Pretreatment method and detection method of malathion biomarker in blood

Abstract

The invention provides a pretreatment method and a detection method of a malathion biomarker in blood, and relates to the technical field of analysis and detection. The method comprises the following steps: separating dimethyl phosphoryl butyrylcholine esterase and methyl phosphoryl butyrylcholine esterase by using antibody magnetic beads, and then carrying out enzymolysis by using pepsin to generate dimethyl phosphoryl nonapeptide and methyl phosphoryl nonapeptide, so as to obtain a nonapeptide solution to be detected; carrying out protein precipitation on the blood subjected to IMS treatment, wherein supernate is to-be-detected liquid of malathion and metabolite (malathion dihydroxy acid) of malathion; according to the method, malathion is taken as a template, protein precipitation is digested by streptavidin to generate dimethyl thiophosphoryl tyrosine, to-be-detected dimethyl thiophosphoryl tyrosine solutions are obtained, and the to-be-detected solutions are subjected to subsequent UPLC-MS detection, so that comprehensive monitoring of malathion at different levels and exposure conditions at different time points is realized, the method has a wide application prospect, and the detection method is high in sensitivity and high in precision.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a pretreatment method and a detection method for malathion biomarkers in blood. Background Art

[0002] Organophosphates (OPs) are widely used as pesticides and chemical warfare agents worldwide. They inhibit acetylcholinesterase (AChE) to block acetylcholine-mediated signal transduction. AChE maintains normal nerve function by hydrolyzing the neurotransmitter acetylcholine (ACh). The inhibition of AChE by organophosphates leads to the accumulation of acetylcholine in synapses and the loss of signal transmission. Organophosphate pesticides are used to protect crops from insects and parasites, but the same mechanism of action can also be toxic to humans, potentially inducing cholinergic crisis, leading to paralysis, suffocation and death.

[0003] In recent years, various biomarkers have been used as indicators of organophosphate poisoning, such as free unbound OPs and their metabolites in blood and urine, albumin and cholinesterase adducts in blood. Different analytical methods have been developed to identify them. Traditionally, inhibition of acetylcholinesterase is detected by the Ellman activity assay, which is simple, rapid and widely used in clinical testing to measure choline production as a measure of enzyme activity. However, it requires individual enzyme baseline levels before exposure (pre-exposure levels) or normal population levels (in the absence of baseline levels) to be compared with post-exposure levels to obtain a true picture of changes in enzyme activity. Because individual enzyme baseline levels are often unavailable or vary, clinical results using normal population levels as a baseline may be inaccurate. The metabolites of hydrolyzed OPs are usually detected by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), which are more specific and sensitive than the Ellman method. However, due to the short half-life of the hydrolysis products, 90% of the hydrolysis products are eliminated from the body within 2 to 3 days after exposure. Therefore, samples must be collected within 48 hours after exposure to obtain significant levels of hydrolysis products.

[0004] Therefore, current research focuses on long-term biomarkers derived from covalent adducts of OPs with endogenous proteins, which have half-lives ranging from days to weeks. Albumin is a common protein biomarker because organophosphorus can covalently bind to Tyr411 and Cys 34 of albumin. Unlike other protein biomarkers, dimethylthiophosphonotyrosine does not age or degrade in the presence of oximes. In addition, albumin is the most abundant protein in plasma (40 mg / mL) and has a long half-life of 20 days. However, albumin reacts 500 times slower than cholinesterase with nerve agents in the blood, which limits its application. Butyylcholinesterase (BChE) is a natural OPs scavenger that is easily extracted from plasma, has a high reaction rate, and is much more abundant in serum than AChE. OPs-BChE adducts have a long half-life (8 to 12 days) and do not bind to cell membranes, so they can be easily extracted from the blood. The relative abundance and longevity of BChE in blood make it a promising target for assessing OPs exposure.

[0005] Malathion (DDVP) is one of the most effective organophosphorus insecticides, a broad-spectrum pesticide and acaricide with contact toxicity. Every year, there are 3 million cases of pesticide poisoning reported worldwide, and about 220,000 people die from pesticide poisoning. Dichlorvos is one of the most commonly used pesticides. The detection of dichlorvos poisoning mainly focuses on the original drug and metabolites. There are no reports on malathion adducts, and it is impossible to monitor long-term exposure to dichlorvos. At present, there is no report on the simultaneous detection method of malathion original drug and its metabolites, butyric acid cholinesterase adducts and dimethylthiophosphonotyrosine. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a pretreatment method and a detection method for malathion biomarkers in blood. The pretreatment method provided by the present invention can separate the malathion original drug and its metabolites, butyric cholinesterase adducts and dimethylthiophosphonotyrosine in the blood, and can achieve simultaneous detection of the malathion original drug and its metabolites, butyric cholinesterase adducts and dimethylthiophosphonotyrosine through subsequent UPLC-MS.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for pre-processing malathion biomarkers in blood, comprising the following steps:

[0009] The blood to be tested and the antibody magnetic beads are mixed, and a first incubation and magnetic separation are performed to obtain antibody-adsorbed magnetic beads and blood treated with IMS, respectively; the antibody magnetic beads are obtained by modifying magnetic beads with antibodies, and the antibodies include butyrylcholinesterase monoclonal antibodies; the antibody-adsorbed magnetic beads contain dimethylphosphonylbutyrylcholinesterase and / or methylphosphonylbutyrylcholinesterase;

[0010] The antibody-adsorbed magnetic beads and the pepsin solution are mixed, and a second incubation is performed, and solid-liquid separation is performed to obtain a nonapeptide test solution; the nonapeptides in the nonapeptide test solution include dimethylphosphonyl nonapeptide and methylphosphonyl nonapeptide;

[0011] The IMS-treated blood is mixed with acetonitrile to perform protein precipitation, thereby obtaining a protein precipitate and a malathion and its metabolite test solution, respectively; the metabolites in the malathion and its metabolite test solution include malathion dihydroxy acid;

[0012] The protein precipitate is mixed with a pronase solution, and solid-liquid separation is performed after a third incubation to obtain a dimethylthiophosphonotyrosine test solution.

[0013] Preferably, the volume ratio of the magnetic beads to the blood to be tested is 1:4-12;

[0014] The first incubation time is 1.5 to 2.5 hours, and the first incubation includes rotary incubation, and the rotation speed of the rotary incubation is 800 to 1200 r / min;

[0015] The magnetic separation obtains solid components and blood after IMS treatment respectively; the solid components are washed with washing buffer to obtain antibody-adsorbed magnetic beads.

[0016] Preferably, the ratio of the volume of the magnetic beads to the mass of pepsin in the pepsin solution is 25 μL: 0.05-0.3 mg;

[0017] The concentration of the pepsin solution is 0.5-1 mg / mL, and the solvent in the pepsin solution includes a formic acid aqueous solution;

[0018] The second incubation time is 1.5 to 2.5 hours, and the second incubation includes rotary incubation, and the rotation speed of the rotary incubation is 800 to 1200 r / min.

[0019] Preferably, the volume ratio of the IMS-treated blood to acetonitrile is 1:2.5-10;

[0020] The protein precipitation is carried out under vortex oscillation conditions, and the protein precipitation time is 5 to 10 minutes;

[0021] The method further comprises solid-liquid separation after the protein precipitation.

[0022] Preferably, the protein precipitate is first washed with acetonitrile and dried in sequence, and then mixed with the pronase solution;

[0023] The ratio of the volume of the blood after the IMS treatment to the mass of the pronase in the pronase solution is 1 mL: 0.375-2.5 mg;

[0024] The third incubation time is 1.5 to 2.5 hours, and the third incubation includes rotary incubation, and the rotation speed of the rotary incubation is 800 to 1200 r / min.

[0025] The present invention also provides a method for detecting malathion biomarkers in blood, comprising the following steps:

[0026] According to the pretreatment method described in the above technical scheme, a nonapeptide test solution, a malathion and its metabolite test solution, and a dimethylthiophosphonotyrosine test solution are obtained respectively;

[0027] The nonapeptide test solution, malathion and its metabolites test solution, and dimethylthiophosphonotyrosine test solution are subjected to UPLC-MS detection respectively to obtain the detection results of the nonapeptide, malathion and its metabolites, and dimethylthiophosphonotyrosine respectively;

[0028] The UPLC-MS detection is ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap mass spectrometry detection, and the detection conditions include ultra-high performance liquid chromatography separation conditions and mass spectrometry detection conditions;

[0029] The ultra-high performance liquid chromatography separation conditions include: the chromatographic column is Hypersil GOLD VANQUISH, the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is 0.1v / v% formic acid aqueous solution, the mobile phase B is acetonitrile, the elution method is gradient elution, and the gradient elution program includes: 0-2min, the volume fraction of the mobile phase B is 5-20%; 2-3min, the volume fraction of the mobile phase B increases from 5-20% to 50-70%; 3-5min, the volume fraction of the mobile phase B is 50-70%; 5-5.2min, the volume fraction of the mobile phase B decreases from 50-70% to 5-20%; 5.2-6min, the volume fraction of the mobile phase B is 5-20%;

[0030] The UPLC-MS detection includes qualitative detection and / or quantitative detection; the quantitative detection method includes an internal standard curve method.

[0031] Preferably, the ultra-high performance liquid chromatography separation conditions also include: column temperature of 40° C.; mobile phase flow rate of 0.3 mL / min; and injection volume of 1 to 2 μL.

[0032] Preferably, the mass spectrometry detection conditions include: using a positive and negative ion switching acquisition mode; the ion source is a heatable electrospray ion source; the positive ion mode spray voltage is 3.0 kV; the negative ion mode spray voltage is 2.5 kV; the ion transfer tube temperature is 320°C; the auxiliary gas heating temperature is 350°C; the auxiliary gas flow rate is 10 arb; the sheath gas flow rate is 35 arb; the purge gas flow rate is 0 arb; the S-lens RF voltage is 50 V; the acquisition mode is a primary mass spectrometry full scan / data-dependent secondary mass spectrometry scan;

[0033] The parameters of the first-stage mass spectrometer full scan include: resolution of 70000, scanning range of 50-300, automatic gain control of 3e6, and maximum injection time of 100ms;

[0034] The parameters of the data-dependent secondary mass spectrometry scan include: resolution of 17500, automatic gain control of 1e5, maximum injection time of 50ms, normalized collision energy of 20%, 30% or 40%; TopN of 10, number of cycles of 10, and separation window of 2.0m / z;

[0035] The mass spectrometry scanning parameters include: apex excitation of 2 to 6 seconds, dynamic exclusion time of 6 seconds, and isotope exclusion of on.

[0036] Preferably, the malathion adopts a positive ion collection mode, and the secondary ions are 98.9845m / z and 176.9889m / z; the malathion dihydroxy acid adopts a negative ion collection mode, and the secondary ions are 140.9776m / z and 156.9546m / z; the dimethylphosphonyl nonapeptide adopts a negative ion collection mode, and the secondary ions are 486.2935m / z and 776.3228m / z; the methylphosphonyl nonapeptide adopts a negative ion collection mode, and the secondary ions are 443.1898m / z and 776.3236m / z; the dimethylthiophosphonotyrosine adopts a positive ion collection mode, and the secondary ions are 289.0295m / z and 260.0504m / z.

[0037] Preferably, the linear range of malathion is 0.1 to 100 ng / mL; the linear range of malathion dihydroxy acid is 0.5 to 100 ng / mL; the linear range of dimethylphosphonyl nonapeptide is 2 to 100 ng / mL; the linear range of methylphosphonyl nonapeptide is 2 to 100 ng / mL; the linear range of dimethylthiophosphonotyrosine is 0.1 to 100 ng / mL.

[0038] The pretreatment method provided by the present invention comprises the following steps: firstly, using antibody magnetic beads (IMS) to separate dimethylphosphonobutyrylcholinesterase (DMP-BChE) and methylphosphonobutyrylcholinesterase (MxP-BChE), and then using pepsin to hydrolyze the two to generate dimethylphosphono nonapeptide (DMP-nonapeptide) and methylphosphono nonapeptide (MxP-nonapeptide), so as to obtain a nonapeptide test solution; protein precipitation is performed on the blood after IMS treatment, and the supernatant is a test solution of malathion and its metabolite (malathion dihydroxy acid, MDA); and the protein precipitate is digested by pronase to generate dimethylthiophosphonotyrosine (DMTP-Tyr), so as to obtain a dimethylthiophosphonotyrosine test solution. The pretreatment method provided by the present invention can separate the malathion original drug and its metabolites malathion dihydroxy acid, butyric acid cholinesterase adduct and dimethylthiophosphonotyrosine in the blood, and can achieve simultaneous detection of the malathion original drug and its metabolites malathion dihydroxy acid, butyric acid cholinesterase adduct and dimethylthiophosphonotyrosine through subsequent UPLC-MS. Moreover, the pretreatment method provided by the present invention is simple to operate and has good extraction and purification effects of various malathion biomarkers.

[0039] The present invention adopts ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap mass spectrometry to quantitatively analyze nonapeptide test liquid, malathion and its metabolite test liquid and dimethylthiophosphonotyrosine test liquid, and can respectively obtain the detection results of nonapeptide (dimethylphosphono nonapeptide and methylphosphono nonapeptide), malathion and its metabolite malathion dihydroxy acid, and dimethylthiophosphonotyrosine, with the characteristics of high resolution, high mass accuracy and data acquisition in two layers. Moreover, the detection method provided by the present invention has a wide linear range and good linear relationship of each malathion biomarker, low detection limit and quantitative limit, high detection sensitivity, good precision, and can detect multiple biomarkers at the same time, realizing comprehensive monitoring of exposure conditions of malathion at different levels and different time points, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the chromatogram of malathion;

[0041] Figure 2 is the chromatogram of malathion dihydroxy acid;

[0042] Figure 3 is the chromatogram of dimethylphosphonyl nonapeptide;

[0043] Figure 4 is the chromatogram of methylphosphonyl nonapeptide;

[0044] Figure 5 is the chromatogram of dimethylthiophosphonotyrosine;

[0045] Figure 6Primary mass spectrum of malathion;

[0046] Figure 7 is the primary mass spectrum of malathion dihydroxy acid;

[0047] Figure 8 is the primary mass spectrum of dimethylphosphonyl nonapeptide;

[0048] Fig. 9 is the primary mass spectrum of methylphosphonyl nonapeptide;

[0049] Fig.10 is the primary mass spectrum of dimethylthiophosphonotyrosine;

[0050] Fig.11 This is the secondary mass spectrum of malathion;

[0051] Fig.12 is the secondary mass spectrum of malathion dihydroxy acid;

[0052] Fig.13 is the secondary mass spectrum of dimethylphosphonyl nonapeptide;

[0053] Fig.14 is the secondary mass spectrum of methylphosphonyl nonapeptide;

[0054] Fig.15 This is the secondary mass spectrum of dimethylthiophosphonotyrosine. DETAILED DESCRIPTION

[0055] The present invention provides a method for pre-processing malathion biomarkers in blood, comprising the following steps:

[0056] The blood to be tested and the antibody magnetic beads are mixed, and a first incubation and magnetic separation are performed to obtain antibody-adsorbed magnetic beads and blood treated with IMS, respectively; the antibody magnetic beads are obtained by modifying magnetic beads with antibodies, and the antibodies include butyrylcholinesterase monoclonal antibodies; the antibody-adsorbed magnetic beads contain dimethylphosphonylbutyrylcholinesterase and / or methylphosphonylbutyrylcholinesterase;

[0057] The antibody-adsorbed magnetic beads and the pepsin solution are mixed, and a second incubation is performed, and solid-liquid separation is performed to obtain a nonapeptide test solution; the nonapeptides in the nonapeptide test solution include dimethylphosphonyl nonapeptide and methylphosphonyl nonapeptide;

[0058] The IMS-treated blood is mixed with acetonitrile to perform protein precipitation, thereby obtaining a protein precipitate and a malathion and its metabolite test solution, respectively; the metabolites in the malathion and its metabolite test solution include malathion dihydroxy acid;

[0059] The protein precipitate is mixed with a pronase solution, and solid-liquid separation is performed after a third incubation to obtain a dimethylthiophosphonotyrosine test solution.

[0060] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0061] The present invention mixes the blood to be tested and the antibody magnetic beads, performs a first incubation, and magnetic separation (referred to as the first magnetic separation) to obtain antibody-adsorbed magnetic beads and blood treated with IMS, respectively; the antibody magnetic beads are obtained by modifying magnetic beads with antibodies, and the antibodies include butyrylcholinesterase monoclonal antibodies; the antibody-adsorbed magnetic beads contain dimethylphosphonylbutyrylcholinesterase and / or methylphosphonylbutyrylcholinesterase.

[0062] In the present invention, the magnetic beads are preferably Protein G, the magnetic beads are preferably used in the form of a magnetic bead mixture, the magnetic bead mixture is preferably obtained by mixing the magnetic beads, AB washing buffer and washing buffer in the magnetic bead kit; the magnetic bead kit is preferably purchased from Thermo Fisher, USA. In the present invention, the volume ratio of the magnetic beads to the blood to be tested is preferably 1:4-12, and in specific embodiments it can be 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11 or 1:12. The volume of the magnetic beads in the present invention is measured by the volume of the magnetic bead mixture.

[0063] In the present invention, the temperature of the first incubation is preferably 26°C, the time of the first incubation is preferably 1.5 to 2.5 hours, and in a specific embodiment it can be 1.5 hours, 2 hours or 2.5 hours; the first incubation preferably includes rotary incubation, and the rotation speed of the rotary incubation is preferably 800 to 1200 r / min, and in a specific embodiment it can be 800 r / min, 900 r / min, 1000 r / min, 1100 r / min or 1200 r / min.

[0064] In the present invention, the first magnetic separation is preferably performed using a DynaMag-2 magnetic stand; the time of the first magnetic separation is preferably 40 to 90 seconds, and in a specific embodiment, it can be 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds or 90 seconds. In the present invention, the first magnetic separation obtains solid components and blood treated with IMS respectively; the solid components are washed with a washing buffer to obtain antibody-adsorbed magnetic beads. In the present invention, the number of washings with the washing buffer is preferably 2 to 4 times, and in a specific embodiment, it can be 2 times, 3 times or 4 times.

[0065] In the present invention, the method for preparing the antibody magnetic beads preferably comprises the following steps: mixing the magnetic beads with a butyrylcholinesterase monoclonal antibody solution, and performing a fourth incubation to obtain the antibody magnetic beads.

[0066] In the present invention, the magnetic beads preferably include before use: fully mixing the magnetic beads, AB washing buffer and washing buffer in the magnetic bead kit on a vortex mixer to obtain a magnetic bead mixture, subjecting the magnetic bead mixture to magnetic separation (referred to as the second magnetic separation), removing the supernatant to obtain magnetic beads, and then mixing the magnetic beads with a butyrylcholinesterase monoclonal antibody solution. In the present invention, the second magnetic separation is preferably performed using a DynaMag-2 magnetic stand; the purpose of the second magnetic separation is to separate the magnetic beads from the supernatant; the time of the second magnetic separation is preferably 30 to 60 seconds, and in a specific embodiment it can be 30 seconds, 40 seconds, 50 seconds or 60 seconds.

[0067] In the present invention, the volume ratio of the magnetic bead mixture to the butyrylcholinesterase monoclonal antibody solution is preferably 1:2-8, and in specific embodiments, it can be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8. In the present invention, the concentration of the butyrylcholinesterase monoclonal antibody solution is preferably 25-50 μg / mL, and in specific embodiments, it can be 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL or 50 μg / mL.

[0068] In the present invention, the temperature of the fourth incubation is preferably 4°C, and the time of the fourth incubation is preferably 1.5 to 2.5 hours, and in a specific embodiment it can be 1.5 hours, 2 hours or 2.5 hours; the fourth incubation preferably includes rotary incubation, and the rotation speed of the rotary incubation is preferably 800 to 1200 r / min, and in a specific embodiment it can be 800 r / min, 900 r / min, 1000 r / min, 1100 r / min or 1200 r / min.

[0069] After completing the fourth incubation, the present invention preferably further comprises washing the incubation system obtained by the fourth incubation with AB washing buffer, removing the supernatant, and obtaining antibody magnetic beads. In the present invention, the washing times of the washing buffer are preferably 2 times. In the present invention, the AB washing buffer is preferably the AB washing buffer in the magnetic bead kit.

[0070] After obtaining the antibody-adsorbed magnetic beads, the present invention mixes the antibody-adsorbed magnetic beads with a pepsin solution, performs a second incubation, and performs solid-liquid separation (referred to as the first solid-liquid separation) to obtain a nonapeptide test solution. The nonapeptides in the nonapeptide test solution include dimethylphosphonyl nonapeptide and methylphosphonyl nonapeptide.

[0071] In the present invention, the ratio of the volume of the magnetic beads (based on the volume of the magnetic bead mixture) to the mass of pepsin in the pepsin solution is preferably 25 μL: 0.05-0.3 mg, and in specific embodiments, it can be 25 μL: 0.05 mg, 25 μL: 0.1 mg, 25 μL: 0.15 mg, 25 μL: 0.2 mg, 25 μL: 0.25 mg or 25 μL: 0.3 mg. In the present invention, the concentration of pepsin in the pepsin solution is preferably 0.5-1.0 mg / mL, and in specific embodiments, it can be 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1 mg / mL; the solvent in the pepsin solution preferably includes an aqueous formic acid solution, and the volume concentration of the aqueous formic acid solution is preferably 0.6%.

[0072] In the present invention, the temperature of the second incubation is preferably 37°C, the time of the second incubation is preferably 1.5 to 2.5 hours, and in a specific embodiment it can be 1.5 hours, 2 hours or 2.5 hours; the second incubation preferably includes rotary incubation, and the rotation speed of the rotary incubation is preferably 800 to 1200 r / min, and in a specific embodiment it can be 800 r / min, 900 r / min, 1000 r / min, 1100 r / min or 1200 r / min.

[0073] In the present invention, the first solid-liquid separation preferably includes centrifugal separation, and the rotation speed of the centrifugal separation is preferably 8000-12000 r / min, and in specific embodiments it can be 8000 r / min, 9000 r / min, 10000 r / min, 11000 r / min or 12000 r / min; the time of the centrifugal separation is preferably 5-10 min, and in specific embodiments it can be 5 min, 6 min, 7 min, 8 min, 9 min or 10 min.

[0074] After completing the first solid-liquid separation, the present invention preferably further comprises: filtering the obtained liquid component through a 0.22 μm filter membrane to obtain a nonapeptide test solution.

[0075] After obtaining the IMS-treated blood, the present invention mixes the IMS-treated blood with acetonitrile to perform protein precipitation, thereby obtaining a protein precipitate and a malathion and its metabolite test solution, respectively; the metabolites in the malathion and its metabolite test solution include malathion dihydroxy acid.

[0076] In the present invention, the volume ratio of the IMS-treated blood to acetonitrile is preferably 1:2.5-10, and in specific embodiments it can be 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10.

[0077] In the present invention, the protein precipitation is preferably carried out under vortex oscillation conditions, and the present invention has no special limitation on the rotation speed of the vortex oscillation; the protein precipitation time is preferably 5 to 10 minutes, and in specific embodiments it can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes.

[0078] After the protein precipitation is completed, the present invention preferably further comprises: subjecting the system after the protein precipitation to solid-liquid separation (referred to as the second solid-liquid separation), respectively obtaining a protein precipitate and a liquid component, and filtering the liquid component through a 0.22 μm filter membrane to obtain a test solution of malathion and its metabolites. In the present invention, the second solid-liquid separation preferably comprises centrifugal separation, and the rotation speed of the centrifugal separation is preferably 8000 to 12000 r / min, and in a specific embodiment, it can be 8000 r / min, 9000 r / min, 10000 r / min, 11000 r / min or 12000 r / min; the time of the centrifugal separation is preferably 5 to 10 min, and in a specific embodiment, it can be 5 min, 6 min, 7 min, 8 min, 9 min or 10 min.

[0079] After obtaining the protein precipitate, the present invention mixes the protein precipitate with a pronase solution, performs a third incubation and then solid-liquid separation (referred to as the third solid-liquid separation), and obtains a dimethylthiophosphonotyrosine test solution.

[0080] In the present invention, the protein precipitate is preferably washed with acetonitrile and dried in sequence before being mixed with the pronase solution. In the present invention, the drying temperature is preferably 20-26° C. The present invention has no particular limitation on the drying time, and the drying time may be to a constant weight.

[0081] In the present invention, the ratio of the volume of the blood after the IMS treatment to the mass of the pronase in the pronase solution is 1 mL: 0.375-2.5 mg, and in specific embodiments can be 1 mL: 0.375 mg, 1 mL: 0.5 mg, 1 mL: 0.75 mg, 1 mL: 1 mg, 1 mL: 1.25 mg, 1 mL: 1.5 mg, 1 mL: 1.75 mg, 1 mL: 2 mg, 1 mL: 2.25 mg or 1 mL: 2.5 mg. In the present invention, the solvent in the pronase solution is preferably a bicarbonate aqueous solution, and the concentration of the bicarbonate aqueous solution is preferably 50 mmol / L.

[0082] In the present invention, the temperature of the third incubation is preferably 37° C., and the time of the third incubation is preferably 1.5 to 2.5 hours, and in a specific embodiment, it can be 1.5 hours, 2 hours or 2.5 hours; the third incubation preferably includes rotary incubation, and the rotation speed of the rotary incubation is preferably 800 to 1200 r / min, and in a specific embodiment, it can be 800 r / min, 900 r / min, 1000 r / min, 1100 r / min or 1200 r / min.

[0083] In the present invention, the third solid-liquid separation preferably includes centrifugal separation, and the rotation speed of the centrifugal separation is preferably 8000-12000 r / min, and in a specific embodiment it can be 8000 r / min, 9000 r / min, 10000 r / min, 11000 r / min or 12000 r / min; the time of the centrifugal separation is preferably 5-10 min, and in a specific embodiment it can be 5 min, 6 min, 7 min, 8 min, 9 min or 10 min.

[0084] After completing the third solid-liquid separation, the present invention preferably further comprises: filtering the obtained liquid component through a 0.22 μm filter membrane to obtain a dimethylthiophosphonotyrosine test solution.

[0085] The pretreatment method provided by the present invention comprises the following steps: firstly, using antibody magnetic beads (IMS) to separate dimethylphosphonobutyrylcholinesterase (DMP-BChE) and methylphosphonobutyrylcholinesterase (MxP-BChE), and then using pepsin to hydrolyze the two to generate dimethylphosphono nonapeptide (DMP-nonapeptide) and methylphosphono nonapeptide (MxP-nonapeptide), so as to obtain a nonapeptide test solution; protein precipitation is performed on the blood after IMS treatment, and the supernatant is a test solution of malathion and its metabolite (malathion dihydroxy acid, MDA); and the protein precipitate is digested by pronase to generate dimethylthiophosphonotyrosine (DMTP-Tyr), so as to obtain a dimethylthiophosphonotyrosine test solution. The pretreatment method provided by the present invention can separate the malathion original drug and its metabolites malathion dihydroxy acid, butyric acid cholinesterase adduct and dimethylthiophosphonotyrosine in the blood, and can achieve simultaneous detection of the malathion original drug and its metabolites malathion dihydroxy acid, butyric acid cholinesterase adduct and dimethylthiophosphonotyrosine through subsequent UPLC-MS. Moreover, the pretreatment method provided by the present invention is simple to operate and has good extraction and purification effects of various malathion biomarkers.

[0086] The present invention also provides a method for detecting malathion biomarkers in blood, comprising the following steps:

[0087] According to the pretreatment method described in the above technical scheme, a nonapeptide test solution, a malathion and its metabolite test solution, and a dimethylthiophosphonotyrosine test solution are obtained respectively;

[0088] The nonapeptide test solution, malathion and its metabolites test solution and dimethylthiophosphonotyrosine test solution were respectively subjected to UPLC-MS detection to obtain the detection results of the nonapeptide, malathion and its metabolites and dimethylthiophosphonotyrosine respectively.

[0089] In the present invention, the UPLC-MS detection is ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap mass spectrometry detection, and the detection conditions include ultra-high performance liquid chromatography separation conditions and mass spectrometry detection conditions.

[0090] In the present invention, the ultra-high performance liquid chromatography (UPLC) separation conditions include: the chromatographic column is Hypersil GOLDVANQUISH; the column temperature is preferably 40°C; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is 0.1v / v% formic acid aqueous solution, and the mobile phase B is acetonitrile; the mobile phase flow rate is preferably 0.3mL / min; the injection volume is preferably 1-2μL, and in a specific embodiment it can be 1μL, 1.5μL or 2μL. In the present invention, the elution method is gradient elution, and the gradient elution procedure includes: 0-2min, the volume fraction of the mobile phase B is 5-20%, and in a specific embodiment it can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%;

[0091] 2-3min, the volume fraction of the mobile phase B is increased from 5-20% to 50-70%. In a specific embodiment, the volume fraction of the mobile phase B can be increased from 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% and 20% to any one of 50%, 51%, 52%, 53%, 4%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70%;

[0092] 3 to 5 min, the volume fraction of the mobile phase B is 50 to 70%, and in specific embodiments can be 50%, 51%, 52%, 53%, 4%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%;

[0093] 5-5.2min, the volume fraction of the mobile phase B is reduced from 50-70% to 5-20%, and in a specific embodiment, it can be reduced from any one of 50%, 51%, 52%, 53%, 4%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70% to any one of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% and 20%;

[0094] 5.2~6min, the volume fraction of the mobile phase B is 5-20%, and in specific embodiments it can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0095] In a specific embodiment of the present invention, the gradient elution program is more preferably as shown in Table 1.

[0096] Table 1 Gradient elution program

[0097] Time (min) Mobile phase A volume fraction (%) Volume fraction of mobile phase B (%) 0.0 95 5 2.0 95 5 3.0 50 50 5.0 50 50 5.2 95 5 6.0 95 5

[0098] The present invention adopts the above-mentioned chromatographic column, mobile phase system and gradient elution program, and the separation degree of 5 kinds of targets is high, the retention time is appropriate, the peak time of the targets is stable, the peak shape is symmetrical and sharp, the response value is generally large, the sensitivity is high, and it meets the detection requirements.

[0099] In the present invention, the mass spectrometry (MS) detection conditions preferably include: using a positive and negative ion switching acquisition mode; the ion source is a heatable electrospray ion source; the positive ion mode spray voltage is 3.0 kV; the negative ion mode spray voltage is 2.5 kV; the ion transfer tube temperature is 320°C; the auxiliary gas heating temperature is 350°C; the auxiliary gas flow rate is 10 arb; the sheath gas flow rate is 35 arb; the purge gas flow rate is 0 arb; the S-lens RF voltage is 50V; the acquisition mode is a primary mass spectrometry full scan / data-dependent secondary mass spectrometry scan; the parameters of the primary mass spectrometry full scan preferably include: a resolution of 70000, a scanning range of 50 to 300, an automatic gain control of 3e6, and a maximum injection time of 100 ms; the parameters of the data-dependent secondary mass spectrometry scan preferably include: a resolution of 17500, an automatic gain control of 1e5, a maximum injection time of 50 ms, and a normalized collision energy of 20%, 30% or 40%; Top N (the first 10 ions to be triggered) is 10, the number of loops is 10, and the separation window is 2.0 m / z; the mass spectrometry scanning parameters preferably include: apex trigger is 2 to 6 s, dynamic exclusion time is 6 s, and isotope exclusion is on.

[0100] In the present invention, the mass spectrometry parameters of malathion, malathion dihydroxy acid, dimethylphosphonate, methylphosphonate nonapeptide and dimethylthiophosphonate tyrosine are preferably as shown in Table 2, and the gradient elution program shown in Table 1 is used. The retention time of each malathion biomarker is shown in Table 2.

[0101] Table 2 Mass spectrometry parameters of 5 target compounds

[0102]

[0103]

[0104] In the present invention, the UPLC-MS detection includes qualitative detection and / or quantitative detection; the quantitative detection method includes the internal standard curve method.

[0105] In the present invention, the internal standard method is used for quantitative detection, and the sample pretreatment of the blood to be tested is different from the pretreatment method described in the above technical solution only in that an internal standard is added during the preparation of the nonapeptide test solution, the malathion and its metabolite test solution, and the dimethylthiophosphonotyrosine test solution. Specifically, the antibody-adsorbed magnetic beads, the pepsin solution, and the nonapeptide internal standard are mixed, a second incubation is performed, and solid-liquid separation is performed to obtain the nonapeptide test solution; the blood treated with IMS, acetonitrile, and the first internal standard SKF 525A Mixing, protein precipitation, respectively obtaining protein precipitate and malathion and its metabolite test solution; drying the protein precipitate and mixing it with pronase solution and second internal standard SKF 525A The mixture is mixed, and solid-liquid separation is performed after the third incubation to obtain a dimethylthiophosphonotyrosine test solution.

[0106] The present invention mixes the antibody-absorbed magnetic beads, pepsin solution and nonapeptide internal standard, performs a second incubation, and solid-liquid separation to obtain a nonapeptide test solution. In the present invention, the ratio of the volume of the magnetic beads to the mass of the nonapeptide internal standard is preferably 25 μL: 0.5-6 ng, and in a specific embodiment, it can be 25 μL: 0.5 mg, 25 μL: 1 mg, 25 μL: 1.5 mg, 25 μL: 2 mg, 25 μL: 2.5 ng, 25 μL: 3 ng, 25 μL: 3.5 ng, 25 μL: 4 ng, 25 μL: 4.5 ng, 25 μL: 5 ng, 25 μL: 5.5 ng or 25 μL: 6 ng. In the present invention, the mixing of the antibody-absorbed magnetic beads, pepsin solution and nonapeptide internal standard preferably includes: mixing the pepsin solution and the nonapeptide internal standard, and mixing the obtained pepsin-internal standard mixed solution with the antibody-absorbed magnetic beads. In the present invention, the concentration of the nonapeptide internal standard in the pepsin-internal standard mixture is preferably 5-20 ng / mL, and in specific embodiments can be 5 ng / mL, 10 ng / mL, or 15 ng / mL, 20 ng / mL.

[0107] The present invention comprises the blood, acetonitrile and the first internal standard SKF treated by IMS. 525A The mixture is mixed and protein precipitation is performed to obtain a protein precipitate and a test solution of malathion and its metabolites. In the present invention, the volume of the blood after IMS treatment and the first internal standard SKF 525AThe mass ratio of is preferably 1mL:25-100ng, and in specific embodiments can be 1mL:25ng, 1mL:30ng, 1mL:35ng, ​​1mL:40ng, 1mL:45ng, 1mL:50ng, 1mL:55ng, 1mL:60ng, 1mL:65ng, 1mL:70ng, 1mL:75ng, 1mL:80ng, 1mL:85ng, 1mL:90ng, 1mL:95ng or 1mL:100ng. In the present invention, the blood, acetonitrile and the first internal standard SKF treated by IMS are mixed. 525A The mixing preferably comprises: mixing acetonitrile and the first internal standard SKF 525A Mix the obtained acetonitrile-internal standard mixed solution with the blood after IMS treatment.

[0108] The protein precipitate is dried and mixed with a pronase solution and a second internal standard SKF 525A The mixture is mixed, and solid-liquid separation is performed after the third incubation to obtain a dimethylthiophosphonotyrosine test solution. In the present invention, the volume of the blood after IMS treatment and the second internal standard SKF 525A The mass ratio of is 1mL:7.5-12.5ng, and in specific embodiments can be 1mL:7.5ng, 1mL:8ng, 1mL:8.5ng, 1mL:9ng, 1mL:9.5ng, 1mL:10ng, 1mL:10.5ng, 1mL:11ng, 1mL:11.5ng, 1mL:12ng or 1mL:12.5ng. In the present invention, the mixing of the protein precipitate with the pronase solution preferably includes: mixing the pronase solution, and mixing the obtained pronase-internal standard mixed solution with the protein precipitate.

[0109] The present invention has no particular limitation on the specific operations of the qualitative detection and the internal standard curve method quantitative detection, and the operations of UPLC-MS qualitative detection and internal standard curve method quantitative detection well known to those skilled in the art can be used.

[0110] In the present invention, the linear range of malathion is preferably 0.1 to 100 ng / mL; the linear range of malathion dihydroxy acid is preferably 0.5 to 100 ng / mL; the linear range of dimethylphosphonyl nonapeptide is preferably 2 to 100 ng / mL; the linear range of methylphosphonyl nonapeptide is preferably 2 to 100 ng / mL; the linear range of dimethylthiophosphonotyrosine is preferably 0.1 to 100 ng / mL.

[0111] To further illustrate the present invention, the pretreatment method and detection method of malathion biomarkers in blood provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0112] In the following examples, the instruments and reagents are as follows:

[0113] Q Exactive ultra-high performance liquid chromatography quadrupole / electrostatic field orbital ion trap high-resolution mass spectrometer (ThermoFisher, USA); CF16RN floor-standing multi-purpose centrifuge (Hitachi, Japan); Hypersll GOLDTMVANQUISH chromatographic column (Thermo Fisher, USA); DynaMag-2 magnetic stand (Thermo Fisher, USA); ThermomixerR constant temperature mixer (Eppendorf, Germany); 10-kDa molecular weight cutoff filter membrane (Sigma, Germany).

[0114] Methanol, acetonitrile, formic acid (chromatographic grade, Thermo Fisher, USA); ammonium acetate (Shanghai Aladdin Biochemical Technology Co., Ltd., purity ≥99%); ammonium bicarbonate (NH4HCO3) (Sigma, Germany, purity ≥99%).

[0115] Malathion, Malathion dihydroxy acid (MDA), SKF 525 (Beijing Bailingwei Technology Co., Ltd., purity ≥99%); nonapeptide internal standard (Beijing Saibaisheng Company, FGESAGAAG-98.48%); dimethylphosphono nonapeptide (DMP-nonapeptide, formula I); methylphosphono nonapeptide (MxP-nonapeptide, formula II); dimethylthiophosphonotyrosine (DMTP-Tyr, formula III); butyrylcholinesterase, pepsin, and pronase were all purchased from Sigma, Germany; butyrylcholinesterase monoclonal antibody (3E8, Thermo Fisher, USA); magnetic bead kit: Protein G (Thermo Fisher, USA, containing AB washing buffer and washing buffer).

[0116]

[0117] Blank blood (provided by the Central Blood Station). Preparation of blank matrix: Blank blood was filtered through a 10 kDa molecular weight cutoff membrane. Malathion poisoning case specimens (provided by the Public Security Bureau).

[0118] The qualitative and quantitative detection conditions of ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap mass spectrometry are as follows:

[0119] The UPLC chromatographic conditions were as follows: the chromatographic column was Hypersil GOLD VANQUISH (100 mm×2.1 mm, 1.9 μm); the column temperature was 40°C; the mobile phase A was 0.1 v / v% formic acid aqueous solution, and the mobile phase B was acetonitrile; the flow rate was 0.3 mL / min; the injection volume was 1 μL; the gradient elution program was shown in Table 1.

[0120] Mass spectrometry conditions: According to the different structures of organophosphorus pesticide protein adducts, the positive and negative ion switching acquisition mode was adopted (see Table 2), the ion source was a heatable electrospray ion source (HESI), and the acquisition mode was a primary mass spectrometry full scan / data-dependent secondary mass spectrometry scan (FullMS / ddMS 2 ). Mass spectrometry ionization parameters: positive ion mode spray voltage is 3.0 kV, negative ion mode spray voltage is 2.5 kV, ion transfer tube temperature is 320 ° C, auxiliary gas (N2) heating temperature is 350 ° C, auxiliary gas (N2) flow rate is 10 arb, sheath gas (N2) flow rate is 35 arb, purge gas flow rate is 0 arb, S-lens RF voltage is 50 V; mass spectrometry scanning Full MS parameters: resolution is 70000, scanning range is 50-300, automatic gain control is 3e6, maximum injection time is 100 ms; mass spectrometry scanning Full MS parameters: resolution is 70000, scanning range is 50-300, automatic gain control is 3e6, maximum injection time is 100 ms; mass spectrometry scanning ddMS 2 Parameters: resolution is 17500, automatic gain control is 1e5, maximum injection time is 50ms, normalized collision energy is 20%, 30%, 40%, TopN is 10, Loop count is 10, separation window is 2.0m / z; mass spectrometry scan dd Settings parameters: Apex trigger is 2~6s, dynamic exclusion time is 6s, and isotope exclusion is on.

[0121] Example 1

[0122] Sample preparation

[0123] 1.1 Purification and extraction of BChE (butyrylcholinesterase)

[0124] Preparation of antibody magnetic beads: Protein G, AB washing buffer and washing buffer were mixed thoroughly on a vortex mixer, and 25 μL of the magnetic bead mixture was transferred to a 1.5 mL conical bottom test tube and placed in a DynaMag-2 magnetic stand for 30 to 60 seconds to separate the magnetic beads from the supernatant. After removing the supernatant, 100 μL of 25 μg / mL butyrylcholinesterase monoclonal antibody solution was added to the magnetic beads, placed on a Thermomixer R constant temperature mixer, and rotated and incubated at 1000 r / min for 2 hours at 4°C, washed twice with 200 μL AB washing buffer, and the supernatant was removed to obtain antibody magnetic beads.

[0125] 250 μL of blood was added to the antibody magnetic beads, and the beads were rotated and incubated at 1000 r / min for 2 h at 26 ° C. After incubation, the beads were placed in a DynaMag-2 magnetic stand for 60 s to separate the beads from the IMS-treated blood, and the antibody-adsorbed magnetic beads and the IMS-treated blood were obtained respectively. The antibody-adsorbed magnetic beads were washed 3 times with 200 μL of washing buffer, the supernatant was removed, 200 μL of a 0.5 mg / mL pepsin-internal standard mixed solution (solvent was 0.6 v / v% formic acid, pepsin concentration was 0.5 mg / mL, and the concentration of the nonapeptide internal standard was 10.0 ng / mL) was added, and the beads were rotated and incubated (enzyme hydrolysis) at 1000 r / min for 2 h at 37 ° C, centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain a nonapeptide test solution, and the dimethylphosphonyl nonapeptide and methylphosphonyl nonapeptide in the nonapeptide test solution were analyzed by UPLC-MS.

[0126] 1.2 Extraction of malathion technical and its metabolites

[0127] Take 200 μL of the blood treated with IMS in 1.1, add 1.0 mL of acetonitrile-internal standard mixed solution (internal standard SKF 525A The mixture was centrifuged at 12000 r / min for 7 minutes to obtain a protein precipitate and a supernatant, respectively. The supernatant was filtered through a 0.22 μm filter membrane to obtain a malathion and metabolite test solution. UPLC-MS was used to analyze the malathion original drug and its metabolite malathion dihydroxy acid in the malathion and metabolite test solution.

[0128] 1.3 Extraction of dimethylthiophosphonotyrosine

[0129] The protein precipitate obtained in 1.2 was washed once with acetonitrile, dried to powder, and 200 μL of 1.0 mg / mL pronase-internal standard mixed solution (solvent: 50 mM ammonium bicarbonate aqueous solution, internal standard SKF 525AThe concentration is 10.0 ng / mL), incubated (enzymatically hydrolyzed) at 37°C for 2 h, centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain a dimethylthiophosphonotyrosine test solution. The dimethylthiophosphonotyrosine in the dimethylthiophosphonotyrosine test solution was analyzed by UPLC-MS.

[0130] Example 2

[0131] Optimization of instrument conditions

[0132] UPLC-MS injection sample solutions: malathion methanol solution, malathion dihydroxy acid methanol solution, dimethylphosphonobutyrylcholinesterase aqueous solution, methylphosphonobutyrylcholinesterase aqueous solution and dimethylthiophosphonotyrosine aqueous solution. The concentrations of the five target solutions were 25.0 ng / mL, 50.0 ng / mL and 100.0 ng / mL.

[0133] 1.1 Optimization of UPLC Chromatographic Conditions

[0134] 1.1.1 Column optimization

[0135] According to the characteristics of the polar structures of the five targets, this experiment investigated HSS T3, BEH C 18 、Hypersll GOLD TM The experimental results show that the use of HypersllGOLD with low ligand density is more effective. TM When using the VANQUISH column, the target substances were separated well and retained at appropriate times.

[0136] 1.1.2 Optimization of mobile phase conditions

[0137] The selection of mobile phase is one of the important factors to improve the separation and peak shape of the target. This experiment investigated four mobile phase systems: water-acetonitrile, water-methanol, 0.1v / v% formic acid water-acetonitrile, and 1mmol / L ammonium acetate water-acetonitrile, and the separation was performed using the gradient elution program shown in Table 1. The study found that when acetonitrile was used as the organic phase, the sensitivity and separation effect of each target were significantly better than methanol. After pure water was used as the aqueous phase or ammonium acetate buffer was added to the aqueous phase, the peak shapes of dimethylphosphonyl nonapeptide and methylphosphonyl nonapeptide became asymmetric, tailing occurred, and there were more burrs. When 0.1v / v% formic acid water was used as the aqueous mobile phase, the peak time of the target was stable, the peak shape was symmetrical and sharp, and the response value was generally large, which met the detection requirements.

[0138] 1.1.3 Elution Elution program optimization

[0139] Gradient elution was used to separate the five target substances. The following gradient elution procedures were investigated in this experiment: ①0-0.5min, 20% B (indicating that the volume fraction of mobile phase B is 20%); 0.5-2min, 20% B→80% B (indicating that the volume fraction of mobile phase B increases from 20% to 80%), 2-4min, 80% B; 4-4.5min, 82%→20% B (indicating that the volume fraction of mobile phase B decreases from 82% to 20%); 4.5-7.5min, 20% B; ②0-0.5min, 10% B; 0.5-2min, 10% B→90% B, 2-4min, 90% B; 4-4.5min, 90% B→10% B; 4.5-7.5min, 10% B; ③0-0.5min, 8% B; 0.5-2min n, 8% B→92% B, 2~4 min, 92% B; 4~4.5 min, 92% B→8% B; 4.5~7.5 min, 8% B; ④0~0.5 min, 8% B; 0.5~1 min, 8% B→75% B, 1~2 min, 75% B→92% B, 2~4 min, 92% B; 4~4.5 min, 92% B→8% B; 4.5~7.5 min, 8% B; ⑤0~0.5 min, 8% B; 0.5~1.5 min, 8% B→25% B, 1.5~2 min, 25% B→92% B, 2~4 min, 92% B; 4~4.5 min, 92% B→8% B; 4.5~7.5 min, 8% B; ⑥ Gradient elution program shown in Table 1. Considering the separation degree, peak shape, sensitivity and other factors of the target chromatographic peak, the experimental results show that the gradient elution program shown in Table 1 has the best overall separation effect.

[0140] 1.1.4 Optimization of flow rate conditions

[0141] If the flow rate is too fast or too slow, it will cause the peak time of the target to be advanced or delayed, and may also affect the separation effect of the target. The appropriate flow rate can well separate the impurity peak and the target peak. This experiment investigated four mobile phase flow rates of 0.2mL / min, 0.3mL / min, 0.4mL / min, and 0.5mL / min. The experimental results show that when the flow rate is 0.3mL / min, the chromatograms of malathion, malathion dihydroxy acid, dimethylphosphonyl nonapeptide, methylphosphonyl nonapeptide, and dimethylthiophosphonotyrosine are shown in 1 to 2. Figure 5 It can be seen that the five malathion poisoning markers can be effectively separated and the peak times are relatively appropriate.

[0142] 1.2 Optimization of mass spectrometry conditions

[0143] 1.2.1 Selection of scanning method

[0144] This experiment investigated the FullMS / ddMS2 、Target-SIM / ddMS 2 、FullMS three scanning modes. FullMS / ddMS 2 After obtaining primary data through full scan of positive and negative ions, the secondary fragment ion information is automatically triggered to obtain qualitative information based on the accurate mass number of the parent ion of the extracted target and the secondary mass spectrum, and the accurate parent ion is used for quantitative analysis. On this basis, the retention time can further improve the accuracy of qualitative analysis. 2 The scanning mode first performs selected ion monitoring on the precursor ions, and then these precursors enter the high-energy collision cell for fragmentation and data-dependent scanning. This mode is more accurate than FullMS / ddMS in the quantitative analysis of the five targets. 2 The FullMS scanning mode only performs qualitative analysis on the target based on retention time and the accurate mass of the parent ion. The detection results are not accurate enough and false positives are prone to occur. In summary: Full MS / ddMS was selected for this experiment. 2 This is the final scan mode.

[0145] 1.2.2 Resolution selection

[0146] The higher the resolution, the better the selectivity of the mass spectrometer for the target ions, and the easier it is to separate ions with similar mass-to-charge ratios. Higher resolution is one of the important factors to ensure accurate identification of trace and ultra-trace targets in complex matrix samples; however, the scanning speed of the mass spectrometer will decrease with the increase of resolution. Too high a resolution will result in too few points for the chromatographic peak collection, affecting the peak shape and making it difficult to quantify. After many experiments, when the full scan resolution of the primary mass spectrometer and the scanning resolution of the secondary mass spectrometer are 70,000 and 17,500 respectively, the impurities in the matrix can be well separated from the target to be measured, and the accuracy of quantification can be guaranteed.

[0147] 1.2.3 Determination of accurate mass

[0148] Through experimental investigation, the four Ops-HSA adducts were characterized by parent ion [M+H + form, with higher response in positive ion scanning mode; the four Ops-BChE adducts were expressed as [M+H] + The form cannot be detected, but the parent ion [MH] - This experiment verified the incubation products of 5 targets. Figure 6 to Figure 10 The primary mass spectra are malathion, malathion dihydroxy acid, dimethylphosphonyl nonapeptide, methylphosphonyl nonapeptide and dimethylthiophosphonyl tyrosine, respectively. The relative mass deviations of the actual and theoretical accurate mass numbers are all less than 10 ppm, meeting the qualitative detection requirements. The accurate mass numbers and relative mass deviations are shown in Table 2.

[0149] 1.2.4 Selection of secondary mass spectrometry conditions

[0150] Compared with quadrupole mass spectrometry, the mass spectrometry parameter settings of high-resolution mass spectrometry are much simpler. When the peak trigger time is optimized to 2-6s, the dynamic exclusion time is 6s and the Top N is 5, the peak shapes of the five targets are good and better secondary fragment ion information can be obtained. In addition, this experiment also compared the three normalized collision energy gradient settings of 20%, 30%, 40%, 25%, 45%, 65%, 50%, 70%, and 90%. The experimental results show that when the normalized collision energy gradient is 20%, 30%, and 40%, the superimposed secondary fragment spectra of the five targets are relatively rich. Figure 11 to Figure 15 They are the secondary mass spectra of malathion, malathion dihydroxy acid, dimethylphosphonyl nonapeptide, methylphosphonyl nonapeptide and dimethylthiophosphonotyrosine, respectively.

[0151] Example 3

[0152] Method validation

[0153] 1.1 Establishment of standard curve and linear range

[0154] Different concentrations of malathion (mass concentrations were 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL, respectively) and malathion dicarboxylic acid (mass concentrations were 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL, respectively) were added to plasma, and the mixed plasma was subjected to protein precipitation, centrifugation, and membrane filtering according to step 1.2 of Example 1 to obtain malathion series working solutions and malathion dicarboxylic acid series working solutions, respectively.

[0155] Preparation of a series of standard working solutions of a single standard: dimethylphosphonyl nonapeptide, methylphosphonyl nonapeptide and dimethylthiophosphonotyrosine were added to the blank matrix respectively to prepare a series of standard working solutions of a single standard. The mass concentrations of dimethylphosphonyl nonapeptide and methylphosphonyl nonapeptide in the series of standard working solutions were 2ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, 50ng / mL, 80ng / mL and 100ng / mL respectively; the mass concentrations of dimethylthiophosphonotyrosine were 0.1ng / mL, 0.5ng / mL, 1ng / mL, 2ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, 50ng / mL and 100ng / mL respectively.

[0156] Preparation of a series of working solutions of dimethylphosphonyl nonapeptide and methylphosphonyl nonapeptide: The antibody magnetic beads in 1.1 of Example 1 were replaced with a single series of standard working solutions of dimethylphosphonyl nonapeptide and methylphosphonyl nonapeptide, respectively, and enzymatic hydrolysis, centrifugation and membrane filtration were performed according to 1.1 of Example 1.

[0157] Preparation of a series of working solutions of dimethylphosphonothioate tyrosine: The protein precipitate in 1.3 of Example 1 was washed and dried, and the powder was replaced with 250 μL of a series of standard working solutions of dimethylphosphonothioate tyrosine, and enzymatic hydrolysis, centrifugation and membrane filtration were performed according to 1.3 of Example 1.

[0158] Each series of standard working solutions were analyzed by UPLC-MS. The mass concentration of the target was used as the independent variable (x) and the peak area ratio of the target to the internal standard was used as the dependent variable (y). The standard curve was drawn to obtain the regression equation and correlation coefficient. The results are shown in Table 3. It can be seen that the five malathion biomarkers have a good linear relationship within their respective ranges, and the correlation coefficient (R 2 ) is greater than 0.996, indicating that the detection method provided by the present invention has a wide linear range and a good linear relationship.

[0159] 1.2 Detection limit and quantification limit of the method

[0160] The concentration corresponding to 3 times the signal-to-noise ratio (S / N=3) is taken as the minimum detection limit (LOD), and the concentration corresponding to 10 times the signal-to-noise ratio (S / N=10) is taken as the minimum quantification limit (LOQ). The results are shown in Table 3. It can be seen that the detection limits of the five targets are all between 0.05 and 1.0 ng / mL, and the quantification limits are all between 0.5 and 2.0 ng / mL, indicating that the detection limit and quantification limit of the detection method provided by the present invention are low, and the detection sensitivity is high.

[0161] Table 3 Linear relationship, detection limit and quantification limit of 5 targets

[0162]

[0163] 1.3 Method precision

[0164] The blank matrix was added with 5 target standard solutions prepared in Example 1.1, all of which were prepared into standard working solutions with three concentration levels of low (25.0 ng / mL), medium (50.0 ng / mL), and high (100.0 ng / mL). Six samples were prepared in parallel for each concentration. Each standard working solution was enzymatically hydrolyzed, centrifuged, and membrane-passed according to Example 1.1, and then UPLC-MS analysis was performed. Two samples of different concentrations were taken in three batches in the morning, noon, and evening of the same day, and the samples were injected and tested and analyzed in sequence from low to high concentrations. Each sample was measured three times. The relative standard deviation of the peak area of ​​each target at three concentrations was calculated to obtain the intra-day precision (RSD%). Two samples of the mixed standard solutions of the three concentrations were taken each day for detection and analysis, and the detection was continued for three days. The relative standard deviation of the peak area of ​​each target at three concentrations was calculated to obtain the inter-day precision (RSD%). The results are shown in Table 4. It can be seen that the intra-day precision RSD of the five malathion biomarkers is ≤10.1%, and the inter-day precision RSD is ≤13.6%, indicating that the detection method provided by the present invention has good precision.

[0165] Table 4 Intra-day and inter-day precision of 5 targets

[0166]

[0167]

[0168] 1.4 Recovery and matrix effect of the method

[0169] The internationally accepted Matuszewski method was used to calculate the recovery rate and matrix effect of malathion and DMA.

[0170] The blank matrix was divided into two parts, and the pre-incubation spike experiment and the post-incubation spike experiment were performed respectively to evaluate the matrix effect and recovery rate of butyrylcholinesterase and dimethylthiophosphonotyrosine. Pre-incubation spike: The five target standard solutions prepared in 1.1 of this example were added to the blank matrix to prepare low (25.0 ng / mL), medium (50.0 ng / mL) and high (100.0 ng / mL) level spike sample solutions, and each spike sample solution was enzymatically hydrolyzed, centrifuged and filtered according to 1.1 of this example to obtain the pre-incubation spike sample solution. Post-incubation spike: The blank matrix was first enzymatically hydrolyzed, centrifuged and filtered according to 1.1 of this example, and then the five target standard solutions prepared in 1.1 of this example were added to prepare low (25.0 ng / mL), medium (50.0 ng / mL) and high (100.0 ng / mL) level samples to obtain the post-incubation spike sample solution. The experiment was repeated three times, and the average values ​​of the spiked response values ​​before incubation and the spiked response values ​​after incubation were compared to evaluate the extraction recovery. Recovery (%) = spiked response value before incubation / spike response value after incubation × 100%. The results are shown in Table 5, and it can be seen that the recovery rates of the five target substances are 76.1% to 107.1%.

[0171] The average value of the spiked response after incubation was compared with the average value of the response of the matrix-free spiked solution to evaluate the matrix effect. Matrix effect (%) = spiked response before incubation / matrix-free spiked response × 100%. The results are shown in Table 5, and it can be seen that the matrix effects of the five targets are 81.2% to 100.6%.

[0172] Table 5 Recovery and matrix effects of 5 target compounds

[0173]

[0174] Example 4

[0175] Case Application

[0176] On June 16, 2022, the police in xx County, xx City received a report that someone found a man dead after drinking medicine in the east of the xx Village Cemetery. Two empty bottles labeled "Malathion" and "Niulanshan Erguotou" were found at the scene. The heart blood of the deceased was extracted and sent for inspection on the same day. Sample pretreatment and UPLC-MS detection were performed according to Example 1. After detection and identification, the content of malathion detected in the heart blood of the deceased was 17274.76 ng / mL, the content of the metabolite malathion dihydroxy acid detected was 1852.33 ng / mL, the content of methylphosphonyl nonapeptide detected was 72.29 ng / mL, and the content of dimethylthiophosphonyl tyrosine detected was 319.51 ng / mL.

[0177] Example 5

[0178] Case Application

[0179] On June 11, 2022, Zhang Moumou took a taxi alone and disappeared after getting off the car on the road near xx village. Later, Zhang Moumou's body was found on the hillside of "Dongshan" in xx village. Near the body was a medicine bottle marked with the words "Malathion" with a small amount of liquid left. The sample pretreatment and UPLC-MS detection of Zhang Moumou's heart blood were carried out according to Example 1. The content of malathion component was detected to be 6581.72 ng / mL, the content of metabolite malathion dihydroxy acid was detected to be 793.31 ng / mL, the content of adduct methylphosphonyl nonapeptide was detected to be 122.42 ng / mL, and the content of dimethylthiophosphonyltyrosine was detected to be 302.12 ng / mL.

[0180] In summary, the present invention has established a sensitive sample preparation method for simultaneously detecting five malathion biomarkers in horse blood. By improving the IMS pretreatment method, the operation is simple, the number of magnetic beads is small, and the extraction and purification effect is good. The entire analysis and detection process is completed within 6 minutes, the mobile phase consumption is small, and the target compound separation effect is good. This method can detect multiple biomarkers at the same time, realize the comprehensive monitoring of malathion exposure at different levels, and has broad application prospects.

[0181] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for pre-treating malathion biomarkers in blood, characterized in that: The following steps are involved: The blood to be tested and the antibody magnetic beads are mixed, and a first incubation and magnetic separation are performed to obtain antibody-adsorbed magnetic beads and blood treated with IMS, respectively; the antibody magnetic beads are obtained by modifying magnetic beads with antibodies, and the antibodies include butyrylcholinesterase monoclonal antibodies; the antibody-adsorbed magnetic beads contain dimethylphosphonylbutyrylcholinesterase and / or methylphosphonylbutyrylcholinesterase; The antibody-adsorbed magnetic beads and the pepsin solution are mixed, and a second incubation is performed, and solid-liquid separation is performed to obtain a nonapeptide test solution; the nonapeptides in the nonapeptide test solution include dimethylphosphonyl nonapeptide and methylphosphonyl nonapeptide; The IMS-treated blood is mixed with acetonitrile to perform protein precipitation, thereby obtaining a protein precipitate and a malathion and its metabolite test solution, respectively; the metabolites in the malathion and its metabolite test solution include malathion dihydroxy acid; The protein precipitate is mixed with a pronase solution, and solid-liquid separation is performed after a third incubation to obtain a dimethylthiophosphonotyrosine test solution.

2. The pre-treatment method according to claim 1, characterized in that: The volume ratio of the magnetic beads to the blood to be tested is 1:4-12; The first incubation time is 1.5 to 2.5 hours, and the first incubation includes rotary incubation, and the rotation speed of the rotary incubation is 800 to 1200 r / min; The magnetic separation obtains a solid component and IMS-treated blood, respectively; The solid component is washed with a washing buffer to obtain antibody-adsorbed magnetic beads.

3. The pre-treatment method according to claim 1, characterized in that: The ratio of the volume of the magnetic beads to the mass of pepsin in the pepsin solution is 25 μL: 0.05-0.3 mg; The concentration of the pepsin solution is 0.5-1 mg / mL, and the solvent in the pepsin solution includes a formic acid aqueous solution; The second incubation time is 1.5 to 2.5 hours, and the second incubation includes rotary incubation, and the rotation speed of the rotary incubation is 800 to 1200 r / min.

4. The pre-treatment method according to claim 1, characterized in that: The volume ratio of the IMS-treated blood to acetonitrile is 1:2.5-10; The protein precipitation is carried out under vortex oscillation conditions, and the protein precipitation time is 5 to 10 minutes; The method further comprises solid-liquid separation after the protein precipitation.

5. The pre-treatment method according to claim 1, characterized in that: The protein precipitate is first washed with acetonitrile and dried in sequence, and then mixed with a pronase solution; The ratio of the volume of the blood after the IMS treatment to the mass of the pronase in the pronase solution is 1 mL: 0.375-2.5 mg; The third incubation time is 1.5 to 2.5 hours, and the third incubation includes rotary incubation, and the rotation speed of the rotary incubation is 800 to 1200 r / min.

6. A method for detecting malathion biomarkers in blood, characterized in that: The following steps are involved: According to the pretreatment method of any one of claims 1 to 5, a nonapeptide test solution, a malathion and its metabolite test solution and a dimethylthiophosphonotyrosine test solution are obtained respectively; The nonapeptide test solution, malathion and its metabolites test solution, and dimethylthiophosphonotyrosine test solution are subjected to UPLC-MS detection respectively to obtain the detection results of the nonapeptide, malathion and its metabolites, and dimethylthiophosphonotyrosine respectively; The UPLC-MS detection is ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap mass spectrometry detection, and the detection conditions include ultra-high performance liquid chromatography separation conditions and mass spectrometry detection conditions; The ultra-high performance liquid chromatography separation conditions include: the chromatographic column is Hypersil GOLD VANQUISH, the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is 0.1v / v% formic acid aqueous solution, the mobile phase B is acetonitrile, the elution method is gradient elution, and the gradient elution program includes: 0-2min, the volume fraction of the mobile phase B is 5-20%; 2-3min, the volume fraction of the mobile phase B increases from 5-20% to 50-70%; 3-5min, the volume fraction of the mobile phase B is 50-70%; 5-5.2min, the volume fraction of the mobile phase B decreases from 50-70% to 5-20%; 5.2-6min, the volume fraction of the mobile phase B is 5-20%; The UPLC-MS detection includes qualitative detection and / or quantitative detection; the quantitative detection method includes an internal standard curve method.

7. The detection method according to claim 6, characterized in that: The ultra-high performance liquid chromatography separation conditions also include: column temperature of 40° C.; mobile phase flow rate of 0.3 mL / min; and injection volume of 1 to 2 μL.

8. The detection method according to claim 6, characterized in that: The mass spectrometry detection conditions include: adopting a positive and negative ion switching acquisition mode; the ion source is a heatable electrospray ion source; the spray voltage in the positive ion mode is 3.0 kV; the spray voltage in the negative ion mode is 2.5 kV; the ion transfer tube temperature is 320°C; the auxiliary gas heating temperature is 350°C; the auxiliary gas flow rate is 10 arb; the sheath gas flow rate is 35 arb; the purge gas flow rate is 0 arb; the S-lens RF voltage is 50 V; the acquisition mode is a primary mass spectrometry full scan / data-dependent secondary mass spectrometry scan; The parameters of the first-stage mass spectrometer full scan include: resolution of 70000, scanning range of 50-300, automatic gain control of 3e6, and maximum injection time of 100ms; The parameters of the data-dependent secondary mass spectrometry scan include: resolution of 17500, automatic gain control of 1e5, maximum injection time of 50ms, normalized collision energy of 20%, 30% or 40%; Top N of 10, number of cycles of 10, and separation window of 2.0m / z; The mass spectrometry scanning parameters include: apex excitation of 2 to 6 seconds, dynamic exclusion time of 6 seconds, and isotope exclusion of on.

9. The detection method according to claim 8, characterized in that: The malathion adopts a positive ion collection mode, and the secondary ions are 98.9845m / z and 176.9889m / z; the malathion dihydroxy acid adopts a negative ion collection mode, and the secondary ions are 140.9776m / z and 156.9546m / z; the dimethylphosphonyl nonapeptide adopts a negative ion collection mode, and the secondary ions are 486.2935m / z and 776.3228m / z; the methylphosphonyl nonapeptide adopts a negative ion collection mode, and the secondary ions are 443.1898m / z and 776.3236m / z; the dimethylthiophosphonotyrosine adopts a positive ion collection mode, and the secondary ions are 289.0295m / z and 260.0504m / z.

10. The detection method according to any one of claims 6 to 9, characterized in that: The linear range of malathion is 0.1 to 100 ng / mL; the linear range of malathion dihydroxy acid is 0.5 to 100 ng / mL; the linear range of dimethylphosphonyl nonapeptide is 2 to 100 ng / mL; the linear range of methylphosphonyl nonapeptide is 2 to 100 ng / mL; the linear range of dimethylthiophosphonotyrosine is 0.1 to 100 ng / mL.

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