograms of the sample to be tested and the blank sample are as shown in Figure 2, where the upper part is the sample to be tested and the lower part is the blank sample; the differential substance The chromatogram is shown in Figure 3.
  • the obtained chromatography and mass spectrometry data of the difference substance are matched and screened with the prototype compound database of step (2).
  • the screening conditions include: the precise molecular mass of the difference substance and the precise molecular mass of the compound in the prototype compound database.
  • the deviation range is ⁇ 10mDa, and the deviation range between the retention time of the difference substance and the retention time of the compound in the prototype compound database is ⁇ 0.5min;
  • the screening conditions for the difference substance include the response intensity of the difference substance ⁇ 100, the total number of product ion fragments of the differential substance is ⁇ 2, and the results of the types of unknown poisons in the sample to be tested are shown in Table 3, where the observed mass is the exact molecular mass of the differential substance, and the mass number deviation is the accurate For molecular mass deviation, the observed retention time is the retention time of the difference compound, and the expected retention time is the retention time of the prototype compound in the database.
  • the top five compounds are diflufenzopyr, oxathiapiprolin, dazomet, pyribenzoxim, and afoxolaner, among which the exact molecular mass deviation of flufenac is the smallest, which is -0.1mDa, and the retention time is consistent with the retention time of the prototype compound, indicating that its matching result is the most accurate, so it is initially judged that the patient is taking flufenac Cause poisoning, and the chromatogram of flufenpyrazone in the sample to be tested is shown in Figure 4.
  • the mass spectra of flufenpyrzone at low channel collision energy and high channel collision energy are shown in Figure 5. Its structural formula is as follows :
  • flufenpyrazone (molecular formula C 15 H 12 F 2 N 4 O 3 ) is a semicarbazide herbicide that can be used to control many broadleaf weeds and grass weeds. Its mechanism of action is auxin.
  • Transfer inhibitor suitable for cereal crops, corn, lawns and non-cultivated land; the pure crystal is an off-white odorless solid with a melting point of 135.5°C, a relative density of 0.24 (25°C), and a solubility in water (25°C, mg/mL) of 63 (PH5 ), 5850 (PH7), 10546 (PH9); Toxicity: LD50 (mg/kg): Rat acute oral exposure is greater than 5000, acute dermal exposure is greater than 5000; fish poison LC50 (96h, mg/L): rainbow trout 106, large Sunfish are larger than 135.
  • the poison ingredients and pesticide ingredient database containing 2302 compounds and their chromatography and mass spectrometry data
  • the amino acid database containing 23 compounds and their chromatography and mass spectrometry data
  • a database of ingredients for sedative drugs, psychotropic drugs and non-steroidal anti-inflammatory drugs including chromatographic and mass spectrometric data of 46 compounds detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry using standards. , construct a prototype compound database.
  • a metabolic reaction model is constructed by setting metabolic reaction pathways.
  • the above-mentioned prototype compounds are deduced through the metabolic reaction model, chromatographic and mass spectrometric data of metabolites are obtained, and a metabolite database is constructed; wherein, the metabolic reaction includes phase I reaction and phase II reaction; so
  • the above-mentioned phase I reaction includes oxidation reactions of hydroxylation, N, O-dealkylation, oxidative deamination, oxidation of N, S, P, alcohol oxidation and aldehyde oxidation, and is dominated by oxidation reactions; including aldehyde reduction, Reduction reactions of azo reduction and nitro reduction; hydrolysis reactions including ester hydrolysis, amide hydrolysis and epoxide hydrolysis;
  • the phase II reaction includes glucuronic acid conjugation, sulfate ester conjugation, acetylation, methylation, glycine binding, glutathione binding, etc. as well as custom metabolic pathways or modification types.
  • the obtained chromatography and mass spectrometry data of the difference substance are matched and screened with the prototype compound database of step (2).
  • the screening conditions include: the precise molecular mass of the difference substance and the precise molecular mass of the compound in the prototype compound database.
  • the deviation range is ⁇ 10mDa, and the deviation range between the retention time of the difference substance and the retention time of the compound in the prototype compound database is ⁇ 0.5min;
  • the screening conditions for the difference substance include the response intensity of the difference substance ⁇ 100, the total number of product ion fragments of the differential substance is ⁇ 2, and the results of the types of unknown poisons in the sample to be tested are shown in Table 4, where the observed mass is the exact molecular mass of the differential substance, and the mass number deviation is the accurate Molecular mass deviation, the observed retention time is the retention time of the difference substance.
  • the gray box at 406.06452 is the isotope mass spectrum peak of scopolamine hydrobromide + Na; the chromatogram obtained for phenobarbital is shown in Figure 11, and the chromatogram of phenobarbital under high channel collision energy is obtained
  • the mass spectrum is shown in Figure 12, including the phenobarbital fragment ion structure and characteristic fragment ion mass number; the mass spectrum of phenobarbital under low channel collision energy is shown in Figure 13, in which the gray box at 255.06467 is Isotope mass spectrum peak of phenobarbital + Na.
  • the obtained chromatography and mass spectrometry data of the differential substance are then matched and screened with the metabolite database of step (2).
  • the screening conditions include: the precise molecular mass of the differential substance and the precise molecular mass of the metabolite in the metabolite database.
  • the deviation range of molecular mass is ⁇ 5mDa;
  • the screening conditions for the differential substance include the response intensity of the differential substance ⁇ 5000, the total number of product ion fragments of the differential substance ⁇ 2, and the results of the types of metabolites in the sample to be tested are obtained
  • the observed mass is the exact molecular mass of the differential substance
  • the mass deviation is the exact molecular mass deviation
  • the observed retention time is the retention time of the differential substance.
  • Example 2 Take the whole blood sample to be tested from the poisoned patient in Example 2 (26 hours after taking the drug), and prepare the sample solution to be tested.
  • the preparation method is the same as in Example 1.
  • the screening conditions include: the deviation range between the precise molecular mass of the differential substance and the precise molecular mass of the metabolite in the metabolite database is ⁇ 10mDa;
  • the screening conditions for the differential substance include the response intensity of the differential substance ⁇ 100, the total number of product ion fragments of the differential substance ⁇ 2, and the results of the types of metabolites in the sample to be tested are shown in Table 6, where the observed quality The number is the precise molecular mass of the differential substance, the mass number deviation is the precise molecular mass deviation, and the observed retention time is the retention time of the differential substance.
  • the gray box at 444.04825 is the isotope mass spectrum peak of scopolamine hydrobromide+2 ⁇ (-H2)+C2H2O; the obtained scopolamine hydrobromide+2
  • the mass spectrum of ⁇ (-H2)+C2H2O under high channel collision energy is shown in Figure 21, including the fragment ion structure and characteristic fragment ion mass number of scopolamine hydrobromide+2 ⁇ (-H2)+C2H2O.
  • this application provides a fast, comprehensive and stable identification method for unknown poisons, based on the constructed prototype compound database including nearly 9000 compounds and their chromatography and mass spectrometry data, and/or including nearly 9000 compounds
  • the poisoning drug database of the metabolite database derived from the metabolic reaction model and its chromatographic and mass spectrometric data is detected and obtained through the ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry detection method of the present application.
  • the chromatography and mass spectrometry data of the compounds in the sample and blank sample are compared to obtain the chromatography and mass spectrometry data of the difference substance.
  • the content of the compounds in the sample to be tested is determined.
  • the type of unknown poisons can be quickly, effectively, comprehensively, accurately and stably identified.
  • the method has the advantages of simplicity, high sensitivity, fast analysis speed, and high accuracy, and can be used in clinical applications to quickly and accurately screen. Detect exogenous substances in the patient's body, provide timely theoretical basis for the selection of clinical treatment plans, and accurately assist patient rescue.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Computing Systems (AREA)
  • Theoretical Computer Science (AREA)
  • Library & Information Science (AREA)
  • Databases & Information Systems (AREA)
  • Software Systems (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

一种快速、全面、稳定的未知毒物的鉴定方法，基于包括近9000种化合物及其色谱、质谱数据的原型化合物数据库，和/或，包括近9000种化合物经代谢反应模型所推导获得的代谢物及其色谱、质谱数据的代谢物数据库的中毒药物数据库，通过超高效液相色谱-四级杆-飞行时间串联质谱的检测方法，检测获得待测样品和空白样品中化合物的色谱、质谱数据，经比对获取差异物的色谱、质谱数据，根据获得的差异物的色谱、质谱数据与中毒药物数据库的匹配筛查，确定待测样品中未知毒物的种类，从而实现快速、有效、全面、准确、稳定地鉴定未知毒物的种类。

Description

一种快速、全面、稳定的未知毒物的鉴定方法
本申请要求于2022年4月8日提交中国专利局、申请号为202210365095.1发明名称为“一种快速、全面、稳定的未知毒物的鉴定方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及毒物分析技术领域，特别是涉及一种快速、全面、稳定的未知毒物的鉴定方法。
背景技术
每年世界各国都会发生数以百万计的药物及毒物中毒事件，中毒的发生在日常生活中具有频率高、来势凶、救治紧急等特点；同时毒物来源广泛，如毒物接触、误食或服药过量等，造成中毒的食物或化学药品成分多数并不单一，分析目标物的不定性以及毒物种类的广泛性导致毒物鉴定更为复杂。目前，临床中毒症状多不典型，有些病人就诊前已经昏迷，临床医生难以诊治，而缺乏快速、有效、全面、准确的中毒物质鉴定技术是中毒患者因误诊或治疗不及时而造成不同程度伤害的重要原因。因此建立快速、全面、准确的未知毒物分析鉴定方法，以帮助临床医生或法医快速准确地筛查出患者体内的外源性中毒物质，直接影响临床对症诊疗方案的选择，在临床急救中至关重要。
以往中毒患者抢救，医生多根据病人家属提供的线索、症状和既往经验确定中毒物质及抢救方案，具有一定的局限性。目前，免疫法、气相色谱、质谱联用法、高效液相色谱法均可以用于毒物筛查，但耗时长、准确度低且可筛查化合物范围窄。以往的中毒检测方法也存在一些弊端，如多为单一成分测定，测定多为已知成分，测定时必须要有标准品；检测耗时较长，如液相色谱法用于中毒物质检测时，因人体血样中成分复杂，为了保证专属性，需要的检测时间较长；若采用免疫方法，其应用范围较窄，且需要事先构建方法学。除此之外，既往的检测手段往往聚焦于化合物本身，而忽略了化合物在体内代谢过程，容易错失真相。在国外，毒检与中毒抢救中心已成为一体，但在我国尚没有引起足够的重视，因此，将药物成分的分析和未知毒物的鉴定技术应用于临床实际，建立临床突发急发未知毒物鉴定技术将为临床应急治疗提供突破口，为临床医生的精准治疗创造最有利的依据。
发明内容
本申请的目的在于提供一种快速、全面、稳定的未知毒物的鉴定方法，实现快速、全 面、准确、稳定地鉴定未知毒物的种类。具体技术方案如下：
本申请第一方面提供了一种快速、全面、稳定的未知毒物的鉴定方法，包括：
(1)获取待测样品和空白样品中化合物的色谱、质谱数据：采用超高效液相色谱-四级杆-飞行时间串联质谱仪或超高效液相色谱-三重四极杆质谱联用仪，检测待测样品溶液和空白样品溶液，获得待测样品和空白样品中化合物的色谱、质谱数据；
(2)鉴定未知毒物：将所述待测样品和空白样品中化合物的色谱、质谱数据进行二元比对分析，去除实时动态背景，获得差异物的色谱、质谱数据；将所述差异物的色谱、质谱数据与所述中毒药物数据库进行匹配筛查，确定待测样品中未知毒物的种类；
其中，所述色谱、质谱数据，包括保留时间、精确分子质量、同位素分布、母离子信息、子离子碎片信息、母离子谱图、子离子谱图、结构式、名称和类别中的至少一种；
所述中毒药物数据库，包括原型化合物数据库，和/或，代谢物数据库；所述原型化合物数据库，包括化合物的色谱、质谱数据；所述代谢物数据库，包括化合物经代谢反应模型推导，所获得的代谢物的色谱、质谱数据。
本申请第二方面提供了根据本申请第一方面提供的鉴定方法在20min内鉴定质量数在50-1200范围内的未知毒物的用途。
本申请提供的一种快速、全面、稳定的未知毒物的鉴定方法，基于包括近9000种化合物及其色谱、质谱数据的原型化合物数据库，和/或，包括近9000种化合物经代谢反应模型所推导获得的代谢物及其色谱、质谱数据的代谢物数据库的中毒药物数据库，通过本申请超高效液相色谱-四级杆-飞行时间串联质谱的检测方法，检测获得待测样品和空白样品中化合物的色谱、质谱数据，经比对获取差异物的色谱、质谱数据，根据获得的差异物的色谱、质谱数据与所述中毒药物数据库的匹配筛查，确定待测样品中未知毒物的种类，能够覆盖质量数在50-1200范围内的小分子化合物，检测范围覆盖性强，能够实现一次进样同时检测数千种化合物，且在20min内即可确定未知毒物的种类，实现快速、有效、全面、准确、稳定地鉴定未知毒物的种类；所述方法具有简便、灵敏度高、分辨率高、分析速度快、准确度高等优势，用于临床能够结合临床症状，缩小未知毒物范围，减少筛查时间，能够快速、准确地筛查出患者体内的外源性物质，及时为临床治疗方案的选择提供理论依据，精准助力患者抢救。
附图说明
为了更清楚地说明本发明实施例和现有技术的技术方案，下面对实施例和现有技术中 所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本申请提供的一种未知毒物的鉴定方法的流程图。
图2为实施例1待测样品和空白样品的色谱图，其中上方为待测样品，下方为空白样品。
图3为实施例1差异物的色谱图。
图4为实施例1待测样品中氟吡草腙的色谱图。
图5为实施例1待测样品中氟吡草腙在低通道碰撞能量和高通道碰撞能量下的质谱图。
图6为实施例2待测样品和空白样品的色谱图，其中上方为待测样品，下方为空白样品。
图7为实施例2差异物的色谱图。
图8为实施例2待测样品中氢溴酸东莨菪碱的色谱图。
图9为实施例2待测样品中氢溴酸东莨菪碱在高通道碰撞能量下的质谱图。
图10为实施例2待测样品中氢溴酸东莨菪碱在低通道碰撞能量下的质谱图。
图11为实施例2待测样品中苯巴比妥的色谱图。
图12为实施例2待测样品中苯巴比妥在高通道碰撞能量下的质谱图。
图13为实施例2待测样品中苯巴比妥在低通道碰撞能量下的质谱图。
图14为实施例2中苯巴比妥的代谢物苯巴比妥+O+H2+SO3的色谱图。
图15为实施例2中苯巴比妥的代谢物苯巴比妥+O+H2+SO3在低通道碰撞能量下的质谱图。
图16为实施例2中苯巴比妥的代谢物苯巴比妥+O+H2+SO3在高通道碰撞能量下的质谱图。
图17为实施例2待测样品中母离子质荷比m/z 233.093的苯巴比妥的母离子色谱图。
图18为实施例2待测样品中母离子质荷比m/z 384.081的氢溴酸东莨菪碱的母离子色谱图。
图19为实施例3中氢溴酸东莨菪碱的代谢物氢溴酸东莨菪碱+2×(-H2)+C2H2O的色谱图。
图20为实施例3中氢溴酸东莨菪碱的代谢物氢溴酸东莨菪碱+2×(-H2)+C2H2O在低 通道碰撞能量下的质谱图。
图21为实施例3中氢溴酸东莨菪碱的代谢物氢溴酸东莨菪碱+2×(-H2)+C2H2O在高通道碰撞能量下的质谱图。
具体实施方式
为使本发明的目的、技术方案、及优点更加清楚明白，以下参照附图并举实施例，对本发明进一步详细说明。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本申请第一方面提供了一种快速、全面、稳定的未知毒物的鉴定方法，包括：
(1)获取待测样品和空白样品中化合物的色谱、质谱数据：采用超高效液相色谱-四级杆-飞行时间串联质谱仪或超高效液相色谱-三重四极杆质谱联用仪，检测待测样品溶液和空白样品溶液，获得待测样品和空白样品中化合物的色谱、质谱数据；
(2)鉴定未知毒物：将所述待测样品和空白样品中化合物的色谱、质谱数据进行二元比对分析，去除实时动态背景，获得差异物的色谱、质谱数据；将所述差异物的色谱、质谱数据与所述中毒药物数据库进行匹配筛查，确定待测样品中未知毒物的种类；
其中，所述色谱、质谱数据，包括保留时间、精确分子质量、同位素分布、母离子信息、子离子碎片信息、母离子谱图、子离子谱图、结构式、名称和类别中的至少一种；
所述中毒药物数据库，包括原型化合物数据库，和/或，代谢物数据库；所述原型化合物数据库，包括化合物的色谱、质谱数据；所述代谢物数据库，包括化合物经代谢反应模型推导，所获得的代谢物的色谱、质谱数据。
在本申请中，所述待测样品溶液的制备包括：取中毒患者的待测全血样品，离心，取上清液，得到血浆样品；取血浆样品，加甲醇涡旋2-5min，离心，取上层溶液，得到待测样品溶液；所述血浆样品与所述甲醇的体积比为1:(2-4)。
优选地，所述待测样品溶液的制备包括：取中毒患者的待测全血样品，经3500-4500rpm离心8-12min，取上清液，得到血浆样品；取血浆样品，加甲醇涡旋2-5min，以12500-14000rpm离心8-12min，取上层溶液，得到待测样品溶液；所述血浆样品与所述甲醇的体积比为1:(2-4)。
示例性地，取中毒患者的待测全血样品，经4000rpm离心10min，移取上清液至一洁净离心管中，得到血浆样品；精密移取血浆样品100μL，置1.5mL离心管中，加甲醇300μL， 涡旋3min，以13200rpm离心10min，取上层溶液，得到待测样品溶液，取100μL待测样品溶液至含内衬管进样瓶中用于进样检测，进样量为2μL。
本申请中，检测待测样品溶液时，均设置了空白样品做空白对照，示例性地，可通过取健康人全血样品，采用与待测样品溶液的制备相同的方法制备健康人全血样品的空白样品溶液；采用与待测样品溶液相同的检测方法检测空白样品溶液。
本申请待测样品溶液的制备方法，采用血浆加甲醇沉淀，即能用于样品检测，且样品使用量较少，例如本申请中可采用100μL血浆即能用于检测，鉴定到未知毒物的种类。本申请待测样品溶液的制备方法简单、快速、便捷，有利于及时检测并鉴定未知毒物的种类。
在本申请中，所述二元比对分析，即开启动态背景扣除，从待测样品中成千上万个化合物中排除空白样品所包含的化合物，去除实时动态背景，获得差异物的色谱、质谱数据；同时结合中毒患者的临床症状，缩小未知毒物范围，进而减少筛查时间，该方法处理快，能够提高待测样品未知毒物鉴定的效率。
本申请中，患者血浆样品中的化合物成分复杂，采用健康人的空白血浆作为空白对照，进行实时动态背景扣除，可以减少患者血浆样品中内源性物质的干扰，缩小未知毒物的范围。
在本申请中，可根据中毒患者的留取样本时间距中毒时间选择不同的匹配筛查方法，以提高匹配效率，本申请对此没有特别的限定，只要能够实现本申请的目的即可。
在本申请第一方面的一些实施方式中，所述待测样品根据中毒患者的留取样本时间距中毒时间选择不同的匹配筛查方法，包括：
对于中毒患者的留取样本时间距中毒时间≤24h内的待测样品，所获得的差异物的色谱、质谱数据，与所述原型化合物数据库进行匹配筛查，初步确定待测样品中未知毒物的种类；再与所述代谢物数据库进行匹配筛查，进一步确定待测样品中未知毒物的种类；
对于中毒患者的留取样本时间距中毒时间＞24h的待测样品，所获得的差异物的色谱、质谱数据，与所述代谢物数据库进行匹配筛查，初步确定待测样品中未知毒物的种类；再与所述原型化合物数据库进行匹配筛查，进一步确定待测样品中未知毒物的种类。
本申请中，采用上述匹配筛查方法，可进一步提高匹配筛查的效率，使得未知毒物的鉴定更加快速、全面、准确。
本申请提供的快速、全面、稳定的未知毒物的鉴定方法，可针对不同患者服药后样品的采集时间进行分类分析，即对于服药后24h内的待测样品，首先基于精确分子质量、保 留时间、同位素分布、子离子碎片等多维度对原型化合物进行筛查，辅以代谢物鉴定进行多方面确证；对于服药后超过24h的待测样品，由于化合物原型此时可能经过代谢，待测样品中化合物原型含量低，此时通过鉴定待测样品中的代谢产物，推断未知毒物的种类。
本申请中，根据差异物的精确分子质量、保留时间、一级质谱、二级质谱信息与已经建立的包含化合物的精确分子质量、保留时间、一级质谱、二级质谱信息的中毒药物数据库进行相似度比较，对化合物碎片结构进行匹配和确认，进而完成目标化合物的筛查和确证，确定待测样品中未知毒物的种类；所述相似度比较为本领域常用的技术手段，本领域技术人员可根据实际需要选择比较的方式。
在本申请第一方面的一些实施方式中，所述鉴定方法还包括：构建所述中毒药物数据库。本申请中，所述中毒药物数据库可进行不断扩充，以增加数据库中化合物或代谢物的数据，使得所述中毒药物数据库能够可持续性的进行更新，能够进一步提高未知毒物鉴定的全面性和准确性。
在本申请第一方面的一些实施方式中，所述化合物，包括6416种中药成分，2302种毒药成分、农药成分，23种氨基酸成分，46种镇静类药物成分、精神类药物成分和非甾体抗炎药物成分。
本申请中，所述原型化合物数据库，包括可导入的商用数据库和自建数据库；本申请通过导入商用数据库和自建数据库，构建本申请的原型化合物数据库。本申请所述商用数据库，为市面上可购买使用的数据库，例如包含6416种化合物及其色谱、质谱数据的中药成分数据库，包含2302种化合物及其色谱、质谱数据的毒药成分、农药成分数据库，包含23种化合物及其色谱、质谱数据的氨基酸数据库；所述自建数据库，可包括采用标准品经超高效液相色谱-四级杆-飞行时间串联质谱仪检测获得的化合物的色谱、质谱数据的数据库，可包括自己所统计的已发表文章中已公开的化合物的色谱、质谱数据，还可采用自定义功能，例如对化合物结构式编辑，以扩充自建数据库。
本申请中，所述原型化合物数据库，可基于临床患者的症状，推断可能的毒物种类，选择性地导入商用数据库和/或自建数据库，使得本申请鉴定待测样品未知毒物的方法更具针对性，进一步提高本申请鉴定待测样品未知毒物的速度和准确性。
在本申请中，所述镇静类药物成分、精神类药物成分和非甾体抗炎药物成分包括以下化合物：奥氮平、氨磺必利、阿立哌唑、阿米替林、阿司匹林、艾司西酞普兰、布洛芬、吡罗昔康、保泰松、丙米嗪、多塞平、地西帕明、对乙酰氨基酚、氟比洛芬、氟伏沙明、 奋乃静、氟哌啶醇、氟西汀、癸氟奋乃静、喹硫平、氯丙嗪、氯氮平、罗非昔布、氯米帕明、利培酮、洛索洛芬、米安色林、吗氯贝胺、马普替林、美洛昔康、奈丁美酮、萘普生、尼美舒利、帕利哌酮、帕罗西汀、去甲替林、曲唑酮、秋水仙碱、舒必利、塞来昔布、舒林酸、舍曲林、五氟利多、异丙嗪、吲哚美辛和育亨宾中的至少一种；所述化合物的数据包括如表1所示的编号、分子量、分子式、正离子模式的母离子质荷比和负离子模式的母离子质荷比：
表1
Figure PCTCN2022091819-appb-000001
Figure PCTCN2022091819-appb-000002
在本申请中，所述自建数据库，包含采用标准品经超高效液相色谱-四级杆-飞行时间串联质谱仪检测获得的化合物的色谱、质谱数据的数据库；示例性地，采用表1中46种化合物的标准品，经超高效液相色谱-四级杆-飞行时间串联质谱仪检测，获得46种化合物的色谱、质谱数据；
所述超高效液相色谱的色谱条件包括：
色谱柱：十八烷基硅烷键合硅胶色谱柱；流动相：A相为0.8-1.2mol/L的乙酸铵水溶液，B相为0.8-1.2mol/L的乙酸铵甲醇溶液；采用体积分数1-98％A相，2-99％B相，梯度洗脱；柱温：43-47℃；流速：0.4-0.5mL/min；进样量：1-5μL；
所述四级杆飞行时间质谱的质谱条件包括：
采用飞行时间质谱MS-TOF模式，电喷雾离子源，正、负离子检测模式，质谱参数包括：
毛细管电压2-3kV；锥孔电压30-50V，离子源温度80-120℃；脱溶剂温度400-500℃；锥孔反吹氮气流速45-55L/h；脱溶剂氮气流速700-900L/h；扫描时间0.1-0.4s；扫描间隔0.01-0.02s；碰撞能量分别为10-20V、25-35V、40-50V，质量扫描为所述标准品的母离子质荷比。
优选地，所述超高效液相色谱的色谱条件包括：
色谱柱：BEH C18柱(100mm×2.1mm，1.8μm)；流动相：A相为1mol/L的乙酸铵水溶液(乙酸铵和水体积比为1:99，溶液pH为5.0)，B相为1mol/L的乙酸铵甲醇溶液(乙酸铵和甲醇体积比为1:99，溶液pH为5.0)；梯度洗脱包括：0-0.25min，2％B；0.25-12.25min， 2-99％B；12.25-13.00min，99％B；13.00-13.01min，99-2％B；13.01-17.00min，2％B；柱温：45℃；流速：0.45mL/min；进样量：2μL；自动进样器温度：4℃；
所述四级杆飞行时间质谱的质谱条件包括：
采用飞行时间质谱MS-TOF模式，电喷雾离子源，正、负离子检测模式，质谱参数包括：
毛细管电压2.5kV；锥孔电压40V，离子源温度100℃；脱溶剂温度450℃；锥孔反吹氮气流速50L/h；脱溶剂氮气流速800L/h；扫描时间0.2s；扫描间隔0.015s；碰撞能量分别为15V、30V、45V，质量扫描为所述标准品的母离子质荷比。
亮氨酸-脑啡肽(5ng/mL)作为校正液随行于整个分析中，用于判断仪器质量轴是否有偏差，控制仪器的准确度。
本申请中，所述代谢物数据库，包括化合物经代谢反应模型推导，所获得的代谢物的色谱、质谱数据；其中，可通过设定代谢反应途径构建所述代谢反应模型；所述代谢物数据库，基于原型化合物-基团-代谢反应的关联关系、代谢反应-代谢产物的关联关系，推导获得原型化合物经代谢反应产生的代谢物及其色谱、质谱数据，包括代谢物的精确分子质量、同位素分布、母离子信息、子离子碎片信息、结构式、名称和类别；基于精确分子质量、同位素分布、子离子碎片等信息多维度的鉴定待测样品中的代谢物种类。
本申请中，所述代谢反应包括Ⅰ相反应和/或Ⅱ相反应；因药物在体内的代谢可能是由Ⅰ相反应后直接排出体外，也可能是由Ⅱ相反应后直接排出体外，也有可能是Ⅰ相反应结合Ⅱ相反应后排出体外，在本申请中综合考虑Ⅰ相反应和Ⅱ相反应在人体中的代谢特征：经过Ⅰ相反应和/或Ⅱ相反应推导得到代谢物，可以进一步佐证所确定的待测样品中未知毒物的种类是确实存在的，减少假阳性的可能。
在本申请第一方面的一些实施方式中，所述设定代谢反应途径，通过预设程序实现，包括：
所述Ⅰ相反应包括氧化反应、还原反应和水解反应中的至少一种；所述氧化反应，包括C-羟化、N-羟化、N-脱烷基化、O-脱烷基化、氧化脱氨、N，S，P的氧化、去硫化、去卤素、醇氧化和醛氧化中的至少一种；所述还原反应，包括醛还原、偶氮还原和硝基还原中的至少一种；所述水解反应，包括酯水解、酰胺水解和环氧化物水解中的至少一种；
所述Ⅱ相反应包括葡糖基化、葡萄糖醛酸结合、硫酸化、甲基化、乙酰化、氨基酸结合、谷胱甘肽结合、脂肪酸结合、缩合反应中的至少一种；所述Ⅱ相反应还可以包括自定 义的代谢途径或修饰类型，本申请对此没有特别的限定，只要能够实现本申请的目的即可。
发明人在研究中发现，Ⅰ相反应后代谢物的结构变化，可根据数据参数的变化反映结构的修饰，Ⅰ相反应的结果对应到数据库数据参数的改变，进而映射到结构修饰，比如质量数增加16Da，可能是氧化反应，质量数增加2Da，可能是还原反应；
Ⅱ相反应也同上，质量数改变值不同，可以推测不同的结合基团；比如葡萄糖醛酸结合，质量数增加176Da。
在本申请的一些实施方式中，原型化合物经Ⅰ相反应后得到第Ⅰ相代谢物，第Ⅰ相代谢物与原型化合物的结构变化以质量数改变值体现；其中，质量数增加16Da，Ⅰ相反应为氧化反应；质量数增加2Da，Ⅰ相反应为还原反应；
原型化合物或第Ⅰ相代谢物经Ⅱ相反应后得到第Ⅱ相代谢物，第Ⅱ相代谢物与原型化合物的结构变化以质量数改变值体现；其中，质量数增加162Da，Ⅱ相反应为葡糖基化；质量数增加176Da，Ⅱ相反应为葡萄糖醛酸结合；质量数增加80Da，Ⅱ相反应为硫酸化；质量数增加42Da，Ⅱ相反应为乙酰化；质量数增加14Da，Ⅱ相反应为甲基化；质量数增加57Da，Ⅱ相反应为甘氨酸结合；质量数增加289Da，Ⅱ相反应为谷胱甘肽结合。
在本申请中，根据获得的差异物的色谱、质谱数据与原型化合物数据库比对，通过匹配筛查，能够快速、准确地得到待测样品中未知毒物原型化合物的种类；根据获得的差异物的色谱、质谱数据与代谢物数据库比对，通过匹配筛查，能够快速、准确地得到待测样品中代谢物的种类，进而确定待测样品中未知毒物原型化合物的种类。
本申请采用原型化合物数据库结合代谢物数据库，对待测样品中未知毒物进行鉴定，能够有效减少假阳性和假阴性的结果，使得鉴定结果更为准确；
本申请中，基于所构建的包括近9000种化合物及其色谱、质谱数据的原型化合物数据库，和/或，包括近9000种化合物经代谢反应模型所推导获得的代谢物及其色谱、质谱数据的代谢物数据库的中毒药物数据库，使得能够更全面地确定待测样品中未知毒物的种类。
在本申请第一方面的一些实施方式中，所述筛查条件包括：所述精确分子质量偏差范围为±10mDa，所述保留时间偏差范围为±0.5min；所述差异物的响应强度≥100，所述差异物的子离子碎片总数≥2。
在本申请中，将所述差异物的色谱、质谱数据与所述中毒药物数据库进行匹配筛查，确定待测样品中未知毒物的种类；对所述差异物的筛选条件包括：所述差异物的响应强度 ≥100，所述差异物的子离子碎片总数≥2；所述筛查的条件包括：所述差异物的精确分子质量与所述中毒药物数据库中化合物或代谢物的精确分子质量的偏差范围为±10mDa，所述差异物的保留时间与所述中毒药物数据库中化合物或代谢物的保留时间的偏差范围为±0.5min；其中，当中毒药物数据库中化合物或代谢物没有保留时间的数据时，所述筛查的条件包括：所述差异物的精确分子质量与所述中毒药物数据库中化合物或代谢物的精确分子质量的偏差范围为±10mDa。
进一步地，所述差异物的响应强度可根据需要进行增加，以缩小未知毒物可能的种类范围，本申请对其没有特别的限定，只要能够实现本申请的目的即可，示例性地，所述差异物的响应强度可依次设置大于等于1000、2000、3000、5000；所述精确分子质量的偏差范围和所述保留时间的偏差范围均可根据需要进行缩小，以缩小未知毒物可能的种类范围。
在本申请第一方面的一些实施方式中，所述筛查条件包括：所述精确分子质量偏差范围为±5mDa，所述保留时间偏差范围为±0.1min；所述差异物的响应强度≥5000；其中，当中毒药物数据库中化合物或代谢物没有保留时间的数据时，所述筛查的条件包括：所述差异物的精确分子质量与所述中毒药物数据库中化合物或代谢物的精确分子质量的偏差范围为±5mDa。
在本申请中，所述待测样品溶液和空白样品溶液，采用超高效液相色谱-四级杆-飞行时间串联质谱仪检测，获得待测样品和空白样品中化合物的色谱、质谱数据；
其中，所述超高效液相色谱的色谱条件包括：
色谱柱：十八烷基硅烷键合硅胶色谱柱；流动相：A相为0.8-1.2mol/L的乙酸铵水溶液，B相为0.8-1.2mol/L的乙酸铵甲醇溶液；采用体积分数1-98％A相，2-99％B相，梯度洗脱；柱温：43-47℃；流速：0.4-0.5mL/min；进样量：1-5μL；
所述四级杆飞行时间质谱的质谱条件包括：
采用全信息串联质谱连续采集模式：MS E continuum模式，电喷雾离子源，正、负离子检测模式，质谱参数包括：
毛细管电压2-3kV；锥孔电压30-50V，离子源温度80-120℃；脱溶剂温度400-500℃；锥孔反吹氮气流速45-55L/h；脱溶剂氮气流速700-900L/h；扫描时间0.1-0.4s；扫描间隔0.01-0.02s；低通道碰撞能量4-8eV；高通道碰撞能量20-30eV；质量扫描范围为50-1200m/z。
优选地，所述超高效液相色谱的色谱条件包括：
色谱柱：BEH C18柱(100mm×2.1mm，1.8μm)；流动相：A相为1mol/L的乙酸铵水溶 液(乙酸铵和水体积比为1:99，溶液pH5.0)，B相为1mol/L的乙酸铵甲醇溶液(乙酸铵和甲醇体积比为1:99，溶液pH5.0)；梯度洗脱包括：0-0.25min，2％B；0.25-12.25min，2-99％B；12.25-13.00min，99％B；13.00-13.01min，99-2％B；13.01-17.00min，2％B；柱温：45℃；流速：0.45mL/min；进样量：2μL；自动进样器温度：4℃；
所述四级杆飞行时间质谱的质谱条件包括：
采用全信息串联质谱连续采集模式：MS E continuum模式，电喷雾离子源，正、负离子检测模式，质谱参数包括：
毛细管电压2.5kV；锥孔电压40V，离子源温度100℃；脱溶剂温度450℃；锥孔反吹氮气流速50L/h；脱溶剂氮气流速800L/h；扫描时间0.2s；扫描间隔0.015s；低通道碰撞能量6eV；高通道碰撞能量20-30eV；质量扫描范围为50-1200m/z。
其中，亮氨酸-脑啡肽(5ng/mL)作为校正液随行于整个分析中，用于判断仪器质量轴是否有偏差，控制仪器的准确度。
本申请采用超高效液相色谱-四级杆-飞行时间串联质谱仪，检测方法确定，不需要额外进行方法摸索，实验操作快，样品采集结果输出稳定，得到化合物的特征信息如精确分子量、保留时间、同位素分布、子离子碎片等色谱、质谱数据专一性强，且一次进样能够同时检测数千种化合物，并能够在20min内就能确定未知毒物的种类，实现全面、快速、稳定、准确地鉴定待测样品中未知毒物的种类。
在本申请中，具体实验仪器也可以选用超高效液相色谱-三重四极杆质谱联用仪，本申请对此没有特别的限定，只要能够实现本申请的目的即可。
本申请提供的一种快速、全面、稳定的未知毒物的鉴定方法，基于本申请的中毒药物数据库，根据获得的差异物的色谱、质谱数据与构建的中毒药物数据库的匹配筛查，实现快速、全面、稳定、准确地鉴定未知毒物的种类；所述中毒药物数据库在用于临床时，可以仅需一次构建，保留该数据库，实现数据库的重复使用，大大提高未知毒物鉴定的效率，提高临床筛查未知毒物种类的速度和准确度。
本申请第二方面提供了根据本申请第一方面提供的鉴定方法在20min内鉴定质量数在50-1200范围内的未知毒物的用途。
本申请提供了一种快速、全面、稳定的未知毒物的鉴定方法，基于包括近9000种化合物及其色谱、质谱数据的原型化合物数据库，与包括近9000种化合物经代谢反应模型所推导获得的代谢物及其色谱、质谱数据的代谢物数据库，所述鉴定方法流程图如图1所 示，通过本申请超高效液相色谱-四级杆-飞行时间串联质谱的检测方法，检测获得待测样品和空白样品中化合物的色谱、质谱数据，经二元比对分析，获得差异物的色谱、质谱数据；根据获得的差异物的色谱、质谱数据与所述原型化合物数据库比对，通过匹配筛查，确定待测样品中未知毒物的种类；根据获得的差异物的色谱、质谱数据与所述代谢物数据库比对，通过匹配筛查，确定待测样品中代谢物的种类，进而确定待测样品中未知毒物的种类。
本申请提供的一种快速、全面、稳定的未知毒物的鉴定方法，服务临床20例，准确鉴定到中毒患者体内的外源性毒物种类，参见表2，及时为临床治疗方案的选择提供了理论依据，精准助力了患者抢救。
表2
Figure PCTCN2022091819-appb-000003
Figure PCTCN2022091819-appb-000004
实施例1
(1)获取待测样品和空白样品中化合物的色谱、质谱数据：
待测样品溶液制备：取未知农药中毒患者的待测全血样品(服药后6h)，经4000rpm离心10min，移取上清液至一洁净离心管中，得到血浆样品；精密移取血浆样品100μL，置1.5mL离心管中，加甲醇300μL，涡旋3min，以13200rpm离心10min，取上层溶液，得到待测样品溶液，取100μL待测样品溶液至含内衬管进样瓶中用于进样检测，进样量为2μL。
空白样品溶液制备：取健康人全血样品，采用与待测样品溶液的制备相同的方法制备空白样品溶液。
取待测样品溶液和空白样品溶液，采用超高效液相色谱-四级杆-飞行时间串联质谱仪检测，获得待测样品和空白样品中化合物的色谱、质谱数据，包括保留时间、精确分子质量、同位素分布、母离子信息、子离子碎片信息、母离子谱图、子离子谱图、结构式、名称和类别；
所述超高效液相色谱的色谱条件包括：
色谱柱：BEH C18柱(100mm×2.1mm，1.8μm)；流动相：A相为1mol/L的乙酸铵水溶液(乙酸铵和水体积比为1:99，溶液pH5.0)，B相为1mol/L的乙酸铵甲醇溶液(乙酸铵和甲醇体积比为1:99，溶液pH5.0)；梯度洗脱包括：0-0.25min，2％B；0.25-12.25min，2-99％B；12.25-13.00min，99％B；13.00-13.01min，99-2％B；13.01-17.00min，2％B；柱温：45℃；流速：0.45mL/min；进样量：2μL；自动进样器温度：4℃；
所述四级杆飞行时间质谱的质谱条件包括：
采用全信息串联质谱连续采集模式：MS E continuum模式，电喷雾离子源，正、负离子检测模式，质谱参数包括：
毛细管电压2.5kV；锥孔电压40V，离子源温度100℃；脱溶剂温度450℃；锥孔反吹氮气流速50L/h；脱溶剂氮气流速800L/h；扫描时间0.2s；扫描间隔0.015s；低通道碰撞能量6eV；高通道碰撞能量20-30eV；质量扫描范围为50-1200m/z。
亮氨酸-脑啡肽(5ng/mL)作为校正液随行于整个分析中，用于判断仪器质量轴是否有偏差。
(2)构建中毒药物数据库：
通过导入包含6416种化合物及其色谱、质谱数据的中药成分数据库，包含2302种化合物及其色谱、质谱数据的毒药成分、农药成分数据库，包含23种化合物及其色谱、质谱数据的氨基酸数据库，以及包含采用标准品经超高效液相色谱-四级杆-飞行时间串联质谱仪检测获得的46种化合物的色谱、质谱数据的镇静类药物成分、精神类药物成分和非甾体抗炎药物成分数据库，构建原型化合物数据库。
(3)鉴定未知毒物：
将步骤(1)得到的待测样品和空白样品中化合物的色谱、质谱数据进行二元比对分析，所述二元比对分析为待测样品组分与空白参比组分，进行数据比较，去除实时动态背景，获得差异物的色谱、质谱数据；其中，所述待测样品和空白样品的色谱图如图2所示，其中上方为待测样品，下方为空白样品；所述差异物的色谱图如图3所示。
将所获得的差异物的色谱、质谱数据与步骤(2)的原型化合物数据库进行匹配筛查，筛查条件包括：所述差异物的精确分子质量与所述原型化合物数据库中化合物的精确分子质量的偏差范围为±10mDa，所述差异物的保留时间与所述原型化合物数据库中化合物的保留时间的偏差范围为±0.5min；对所述差异物的筛选条件包括所述差异物的响应强度≥100，所述差异物的子离子碎片总数≥2，得到待测样品中未知毒物的种类结果如表3所示，其中，观测质量数即为差异物的精确分子质量，质量数偏差即为精确分子质量偏差，观测保留时间即为差异物的保留时间，预期保留时间即为数据库中原型化合物的保留时间。
表3
Figure PCTCN2022091819-appb-000005
从表3可以看到，按响应强度排序，前五的化合物分别为氟吡草腙(diflufenzopyr)、氟噻唑吡乙酮(oxathiapiprolin)、棉隆(dazomet)、嘧啶肟草醚(pyribenzoxim)和阿福拉纳(afoxolaner)，其中氟吡草腙的精确分子质量偏差最小，为-0.1mDa，且保留时间与原型 化合物保留时间一致，说明其匹配结果最准确，因此初步判断患者服用氟吡草腙致中毒，得到待测样品中氟吡草腙的色谱图如图4所示，氟吡草腙在低通道碰撞能量和高通道碰撞能量下的质谱图如图5所示，其结构式如下所示：
Figure PCTCN2022091819-appb-000006
查询资料可知，氟吡草腙(分子式C 15H 12F 2N 4O 3)是一种氨基脲类除草剂，可用于防除众多的阔叶杂草和禾本科杂草，作用机理为生长素转移抑制剂，适宜禾谷类作物、玉米、草坪和非耕地；纯晶为灰白色无嗅固体，熔点135.5℃，相对密度0.24(25℃)，水中溶解度(25℃，mg/mL)为63(PH5)、5850(PH7)、10546(PH9)；毒性：LD50(mg/kg)：大鼠急性经口大于5000，急性经皮大于5000；鱼毒LC50(96h，mg/L)：虹鳟106、大翻车鱼大于135。
通过与患者家属和患者本人的交流问话，进一步确证了氟吡草腙的服用，为患者抢救节省了宝贵的时间，使临床医生在抢救中有了客观依据，通过精准用药治疗，大大提高了抢救的成功率。
实施例2
(1)获取待测样品和空白样品中化合物的色谱、质谱数据：
取未知服药种类的中毒患者待测全血样品(服药后2h)，制备待测样品溶液，制备方法同实施例1。
取健康人全血样品，制备空白样品溶液，制备方法同实施例1。
取待测样品溶液和空白样品溶液，采用超高效液相色谱-四级杆-飞行时间串联质谱仪检测，所述超高效液相色谱的色谱条件和所述四级杆飞行时间质谱的质谱条件同实施例1，获得待测样品和空白样品中化合物的色谱、质谱数据，包括保留时间、精确分子质量、同位素分布、母离子信息、子离子碎片信息、母离子谱图、子离子谱图、结构式、名称和类别。
(2)构建中毒药物数据库：
通过导入包含6416种化合物及其色谱、质谱数据的中药成分数据库，包含2302种化合物及其色谱、质谱数据的毒药成分、农药成分数据库，包含23种化合物及其色谱、质 谱数据的氨基酸数据库，以及包含采用标准品经超高效液相色谱-四级杆-飞行时间串联质谱仪检测获得的46种化合物的色谱、质谱数据的镇静类药物成分、精神类药物成分和非甾体抗炎药物成分数据库，构建原型化合物数据库。
通过设定代谢反应途径构建代谢反应模型，上述原型化合物经代谢反应模型推导，获得代谢物的色谱、质谱数据，构建代谢物数据库；其中，所述代谢反应包括Ⅰ相反应和Ⅱ相反应；所述Ⅰ相反应，包括羟化、N，O-脱烷基化、氧化脱氨、N，S，P的氧化、醇氧化和醛氧化的氧化反应，并以氧化反应为主；包括醛还原、偶氮还原和硝基还原的还原反应；包括酯水解、酰胺水解和环氧化物水解的水解反应；所述Ⅱ相反应包括葡糖醛酸结合、硫酸酯结合、乙酰化、甲基化、甘氨酸结合、谷胱甘肽结合等以及自定义的代谢途径或修饰类型。
(3)鉴定未知毒物：
将步骤(1)得到的待测样品和空白样品中化合物的色谱、质谱数据进行二元比对分析，去除实时动态背景，获得差异物的色谱、质谱数据；其中，所述待测样品和空白样品的色谱图如图6所示，其中上方为待测样品，下方为空白样品；所述差异物的色谱图如图7所示。
将所获得的差异物的色谱、质谱数据与步骤(2)的原型化合物数据库进行匹配筛查，筛查条件包括：所述差异物的精确分子质量与所述原型化合物数据库中化合物的精确分子质量的偏差范围为±10mDa，所述差异物的保留时间与所述原型化合物数据库中化合物的保留时间的偏差范围为±0.5min；对所述差异物的筛选条件包括所述差异物的响应强度≥100，所述差异物的子离子碎片总数≥2，得到待测样品中未知毒物的种类结果如表4所示，其中，观测质量数即为差异物的精确分子质量，质量数偏差即为精确分子质量偏差，观测保留时间即为差异物的保留时间。根据表4可知，氢溴酸东莨菪碱的响应强度最高，为37073，观测到氢溴酸东莨菪碱的碎片离子总数为16个，同位素匹配偏差值3.17ppm，推测未知毒物的种类包括氢溴酸东莨菪碱；得到的待测样品中氢溴酸东莨菪碱的色谱图如图8所示，可见氢溴酸东莨菪碱的色谱峰对称性良好，分离度高；得到氢溴酸东莨菪碱在高通道碰撞能量下的质谱图如图9所示，包括氢溴酸东莨菪碱碎片离子结构及特征碎片离子质量数；得到氢溴酸东莨菪碱在低通道碰撞能量下的质谱图如图10所示，其中384.08251处灰色框内是氢溴酸东莨菪碱+H的同位素质谱峰，406.06452处灰色框内是氢溴酸东莨菪碱+Na的同位素质谱峰；获得苯巴比妥的色谱图如图11所示，得到苯巴比妥在高通道碰撞能量下的 质谱图如图12所示，包括苯巴比妥碎片离子结构及特征碎片离子质量数；得到苯巴比妥在低通道碰撞能量下的质谱图如图13所示，其中255.06467处灰色框内是苯巴比妥+Na的同位素质谱峰。
表4
Figure PCTCN2022091819-appb-000007
再将所获得的差异物的色谱、质谱数据与步骤(2)的代谢物数据库进行匹配筛查，筛查条件包括：所述差异物的精确分子质量与所述代谢物数据库中代谢物的精确分子质量的偏差范围为±5mDa；对所述差异物的筛选条件包括所述差异物的响应强度≥5000，所述差异物的子离子碎片总数≥2，得到待测样品中代谢物的种类结果如表5所示，其中，观测质量数即为差异物的精确分子质量，质量数偏差即为精确分子质量偏差，观测保留时间即为差异物的保留时间。根据表5，可见编号为7的苯巴比妥的代谢物响应强度最高，为857150，同时观测出编号为3、4、7的3种苯巴比妥代谢物，各苯巴比妥代谢物均观测到多种子离子碎片；可见编号为1、2、5、6、9的5种氢溴酸东莨菪碱的代谢物，响应强度均大于10000，各氢溴酸东莨菪碱代谢物均观测到多种子离子碎片；其中表5中苯巴比妥的代谢物苯巴比妥+O+H2+SO3的色谱图如图14所示，可见其色谱峰对称性良好，分离度高；得到苯巴比妥+O+H2+SO3在低通道碰撞能量下的质谱图如图15所示，其中331.05286处灰色框内是苯巴比妥+O+H2+SO3+H的同位素质谱峰；得到苯巴比妥+O+H2+SO3在高通道碰撞能量下的质谱图如图16所示，包括苯巴比妥+O+H2+SO3的碎片离子结构及特征碎片离子质量数。
表5
Figure PCTCN2022091819-appb-000008
根据以上分析，最终确认该患者服用了包括苯巴比妥和氢溴酸东莨菪碱的名为“晕动片”的苯巴比妥东莨菪碱，最终获得的中毒物质的临床特征与该患者实际的临床症状进行比对，结果一致，再次论证结果的准确性；其中待测样品中母离子质荷比m/z 233.093的苯巴比妥的母离子色谱图如图17所示，母离子质荷比m/z 384.081的氢溴酸东莨菪碱的母离子色谱图如图18所示。
实施例3
(1)获取待测样品和空白样品中化合物的色谱、质谱数据：
取实施例2中的中毒患者待测全血样品(服药后26h)，制备待测样品溶液，制备方法同实施例1。
取健康人全血样品，制备空白样品溶液，制备方法同实施例1。
取待测样品溶液和空白样品溶液，采用超高效液相色谱-四级杆-飞行时间串联质谱仪检测，所述超高效液相色谱的色谱条件和所述四级杆飞行时间质谱的质谱条件同实施例1，获得待测样品和空白样品中化合物的色谱、质谱数据，包括保留时间、精确分子质量、同位素分布、母离子信息、子离子碎片信息、母离子谱图、子离子谱图、结构式、名称和类别。
(2)保留实施例2中构建的代谢物数据库。
(3)鉴定未知毒物：
将步骤(1)得到的待测样品和空白样品中化合物的色谱、质谱数据进行二元比对分析，去除实时动态背景，获得差异物的色谱、质谱数据；将所获得的差异物的色谱、质谱数据与步骤(2)的代谢物数据库进行匹配筛查，筛查条件包括：所述差异物的精确分子质量与所述代谢物数据库中代谢物的精确分子质量的偏差范围为±10mDa；对所述差异物的筛选条件包括所述差异物的响应强度≥100，所述差异物的子离子碎片总数≥2，得到待测样品中代谢物的种类结果如表6所示，其中，观测质量数即为差异物的精确分子质量，质量数偏差即为精确分子质量偏差，观测保留时间即为差异物的保留时间。根据表6，可见观测出编号1-5的氢溴酸东莨菪碱的代谢物，编号6-8的苯巴比妥的代谢物，各代谢物均观测到多种子离子碎片；其中表6中氢溴酸东莨菪碱的代谢物氢溴酸东莨菪碱+2×(-H2)+C2H2O的色谱图如图19所示，可见其色谱峰对称性良好，分离度高；得到氢溴酸东莨菪碱+2×(-H2)+C2H2O在低通道碰撞能量下的质谱图如图20所示，其中444.04825处灰色框内是氢溴酸东莨菪碱+2×(-H2)+C2H2O的同位素质谱峰；得到氢溴酸东莨菪碱+2×(-H2)+C2H2O在高通道碰撞能量下的质谱图如图21所示，包括氢溴酸东莨菪碱+2×(-H2)+C2H2O的碎片离子结构及特征碎片离子质量数。
表6
Figure PCTCN2022091819-appb-000009
根据所获得的氢溴酸东莨菪碱代谢物和苯巴比妥代谢物的鉴定结果，确定该患者服用 了包括苯巴比妥和氢溴酸东莨菪碱的名为“晕动片”的苯巴比妥东莨菪碱。
综上，本申请提供的一种快速、全面、稳定的未知毒物的鉴定方法，基于所构建的包括近9000种化合物及其色谱、质谱数据的原型化合物数据库，和/或，包括近9000种化合物经代谢反应模型所推导获得的代谢物及其色谱、质谱数据的代谢物数据库的中毒药物数据库，通过本申请超高效液相色谱-四级杆-飞行时间串联质谱的检测方法，检测获得待测样品和空白样品中化合物的色谱、质谱数据，经比对获取差异物的色谱、质谱数据，根据获得的差异物的色谱、质谱数据与构建的中毒药物数据库的匹配筛查，确定待测样品中未知毒物的种类，从而实现快速、有效、全面、准确、稳定地鉴定未知毒物的种类，所述方法具有简便、灵敏度高、分析速度快、准确度高等优势，用于临床能够快速、准确地筛查出患者体内的外源性物质，及时为临床治疗方案的选择提供理论依据，精准助力患者抢救。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明保护的范围之内。

Claims (12)

  1. 一种快速、全面、稳定的未知毒物的鉴定方法，包括：
    (1)获取待测样品和空白样品中化合物的色谱、质谱数据：采用超高效液相色谱-四级杆-飞行时间串联质谱仪或超高效液相色谱-三重四极杆质谱联用仪，检测待测样品溶液和空白样品溶液，获得待测样品和空白样品中化合物的色谱、质谱数据；
    (2)鉴定未知毒物：将所述待测样品和空白样品中化合物的色谱、质谱数据进行二元比对分析，去除实时动态背景，获得差异物的色谱、质谱数据；将所述差异物的色谱、质谱数据与中毒药物数据库进行匹配筛查，确定待测样品中未知毒物的种类；
    其中，所述色谱、质谱数据，包括保留时间、精确分子质量、同位素分布、母离子信息、子离子碎片信息、母离子谱图、子离子谱图、结构式、名称和类别中的至少一种；
    所述中毒药物数据库，包括原型化合物数据库，和/或，代谢物数据库；所述原型化合物数据库，包括化合物的色谱、质谱数据；所述代谢物数据库，包括化合物经代谢反应模型推导，所获得的代谢物的色谱、质谱数据。
  2. 根据权利要求1所述的鉴定方法，其中，所述待测样品根据中毒患者的留取样本时间距中毒时间选择不同的匹配筛查方法，包括：
    对于中毒患者的留取样本时间距中毒时间≤24h内的待测样品，所获得的差异物的色谱、质谱数据，与所述原型化合物数据库进行匹配筛查，初步确定待测样品中未知毒物的种类；再与所述代谢物数据库进行匹配筛查，进一步确定待测样品中未知毒物的种类；
    对于中毒患者的留取样本时间距中毒时间＞24h的待测样品，所获得的差异物的色谱、质谱数据，与所述代谢物数据库进行匹配筛查，初步确定待测样品中未知毒物的种类；再与所述原型化合物数据库进行匹配筛查，进一步确定待测样品中未知毒物的种类。
  3. 根据权利要求1所述的鉴定方法，其中，所述鉴定方法还包括：构建所述中毒药物数据库。
  4. 根据权利要求1-3中任一项所述的鉴定方法，其中，所述化合物，包括6416种中药成分，2302种毒药成分、农药成分，23种氨基酸成分，46种镇静类药物成分、精神类药物成分和非甾体抗炎药物成分。
  5. 根据权利要求1-3中任一项所述的鉴定方法，其中，通过设定代谢反应途径构建所述代谢反应模型；所述代谢反应包括Ⅰ相反应和/或Ⅱ相反应。
  6. 根据权利要求5所述的鉴定方法，其中，所述设定代谢反应途径，通过预设程序 实现，包括：
    所述Ⅰ相反应包括氧化反应、还原反应和水解反应中的至少一种；所述氧化反应，包括C-羟化、N-羟化、N-脱烷基化、O-脱烷基化、氧化脱氨、N，S，P的氧化、去硫化、去卤素、醇氧化和醛氧化中的至少一种；所述还原反应，包括醛还原、偶氮还原和硝基还原中的至少一种；所述水解反应，包括酯水解、酰胺水解和环氧化物水解中的至少一种；
    所述Ⅱ相反应包括葡糖基化、葡萄糖醛酸结合、硫酸化、甲基化、乙酰化、氨基酸结合、谷胱甘肽结合、脂肪酸结合、缩合反应中的至少一种。
  7. 根据权利要求5所述的鉴定方法，其中，原型化合物经Ⅰ相反应后得到第Ⅰ相代谢物，第Ⅰ相代谢物与原型化合物的结构变化以质量数改变值体现；其中，质量数增加16Da，Ⅰ相反应为氧化反应；质量数增加2Da，Ⅰ相反应为还原反应；
    原型化合物或第Ⅰ相代谢物经Ⅱ相反应后得到第Ⅱ相代谢物，第Ⅱ相代谢物与原型化合物的结构变化以质量数改变值体现；其中，质量数增加162Da，Ⅱ相反应为葡糖基化；质量数增加176Da，Ⅱ相反应为葡萄糖醛酸结合；质量数增加80Da，Ⅱ相反应为硫酸化；质量数增加42Da，Ⅱ相反应为乙酰化；质量数增加14Da，Ⅱ相反应为甲基化；质量数增加57Da，Ⅱ相反应为甘氨酸结合；质量数增加289Da，Ⅱ相反应为谷胱甘肽结合。
  8. 根据权利要求1-3中任一项所述的鉴定方法，其中，所述筛查条件包括：所述精确分子质量偏差范围为±10mDa，所述保留时间偏差范围为±0.5min；所述差异物的响应强度≥100，所述差异物的子离子碎片总数≥2。
  9. 根据权利要求1-3中任一项所述的鉴定方法，其中，所述筛查条件包括：所述精确分子质量偏差范围为±5mDa，所述保留时间偏差范围为±0.1min；所述差异物的响应强度≥5000。
  10. 根据权利要求1-3中任一项所述的鉴定方法，其中，所述超高效液相色谱的色谱条件包括：
    色谱柱：十八烷基硅烷键合硅胶色谱柱；流动相：A相为0.8-1.2mol/L的乙酸铵水溶液，B相为0.8-1.2mol/L的乙酸铵甲醇溶液；采用体积分数1-98％A相，2-99％B相，梯度洗脱；柱温：43-47℃；流速：0.4-0.5mL/min；进样量：1-5μL；
    其中，所述梯度洗脱包括：0-0.25min，2％B；0.25-12.25min，2-99％B；12.25-13.00min，99％B；13.00-13.01min，99-2％B；13.01-17.00min，2％B。
  11. 根据权利要求1-3中任一项所述的鉴定方法，其中，所述四级杆飞行时间质谱的 质谱条件包括：
    采用全信息串联质谱连续采集模式：MS Econtinuum模式，电喷雾离子源，正、负离子检测模式，质谱参数包括：
    毛细管电压2-3kV；锥孔电压30-50V，离子源温度80-120℃；脱溶剂温度400-500℃；锥孔反吹氮气流速45-55L/h；脱溶剂氮气流速700-900L/h；扫描时间0.1-0.4s；扫描间隔0.01-0.02s；低通道碰撞能量4-8eV；高通道碰撞能量20-30eV；质量扫描范围为50-1200m/z。
  12. 根据权利要求1-11中任一项所述的鉴定方法在20min内鉴定质量数在50-1200范围内的未知毒物的用途。
PCT/CN2022/091819 2022-04-08 2022-05-10 一种快速、全面、稳定的未知毒物的鉴定方法 WO2023193323A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210365095.1A CN115015403B (zh) 2022-04-08 2022-04-08 一种快速、全面、稳定的未知毒物的鉴定方法
CN202210365095.1 2022-04-08

Publications (1)

Publication Number Publication Date
WO2023193323A1 true WO2023193323A1 (zh) 2023-10-12

Family

ID=83067560

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/091819 WO2023193323A1 (zh) 2022-04-08 2022-05-10 一种快速、全面、稳定的未知毒物的鉴定方法

Country Status (2)

Country Link
CN (1) CN115015403B (zh)
WO (1) WO2023193323A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117250300A (zh) * 2023-11-17 2023-12-19 江西中医药大学 藏药二十五味大汤丸中多成分的检测方法
CN117686622A (zh) * 2024-02-02 2024-03-12 江西中医药大学 八味齐薷散中多成分的分析方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103823008A (zh) * 2014-03-14 2014-05-28 北京市疾病预防控制中心 构建液相色谱-质谱数据库检测未知毒物的方法
JP2016003865A (ja) * 2014-06-13 2016-01-12 株式会社島津製作所 代謝物解析システム及び代謝物解析方法
CN107543874A (zh) * 2017-03-29 2018-01-05 中国检验检疫科学研究院 一种洗护用品中硝基咪唑类抗生素化学风险物质的筛查方法
CN108333286A (zh) * 2018-01-22 2018-07-27 济南市疾病预防控制中心 一种复杂基质的中毒样品中常见农药和鼠药的快速筛查和确证的方法
CN110715999A (zh) * 2019-11-08 2020-01-21 中国检验检疫科学研究院 一种玩具中200种化学风险物质的高通量筛查方法
CN113406244A (zh) * 2021-08-03 2021-09-17 四川大学 一种常见毒物筛查数据库及基于液相色谱-杆轨道阱质谱的快速筛查方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009175141A (ja) * 2007-12-27 2009-08-06 Ono Pharmaceut Co Ltd 化合物のスクリーニング方法
CN101750458B (zh) * 2009-11-05 2013-04-17 香港科技大学 一种检测人体中毒品残留的方法
CN105699565B (zh) * 2015-06-23 2017-09-22 山东出入境检验检疫局检验检疫技术中心 检测动物源食品中残留药物的方法及液质数据库
CN108593828A (zh) * 2018-02-23 2018-09-28 李水军 血浆制备卡中药物和毒物含量的检测方法
CN110658268A (zh) * 2018-06-28 2020-01-07 复旦大学 同时检测血斑中多种乙醇非氧化代谢物的方法
RU2722657C1 (ru) * 2019-06-11 2020-06-02 Автономная некоммерческая образовательная организация высшего образования Сколковский институт науки и технологий Способ идентификации веществ с использованием масс-спектрометра
CN111413444B (zh) * 2020-05-22 2021-10-22 南京大学 基于特征液质碎片匹配的环境样品中雄激素活性物质的鉴别方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103823008A (zh) * 2014-03-14 2014-05-28 北京市疾病预防控制中心 构建液相色谱-质谱数据库检测未知毒物的方法
JP2016003865A (ja) * 2014-06-13 2016-01-12 株式会社島津製作所 代謝物解析システム及び代謝物解析方法
CN107543874A (zh) * 2017-03-29 2018-01-05 中国检验检疫科学研究院 一种洗护用品中硝基咪唑类抗生素化学风险物质的筛查方法
CN108333286A (zh) * 2018-01-22 2018-07-27 济南市疾病预防控制中心 一种复杂基质的中毒样品中常见农药和鼠药的快速筛查和确证的方法
CN110715999A (zh) * 2019-11-08 2020-01-21 中国检验检疫科学研究院 一种玩具中200种化学风险物质的高通量筛查方法
CN113406244A (zh) * 2021-08-03 2021-09-17 四川大学 一种常见毒物筛查数据库及基于液相色谱-杆轨道阱质谱的快速筛查方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117250300A (zh) * 2023-11-17 2023-12-19 江西中医药大学 藏药二十五味大汤丸中多成分的检测方法
CN117250300B (zh) * 2023-11-17 2024-02-20 江西中医药大学 藏药二十五味大汤丸中多成分的检测方法
CN117686622A (zh) * 2024-02-02 2024-03-12 江西中医药大学 八味齐薷散中多成分的分析方法
CN117686622B (zh) * 2024-02-02 2024-05-14 江西中医药大学 八味齐薷散中多成分的分析方法

Also Published As

Publication number Publication date
CN115015403A (zh) 2022-09-06
CN115015403B (zh) 2023-04-18

Similar Documents

Publication Publication Date Title
WO2023193323A1 (zh) 一种快速、全面、稳定的未知毒物的鉴定方法
Gonzalez et al. LC–MS/MS method for the determination of several drugs used in combined cardiovascular therapy in human plasma
Wang et al. Analysis of six synthetic adulterants in herbal weight-reducing dietary supplements by LC electrospray ionization-MS
Langman et al. Toxicology: then and now
Adamowicz et al. A case of intoxication with a new cathinone derivative α-PiHP–A presentation of concentrations in biological specimens
Kuwayama et al. Effectiveness of saliva and fingerprints as alternative specimens to urine and blood in forensic drug testing
Kuwayama et al. Strong evidence of drug-facilitated crimes by hair analysis using LC–MS/MS after micro-segmentation
Kuwayama et al. Time-course measurements of drugs and metabolites transferred from fingertips after drug administration: usefulness of fingerprints for drug testing
Pietsch et al. A non-fatal oleander poisoning
Carlsson et al. Application of drug testing using exhaled breath for compliance monitoring of drug addicts in treatment
Skopikova et al. Matrix-free laser desorption ionization mass spectrometry as an efficient tool for the rapid detection of opiates in crude extracts of Papaver somniferum
Binder Analysis of drugs of abuse in biological fluids by liquid chromatography
Chan et al. Gas chromatographic/mass spectrometric analysis of methylphenidate (Ritalin) in serum
Liew et al. Simultaneous quantification of sildenafil and N‐desmethyl sildenafil in human plasma by UFLC coupled with ESI‐MS/MS and pharmacokinetic and bioequivalence studies in Malay population
Melnik et al. Veratrum alkaloid determination in four cases of Veratrum Aqua poisonings
Rentsch Laboratory diagnostics in acute poisoning: critical overview
Viala et al. Determination of chloroquine and monodesethylchloroquine in hair
Retzl et al. Exploring immune modulatory effects of cyclotide-enriched Viola tricolor preparations
Laurito et al. Bromazepam determination in human plasma by high‐performance liquid chromatography coupled to tandem mass spectrometry: a highly sensitive and specific tool for bioequivalence studies
Wang et al. Metabolomics study of type 2 diabetes and therapeutic effects of Tianqijiangtang-capsule using ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry
Tanna et al. LC-HRMS analysis of dried blood spot samples for assessing adherence to cardiovascular medications
CA2501861C (en) Methods and apparatus for identifying compounds in a sample
Hughs et al. Analysis of Mitragynine and Speciociliatine in Umbilical Cord by LC–MS-MS for Detecting Prenatal Exposure to Kratom
Ainiyah et al. Review of adulterated herbal products and supplements and methods of analysis
Yang et al. Determination of ten antipsychotics in blood, hair and nails: Validation of a LC–MS/MS method and forensic application of keratinized matrix analysis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22936258

Country of ref document: EP

Kind code of ref document: A1