CN114544844A - Method for detecting dichloromethane in blood - Google Patents

Abstract

The invention discloses a method for detecting dichloromethane in blood, which belongs to the technical field of detection and detection, wherein a blood sample is taken as a detection object, qualitative and/or quantitative detection is respectively carried out by adopting a gas chromatography-mass spectrometer and a headspace gas chromatography, and pretreatment on a sample to be detected before detection is optimized; the method not only increases the qualitative confirmation of mass spectrum, but also optimizes the pretreatment condition, has better separation degree and more reasonable quantitative curve, takes the application of the invention as an example, the relative difference RD values of the quantitative results of the blood samples of two deceased on double columns are 0.05 percent and 0.3 percent respectively, and the quantitative method is stable and reliable and can be used for the toxicological detection and analysis of suspected dichloromethane poisoning or lethal cases.

Description

Method for detecting dichloromethane in blood

Technical Field

The invention relates to the technical field of detection, in particular to a method for detecting dichloromethane in blood.

Background

Dichloromethane (DCM; CH2Cl 2; CAS No.75-09-2) belongs to the methyl chloride family, is a colorless, transparent, volatile liquid, slightly soluble in water, easily soluble in organic solvents, toxic to organisms, and currently classified as two carcinogens. In addition to the small amount of dichloromethane which can be produced in the nature, more than 100 million tons of dichloromethane are produced industrially by human every year in the world, and the dichloromethane serving as an organic solvent is widely applied to chemical synthesis, extraction, purification and metal cleaning in the fields of aerospace, electronics, films, medicines and the like. Methylene chloride can enter the human body through three routes of breathing, swallowing and skin absorption, and due to its instability, methylene chloride is mainly present in the air in the environment, and thus inhalation is the main route. After a human body inhales dichloromethane, respiratory and central nervous systems are damaged, symptoms such as dizziness, nausea, vomiting and the like are caused, and high-concentration occupational exposure can cause ataxia, loss of consciousness and even death.

With the increasing accumulation and manifestation of the environmental and human hazards of methylene chloride, and in view of the constant reports of the chloromethane family in terms of abnormal deaths, sexual assaults.

Before the eighties of the twentieth century, dichloromethane is mainly used in the medical field as an anesthetic and a sedative, is gradually widely used in industries such as medicine, chemical engineering and the like due to good organic matter solubility, causes serious pollution to the environment, and simultaneously has continuously reported poisoning and lethal cases caused by occupational exposure and aspiration, and the toxicity of dichloromethane gradually appears and has little harm. According to the American Association of the control of toxicants (AAPPC), 37201 methylene chloride poisoning events were recorded in the United states in 1985-2017; in 1980 and 2018, 85 cases of death are reported, and most of the cases are caused by occupational exposure, such as dichloromethane serving as a paint remover, a degreasing agent, an adhesive and the like. Retrospective statistical analysis aiming at dichloromethane poisoning and lethal events does not exist in China, but acute and chronic poisoning accidents are continuously reported, such as poisoning or death caused by using dichloromethane as a rust remover, aspiration and the like. Therefore, the exploration optimization and the application of the biological test material detection method in suspected dichloromethane poisoning or death cases have forensic medical practice significance.

The dichloromethane detection methods reported at present are all headspace gas chromatography methods, the application is concentrated in the fields of environmental monitoring and health and hygiene, and the forensic medicine detection application based on gas chromatography-mass spectrometry qualitative and gas chromatography quantitative is not seen. The headspace gas chromatography reported at present is applied to qualitative and quantitative detection of dichloromethane in blood, the result specificity is poor, the detection efficiency is low, for example, the required sample amount is large, at least 1ml is required, and the pretreatment time is long; the balance time is at least 20 min; the separation degree is not enough, the separation degrees of dichloromethane and trichloromethane are respectively 5.02 and 4.56, and the specificity is not enough, because the method only depends on gas chromatography for qualitative analysis, and the method uses more accurate mass spectrometry for qualitative analysis.

Disclosure of Invention

The invention aims to provide a method for quickly and accurately detecting dichloromethane in blood so as to solve the problems.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a method for detecting dichloromethane in blood takes a blood sample as a detection object, and adopts a gas chromatography-mass spectrometer and a headspace gas chromatography to carry out qualitative and/or quantitative detection respectively.

As a preferred technical scheme: the sample is pretreated before detection, and the treatment method comprises the following steps: adding blood to be detected into a headspace bottle containing an internal standard working solution, immediately capping, slightly shaking, and placing in a water bath at 60-70 ℃ for 5-15min to complete pretreatment.

As a preferred technical scheme: the headspace conditions for the headspace gas chromatography are: the heating box temperature is 65 ℃, the quantitative ring temperature is 105 ℃, the transmission line temperature is 110 ℃, the sample bottle heating balance time is 10min, the sample bottle pressurization time is 0.1min, the quantitative ring filling time is 0.1min, the quantitative ring balance time is 0.05min, and the sample introduction time is 1 min; the headspace conditions described above are used primarily for quantification.

The chromatographic conditions of the headspace gas chromatography are as follows: double chromatographic columns, the column temperature is 40 ℃; high-purity helium is used as carrier gas, the flow rate is 3mL/min, and the split ratio is 20: 1; the temperature of a sample inlet is 200 ℃; the detector temperature was 250 ℃ for the dual FID detectors. The aforementioned chromatographic conditions are mainly used for quantification.

The inventor finds through experiments that one chromatographic column may have peaks of different substances in the same time period, so that effective separation and identification cannot be achieved, and preferably two chromatographic columns can avoid the peaks, so that qualitative results are more accurate.

As a preferred technical scheme: the gas chromatography-mass spectrometry combined gas chromatography conditions are as follows:

column temperature procedure: maintaining at 100 deg.C for 0 min; heating to 220 deg.C at a rate of 15 deg.C/min, and maintaining for 4 min; high-purity helium is used as carrier gas, the flow rate is 1mL/min, and the split ratio is 20: 1; the injection port temperature was 200 ℃. The aforementioned gas chromatography conditions were used mainly for qualitative purposes.

As a further preferable technical scheme: the mass spectrum conditions of the gas chromatography-mass spectrum combination are as follows:

the transmission line temperature is 230 ℃; electron bombardment of the ion source, the electron energy is 70 eV; the ion source temperature is 230 ℃; full scan mode, scan mass number range 10-200 amu. The foregoing mass spectrometry conditions were used primarily for characterization.

Compared with the prior art, the invention has the advantages that:

the invention relates to a headspace gas chromatography-mass spectrometry combined method, establishes a rapid and stable qualitative and quantitative detection method for dichloromethane in blood, and is successfully applied to forensic toxicology detection and analysis of two dichloromethane lethal cases.

The invention provides a rapid and accurate gas chromatography-mass spectrometry (GC-MS) qualitative method based on the retention time, characteristic fragment ions and spectrum library analysis by using a common blood sample in forensic identification practice as a detection object, exploring and optimizing instrument and equipment parameters, and providing a matched headspace gas chromatography (HS-GC) quantitative method, thereby obtaining methodological verification on different chromatographic columns. Compared with the prior art, the method not only increases the qualitative confirmation of mass spectrum, but also optimizes the pretreatment conditions so that when the method is adopted, the required sample volume is reduced to 0.5ml, the balance time is less, only about 10min is needed, the separation degree is better, the separation degrees of dichloromethane and chloroform respectively reach 46.4 and 33.2 on the double columns, and the quantitative curve is more reasonable (an internal standard-calibration curve method), taking the application of the method as an example, the relative difference RD values of the quantitative results of the blood samples of two deceased on the double columns are respectively 0.05 percent and 0.3 percent, which shows that the quantitative method is stable and reliable, and can be used for the toxicological detection and analysis of suspected dichloromethane poisoning or lethal cases.

Drawings

FIG. 1 is a chromatogram of total ion current of blank blood with dichloromethane added;

FIG. 2 is a mass spectrum of a blank blood supplement;

FIG. 3 is a library search for methylene chloride;

FIG. 4 is a chromatogram of total ion current of blank blood;

FIG. 5 shows the separation of 8 volatile compounds such as dichloromethane and ethanol on an HS-GC two-color chromatographic column;

FIG. 6 is an A-column HS-GC calibration curve for methylene chloride in blood;

FIG. 7 is a B-column HS-GC calibration curve for methylene chloride in blood.

Detailed Description

The invention will be further explained with reference to the drawings.

The following examples used the main instruments and reagents:

trace 1300/ISQQD gas chromatography-Mass Spectrometry (ThermoFisher, USA); 7697A/8860 headspace gas chromatography (Agilent, USA); scales (mertler-toledo instruments shanghai ltd); ultrapure water systems (Sichuan Yopu ultra pure technologies, Inc.); methylene chloride standards (dr. ehrenstorfer, germany); methanol, ethanol, acetaldehyde, acetone, n-propanol, isopropanol, n-butanol and chloroform, analytically pure (Doctorong Chemicals Co., Ltd.); high purity helium (99.999%, grand source gas Co., Ltd.)

Example 1:

a method of detecting methylene chloride in blood comprising the steps of:

(1) sample pretreatment:

taking 0.5mL of blood, adding into a 10mL headspace bottle containing 0.5mL of internal standard working solution (chloroform: 0.2mg/mL), immediately capping, slightly shaking up, placing in a water bath at 65 ℃ for 10min, and finishing the pretreatment;

(2) and (3) qualitative detection:

and (3) manually extracting 100 mu L of middle-layer gas in the bottle by using a sample injection needle, and performing GC-MS (gas chromatography-mass spectrometry) qualitative detection under the following detection conditions:

gas chromatography conditions: agilent GS-CarbonPLOT chromatography column (30 m.times.0.32 mm. times.3 μm), column temperature program: heating to 220 deg.C at a rate of 15 deg.C/min (keeping for 4min) at 100 deg.C (keeping for 0 min); high-purity helium is used as carrier gas, the flow rate is 1mL/min, and the split ratio is 20: 1; the temperature of a sample inlet is 200 ℃;

mass spectrum conditions: the transmission line temperature is 230 ℃; electron impact ion source (EI), electron energy 70 eV; the ion source temperature is 230 ℃; full SCAN mode (SCAN), SCAN mass number range 10-200 amu;

(3) and (3) quantitative detection:

after being capped, the headspace bottle is placed in a headspace automatic sample injector, and quantitative detection is carried out by Agilent7697A/8860HS-GC under the following detection conditions:

headspace conditions: the heating box temperature is 65 ℃, the quantitative ring temperature is 105 ℃, the transmission line temperature is 110 ℃, the sample bottle heating balance time is 10min, the sample bottle pressurization time is 0.1min, the quantitative ring filling time is 0.1min, the quantitative ring balance time is 0.05min, and the sample introduction time is 1 min;

chromatographic conditions are as follows: column A: column DB-ALC1(30m × 0.32mm × 1.8 μm), B column: column DB-ALC2(30m × 0.32mm × 1.2 μm), column temperature 40 deg.C; high-purity helium is used as carrier gas, the flow rate is 3mL/min, and the split ratio is 20: 1; the temperature of a sample inlet is 200 ℃; the detector temperature was 250 ℃ for the dual FID detectors.

Example 2:

methodology validation

2.1 Selectivity

2.1.1 verification method

Taking 10 parts of blank blood (from healthy people and non-dichloromethane poisoning cases) and 3 parts of blank blood (dichloromethane is added to 1mg/mL of the blood of healthy people or dichloromethane, methanol, ethanol, acetaldehyde, acetone, n-propanol, isopropanol, n-butanol and chloroform are added to 1mg/mL) to carry out pretreatment and GC-MS or HS-GC detection according to the method of example 1;

2.1.2 criteria:

and (3) GC-MS: blank blood is free of interference, meanwhile, methylene dichloride chromatographic peaks and mass spectrum characteristic fragment ions are added in the blank, retention time is stable, and the selectivity is considered to be good;

HS-GC: the blank blood has no interference, simultaneously, the blank addition of dichloromethane, methanol, ethanol, acetaldehyde, acetone, n-propanol, isopropanol, n-butanol and chloroform has good chromatographic peak pattern and stable retention time, and the separation degree R of 8 compounds such as dichloromethane, ethanol and the like is more than or equal to 1.5, and the selectivity is considered to be good.

2.1.3 validation results:

2.1.3.1GC-MS

the total ion current chromatogram of blank blood added with dichloromethane is shown in figure 1, the retention time (Rt) of a target dichloromethane is 6.395min, the peak shape is symmetrical, no interference of a foreign peak is caused, and the separation from internal standard chloroform (Rt: 9.674min) is good; the mass spectrum is shown in FIG. 2, the dichloromethane characteristic fragment ions are m/ z 84, 86, 49, 51 and the like, and the matching degree is more than 900 after the spectral library searching and comparison confirmation, as shown in FIG. 3;

the total ion current chromatogram of the blank blood is shown in fig. 4, the internal standard chloroform generates peaks normally, and the dichloromethane generating peak time period has no peak, which shows that the method has strong specificity and good specificity, and endogenous substances in the blood have no interference to the detection of the dichloromethane.

2.1.3.2HS-GC

The blank matrix is free of interference, the retention time (Rt) of blank dichloromethane addition is 2.965min (A column) and 2.842min (B column), the peaks are symmetrical, the R values of the degrees of separation of the blank dichloromethane addition from methanol, ethanol, acetaldehyde, acetone, n-propanol, isopropanol, n-butanol and chloroform are between 3.1 and 86.1 and are all more than 1.5, and the detection of dichloromethane is not interfered by common volatile compounds, as shown in figure 5.

2.2 detection and quantitation limits

2.2.1 validation method

Dichloromethane was added to the blank blood to prepare a series of samples with mass concentrations of 1, 2, 5, 10, 20 μ g/mL, and pretreatment and GC-MS detection (detection limit) or HS-GC detection (quantification limit) were performed according to methods of 1.2 and 1.3, with 3 replicates for each concentration.

2.2.2 criteria of judgment

Taking the lowest concentration of GC-MS signal-to-noise ratio (S/N) being more than or equal to 3 as a method detection Limit (LOD); the lowest concentration with HS-GC signal-to-noise ratio (S/N) being more than or equal to 10, precision (RSD) being less than 15% and accuracy (relative error delta) being less than 20% is taken as the quantitative Limit (LOQ) of the method.

2.2.3 validation results

The dichloromethane blank blood with the series concentrations is added and respectively subjected to GC-MS detection, the signal-to-noise ratio (S/N) is more than or equal to 3, and the stable minimum concentration is 5 mu g/mL, namely the detection Limit (LOD) of the method is determined; HS-GC detection is carried out, the lowest concentration of a signal-to-noise ratio (S/N) ≥ 10 is 20 mug/mL, the precision is respectively 0.29% (A column) and 0.64% (B column) which are both less than 15%, and the accuracy is respectively 15.73% (A column) and 15.83% (B column) which are both less than 20%, so that the concentration is determined as the method quantitative Limit (LOQ).

As shown in fig. 6, the linear relationship of methylene chloride in blood was good in the concentration range of 20-1000 μ g/mL as measured by HS-GC, and the linear equations were y ═ 0.0179x +0.0623(R ═ 0.0179x +0.0623, respectively (R)²0.9985, a column), y 0.018x +0.0656 (R)²0.9984, B bar).

2.3 Linear Range

2.3.1 verification method

Dichloromethane is added into blank blood to respectively prepare a series of samples with mass concentrations of 20, 50, 100, 200, 500, 800 and 1000 mu g/mL, pretreatment and HS-GC detection are carried out according to methods of 1.2 and 1.3, and each concentration is carried out for 3 times.

2.3.2 criteria of judgment

And (3) establishing a unary linear regression equation by taking the mass concentration of the dichloromethane as a horizontal coordinate and the peak area ratio of the dichloromethane to the chloroform chromatographic peak as a vertical coordinate, wherein the correlation coefficient R is more than or equal to 0.999 (or R2 is more than or equal to 0.998) and the linearity is considered to be good.

2.4 precision and accuracy

2.4.1 verification method

Dichloromethane was added to the blank blood to prepare samples with low, medium and high quality concentrations (50, 200, 800. mu.g/mL), and pretreatment and HS-GC detection were performed according to methods 1.2 and 1.3 on the same day, with 6 replicates of each concentration, repeated for 3 consecutive days.

2.4.2 criteria

And substituting the peak area ratio of dichloromethane and chloroform chromatographic peak into a linear equation to calculate the measured value, and calculating the precision (represented by RSD) of each concentration within day and during day respectively, wherein the RSD of less than 15 percent is regarded as qualified. And substituting the peak area ratio of dichloromethane and chloroform chromatographic peak of each concentration into a linear equation to calculate the measured value, and calculating the accuracy (expressed by relative error delta), wherein delta less than 15 percent is regarded as qualified.

2.4.3 validation results

The low, medium and high concentration dichloromethane additions were repeatedly and continuously checked by HS-GC as shown in table 1, with an intra-day/inter-day precision and accuracy of the three concentrations, all < 15% on A, B column.

Precision and accuracy of the methods of Table 1

Example 3:

according to the detection method of the example 1, heart blood is extracted from two chemical plant workers suspected to be killed by occupational exposure poisoning, methylene dichloride is qualitatively and quantitatively detected, and calculation is carried out according to the calibration curve in the example 2, methylene dichloride components are detected in the blood of two deceased persons, the content of the methylene dichloride components is 470 mu g/mL and 915 mu g/mL respectively, and by combining forensic pathology analysis, the death caused by anoxic asphyxiation due to methylene dichloride poisoning is met. The quantitative method of the calibration curve is accurate and reliable, is superior to a single-point quantitative method, and has good linear relation, R value is more than 0.999

Comparative example

The technical effects brought by the special pretreatment method of the invention are verified as follows:

1. the methods reported in the prior art are as follows: taking 1ml of blood, adding into a headspace bottle containing 1ml of internal standard working solution, immediately capping, slightly shaking up, and keeping the temperature at 70 ℃ for 20 min;

pretreating standard 2ml blood according to the method, repeating the experiment for 2 times, and averaging pretreatment time for 25 min;

2. the method comprises the following steps: taking 0.5ml of blood, adding into a headspace bottle containing 0.5ml of internal standard working solution, immediately capping, slightly shaking, and keeping the temperature at 65 ℃ for 10 min;

pretreating standard 2ml blood according to the method, repeating the experiment for 4 times, and averaging pretreatment time for 10 min;

therefore, the pretreatment efficiency can be improved by more than 50% by adopting the method.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (6)

1. A method for detecting methylene chloride in blood, comprising: and (3) taking the blood sample as a detection object, and respectively carrying out qualitative and/or quantitative detection by adopting a gas chromatography-mass spectrometer and a headspace gas chromatography.

2. The method for detecting dichloromethane in blood as claimed in claim 1, wherein: the sample is pretreated before detection, and the treatment method comprises the following steps: adding blood to be detected into a headspace bottle containing an internal standard working solution, immediately capping, slightly shaking, and placing in a water bath at 60-70 ℃ for 5-15min to complete pretreatment.

3. The method for detecting dichloromethane in blood as claimed in claim 1, wherein: the headspace conditions for the headspace gas chromatography are: the heating box temperature is 65 ℃, the quantitative ring temperature is 105 ℃, the transmission line temperature is 110 ℃, the sample bottle heating balance time is 10min, the sample bottle pressurization time is 0.1min, the quantitative ring filling time is 0.1min, the quantitative ring balance time is 0.05min, and the sample introduction time is 1 min.

4. The method of claim 3, wherein the detection of dichloromethane in blood comprises: the chromatographic conditions of the headspace gas chromatography are as follows: double chromatographic columns, the column temperature is 40 ℃; high-purity helium is used as carrier gas, the flow rate is 3mL/min, and the split ratio is 20: 1; the temperature of a sample inlet is 200 ℃; the detector temperature was 250 ℃ for the dual FID detectors.

5. The method for detecting dichloromethane in blood as claimed in claim 1, wherein: the gas chromatography-mass spectrometry combined gas chromatography conditions are as follows:

column temperature procedure: maintaining at 100 deg.C for 0 min; heating to 220 deg.C at a rate of 15 deg.C/min, and maintaining for 4 min; high-purity helium is used as carrier gas, the flow rate is 1mL/min, and the split ratio is 20: 1; the injection port temperature was 200 ℃.

6. The method of claim 5, wherein the detection of dichloromethane in blood comprises: the mass spectrum conditions of the gas chromatography-mass spectrum combination are as follows:

the transmission line temperature is 230 ℃; electron bombardment of the ion source, the electron energy is 70 eV; the ion source temperature is 230 ℃; full scan mode, scan mass number range 10-200 amu.

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