CN113237975B - Method for preliminarily inferring cause of death and method for detecting endogenous metabolites - Google Patents
Method for preliminarily inferring cause of death and method for detecting endogenous metabolites Download PDFInfo
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Abstract
The invention belongs to the technical field of forensic medicine, and particularly relates to a method for preliminarily inferring cause of death based on endogenous metabolites. The method uses acetyl carnitine, propionyl carnitine and succinic acid in blood samples as differential metabolites, realizes the preliminary inference of the cause of death of a main body of an unknown blood sample by quantitative analysis and mathematical modeling of the differential metabolites, can be used for preliminarily judging whether the death factor of a death case is poisoned by excessive use of antipsychotic drugs, and the involved antipsychotic drugs comprise one or more of chlorpromazine, perphenazine, olanzapine and clozapine. The invention also relates to a method for detecting endogenous metabolites, which is used for detecting the endogenous metabolites generated after the chlorpromazine, perphenazine, olanzapine and clozapine are taken in a blood sample.
Description
Technical Field
The invention belongs to the technical field of forensic medicine, and particularly relates to a non-diagnosis purpose method for preliminarily deducing cause of death based on endogenous metabolites and a method for detecting the endogenous metabolites.
Background
Chlorpromazine is a traditional typical antipsychotic, and is mainly used for treating refractory schizophrenia and major depression clinically; it has also been found that chlorpromazine inhibits the growth of certain coronaviruses (RNA viruses), cryptococcus (fungi), escherichia coli and Salmonella (bacteria) to some extent. However, annual report of the U.S. poison control center in 2018 shows that lethal cases of sedative-hypnotic/antipsychotic intoxication are the first. As a therapeutic drug with strong central sedation and easily available prescription, the toxic or toxic lethal blood concentration threshold has a large overlapping area, and besides the detection method and the accuracy, the health condition of the victim is another important reason among influencing factors. Also, unlike poisoning by corrosive toxins (organophosphorous pesticides, nitrites), therapeutic drugs lack characteristic morphological lesions and specific metabolites. Therefore, in the case of lacking other characteristic morphological characteristics, the death cause of the death case is not easy to be accurately judged by the forensic, and the identification process is more complicated when various drugs are poisoned.
Disclosure of Invention
Aiming at the technical problem that the death cause is difficult to be accurately judged for the case lacking other characteristic morphological characteristics in the prior art, the invention provides a method for preliminarily deducing the death cause based on endogenous metabolites with a non-diagnosis purpose. The method can preliminarily judge the death cause of death cases, and is suitable for preliminarily distinguishing the death causes of chlorpromazine, perphenazine, olanzapine and clozapine overdue from death causes of non-antipsychotic drug poisoning in the forensic field. The invention also provides a method for detecting endogenous metabolites, which can be used for detecting the metabolites produced after chlorpromazine, perphenazine, olanzapine and clozapine are taken in a blood sample.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
in a first aspect, the embodiments of the present invention provide a method for preliminarily inferring cause of death based on endogenous metabolites, including the following steps:
detecting the content of acetyl carnitine, propionyl carnitine and succinic acid in blood samples with different lethal reasons;
constructing a reference standard library of the secondary classification features according to the absolute content;
and detecting the absolute content of acetyl carnitine, propionyl carnitine and succinic acid in the blood sample to be detected, bringing the absolute content into the reference standard library, and performing preliminary inference on the cause of death of the main body of the blood sample.
According to the invention, through researches, the endogenous metabolites of acetyl carnitine, propionyl carnitine and succinic acid are characteristic differences existing in blood in the case of chlorpromazine poisoning death and are also characteristic differences existing in blood in the case of death of other antipsychotic drugs with the same drug effect, such as perphenazine, olanzapine and clozapine, therefore, the method uses the acetyl carnitine, the propionyl carnitine and the succinic acid for predicting the death cause of death of the drugs which are taken independently or taken in a mixed mode, and establishes a prediction model by combining quantitative analysis of the acetyl carnitine, the propionyl carnitine and the succinic acid with mathematical two-classification probability to realize preliminary inference of the death cause of a main body of an unknown blood sample, and the prediction model has stable result and high reliability. When compared with other common causes of death: this approach is applicable to chlorpromazine and other antipsychotics with similar receptor of action and affinity when drowning, asphyxia, hemorrhagic shock, high cervical myelolysis are distinguished: perphenazine, olanzapine, clozapine are distinguished from the above causes of death. This method provides direct and objective laboratory test evidence for the determination of mortality from antipsychotic intoxication in cases where the suspected antipsychotic is overdose and lacks other characteristic morphological features. In addition, in the case of death caused by co-administration of a plurality of medicaments with similar receptor affinity to chlorpromazine, the method also provides direct laboratory basis for explaining that the blood concentration of a single medicament is lower than a threshold value.
Because the result judgment is inaccurate due to human body difference (for example, the health condition causes the abnormality of the metabolites in the blood), the method can only be used for preliminary judgment and auxiliary judgment in the process of judging the death cause of the death case by forensics, the death cause and the health condition of the blood sample body cannot be directly obtained, and the condition that the drug information, the body state and the like are required to be combined in the final judgment is still required.
Preferably, the operation of detecting the content of acetyl carnitine, propionyl carnitine and succinic acid in blood samples with different lethal causes comprises the following steps:
adding anticoagulant into blood sample to be tested, and centrifuging at 4 deg.C at 3000-5000r/min for 5-10min to remove blood cells (mainly red blood cells) to obtain first supernatant; centrifuging the first supernatant at 12000r/min for 5-10min to remove part of protein to obtain a second supernatant; adding chromatographic pure methanol with the volume being 3 times of the volume of the second supernatant into the second supernatant, uniformly mixing, performing ultrasonic treatment to sufficiently mix and precipitate macromolecules such as protein, standing for 15-20min at-20 ℃, and performing high-speed centrifugation for 10-15min at a rotating speed of not less than 12000r/min to obtain a third supernatant; drying the third supernatant, adding ultrapure water with the volume 5 times that of the third supernatant for redissolution, and finally filtering by using a 0.22-micron biological membrane to serve as a liquid to be detected;
and detecting the endogenous metabolites in the solution to be detected by using an ultra-performance liquid chromatography-mass spectrometry technology.
Wherein the centrifugation speed for obtaining the first supernatant is preferably 5000r/min to ensure complete removal of red blood cells.
And (4) uniformly mixing the second supernatant with chromatographic pure methanol, and setting the ultrasonic time to be about 10 min.
In the detection process, the volume ratio of the second supernatant to the chromatographic pure methanol and the volume ratio of the third supernatant to the ultrapure water ensure good peak shapes and proper peak emergence time of the acetyl carnitine, the propionyl carnitine and the succinic acid.
Preferably, the third supernatant is dried by a nitrogen-blowing treatment.
Preferably, in the ultra performance liquid chromatography-mass spectrometry combined technology, the chromatographic conditions are as follows:
a chromatographic column: an ultra-high pressure T3 chromatography column;
mobile phase A:0.1% aqueous formic acid; and (3) mobile phase B: acetonitrile;
gradient elution was performed according to the following procedure:
the flow rate is 0.3ml/min;
the above gradient elution procedure represents:
the volume percentage of the mobile phase A is 98 percent for 0 to 1 min; 1-3 min, reducing the volume percentage of the mobile phase A from 98% to 85% at a constant speed;
the volume percentage of the mobile phase A is reduced from 85 percent to 50 percent at a constant speed within 3-6 min;
the volume percentage of the mobile phase A is reduced from 50 percent to 2 percent at a constant speed within 6-9 min;
the volume percentage of the mobile phase A is 2 percent between 9min and 16.0 min;
the volume percentage of the mobile phase A is increased from 2 percent to 98 percent within 16 min-16.1 min;
the volume percentage of the mobile phase A is 2 percent between 16.1min and 20 min.
The column is preferably a UPLC HSS T3 column (2.1 mm. Times.100mm, 1.8 μm, waters).
The amount of the sample is preferably 5. Mu.l.
The preferred chromatographic column, mobile phase and elution program can ensure that the detection method has higher sensitivity, accuracy and repeatability.
The mass spectrum conditions are as follows:
adopting an HESI ionization mode; spraying voltage: positive electrode, 3.0kV; negative electrode, 2.7kV; the capillary temperature is 320 ℃; flow rate of sheath gas: 30arb, assist gas flow rate: 15arb; the scanning mode is Full Scan/dd-MS2, the acquisition range is m/z 70-1500, and the positive and negative ions are switched to the acquisition mode; resolution was obtained using MS Full Scan35000FWHM, MS2:17500FWHM, NCE in gradient modes of 12.5eV,25eV and 37.5 eV.
In a second aspect, embodiments of the present invention also provide a method of detecting endogenous metabolites including acetyl carnitine, propionyl carnitine, and succinic acid, the method comprising the steps of: adding anticoagulant into blood to be detected, and centrifuging at 5000r/min for 5min at 4 ℃ to obtain a first supernatant; centrifuging the first supernatant for 10min at 12000r/min to obtain a second supernatant; adding chromatographic pure methanol with the volume equivalent to 3 times that of the second supernatant into the second supernatant, uniformly mixing, performing ultrasonic treatment for 10min, standing at-20 ℃ for 15min, and centrifuging at 12000r/min for 10min to obtain a third supernatant; drying the third supernatant, adding ultrapure water with the volume 5 times that of the third supernatant for redissolving, and filtering to obtain a solution to be detected;
detecting the endogenous metabolites in the solution to be detected by using an ultra-high performance liquid chromatography-mass spectrometry technology:
the chromatographic conditions are as follows:
a chromatographic column: an ultra-high pressure T3 chromatography column;
mobile phase A:0.1% aqueous formic acid; mobile phase B: acetonitrile;
gradient elution was performed according to the following procedure:
the flow rate is 0.3ml/min;
the mass spectrum conditions are as follows:
adopting an HESI ionization mode; spraying voltage: positive electrode, 3.0kV; negative electrode, 2.7kV; the capillary temperature is 320 ℃; flow rate of sheath gas: 30arb, assist gas flow rate: 15arb; the scanning mode is Full Scan/dd-MS2, the acquisition range is m/z 70-1500, and the positive and negative ions are switched to the acquisition mode; resolution was obtained using MS Full Scan35000FWHM, MS2:17500FWHM, NCE in gradient modes of 12.5eV,25eV and 37.5 eV.
Drawings
FIG. 1 is a chromatogram of a test solution and an acetyl-carnitine standard in example 1;
FIG. 2 is a chromatogram of the test solution and propionyl carnitine standard in example 1;
FIG. 3 is a chromatogram of the test solution and succinic acid standard in example 1;
FIG. 4 is a second-order mass spectrum of the solution to be tested and the acetyl-carnitine standard in example 1;
FIG. 5 is a secondary mass spectrum of the test solution and propionyl carnitine standard in example 1;
FIG. 6 is a second-order mass spectrum of the solution to be tested and a succinic acid standard substance in example 1;
FIG. 7 is a graph of a confusion matrix of the two classification probabilities of blood samples from mice that are poisoned and killed versus non-poisoned and killed in example 2;
FIG. 8 is a ROC curve for the discrimination ability between propionyl-carnitine, acetyl-carnitine and succinic acid in a mouse blood sample in example 2;
FIG. 9 shows the stability and prediction capability of the displacement testing model in example 2;
FIG. 10 is a confusion matrix of the two classification probabilities of human blood samples from toxic lethality versus non-toxic lethality in example 3;
FIG. 11 is a ROC curve for the discrimination ability between propionyl-carnitine, acetyl-carnitine and succinic acid in human blood samples of example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the following examples, the hplc in the hplc-MS coupling technology is Ultimate 3000UPLC and the mass spectrometer is Q active (Orbitrap MS, thermo, germany).
Example 1
The embodiment of the invention provides a method for detecting endogenous metabolites of acetyl carnitine, propionyl carnitine and succinic acid, which comprises the following specific operations:
1.1 preparation of the test solution
Taking 1.5mL of blood to be detected, which is added with an anticoagulant, in an EP tube, and centrifuging at 5000r/min for 5min at 4 ℃ to obtain a first supernatant; centrifuging the first supernatant for 10min at 12000r/min to obtain a second supernatant; adding chromatographic pure methanol with volume equivalent to 3 times of the second supernatant, uniformly mixing, performing ultrasonic treatment for 10min, standing at-20 ℃ for 15min, and centrifuging at 12000r/min for 10min to obtain a third supernatant; drying the third supernatant by nitrogen blowing, adding ultrapure water (milliQ water) with the volume 5 times that of the third supernatant for redissolving, filtering, and taking the filtrate as a liquid to be detected for later detection.
1.2 sample detection
Detecting acetyl carnitine, propionyl carnitine and succinic acid in the solution to be detected by using an ultra-performance liquid chromatography-mass spectrometry technology:
1.2.1 chromatographic conditions
And (3) chromatographic column: a UPLC HSS T3 column (2.1 mm. Times.100mm, 1.8 μm, waters);
a mobile phase A:0.1% aqueous formic acid; mobile phase B: acetonitrile;
gradient elution was performed according to the following procedure:
the flow rate is 0.3ml/min; the sample amount is 5 mul;
1.2.2 Mass Spectrometry conditions
Adopting an HESI ionization mode; spraying voltage: positive electrode, 3.0kV; negative electrode, 2.7kV; the capillary temperature is 320 ℃; flow rate of sheath gas: 30arb, assist gas flow rate: 15arb; the scanning mode is Full Scan/dd-MS2, the acquisition range is m/z 70-1500, and the positive and negative ions are switched to the acquisition mode; resolution was measured using MS Full Scan35000fwhm, MS2:17500FWHM, NCE in gradient modes of 12.5eV,25eV and 37.5 eV.
1.2.3 test results
(1) Comparing the tested liquid with the standard product
The identification information obtained for the propionyl-carnitine, acetyl-carnitine and succinic acid standards under the above chromatographic and mass spectrometric conditions is shown in table 1.
TABLE 1 identification information for propionyl-carnitine, acetyl-carnitine and succinic acid standards
| Name(s) | RT(min) | m/z | Polarity | HMDB |
| Propionyl carnitine | 2.6 | 218.1386 | Positive signal | HMDB0000824 |
| Acetyl carnitine | 1.3 | 203.1156 | Positive signal | HMDB0000201 |
| Succinic acid | 2.0 | 151.0259 | Negative signal | HMDB0000254 |
The results of comparison of Retention Time (RT) and molecular weight of the solution to be tested with the propionyl-carnitine, acetyl-carnitine and succinic acid standard under the above chromatographic conditions and mass spectrometry are shown in fig. 1-3 (in each drawing, the upper graph is the solution to be tested, and the lower graph is the standard), and the results of comparison of the secondary mass spectrometry (the solution to be tested and the self-constructed library) are shown in fig. 4-6 (in each drawing, the upper graph is the solution to be tested, and the lower graph is the self-constructed library).
(2) Linearity
Under the above chromatographic and mass spectrometric conditions, the linear equations and quantitative limits of acetylcarnitine (0.21-4. Mu.g/ml), propionylcarnitine (0.02-0.1. Mu.g/ml) and succinic acid (0.33-4. Mu.g/ml) in the respective ranges are shown in Table 2. It can be seen that acetyl-carnitine, propionyl-carnitine and succinic acid are well quantified (R) within the respective ranges 2 >0.96)。
TABLE 2 Linear equation and quantitative limits
| Linear equation of equations | R 2 | Limit of quantitation (ug/ml) | |
| Acetyl carnitine | Y＝2.12516 e8 +9.21684 e8 *X | 0.99 | 0.21 |
| Propionyl carnitine | Y＝3.18819 e6 +3.17401 e9 *X | 0.96 | 0.02 |
| Succinic acid | Y＝-4.38729 e6 +2.0243 e8 *X | 0.99 | 0.33 |
(3) Precision degree
The day-to-day and day-to-day precision measurements were performed on the chlorpromazine poisoning lethal plasma samples according to the above linear equation, and the Coefficient of Variation (CV) was calculated according to the following formula:
coefficient of variation CV (%) = σ/μ × 100.
Where σ is the standard deviation and μ is the mean concentration.
The results are shown in Table 3.
TABLE 3 precision
Example 2
This example provides a method for preliminary inference of the cause of death in a sample of mouse deaths based on endogenous metabolites. The specific operation steps are as follows:
2.1 preparation of blood samples
Collecting blood samples of chlorpromazine, perphenazine, olanzapine and clozapine poisoning lethal mice, and preparing a poisoning lethal test solution according to the operation process of 1.1 in example 1;
collecting blood samples of mice which are drowned, suffocated, hemorrhagic shock and lethal to high cervical marrow separation, and preparing a non-poisoning lethal solution to be tested according to the operation process of 1.1 in the embodiment 1;
2.2 testing of blood samples
Respectively detecting the contents of acetyl carnitine, propionyl carnitine and succinic acid in a poisoning and lethal test solution and a non-poisoning and lethal test solution by using an ultra-high performance liquid chromatography-mass spectrometry technology, wherein the chromatographic conditions are the same as 1.2.1 in example 1, and the mass spectrometry conditions are the same as 1.2.2 in example 1.
The absolute levels of acetyl-carnitine, propionyl-carnitine, and succinic acid in the blood samples were estimated using calibur software.
2.3 calculation of the probability of two-Classification
Using the "marker analysis" module in the analysis software on Metabioanalyst line, the method calculates the classification probability of the acetylcarnitine, propionyl-carnitine and succinic acid content in the blood samples of mice with different causes of death by the SVM algorithm, and obtains a confusion matrix (as shown in FIG. 7) and ROC curve of the discrimination ability of acetylcarnitine, propionyl-carnitine and succinic acid (as shown in FIG. 8). The abscissa of fig. 7 is the probability that each sample was classified as a different blood sample in the binary scenario, the open circles represent toxic lethal blood samples (CPZ: chlorpromazine; PER: perphenazine; OLA: olanzapine; CLO: clozapine), and the filled circles represent Non-toxic lethal blood samples (i.e., non-toxic fatalities in the figure). The ROC curve shows the predictive power of acetyl-carnitine, propionyl-carnitine and succinic acid on the sample (AUC = 0.977).
As can be seen from fig. 7 and 8, the content of acetyl carnitine, propionyl carnitine and succinic acid in blood has strong ability to distinguish toxic lethality from non-toxic lethality.
2.4 model predictive power analysis based on permutation test statistical methods
In order to prevent the overfitting phenomenon occurring in the modeling process (namely, the situation that the model is only suitable for the samples of the batch and has low prediction capability on other batches of samples) the invention further verifies the prediction capability of the model through replacement test in the research process. The results are shown in FIG. 9. The verification result shows that the prediction results of the acetylcarnitine, the propionyl carnitine and the succinic acid against the intoxication and the lethal of the psychosis medicament are reliable (the accuracy rate is greater than 0.9), the model stability is strong (p is less than 0.05), and the hypothesis that the intoxication lethal group and the non-intoxication lethal group are from the same whole is not established.
2.5 determination of classification of unknown samples
Collecting blood samples of mice died of unknown reasons, and preparing a solution to be tested according to the operation process 1.1 in the example 1; respectively detecting the contents of acetyl carnitine, propionyl carnitine and succinic acid in a poisoning and lethal test solution and a non-poisoning and lethal test solution by using an ultra-high performance liquid chromatography-mass spectrometry technology, wherein the chromatographic conditions are the same as 1.2.1 in example 1, and the mass spectrometry conditions are the same as 1.2.2 in example 1. Calibur software was used to estimate the absolute levels of acetyl-carnitine, propionyl-carnitine and succinic acid in blood samples. Using the ROC curve shown in fig. 8, the cause of death of the mouse was preliminarily determined: if the absolute content is in a poisoning and lethal area, preliminarily judging that the death cause of the mouse is poisoning caused by excessive dosage of one or more of chlorpromazine, perphenazine, olanzapine and clozapine; if the absolute content is in a non-toxic lethal area, the cause of death of the mouse is preliminarily judged to be non-toxic death.
Based on the results of the preliminary assessment, the tissues, fluids, etc. of the mice were further examined to determine the specific cause of death.
Example 3
This example provides a method for forensics to preliminarily infer the cause of death in a human death sample based on endogenous metabolites. The specific operation steps are as follows:
3.1 preparation of blood samples
Collecting blood samples of chlorpromazine, perphenazine, olanzapine and clozapine in death cases from poisoning, and preparing a poisoning and death solution to be tested according to the operation process 1.1 in the example 1;
collecting a blood sample which is not lethal to the drug, and preparing a solution to be tested which is not lethal to the drug according to the operation process 1.1 in the example 1;
3.2 detection of blood samples
Respectively detecting the contents of acetyl carnitine, propionyl carnitine and succinic acid in a poisoning and lethal test solution and a non-poisoning and lethal test solution by using an ultra-high performance liquid chromatography-mass spectrometry technology, wherein the chromatographic conditions are the same as 1.2.1 in example 1, and the mass spectrometry conditions are the same as 1.2.2 in example 1.
Calibur software was used to estimate the absolute levels of acetyl-carnitine, propionyl-carnitine and succinic acid in blood samples.
3.3 calculation of the probability of two-Classification
Using the "marker analysis" module in the analysis software on Metabioanalyst line, the method calculates the classification probability of the acetylcarnitine, propionyl-carnitine and succinic acid content in the human blood samples of different causes of death by the SVM algorithm, and obtains a confusion matrix chart (as shown in FIG. 10) and a ROC curve (as shown in FIG. 11) of the discrimination ability of acetylcarnitine, propionyl-carnitine and succinic acid. The abscissa of fig. 10 is the probability that each sample falls under the binary scenario into a different blood sample, the open circles representing toxic lethal blood samples (OLA: olanzapine; CLO: clozapine), and the filled circles representing non-toxic lethal blood samples (i.e. Control in the figure). The ROC curve shows that the levels of acetyl-carnitine, propionyl-carnitine and succinic acid in human blood samples are also highly discriminative between toxic and non-toxic lethal (AUC = 0.953).
3.4 determination of sample Classification
The ROC curve of 3.3 was used to verify the causes of death between the blood samples of cases of known toxic causes and the subjects of blood samples of cases of known normal non-drug-taking populations, and the results are shown in Table 4.
TABLE 4 preliminary assessment results for death cases
| Case number | Case situation | Two class prediction probability | |
| 1 | Death from antipsychotic intoxication | 0.9784 | Death from |
| 2 | Death from antipsychotic intoxication | 0.73309 | Death from |
| 3 | Death from antipsychotic intoxication | 0.77717 | Death from |
| 4 | Non-drug related death | 0.71172 | Non-drug related |
| 5 | Non-drug related death | 0.54675 | Non-drug related |
| 6 | Non-drug related death | 0.73996 | Non-drug related |
| 7 | Non-drug related death | 0.57719 | Non-drug related death |
The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Claims (6)
1. A method for preliminarily inferring cause of death based on endogenous metabolites for non-diagnostic purposes comprising the steps of:
detecting the content of acetyl carnitine, propionyl carnitine and succinic acid in blood samples with different lethal reasons;
constructing a reference standard library of secondary classification features according to the absolute contents of the acetyl carnitine, propionyl carnitine and succinic acid;
and detecting the absolute content of acetyl carnitine, propionyl carnitine and succinic acid in the blood sample to be detected, and bringing the absolute content into the reference standard library to preliminarily infer the death of the antipsychotic drugs for the main body of the blood sample.
2. The method for preliminary inference of cause of death based on endogenous metabolites of non-diagnostic interest as claimed in claim 1, wherein the operation of detecting the content of acetyl-carnitine, propionyl-carnitine and succinic acid in blood samples of different causes of death comprises the following steps:
adding an anticoagulant into a blood sample to be detected, and centrifuging at the temperature of 4 ℃ at 3000-5000r/min for 5-10min to obtain a first supernatant; centrifuging the first supernatant for 5-10min at 12000r/min to obtain a second supernatant; adding chromatographic pure methanol with the volume which is 3 times that of the second supernatant into the second supernatant, uniformly mixing, performing ultrasonic treatment, standing at-20 ℃ for 15-20min, and centrifuging at the rotating speed of not less than 12000r/min for 10-15min to obtain a third supernatant; drying the third supernatant, adding ultrapure water with the volume 5 times that of the third supernatant for redissolution, and finally filtering by using a 0.22-micron biological membrane to serve as a liquid to be detected;
and detecting the endogenous metabolites in the solution to be detected by using an ultra-performance liquid chromatography-mass spectrometry technology.
3. The method for preliminary inference of mortality based on endogenous metabolites of non-diagnostic purpose according to claim 2, wherein the third supernatant is dried by nitrogen blowing treatment.
4. The method for preliminary inference of cause of death based on endogenous metabolites for non-diagnostic purposes as claimed in claim 2, wherein in said hplc-ms technique the chromatographic conditions are:
a chromatographic column: an ultra-high pressure T3 chromatography column;
mobile phase A:0.1% aqueous formic acid; mobile phase B: acetonitrile;
gradient elution was performed according to the following procedure:
The flow rate was 0.3ml/min.
5. The method of claim 4, wherein the chromatography column is a Waters UPLC HSS T3 chromatography column with a size of 2.1mm x 100mm and 1.8 μm.
6. The method for preliminary inference of cause of death based on endogenous metabolites for non-diagnostic purposes as claimed in claim 2, wherein in the ultra performance liquid chromatography-mass spectrometry technique, the mass spectrometry conditions are:
adopting an HESI ionization mode; spraying voltage: positive electrode, 3.0kV; negative electrode, 2.7kV; the capillary temperature is 320 ℃; flow rate of sheath gas: 30arb, assist gas flow rate: 15arb; the scanning mode is Full Scan/dd-MS2, the acquisition range is m/z 70-1500, and the acquisition mode is switched between positive and negative ions; resolution was measured using MS Full Scan35000fwhm, MS2:17500FWHM, NCE in gradient modes of 12.5eV,25eV and 37.5 eV.
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