Kit and method for screening 74 toxic drugs in blood

Abstract

The invention provides a kit and a method for establishing a local poison substance spectrum database and screening 74 poison drugs in blood. The kit comprises a mobile phase solution and a detection reagent, wherein the mobile phase solution comprises a mobile phase solution A and a mobile phase solution B which are separately stored; the standard substance solution is a standard stock solution prepared from 74 toxic drug standard substances respectively; the internal standard solution is a mixed internal standard stock solution prepared from isotope standard substances of the two toxic drugs; the quality control product is a mixed quality control stock solution prepared from 74 toxic drug standard substances; and blank serum. The kit and the method have the advantages that for 74 toxins (drugs), the drugs / toxins have high linearity (Rgt; 0.9), high sensitivity, accuracy and precision are achieved, and the detection performance is excellent.

Description

Technical Field

[0001] The invention belongs to the field of detection technology and relates to a kit and a method for screening 74 kinds of toxic drugs in blood. Background Art

[0002] Acute poisoning by drugs is a one-time or short-term exposure of the human body to poisons, which leads to pathological or functional changes in the body and corresponding clinical manifestations. The poisons of acute poisoning mainly include various therapeutic drugs, alkaloids, pesticides, rat poisons, etc. There are many types of drug poisons with different properties, and the symptoms of poisoning are often atypical, which increases the difficulty of clinical diagnosis and treatment. With the development of modern detection technology and in-depth research on poison determination, qualitative and quantitative analysis of the poisonous components of the poisoned person has become possible, which provides clinicians with an objective basis for rescue and greatly improves the success rate of rescue. In emergency poisoning detection, liquid chromatography-high resolution mass spectrometry (LC-HRMS) is a detection technology that uses high-performance liquid chromatography and high-resolution mass spectrometry in series. Among the methods for extensive screening of various drug poisons, the LC-HRMS technology for toxic drug screening and quantification has the highest accuracy, sensitivity and throughput. However, at present, on the one hand, there is a lack of mass spectrometry information database for the full spectrum characteristics of various poisons (drugs) on the market, and on the other hand, there is no kit and preparation method for extensive screening and quantification of various poisons (drugs) based on LC-HRMS.

[0003] Therefore, it is necessary to develop a kit and method with high sensitivity, accuracy, precision and excellent detection performance for a wide range of screening of various toxic drugs. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a kit for screening 74 toxic drugs in blood. For the 74 poisons (drugs), the drugs / poisons are highly linear (R>0.9) at concentration levels of 0.625-20 ng / mL or 6.25-200 ng / mL, with high sensitivity, accuracy and precision, and excellent detection performance.

[0005] The present invention is achieved through the following technical solutions:

[0006] In a first aspect of the present invention, a kit for screening 74 toxic drugs in blood is provided, the kit comprising:

[0007] Mobile phase solution: including separately stored mobile phase A solution and mobile phase B solution;

[0008] Standard solution: Standard stock solution prepared from 74 kinds of toxic drug standard substances;

[0009] Internal standard solution: a mixed internal standard stock solution prepared for toxic drug isotope standard substances;

[0010] Quality control products: mixed quality control stock solutions prepared from 74 kinds of toxic drug standard substances;

[0011] Blank serum;

[0012] Among them, the 74 toxic drugs include:

[0013] Pesticides and rodenticides: propanil, carbofuran, monocrotophos, dimethoate, isothiocyanate, methamidophos, isoproturon, oxydemeton, atrazine, acetochlor, alachlor, metsulfuron-methyl, bensulfuron-methyl, avermectin, paraquat dichloride, methomyl, ethoxyprolin, methyl parathion, coumatetrachloride, brodifacoum, brodifacoum, diphacin;

[0014] Alkaloid drugs: scopolamine, anisodamine, scopolamine, physostigmine, atropine, aconitine, hypoaconitine, mesaconitine, artemisinin, artemisinin A, veratramine, dianconitine, colchicine, sinomenine, strychnine, dimethylstrychnine, deoxyaconitine, huperzine A, camptothecin and orthoopine;

[0015] Other drugs: ondansetron, nifedipine, prazosin, nisoldipine, dexamethasone, amlodipine, nikethamide, mexiletine, ethambutol, metoprolol, promethazine, nimodipine, domperidone, sildenafil, cimetidine, propranolol, labetalol, famotidine, nitrendipine, amantadine, galantamine, phentolamine, amiodarone, propafenone, diltiazem, verapamil, warfarin, clonidine, salbutamol, esmolol, bisoprolol, dextromethorphan.

[0016] In the above technical solution, the blank serum is blank bovine serum.

[0017] In a second aspect of the present invention, a method for establishing a mass spectrometry database of 74 toxic drugs using the kit is provided, the method comprising:

[0018] Step S1, diluting the standard solution to 1±0.2 ug / mL with methanol, and collecting primary mass spectrum and secondary mass spectrum full spectrum data on a high-resolution mass spectrometer in Full MS-ddMS2 mode;

[0019] Step S2: Input the retention time, mass-to-charge ratio, isotope peak and full secondary mass spectrum of each toxic drug in the mass spectrum data into TraceFinder software to establish a local mass spectrum database of 74 toxic drugs.

[0020] Furthermore, in step S1, the chromatographic conditions and mass spectrometry conditions used in collecting the primary mass spectrometry and the secondary mass spectrometry full spectrum data include:

[0021] Chromatographic conditions: Mobile phase: A: 0.1±0.02% formic acid, 2±0.2mM ammonium formate and water, B: 0.1±0.02% formic acid, 2±0.2mM ammonium formate, volume ratio of 50±2:50±2:1 methanol / acetonitrile / water; flow rate: 0.2-0.4mL / min; injection volume: 4-6ul, chromatographic column specifications are ACQUITY UPLC BEH C18 Column, 1.7μm,2.1mm X 100mm;

[0022] Mass spectrometry conditions: Thermo Fisher Scientific liquid chromatography / quadrupole orbital trap high-resolution mass spectrometer, using ESI source, heater temperature 300°C, sheath gas flow rate 40arb, auxiliary gas flow rate 15arb, spray voltage 3.8kV, capillary temperature 320°C, s-lens RF level 60%, primary mass spectrometry scanning range: 70-1200m / z.

[0023] Furthermore, in step S2, the method for establishing a mass spectrometry database of 74 toxic drugs specifically includes:

[0024] Firstly, the compound name, CAS number, molecular formula, precise molecular weight and other information of 74 toxic drugs were entered into the database editing compound information entry in TraceFinder software; then, the retention time, mass-to-charge ratio and isotope peak unique to each of the 74 toxic drugs were obtained through the collected primary mass spectrometry data, and the retention time, mass-to-charge ratio and isotope peak were input into TraceFinder software; at the same time, the RAW file containing the primary mass spectrum and secondary mass spectrum full spectrum data was imported into mzVault software to obtain the secondary mass spectrum full spectrum unique to each toxic drug; finally, the local mass spectrum database of 74 toxic drugs was established by integrating the above retention time, mass-to-charge ratio, isotope peak, secondary mass spectrum full spectrum and other information.

[0025] In the third aspect of the present invention, a mass spectrum database obtained by the method is provided for screening 74 toxic drugs in blood.

[0026] In a fourth aspect of the present invention, a method for screening blood for 74 toxic drugs is provided, the method comprising:

[0027] The kit is used to pre-treat the sample to be tested, and mass spectrometry data is collected according to the chromatographic conditions and mass spectrometry conditions. The toxic and drug mass spectrometry database is used for comparison to determine whether the sample screening result is "positive". For the "positive" sample, further quantitative analysis is performed to determine its content.

[0028] Furthermore, the sample pretreatment step includes:

[0029] Using the kit, the quality control product is diluted with blank serum to a quality control working solution of 10-100 ng / mL, the blood sample to be tested is centrifuged, and the supernatant is taken; methanol is added to the blank serum control, the quality control working solution, and the sample supernatant, respectively, and the mixture is fully mixed and centrifuged, and the supernatant is taken and filtered through a filter membrane, and analyzed on the machine in turn.

[0030] Furthermore, the centrifugation conditions include: 10000±200g, 10±2min.

[0031] For blank serum control and quality control working solution, the volume of sample supernatant was 100ul and that of methanol was 300ul.

[0032] Furthermore, the database comparison step includes:

[0033] The mass spectrometry data collected by the on-machine analysis are imported into the TraceFinder software and compared with the toxic drug mass spectrum database. When the target toxic drug meets the following conditions: molecular weight match, isotope peak match, retention time match, and secondary mass spectrum match, it is considered to be detected in the target toxic drug sample.

[0034] Furthermore, the step of interpreting the results includes:

[0035] When the blank serum control is not detected, the quality control is detected, and the sample is detected, the screening result is considered "positive"; when the blank serum control is not detected, the quality control is detected, and the sample is not detected, the screening result is considered "negative".

[0036] Furthermore, the quantitative analysis steps of the "positive" samples include:

[0037] Step S1, using the kit, diluting the standard solution corresponding to the detected toxic drug with blank serum to prepare standard curve working solutions of four concentrations, namely, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL;

[0038] Step S2, take 100ul of each of the blank serum control, the supernatant of the sample to be tested, the quality control working solution, and the standard curve working solution, add 2ul of the internal standard solution, mix well, add 300ul of methanol, mix well and centrifuge, take the supernatant and filter through a filter membrane, analyze on the machine, set the mass-to-charge ratio of the detected toxic drugs in the Inclusion list, and collect mass spectrometry data in the Full MS-PRM mode;

[0039] Step S3: Import the collected mass spectrometry data into TraceFinder software and draw a standard curve based on the chromatographic peak area ( Fig.11 , Fig.12 , Fig.13 , Fig.14The standard working curve of 74 toxic drugs was used to calculate the concentration of the target toxic drug.

[0040] In the above technical solution, 100ul of the supernatant of the sample to be tested, the quality control working solution, and the standard curve working solution are taken respectively. The volume of the internal standard solution is 2ul. The volume of methanol is 300ul.

[0041] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0042] 1. The present invention provides a kit and method for screening 74 toxic drugs in blood, with excellent quantitative detection performance:

[0043] For 74 poisons (drugs), the drugs / poisons have high linearity (R>0.9) at the concentration level of 0.625-20ng / mL or 6.25-200ng / mL. The linearity of this project can meet the screening needs.

[0044] In addition, in the detection of quality control products spiked with three concentration levels (20, 10, and 5 ng / mL for pesticides / rat poisons / alkaloids, and 200, 100, and 50 ng / mL for other therapeutic drugs), greater than 87% of the results were between 80% and 120%; greater than 93% of the results had a recovery rate between 75% and 125%. The quantitative accuracy of this project can meet the requirements.

[0045] In addition, in the detection of quality control products spiked at three concentration levels (20, 10, and 5 ng / mL for pesticides / rat poisons / alkaloids, and 200, 100, and 50 ng / mL for other therapeutic drugs), the precision of results greater than 83% was less than 20%; the precision of results greater than 73% was less than 15%; the quantitative accuracy of this project can meet the requirements.

[0046] Since there are many types of poisons (drugs) with different properties, this kit has high sensitivity, accuracy and precision, and excellent detection performance.

[0047] 2. The kit is simple to prepare and low in cost. The kit consists of: mobile phase solution, standard solution, internal standard solution, quality control product, and blank serum. The kit is relatively simple to prepare, and the cost mainly comes from the toxic (drug) standard product, which is relatively low in cost.

[0048] 3. Simple sample pretreatment and short detection cycle. This kit has simple sample pretreatment and uses a one-step method to extract poisons (drugs). The sample pretreatment is simple and the detection cycle is short.

[0049] 4. The kit provided by the present invention is suitable for the rapid screening and quantification of 74 kinds of poisons (drugs) in blood.

[0050] At present, there is a lack of a kit and a preparation method for extensive screening and quantification of various poisons (drugs) based on liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS) in the market. The present invention relates to a mass spectrometry database for 74 kinds of poisons (drugs) and a kit for rapid screening and quantification and a preparation method thereof, which can screen and quantitatively analyze the components of poisons (drugs), which provides an objective basis for clinicians in rescue and helps to improve the success rate of poisoning treatment.

[0051] 5. The mass spectrometry database for detecting 74 kinds of toxic drugs provided by the present invention has comprehensive and rich information, covering retention time, mass-to-charge ratio, isotope peak distribution, and secondary mass spectrometry full spectrum. Currently, there is a lack of secondary mass spectrometry full spectrum database for the complete characteristics of various types of toxic (drug) substances on the market. We purchased standard products of various types of drugs and poisons, optimized the retention time, peak area, and isotope peak distribution, and established a local mass spectrometry database, and further evaluated the above indicators in actual samples: retention time, peak area, isotope peak distribution, and secondary spectra. It can be seen that the retention time deviation of actual sample detection is small, the variation of detection peak area is small, the isotope peak distribution is stable, the fragment ions are stable, and the secondary spectra are accurate and reliable, which can meet clinical screening needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a box plot of the mass spectrometry retention time distribution of 74 poisons (drugs);

[0053] Figure 2 It is a box plot of the peak area distribution of 74 poisons (drugs);

[0054] Figure 3 The box plot is the isotope peak distribution score of 74 poisons (drugs);

[0055] Figure 4 Box plot of the secondary spectra scores of 74 poisons (drugs);

[0056] Figure 5 , Figure 6 , Figure 7 Standard working curves for 74 poisons (drugs) (22 pesticides and rodenticides);

[0057] Figure 8 , Fig. 9 , Fig.10 Standard working curves for 74 poisons (drugs) (20 alkaloids);

[0058] Fig.11 , Fig.12 , Fig.13 , Fig.14 It is the standard working curve of 74 poisons (drugs) (32 therapeutic drugs).

[0059] Fig.15 The following are the chromatograms and secondary full spectra of 4 representative toxic drugs. DETAILED DESCRIPTION

[0060] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0061] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0062] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0063] The technical solution provided by the embodiment of the present invention is to solve the above technical problems, and the overall idea is as follows:

[0064] According to a typical embodiment of the present invention, a kit for screening 74 toxic drugs in blood is provided, the kit comprising:

[0065] Mobile phase solution: including separately stored mobile phase A solution and mobile phase B solution;

[0066] Standard solution: Standard stock solution prepared from 74 kinds of toxic drug standard substances;

[0067] Internal standard solution: a mixed internal standard stock solution prepared for toxic drug isotope standard substances;

[0068] Quality control products: mixed quality control stock solutions prepared from 74 kinds of toxic drug standard substances;

[0069] Blank serum: blank bovine serum;

[0070] Among them, the 74 toxic drugs include:

[0071] Pesticides and rodenticides: propanil, carbofuran, monocrotophos, dimethoate, isothiocyanate, methamidophos, isoproturon, oxydemeton, atrazine, acetochlor, alachlor, metsulfuron-methyl, bensulfuron-methyl, avermectin, paraquat dichloride, methomyl, ethoxyprolin, methyl parathion, coumatetrachloride, brodifacoum, brodifacoum, diphacin;

[0072] Alkaloid drugs: scopolamine, anisodamine, scopolamine, physostigmine, atropine, aconitine, hypoaconitine, mesaconitine, artemisinin, artemisinin A, veratramine, dianconitine, colchicine, sinomenine, strychnine, dimethylstrychnine, deoxyaconitine, huperzine A, camptothecin and orthoopine;

[0073] Other drugs: ondansetron, nifedipine, prazosin, nisoldipine, dexamethasone, amlodipine, nikethamide, mexiletine, ethambutol, metoprolol, promethazine, nimodipine, domperidone, sildenafil, cimetidine, propranolol, labetalol, famotidine, nitrendipine, amantadine, galantamine, phentolamine, amiodarone, propafenone, diltiazem, verapamil, warfarin, clonidine, salbutamol, esmolol, bisoprolol, dextromethorphan.

[0074] The present invention provides a kit for screening 74 toxic drugs in blood. By integrating standard reagents of 74 toxic drugs such as pesticides, rodenticides, alkaloids, and other types into one kit, the kit can be used to construct a local mass spectrum information database containing the full spectrum characteristics of primary and secondary mass spectra of toxic drugs for extensive screening, and can also be used to configure standard curves for subsequent accurate quantitative analysis. At the same time, all 74 toxic drug components are integrated in the quality control product of the kit, and quality control of each batch of screening and quantitative analysis can be achieved. In addition, the internal standard solution for error correction and the blank serum for matrix matching contained in the kit make screening and quantitative analysis more reliable.

[0075] The specific solution formulas are as follows:

[0076] The formula of mobile phase A was 0.1% formic acid, 2 mM ammonium formate, and water;

[0077] The formula of mobile phase B was 0.1% formic acid, 2 mM ammonium formate, and methanol / acetonitrile / water in a volume ratio of 50:50:1;

[0078] The formula of the standard solution is a single standard stock solution prepared from 74 toxic drug standard substances in methanol, with concentrations ranging from 100 to 2000ug / mL;

[0079] The formula of the internal standard solution is a mixed internal standard stock solution of atropine-d5 and warfarin-d5 isotope standard substances prepared with methanol, with a concentration of 10ug / mL;

[0080] The formula of the quality control product is a mixed quality control stock solution of 74 toxic drug standard substances prepared with methanol, with concentrations ranging from 1 to 10ug / mL;

[0081] The formula of blank serum is blank bovine serum;

[0082] Among them, the basic information of 74 toxic drugs and their specific concentrations in standard solutions and quality control products are shown in Table 1 below:

[0083] Table 1

[0084]

[0085]

[0086]

[0087]

[0088] The detection principle of the kit of the present invention is to adopt liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS) detection equipment, use the separation ability of chromatography to achieve efficient separation of target compounds, use a high-resolution mass spectrometer to collect full spectra of primary and secondary mass spectra, and establish a chromatographic mass spectrum full spectrum database containing the characteristics of 74 toxic drugs. The database is used to achieve extensive screening and accurate quantification of blood toxic drugs.

[0089] According to another typical embodiment of the present invention, a method for constructing a mass spectrometry database for screening 74 toxic drugs in blood is provided, and the specific steps include:

[0090] Step S1, after the standard solution is diluted to 1ug / mL with methanol, the primary mass spectrometry and secondary mass spectrometry full spectrum data are collected on a high-resolution mass spectrometer in FullMS-ddMS2 mode; chromatographic conditions: mobile phase: A: 0.1% formic acid, 2mM ammonium formate and water, B: 0.1% formic acid, 2mM ammonium formate, methanol / acetonitrile / water with a volume ratio of 50:50:1; flow rate: 0.3mL / min; injection volume: 5ul. Mass spectrometry conditions: Thermo Fisher Scientific liquid chromatography / quadrupole orbital trap high-resolution mass spectrometer (LC-Q-HRMS), using ESI source, heater temperature 300°C, sheath gas flow rate 40arb, auxiliary gas flow rate 15arb, spray voltage 3.8kv, capillary temperature 320°C, s-lens RF level 60%, primary mass spectrometry scanning range: 70-1200m / z;

[0091] Step S2, first, enter the compound name, CAS number, molecular formula, accurate molecular weight and other information of the 74 poisons into the database editing compound information entry in the TraceFinder software; then, obtain the retention time, mass-to-charge ratio and isotope peak unique to each of the 74 poisons through the collected primary mass spectrometry data, and input the retention time, mass-to-charge ratio and isotope peak into the TraceFinder software; at the same time, import the RAW file containing the primary mass spectrum and the secondary mass spectrum full spectrum data into the mzVault software to obtain the secondary mass spectrum full spectrum map unique to each poison; finally, based on the above retention time, mass-to-charge ratio, isotope peak, secondary mass spectrum full spectrum map and other information, a local mass spectrum database of the 74 poisons is established.

[0092] According to another typical embodiment of the present invention, a method for detecting 74 toxic drugs is provided, and the specific steps include:

[0093] Step S1, using the kit, diluting the quality control product with blank serum to a quality control working solution of 10-100 ng / mL, centrifuging the blood sample to be tested at 10000g for 10 min, and taking the supernatant; taking 100ul of the blank serum control, the quality control working solution, and the sample supernatant, respectively, adding 300ul of methanol, mixing thoroughly and centrifuging at 10000g for 10min, taking the supernatant and filtering it through a 0.22um filter membrane, and analyzing it in turn.

[0094] Step S2, importing the mass spectrometry data collected by the on-machine analysis into the TraceFinder software, and using the toxic drug mass spectrum database for comparison. When the target toxic drug meets the following conditions: molecular weight matching, isotope peak matching, retention time matching, and secondary mass spectrum matching, it is considered to be detected in the target toxic drug sample.

[0095] Step S3, interpretation of screening results: when the blank serum control is not detected, the quality control is detected, and the sample is detected, the screening result is considered "positive"; when the blank serum control is not detected, the quality control is detected, and the sample is not detected, the screening result is considered "negative".

[0096] Step S4, quantitative analysis: dilute the standard solution corresponding to the detected toxic drugs with blank serum to prepare standard curve working solutions of four concentrations, namely 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL; take 100 ul of blank serum control, supernatant of the sample to be tested, quality control working solution, and standard curve working solution, add 2 ul of internal standard solution, mix well, add 300 ul of methanol, mix well, centrifuge at 10000 g for 10 min, take the supernatant and filter it through a 0.22 um filter membrane, analyze it on the machine, set the mass-to-charge ratio of the detected toxic drugs in the Inclusion list, and collect mass spectrometry data in the Full MS-PRM mode; import the collected mass spectrometry data into TraceFinder software, draw a standard curve according to the chromatographic peak area, and calculate the concentration of the target toxic drugs.

[0097] The following is a detailed description of a kit for detecting 74 kinds of toxic drugs in the present application in conjunction with embodiments.

[0098] Example 1. Kit and method for screening 74 toxic drugs in blood

[0099] 1. Detection principle

[0100] Based on liquid chromatography-quadrupole high-resolution orbitrap mass spectrometer (LC-Q-HRMS), a full mass spectrum database of poisons (drugs) was established, and various types of drug poisons were extracted, mass spectrum data were collected and compared with the database for unknown samples. Blank samples and spiked samples were used as controls, and mass-to-charge ratio, retention time, isotope peak distribution, and secondary fragment ion / secondary mass spectrum were used as the basis for judgment. Rapid screening and quantification of 74 poisoning drugs including pesticides, rodenticides, alkaloids, therapeutic drugs, etc. were carried out.

[0101] 2. Testing instruments and software

[0102] The detection instrument was a Thermo Fisher liquid chromatography-quadrupole high-resolution orbitrap mass spectrometer (LC-Q-HRMS), the chromatograph model was Utimate 3000, and the mass spectrometer model was Q-Exactive;

[0103] The chromatographic acquisition conditions were as follows: mobile phase: A: 0.1% formic acid, 2 mM ammonium formate and water, B: 0.1% formic acid, 2 mM ammonium formate, methanol / acetonitrile / water with a volume ratio of 50:50:1; flow rate: 0.3 mL / min; injection volume: 5 ul; chromatographic column specifications: ACQUITY UPLC BEH C18 Column, 1.7μm,2.1mm X 100mm;

[0104] The mass spectrometry conditions were: Thermo Fisher Scientific liquid chromatography / quadrupole orbital trap high-resolution mass spectrometer, using ESI source, heater temperature 300°C, sheath gas flow rate 40arb, auxiliary gas flow rate 15arb, spray voltage 3.8kv, capillary temperature 320°C, s-lens RF level 60%, primary mass spectrometry scanning range: 70-1200m / z;

[0105] The database comparison software was Tracefinder 5.2, and the secondary mass spectrometry full spectrum database construction software was mzVault;

[0106] 3. A test kit for screening 74 toxic drugs in blood

[0107] The kit consists of:

[0108] The formula of mobile phase A was 0.1% formic acid, 2 mM ammonium formate, and water;

[0109] The formula of mobile phase B was 0.1% formic acid, 2 mM ammonium formate, and methanol / acetonitrile / water in a volume ratio of 50:50:1;

[0110] The formula of the standard solution is a single standard stock solution prepared from 74 toxic drug standard substances in methanol, with concentrations ranging from 100 to 2000ug / mL;

[0111] The formula of the internal standard solution is a mixed internal standard stock solution of atropine-d5 and warfarin-d5 isotope standard substances prepared with methanol, with a concentration of 10ug / mL;

[0112] The formula of the quality control product is a mixed quality control stock solution of 74 toxic drug standard substances prepared with methanol, with concentrations ranging from 1 to 10ug / mL;

[0113] The formula of blank serum is blank bovine serum;

[0114] 4. A method for constructing a mass spectrometry database for screening 74 toxic drugs in blood

[0115] 4.1 Sample preparation: dilute each standard solution in the kit to 1ug / mL with methanol;

[0116] 4.2 Full mass spectrometry acquisition: On the LC-Q-HRMS mass spectrometer, spot 50ul of sample and inject 1ul. According to the above chromatographic and mass spectrometric conditions, collect the primary mass spectrometry and secondary mass spectrometry full spectrum data in the Full MS-ddMS2 mode;

[0117] 4.3 Database construction: The compound name, CAS number, molecular formula, precise molecular weight and other information of the 74 toxic drugs were entered into the database editing compound information entry in the TraceFinder software. Then, the retention time, mass-to-charge ratio and isotope peak unique to each of the 74 toxic drugs were obtained through the collected primary mass spectrometry data, and the retention time, mass-to-charge ratio and isotope peak were entered into the TraceFinder software. At the same time, the RAW file containing the primary mass spectrum and secondary mass spectrum full spectrum data was imported into the mzVault software to obtain the secondary mass spectrum full spectrum unique to each poison. Finally, the local mass spectrum database of the 74 toxic drugs was established by integrating the above retention time, mass-to-charge ratio, isotope peak, secondary mass spectrum full spectrum and other information.

[0118] 4.4 Main information of the database: covering retention time, mass-to-charge ratio, isotope peak distribution, full secondary mass spectrometry, etc., CAS number, molecular weight, retention time range, isotope distribution score, fragment ion score and other information of all 74 drugs and poisons, see Appendix 2.

[0119] Table 2. Basic information of 74 poisons (drugs) and their retention time, peak area, isotope peak score, and secondary spectrum score.

[0120]

[0121]

[0122]

[0123]

[0124] The secondary mass spectrometry database is a database of characteristic spectra of 74 therapeutic drugs, pesticides, rodenticides and alkaloids, which is suitable for the detection of clinical high-throughput samples. Among them, the chromatograms and secondary full spectra of 4 representative toxic drugs are as follows: Fig.15 As shown:

[0125] 5A method for screening 74 toxic drugs in blood

[0126] 5.1 Specimen preparation

[0127] Collect 3-5mL of blood sample in a heparin anticoagulation tube, gently invert and mix, and send to the laboratory as soon as possible. The specimen is transported in a special sealed transport box; after confirming that the sample information is complete, number the sample, centrifuge at 3000rpm for 10min, and take the supernatant. Note: For pesticide samples, whole blood and centrifuged plasma are processed and analyzed at the same time;

[0128] Take 100ul of blank serum control, quality control product, and sample to be tested, add 300ul of methanol, shake well for about 5min, centrifuge at 12000g for 10min; take the supernatant and filter it through a 0.22um filter membrane, and the filtrate is tested.

[0129] 5.2 Loading specimens

[0130] Before analysis, the column was equilibrated with 95% mobile phase A, 5% mobile phase B, 0.3 ml / min for 10 min;

[0131] Spot 100ul of sample, place the sample bottle, edit the sample queue, load the sample in the order of blank serum control-test sample-quality control product, ensure that both the mass spectrometer and the liquid phase are in the ready state, inject 5ul of sample, submit the queue or specific row, and collect the primary mass spectrometry and secondary mass spectrometry full spectrum data in the Full MS-ddMS2 mode according to the above chromatographic and mass spectrometry conditions;

[0132] 5.3 Database comparison:

[0133] The test results were compared with the database using TraceFinder 5.2 and the results were screened. When interpreting the results, the preliminary screening results were divided into: "positive" and "negative".

[0134] When the preliminary screening result is "positive": the blank serum control is not detected, and the quality control product is detected. And the sample to be tested must meet the following conditions: ① Molecular weight matching; complete peak profile; signal-to-noise ratio>10; isotope peak distribution matching (IP(%) greater than 50); retention time matching; secondary mass spectrometry full spectrum matching (Library matching).

[0135] When the preliminary screening result is "negative": the blank serum control is not detected, the quality control product is detected. And the sample to be tested is not detected, and the following conditions are met: the molecular weight does not match, or the molecular weight matches but the chromatographic peak has no complete outline, and the signal-to-noise ratio is poor.

[0136] In order to evaluate the stability and accuracy of database comparison of blood specimens, we used the standard solution to prepare low-concentration samples with blank serum for the above 74 kinds of pesticides, rodenticides, alkaloids, therapeutic drugs, etc., of which pesticides / rodenticides / alkaloids were 20ng / mL and other therapeutic drugs were 200ng / mL. Samples were processed and tested on the machine on 7 testing days, and retention time, mass-to-charge ratio, isotope peak distribution, and secondary spectrum score were evaluated. The results are as follows:

[0137] Retention time: as attached Figure 1 As shown, the retention times of various drugs / poisons are relatively stable, ranging from 0.86 to 11.11 minutes, and the deviations are all within 30 seconds.

[0138] Peak area: as attached Figure 2 As shown, it can be seen that the peak areas of various drugs / poisons are relatively stable, and the Log2 values ​​are distributed between 21.17-29.81.

[0139] Isotope distribution: as attached Figure 3 As shown, it can be seen that the fragment ions of various drugs / poisons are relatively stable, and the fragment scores are between 98.5-100.

[0140] Secondary spectrum: as attached Figure 4 As shown in the figure, it can be seen that the full spectrum collection of the secondary spectra of various drugs / poisons is relatively stable. The secondary spectrum scores are between 83-100.

[0141] It can be seen that the retention time, peak area, isotope distribution and secondary spectra of all 74 drug poisons in this project are relatively stable and can meet the needs of clinical screening.

[0142] 5.4 Quantitative analysis

[0143] Database comparison of "positive" samples, further quantitative analysis steps include:

[0144] Dilute the standard solution corresponding to the detected toxic drug with blank serum to prepare four concentrations of standard curve working solution: 50ng / mL, 100ng / mL, 200ng / mL, 500ng / mL;

[0145] Take 100ul of blank serum control, supernatant of sample to be tested, quality control working solution, and standard curve working solution respectively, add 2ul internal standard solution, mix well, add 300ul methanol, mix well, centrifuge at 10000g for 10min, take supernatant and filter through 0.22um filter membrane, analyze on the machine, set the mass-to-charge ratio of the detected toxic drugs in the Inclusion list, and collect mass spectrometry data in the Full MS-PRM mode according to the above chromatographic and mass spectrometry conditions;

[0146] The collected mass spectrometry data were imported into TraceFinder software, and a standard curve was drawn according to the chromatographic peak area to calculate the concentration of the target toxic drug.

[0147] Test Example 1: Performance Measurement

[0148] 1. Quantitative accuracy

[0149] Blank serum was used and various drug and toxicant standards were added at three concentration levels: 20, 10, and 5 ng / mL for pesticides / rodent poisons / alkaloids, and 200, 100, and 50 ng / mL for other therapeutic drugs. Repeated tests were performed within 9 test days, and the mean of repeated tests was calculated. The percentage of the mean to the added concentration was evaluated, and the recovery rate was calculated. The results are shown in Appendix 3.

[0150] Table 3. Mean, precision and spike recovery of 74 poisons (drugs) at three concentration levels.

[0151]

[0152]

[0153]

[0154]

[0155] From Table 3, we can see that:

[0156] Level 1 (high concentration): 87.8% (65 / 74) of the results had a recovery rate between 80% and 120%; 94.6% (70 / 74) of the results had a recovery rate between 75% and 125%;

[0157] Level 2 (medium concentration): 89.2% (66 / 74) of the results had a recovery rate between 80% and 120%; 93.2% (69 / 74) of the results had a recovery rate between 75% and 125%;

[0158] Level 3 (low concentration): 89.2% (66 / 74) of the results had a recovery rate between 80% and 120%; 95.9% (71 / 74) of the results had a recovery rate between 75% and 125%;

[0159] As a screening project, the quantitative accuracy of this project can basically meet the screening needs.

[0160] 2. Quantitative precision

[0161] Using blank serum, add various drug and toxicant standards at three concentration levels, pesticides / rat poisons / alkaloids at 20, 10, 5 ng / mL, other therapeutic drugs at 200, 100, 50 ng / mL, repeat the test within 9 test days, calculate the precision of quantitative analysis, the results are shown in Appendix 3. It can be seen that:

[0162] At level 1, 83.8% (62 / 74) of the results had a precision less than 20%; 73.0% (54 / 74) of the results had a precision less than 15%;

[0163] At the second level, 85.1% (63 / 74) of the results had a precision less than 20%; 77.0% (57 / 74) of the results had a precision less than 15%;

[0164] The precision of 85.1% (63 / 74) of the results at three levels was less than 20%; the precision of 77.0% (57 / 74) of the results was less than 15%;

[0165] It can be seen that as a screening project, the quantitative accuracy of this project can meet the screening needs.

[0166] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0167] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0168] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A kit for screening 74 toxic drugs in blood, characterized in that: The kit comprises: Mobile phase solution: including separately stored mobile phase A solution and mobile phase B solution; Standard solution: Standard stock solution prepared from 74 kinds of toxic drug standard substances; Internal standard solution: a mixed internal standard stock solution prepared for toxic drug isotope standard substances; Quality control products: mixed quality control stock solutions prepared from 74 kinds of toxic drug standard substances; Blank serum; Among them, the 74 toxic drugs include: Pesticides and rodenticides: propanil, carbofuran, monocrotophos, dimethoate, isothiocyanate, methamidophos, isoproturon, oxydemeton, atrazine, acetochlor, alachlor, metsulfuron-methyl, bensulfuron-methyl, avermectin, paraquat dichloride, methomyl, ethoxyprolin, methyl parathion, coumatetrachloride, brodifacoum, brodifacoum, diphacin; Alkaloid drugs: scopolamine, anisodamine, scopolamine, physostigmine, atropine, aconitine, hypoaconitine, mesaconitine, artemisinin, artemisinin A, veratramine, dianconitine, colchicine, sinomenine, strychnine, dimethylstrychnine, deoxyaconitine, huperzine A, camptothecin and orthoopine; Other drugs: ondansetron, nifedipine, prazosin, nisoldipine, dexamethasone, amlodipine, nikethamide, mexiletine, ethambutol, metoprolol, promethazine, nimodipine, domperidone, sildenafil, cimetidine, propranolol, labetalol, famotidine, nitrendipine, amantadine, galantamine, phentolamine, amiodarone, propafenone, diltiazem, verapamil, warfarin, clonidine, salbutamol, esmolol, bisoprolol, dextromethorphan.

2. A kit for detecting 74 kinds of toxic drugs according to claim 1, characterized in that: The formula of the mobile phase A liquid is 0.1±0.02% formic acid, 2±0.2mM ammonium formate, and water; the formula of the mobile phase B liquid is 0.1±0.02% formic acid, 2mM±0.2 ammonium formate, and a volume ratio of 50±2:50±2:1 of methanol, acetonitrile, and water.

3. A kit for detecting 74 kinds of toxic drugs according to claim 1, characterized in that: The standard solution is a single standard stock solution prepared by using methanol for 74 kinds of toxic drug standard substances, and the concentration is in the range of 100-2000ug / mL; the internal standard solution is a mixed internal standard stock solution prepared by using methanol for atropine-d5 and warfarin-d5 isotope standard substances, and the concentration is 8-12ug / mL; the quality control product is a mixed quality control stock solution prepared by using methanol for 74 kinds of toxic drug standard substances, and the concentration is in the range of 1-10ug / mL.

4. A method for establishing a drug mass spectrum database using the kit according to claims 1-3, characterized in that: The method comprises: Step S1, diluting the standard solution to 1±0.2 ug / mL with methanol, and collecting primary mass spectrum and secondary mass spectrum full spectrum data on a high-resolution mass spectrometer in FullMS-ddMS2 mode; Step S2, inputting the retention time, mass-to-charge ratio, isotope peak and secondary mass spectrum of each toxic drug in the mass spectrum data into TraceFinder software to establish a local mass spectrum database of 74 toxic drugs; Wherein, in the step S1, the chromatographic conditions and mass spectrometry conditions used in collecting the primary mass spectrometry and the secondary mass spectrometry full spectrum data include: Chromatographic conditions: Mobile phase: A: 0.1±0.02% formic acid, 2±0.2mM ammonium formate and water, B: 0.1±0.02% formic acid, 2±0.2mM ammonium formate, volume ratio of 50±2:50±2:1 methanol / acetonitrile / water; flow rate: 0.2-0.4mL / min; injection volume: 4-6ul, chromatographic column specifications are ACQUITY UPLC BEH C18 Column, 1.7μm, 2.1mm X 100mm; Mass spectrometry conditions: Thermo Fisher Scientific liquid chromatography / quadrupole orbital trap high-resolution mass spectrometer LC-Q-HRMS, using ESI source, heater temperature 300°C, sheath gas flow rate 40arb, auxiliary gas flow rate 15arb, spray voltage 3.8kv, capillary temperature 320°C, s-lens RF level 60%, primary mass spectrometry scanning range: 70-1200m / z.

5. The method according to claim 4, characterized in that In step S2, the method for establishing a mass spectrum database of 74 toxic drugs specifically includes: Enter the compound names, CAS numbers, molecular formulas, and precise molecular weights of 74 toxic drugs into the database editing compound information entry in the TraceFinder software; The retention time, mass-to-charge ratio and isotope peak of each of the 74 poisons were obtained through the collected primary mass spectrometry data, and the retention time, mass-to-charge ratio and isotope peak were input into the TraceFinder software; at the same time, the RAW file containing the primary mass spectrum and the secondary mass spectrum full spectrum data was imported into the mzVault software to obtain the secondary mass spectrum full spectrum of each poison; Based on the above information such as retention time, mass-to-charge ratio, isotope peak, and full secondary mass spectrum, a local mass spectrometry database of 74 toxic drugs was established.

6. A local mass spectrometry database for screening 74 toxic drugs in blood obtained according to the method of claims 4-5.

7. A method for screening 74 toxic drugs in blood, characterized in that: The method comprises: After pre-treatment of the sample to be tested using the kit described in claims 1-3, mass spectrometry data is collected according to the chromatographic conditions and mass spectrometry conditions used in the method for establishing a toxic drug mass spectrometry database according to claim 4, and the toxic drug mass spectrometry database obtained by the method according to claim 4 is used for comparison to determine whether the sample screening result is "positive", and for samples with a "positive" screening result, further quantitative analysis is performed to determine their content.

8. The method according to claim 7, characterized in that The sample pretreatment steps include: Using the kit described in any one of claims 1 to 3, the quality control product is diluted with blank serum to a quality control working solution of 10-100 ng / mL, the blood sample to be tested is centrifuged and the supernatant is taken; methanol is added to the blank serum control, the quality control working solution, and the sample supernatant, respectively, and the mixture is fully mixed and centrifuged, the supernatant is taken and filtered through a filter membrane, and the samples are analyzed in sequence.

9. The method according to claim 7, characterized in that: The step of comparing the database comprises: The mass spectrometry data collected by the on-machine analysis is imported into the TraceFinder software, and compared with the toxic drug mass spectrum database described in claim 6. When the target toxic drug meets the following conditions: molecular weight matching, isotope peak matching, retention time matching, and secondary mass spectrum matching, it is considered to be detected in the target toxic drug sample.

10. The method according to claim 7, characterized in that The quantitative analysis step of detecting toxic drugs comprises: Step S1, using the kit described in any one of claims 1 to 3, diluting the standard solution corresponding to the detected toxic drug with blank serum to prepare standard curve working solutions of four concentrations: 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL; Step S2, adding internal standard solution to blank serum control, supernatant of sample to be tested, quality control working solution, standard curve working solution, respectively, mixing, adding methanol, mixing thoroughly, centrifuging, taking supernatant, filtering through filter membrane, analyzing on machine, setting mass-to-charge ratio of detected toxic drugs in Inclusion list, and collecting mass spectrometry data in Full MS-PRM mode; Step S3: import the collected mass spectrometry data into TraceFinder software, draw a standard curve based on the chromatographic peak area, and calculate the concentration of the target toxic drug.

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