CN106950303B - Method for measuring benzene series in biological sample blood - Google Patents
Method for measuring benzene series in biological sample blood Download PDFInfo
- Publication number
- CN106950303B CN106950303B CN201710169700.7A CN201710169700A CN106950303B CN 106950303 B CN106950303 B CN 106950303B CN 201710169700 A CN201710169700 A CN 201710169700A CN 106950303 B CN106950303 B CN 106950303B
- Authority
- CN
- China
- Prior art keywords
- blood
- standard
- sample
- series
- benzene series
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a method for measuring benzene series in biological sample blood, in particular to a method for simultaneously measuring the content of 6 benzene series in blood by a purging and trapping/gas chromatography/mass spectrometry combined technology. The invention optimizes the purging and trapping experimental conditions to enrich and resolve 6 benzene series in blood, and then uses gas chromatography/mass spectrometry and an isotope internal standard method to select characteristic ions for qualitative and quantitative analysis. The method has the advantages of simple operation, short analysis period, good separation degree, and accuracy and precision meeting the requirements of analysis and test, and is suitable for simultaneous detection and analysis of the benzene series in the blood of the biological sample.
Description
Technical Field
The invention relates to the technical field of chemical analysis and detection, in particular to a method for determining benzene series in biological sample blood.
Background
The benzene series is an important raw material and solvent in the industries of chemical industry, medicine, pesticide, leather making and the like. Because benzene series has stronger volatility and widely exists in the environment, the benzene series becomes an important occupational hazard factor due to wide contact routes. Benzene series has great harm to human health, such as damage to hematopoietic system and teratogenesis. Therefore, how to effectively measure the exposure content in the benzene series is a hot spot of current research in the environmental medical field and an urgent problem to be solved.
At present, many methods for detecting benzene compounds in air and water are known, and various methods are known, and blood components and physicochemical properties are very complicated compared with water, air, and the like, and the method for detecting benzene compounds in water and air is not suitable for detecting blood. At present, no report about the detection technology of benzene series in the blood of biological samples is found.
Disclosure of Invention
The invention provides a method for measuring benzene series in biological sample blood, which at least achieves good separation degree, and the accuracy and precision can meet the requirements of analysis and test.
Aiming at the technical problems, the method for measuring the benzene series in the blood of the biological sample provided by the invention adopts a sweeping trapping/gas chromatography/isotope internal standard mass spectrometry combined method to measure the content of the benzene series in the blood, and the measuring method specifically comprises the following steps:
preparing a mixed standard solution: weighing pure substances of benzene series, and preparing a mixed standard solution by using methanol;
preparing isotope internal standard solution: weighing isotope substances of benzene series, and preparing isotope internal standard solution by using methanol;
preparation of standard series of solutions: taking the mixed standard solution, and preparing a standard series solution with a concentration gradient of more than 5 grades by using methanol; respectively adding standard series solutions with different concentrations into corresponding sample injection bottles, sequentially adding an isotope internal standard solution, blank blood and a defoaming agent into each sample injection bottle, and finally fixing the volume by using pure water;
detection of standard series solution and drawing of standard curve: measuring the standard series solutions with different concentrations by a purging and trapping instrument and a gas chromatograph, drawing a standard curve of each substance by taking the concentration as a horizontal coordinate and taking the ratio of the peak areas of the benzene series in the standard series solutions and the benzene series in the isotope internal standard solutions as a vertical coordinate, and calculating to obtain a standard curve equation;
measurement of blood samples: adding a blood sample into a sample feeding bottle, sequentially adding an isotope internal standard solution and a defoaming agent, finally fixing the volume by pure water, then measuring by a purging and trapping instrument and a gas chromatograph-mass spectrometer to obtain the peak area ratio of the benzene series in the blood sample and the benzene series in the isotope internal standard solution, and respectively substituting the peak area ratio into corresponding standard curve equations to obtain the content of the benzene series in the blood sample.
The invention adopts an isotope internal standard method of P & T/GC/MS combined technology to carry out accurate qualitative and quantitative analysis on the benzene series content in the biological sample blood, provides scientific basis for the detection method of the human benzene series internal exposure, and has good separation degree of the detection result and accuracy and precision which can meet the requirements of analysis and test. The invention uses an isotope internal standard method. In the prior art, the quantitative method is mainly an external standard method or a normalization method, but the normalization method needs to know the correction factors of all components and is not suitable for measuring the content of trace impurities; the external standard method requires that the concentration of a reference solution is similar to the concentration of components in a sample, and the accuracy of the method is influenced by the repeatability of sample injection and the stability of experimental conditions. The internal standard method is a method for determining the content of the component to be measured by taking peak areas of the component to be measured and the internal standard as a ratio by adding pure substances which are not contained in the sample as the internal standard into the solution of the sample to be measured. The internal standard method has the advantages that: in the range of no overload of the chromatographic column, the quantitative result is irrelevant to the repeatability of the sample injection amount; the quantitative determination can be carried out as long as the measured substance and the internal standard substance generate peaks and can be completely separated, and the quantitative determination is independent of other components; can avoid inaccurate quantification caused by the loss of the components to be measured in each step of treatment operation. The internal standard method has the following defects: internal standards are not readily available. Requirements for internal standards: the internal standard substance is a component which is not contained in the original sample, otherwise, peaks are overlapped and the peak area of the internal standard substance cannot be accurately measured; the internal standard substance and the compound to be detected have the same or similar physicochemical properties, and the retention time of the internal standard substance and the compound to be detected is similar, but the internal standard substance and the compound to be detected can be completely separated from each other (the separation degree is more than or equal to 1.5); the internal standard substance is required to be not interfered and not reacted with the sample components; the internal standard must be pure and of a desired purity. The invention uses isotope substitution substance as internal standard, namely, the isotope is used for substituting C or H of the compound to be detected, so that the internal standard substance and the compound to be detected have similar physicochemical properties and can be completely separated from each other. The chromatographic behavior of the isotope internal standard is similar to that of the retention time and ion abundance of the compound to be detected.
Further, parameter setting of the purge trap instrument: taking 99.999% helium as purge gas, wherein the purge time is 11min, and the purge flow is 40 mL/min; the resolving temperature is 250 ℃, the resolving flow is 300 mL/min, and the resolving time is 2 min; the baking temperature is 280 ℃, the baking flow is 200 mL/min, and the baking time is 2 min. About 35% to 70% of blood test results were erroneous from the blood sample prior to testing. In order to obtain reliable detection data and provide real and accurate experimental data for a disease prevention control center and an environmental protection department, interference in a series of processes of blood collection, blood preservation, detection and the like must be strictly controlled. Benzene series in blood belongs to trace toxic and harmful organic pollutants, and needs to be detected by adopting an analysis and test technology with high enrichment efficiency and high sensitivity. Therefore, the invention adopts the purging and trapping technology (P & T) to efficiently enrich the volatile benzene series (benzene, toluene, ethylbenzene, m/P-xylene and o-xylene) in the blood of the biological sample, which not only can reduce the detection limit of the analysis method, but also does not need organic solvent and extraction matrix, and can effectively avoid secondary pollution caused by the interaction of organic reagent and certain components in the blood sample. The invention aims at the characteristics of blood, performs targeted optimization on the condition parameters of purging and trapping, and the like, for example, analyzes the influence of the purging temperature, the purging time, the desorption temperature and the desorption time on the purging and trapping efficiency, and finally determines the suitable parameters.
Further, the parameters of the gas chromatograph-mass spectrometer are set as follows: the chromatographic column is a DB-624 capillary chromatographic column, and the specification of the chromatographic column is 30m multiplied by 0.25mm multiplied by 1.4 mu m; keeping the column temperature at 40 deg.C for 3min, heating to 150 deg.C at 10 deg.C/min, keeping for 0.5min, heating to 210 deg.C at 15 deg.C/min, and keeping for 4 min; split-flow sample injection, the split-flow ratio is 20: 1; the temperature of a sample inlet is 250 ℃; the carrier gas is 99.999% helium; in the constant-current mode, the carrier gas flow is 1.2 mL/min; the ion source type is EI, and the ion source temperature is 250 ℃; the temperature of the transmission line is 280 ℃; the electron bombardment energy was 70 eV. The gas chromatography-mass spectrometry (GC/MS) can be used for qualitatively and quantitatively analyzing trace unknown compounds, has high sensitivity and wide application range, is one of the most important analysis and test technologies at present, and is widely applied to analyzing VOCs in water and air. The chemical properties of the isotope internal standard compound and the target compound are almost the same, so that the enrichment efficiency, the loss degree, the matrix influence and the like in the test process are completely consistent, and the isotope internal standard compound can be used for correcting the test deviation of the analysis method.
Further, step 1), a mixed standard solution with each single standard concentration of 100. mu.g/mL is prepared.
Further, step 2), isotope internal standard solutions with each single standard concentration of 200ng/mL were prepared.
Further, the preparation process of the blood sample comprises collecting the blood sample in a blood collection tube by venipuncture, pouring the blood sample in the vacuum blood collection tube into a sample bottle after sampling, and preserving the blood sample in a refrigerated manner at 4 ℃.
In summary, the method for simultaneously determining the content of the benzene series in the blood of the biological sample by using the blowing and trapping/gas chromatography/mass spectrometry combined technology provided by the invention optimizes the blowing and trapping experimental conditions to enrich and analyze the benzene series in the blood of the biological sample, and then uses the gas chromatography/mass spectrometry combined technology and an isotope internal standard method to select characteristic ions to perform qualitative and quantitative analysis, through optimizing the blowing and trapping parameters, the method realizes the simultaneous enrichment, analysis and accurate determination of 6 benzene series of benzene, toluene, ethylbenzene, m/p-xylene and o-xylene in the blood of the biological sample, the detection limit of the method is 0.002 ~ 0.011 mu g/L, the linear range is 0.043 ~ 10.99 mu g/L, the precision ranges of low and high concentration are 72.78% ~.03%, 82.27% ~ 109.09%, the same sample is repeatedly analyzed for 6 times, the RSD range is 3.47% 367.36%, the operation is simple and convenient, the separation cycle is good, the separation recovery rate is good, and the method is suitable for simultaneous detection of the benzene series in the blood, and can meet the requirements of biological sample detection.
Drawings
FIG. 1 shows the trend of the spectral peak area for different purge flow rates.
FIG. 2 shows the trend of the area of each spectral peak at different purge temperatures.
FIG. 3 shows the trend of the peak area of each spectrum at different analysis temperatures.
FIG. 4 shows the trend of the peak area of each spectrum at different analysis times.
FIG. 5 is a selected ion spectrum of a mixed standard solution with a concentration of 1.563. mu.g/L.
Detailed Description
The method adopts a combined method of purging trapping/gas chromatography/isotope internal standard mass spectrometry to determine the content of the benzene series in the blood, and in a typical embodiment, the determination method specifically comprises the following steps:
preparing a mixed standard solution: weighing pure substances of benzene series, and preparing a mixed standard solution by using methanol;
preparing isotope internal standard solution: weighing isotope substances of benzene series, and preparing isotope internal standard solution by using methanol;
preparation of standard series of solutions: taking the mixed standard solution, and preparing a standard series solution with a concentration gradient of more than 5 grades by using methanol; respectively adding standard series solutions with different concentrations into corresponding sample injection bottles, sequentially adding an isotope internal standard solution, blank blood and a defoaming agent into each sample injection bottle, and finally fixing the volume by using pure water;
detection of standard series solution and drawing of standard curve: measuring the standard series solutions with different concentrations by a purging and trapping instrument and a gas chromatograph, drawing a standard curve of each substance by taking the concentration as a horizontal coordinate and taking the ratio of the peak areas of the benzene series in the standard series solutions and the benzene series in the isotope internal standard solutions as a vertical coordinate, and calculating to obtain a standard curve equation;
measurement of blood samples: adding a blood sample into a sample feeding bottle, sequentially adding an isotope internal standard solution and a defoaming agent, finally fixing the volume by pure water, then measuring by a purging and trapping instrument and a gas chromatograph-mass spectrometer to obtain the peak area ratio of the benzene series in the blood sample and the benzene series in the isotope internal standard solution, and respectively substituting the peak area ratio into corresponding standard curve equations to obtain the content of the benzene series in the blood sample.
In a relatively specific embodiment, the method comprises the steps of:
1) first, a high purity nitrogen mask was opened and the atmosphere was vented for 15 min to fill the operating environment with nitrogen.
2) Preparing a mixed standard solution: pure benzene series substances are weighed out, and are preferably prepared by methanol to obtain mixed standard solutions with each single standard concentration of 100 mu g/mL.
The method specifically comprises the following steps: a. 0.0281g of benzene, 0.0344g of toluene, 0.0248g of ethylbenzene, 0.0283g of m-xylene, 0.0283g of p-xylene and 0.0277 g of o-xylene are accurately weighed in a 10.0mL volumetric flask, and methanol is used for fixing the volume to a scale mark, so that standard solutions of benzene 2.81mg/mL, toluene 3.44mg/mL, ethylbenzene 2.48mg/mL, m-xylene 2.83mg/mL, p-xylene 2.83mg/mL and o-xylene 2.77mg/mL are obtained; b. accurately placing 356 mu L of benzene, 291 mu L of toluene, 403 mu L of ethylbenzene, 354 mu L of m-xylene, 354 mu L of p-xylene and 361 mu L of o-xylene in the prepared standard solutions in a 10.0mL volumetric flask, and metering the volume to a scale mark by using methanol to obtain mixed standard solutions with the single standard concentration of 100.0 mu g/mL; c. the mixture is respectively put into 1mL brown ampoule bottles, sealed and placed in a refrigerator for refrigeration storage at 4 ℃.
3) Preparing isotope internal standard solution: isotope substances of the benzene series are weighed, and preferably methanol is used for preparing isotope internal standard solutions with the single standard concentration of 200 ng/mL.
The method specifically comprises the following steps: a. accurately weighing 0.0421g of benzene (D5), 0.0435g of toluene (D5), 0.0451g of ethylbenzene (D10), 0.0452g of m/p-xylene (D10) and 0.0357g of o-xylene (D4) in a 10.0mL volumetric flask, and metering the volume to a scale mark by using methanol to obtain an isotope single-label stock solution; b. accurately taking 2.375 mL of the prepared benzene (D5), 2.299mL of the prepared toluene (D5), 2.217 mL of ethylbenzene (D10), 2.212mL of m/p-xylene (D10) and 2.801mL of o-xylene (D4) in a 50.0mL volumetric flask, and metering the volume to a scale mark by using methanol to obtain an isotope mixed stock solution with the concentration of 200 mu g/mL; c. respectively filling into 1mL brown ampoule bottles, sealing, placing in a refrigerator, and refrigerating at 4 deg.C for storage; d. taking 1.0mL of isotope mixed label stock solution, diluting to 10.00mL with methanol to obtain 20.0 μ g/mL isotope mixed label intermediate solution; e. taking 0.1mL of the isotope mixed standard intermediate solution, and diluting the isotope mixed standard intermediate solution to 10.00mL by using methanol to obtain an isotope internal standard solution with the concentration of 200.0 ng/mL; f. the mixture is respectively put into a 10 mL brown solvent bottle, sealed and placed in a refrigerator, and refrigerated at 4 ℃.
4) Parameter setting of the measuring instrument: the apparatus used in the measurement process is TQU03947 GC from Thermo Fisher scientific, USA; purge and trap instruments, purge and trap instrument autosampler, Tekmar, usa; DB-624 elastic quartz capillary chromatography column from Agilent, USA; 25 mL of sand core type purging pipe; 40mL of a brown sample bottle lined with a polytetrafluoroethylene film; 7 mL of glass vacuum blood collection tube containing heparin sodium and sodium fluoride; 5mL brown glass sample bottles.
Setting parameters of the purging and trapping instrument: taking 99.999% helium as purge gas, wherein the purge time is 11min, and the purge flow is 40 mL/min; the resolving temperature is 250 ℃, the resolving flow is 300 mL/min, and the resolving time is 2 min; the baking temperature is 280 ℃, the baking flow is 200 mL/min, and the baking time is 2 min.
a. Determination of purge time and purge flow rate
The purge time and the purge flow rate are important parameters of the purge trapping technology, and the product of the purge time and the purge flow rate is the total volume of the purge gas. The larger the total volume of purge gas, the higher the purge efficiency. However, the total volume of the purge gas is too large, and the analyzed components trapped in the cold trap are blown off or scattered, so that the purge efficiency and the reproducibility of the result are reduced. Aiming at blood characteristics, saving analysis time and improving working efficiency, the method selects 11min as purging time and optimizes the purging flow rate. Selecting 34, 36, 38, 40 and 42 mL/min as time gradients, taking the mixed standard solution with the same concentration for analysis, and comparing the change of the area of each chromatographic peak under different purge flow rates.
The concentration of six benzene series in the mixed standard solution is 200 ng/L, the change trend of the chromatographic peak area under different purging flow rates is shown in figure 1. it can be seen that the chromatographic peak area is gradually increased along with the increase of the purging flow rate in the range of 36 ~ 44mL/min, the chromatographic peak area reaches the maximum value when the purging flow rate is 40mL/min, the chromatographic peak area is reduced after the purging flow rate is more than 40mL/min, and the purging flow rate is selected to be 40mL/min according to the blood characteristics.
b. Determination of purge temperature
The method has the advantages that the purging efficiency is improved by increasing the purging temperature, the purging time is shortened, particularly when high-water-solubility compounds are purged, the purging temperature has a larger influence on the purging efficiency, if the purging temperature is too high, too much water vapor is purged, the adsorption of components to be detected in a cold trap is not facilitated, in addition, the separation efficiency of the middle-polarity gas chromatographic column is reduced due to high moisture, the service life of the middle-polarity gas chromatographic column is damaged, the common temperature of 20 ~ 50 ℃ is generally selected for the VOC in water, the matrix in blood is complicated, and partial protein is deformed and even blocks a pipeline due to the temperature of more than 40 ℃, so the temperature gradients of 20 ℃, 25 ℃, 30, 35 and 40 ℃ are selected, the mixed standard solution with the same concentration is used for analysis, and the change of the area of each chromatographic peak under different purging temperatures is compared.
The concentrations of six benzene series in the mixed standard solution are all 200 ng/L, and the change trend of the area of each chromatographic peak under different blowing temperatures is shown in figure 2. It can be seen that the chromatographic peak area increases with increasing purge temperature. However, the present invention selects a purge temperature of 40 deg.C in consideration of the characteristics of blood.
c. Determination of analytical temperature
The desorption temperature is another key parameter of the purge trap technique. When the purge is complete, the resolver rapidly heats the trap to desorb compounds adsorbed on the trap surface and blows the compounds with the carrier gas into the gas phase system. The higher the resolution temperature, the shorter the heating time of the trap, the more complete the resolution, i.e., the higher the purging efficiency, and the higher the resolution temperature is in favor of forming sharp and symmetrical chromatographic peaks. However, too high a desorption temperature may reduce the life of the trap and the adsorbent. According to the characteristics of blood, the invention selects 220 ℃, 230, 240, 250 and 260 ℃ as temperature gradients, takes the mixed standard solution with the same concentration for analysis, and compares the change of the area of each spectrum peak at different analysis temperatures.
The concentrations of six benzene series in the mixed standard solution are all 200 ng/L, the change trend of the chromatographic peak areas at different analysis temperatures is shown in figure 3. it can be seen that the chromatographic peak areas are gradually increased along with the increase of the analysis temperatures in the temperature range of 210 ~ 250 ℃, the chromatographic peak areas are stable after 250 ℃, and the analysis temperature is selected to be 250 ℃ aiming at the blood characteristics and in order to protect the trap and the adsorbent.
d. Determination of resolution time
The longer the desorption time, the more complete the desorption and the higher the purge efficiency. However, too long a resolving time may reduce the adsorbent lifetime and cause the chromatographic peak to be smeared. Aiming at blood characteristics, 1.0, 1.5, 2.0, 2.5 and 3.0 min are selected as time gradients, mixed standard solutions with the same concentration are taken for analysis, and the change of the area of each chromatographic peak under different analysis times is compared.
The concentrations of six benzene series in the mixed standard solution are all 200 ng/L, the change trend of the chromatographic peak areas under different analysis times is shown in figure 4. it can be seen that the chromatographic peak areas gradually increase along with the increase of the analysis time within the temperature range of 1.0 ~ 2.0.0 min, the chromatographic peak areas tend to be stable after 2.0 min, and the analysis time is selected to be 2.0 min for the blood characteristics and the adsorbent protection.
e. Determination of cleaning and baking parameters
Typically the wash cycle in water is set to 1. Blood is complicated in composition and contains a large amount of blood clots, flocculent proteins, blood cells, and the like. In order to reduce the loss of volatile substances to the maximum extent, the pretreatment step of the invention is simple, so that various substances in the blood are easy to block the transmission pipeline and each electromagnetic valve of the purging and trapping instrument. In the test, the phenomenon of instrument blockage is obviously reduced after the cleaning cycle is set to be 3 times.
Higher concentrations of the component to be measured can result in higher instrument background values. Therefore, a higher baking temperature and a longer baking time should be set to reduce the volatile substances remaining in the instrument. The baking temperature is 280 ℃ and the baking time is 2 min.
Setting parameters of the gas chromatograph-mass spectrometer: the chromatographic column is a DB-624 capillary chromatographic column, and the specification of the chromatographic column is 30m multiplied by 0.25mm multiplied by 1.4 mu m; keeping the column temperature at 40 deg.C for 3min, heating to 150 deg.C at 10 deg.C/min, keeping for 0.5min, heating to 210 deg.C at 15 deg.C/min, and keeping for 4 min; split-flow sample injection, the split-flow ratio is 20: 1; the temperature of a sample inlet is 250 ℃; the carrier gas is 99.999% helium; in the constant-current mode, the carrier gas flow is 1.2 mL/min; the ion source type is EI, and the ion source temperature is 250 ℃; the temperature of the transmission line is 280 ℃; the electron bombardment energy was 70 eV. The retention time for each compound was determined by full scan mode, the mass spectrum peak top value was determined by SIM mode, and the ions selected are shown in table 1.
5) Preparation of standard series of solutions: taking the mixed standard solution, and preparing a standard series solution with a concentration gradient of more than 5 grades by using methanol; respectively adding standard series solutions with different concentrations into corresponding sample injection bottles, sequentially adding an isotope internal standard solution, blank blood and a defoaming agent into each sample injection bottle, and finally fixing the volume by using pure water; when VOC in water is measured, the prepared standard series solution is directly prepared by water without adding a matrix; the standard series of solutions prepared by the invention is added with blank blood, thereby eliminating matrix effect.
The method specifically comprises the following steps: a. recovering the mixed standard solution and the isotope internal standard solution to room temperature; b. adding 0.40mL of methanol into 15 sample injection vials respectively, taking 0.40mL and 100.0 mu g/mL of mixed standard solution (A) to the first vial, and mixing uniformly to obtain mixed standard solution (B) with the concentration of 50.0 mu g/mL; similarly, 0.40mL of mixed standard solution (B) is taken to a second vial to obtain mixed standard solution (C) … … with the concentration of 25.0 mug/mL, and the like, and the mixed standard solution (C) … … is sequentially diluted step by the multiple ratio, wherein the standard series solution with the concentration of 2500, 1250, 625, 313, 156, 78.1, 39.1, 19.5, 9.77, 4.89 and 0ng/mL is obtained firstly (a blank is added), and then the mixed standard solution is continuously diluted step by methanol, and finally the standard series solution with the concentration gradient of 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.10, 0.05, 0.025 and 0ng/mL is obtained; c. adding 200 μ L of standard series solution into 40mL sample bottle, adding 5mL blank blood, sequentially adding 200 μ L isotope internal standard solution, one drop of defoamer (about 0.039 g) and 20mL pure water, sealing the sample bottle (injecting: the polytetrafluoroethylene surface of the gasket faces downwards), shaking up and down for 20 times, filling pure water into the sample bottle, and sealing the sample bottle.
6) Detection of standard series solution and drawing of standard curve: measuring the standard series solutions with different concentrations by a purging and trapping instrument and a gas chromatograph, drawing a standard curve of each substance by taking the concentration as a horizontal coordinate and taking the ratio of the peak areas of the benzene series in the standard series solutions and the benzene series in the isotope internal standard solutions as a vertical coordinate, and calculating to obtain a standard curve equation;
a. result of separation
The selective ion spectrum of the mixed standard solution with the concentration of 1.563. mu.g/L is shown in FIG. 5, in which, 1-benzene; 2-toluene; 3-ethylbenzene; 4-m/p-xylene; 5-o-xylene, it can be seen from the figure that all 5 benzene series are well separated (m/p-xylene is not separated).
b. Linear range and detection limit
6 concentration standard series solutions were prepared as described in step 5), and were tested in parallel according to the experimental conditions described above. The concentration C (. mu.g/L) was plotted on the abscissa and the peak area ratio (Y) was plotted on the ordinate to prepare a standard curve of each substance. The detection limit of each compound was measured as the signal-to-noise ratio response value. The results are shown in Table 2.
c. Recovery and precision
Blank blood samples were taken for blank spiking recoveries at two concentration levels, high (1.563. mu.g/L) and low (0.195. mu.g/L), with six replicates per concentration being determined for precision. The results are shown in Table 3.
RSD meets the requirement of less than 20 percent, the recovery rate meets the requirement of 70 percent ~ 130 percent, and the detection range of each compound is within the allowable range when the standard sample is analyzed, so that the method can meet the monitoring and analysis requirements for detecting 6 benzene series.
7) Preparation of blood samples: collecting a blood sample in a vacuum blood collection tube through venipuncture, wherein the blood collection tube is required to be full, pouring the blood sample in the vacuum blood collection tube into a sample bottle after sampling, and refrigerating and storing at 4 ℃;
the method specifically comprises the following steps: blood samples were collected by venipuncture in 7 mL vacuum blood collection tubes and mixed gently by inversion 20 times to dissolve the crystalline heparin sodium/sodium fluoride in the tubes sufficiently to minimize clotting. After sampling, the sample in the vacuum blood collection tube is gently poured into a 5mL brown screw sample bottle to completely fill the sample bottle, and the bottle is screwed down by a bottle cap with a white Teflon gasket. The samples were stored refrigerated at 4 ℃ and the analysis was completed within 10 weeks of sampling.
8) Measurement of blood samples: adding a blood sample into a sample feeding bottle, sequentially adding an isotope internal standard solution and a defoaming agent, finally fixing the volume by pure water, then measuring by a purging and trapping instrument and a gas chromatograph-mass spectrometer to obtain the peak area ratio of the benzene series in the blood sample and the benzene series in the isotope internal standard solution, and respectively substituting the peak area ratio into corresponding standard curve equations to obtain the content of the benzene series in the blood sample.
The method specifically comprises the following steps: and adding 5mL of blood sample into a 40mL sample injection bottle, adding 200 mu L of isotope internal standard solution and one drop of defoaming agent, adding 20mL of pure water, shaking up and down for 20 times, filling the pure water, and sealing the sample bottle. Shaking in ultrasonic oscillator for 1min, and testing on computer.
The formula of the concentration of benzene series in blood is as follows: ci ═ Cis × 40/5, wherein: ci is the concentration of the component to be measured in the actual sample, mu g/L; cis is the concentration of the measured component, μ g/L, calculated from the standard curve.
The benzene-based component in a specific blood sample was measured by the measurement method described above.
By adopting the method, the benzene series content in the blood of biological samples of 300 persons in a polluted area and 200 persons in a clean control area is measured. The content range of each benzene series in the blood samples tested in the heavily contaminated area and the clean control area is shown in Table 4. As can be seen from the table, the concentration of each benzene series in the blood samples in the heavily contaminated areas is higher than that in the cleaner control areas.
The method has high sensitivity, good specificity and simple operation, so the method can be used for analyzing the blood samples of a large batch of biological samples (particularly for measuring the exposure level content in the benzene series of residents in heavily polluted areas).
The establishment of the method has important guiding significance for the prevention and control of related diseases caused by environmental pollution.
Example 1:
5mL of Wangzhi blood of residents in key polluted areas is taken, added into a 40mL sample bottle according to the method, and then added with 200 mu L of isotope internal standard solution, one drop of antifoaming agent and 20mL of pure water in sequence. The sample vial was sealed and shaken up and down 20 times. And filling pure water into the sample injection bottle, and sealing the sample bottle. Shaking in ultrasonic oscillator for 1min, and testing on computer. The results are shown in Table 5.
Six benzene series in Wangzhi blood are detected, wherein the concentrations of benzene and toluene in the sample are higher, and the concentrations of ethylbenzene, m/p-xylene and o-xylene are lower. The resident is prompted to pay attention to prevent the resident from directly or indirectly contacting the benzene series for a long time, and meanwhile, a series of prevention and treatment measures are taken to ensure the physical and psychological health of the resident.
Example 2:
5mL of blood of a resident in the key polluted area is taken, added into a 40mL sample bottle according to the method, and then added with 200 mu L of isotope internal standard solution, one drop of antifoaming agent and 20mL of pure water in sequence. The sample vial was sealed and shaken up and down 20 times. And filling pure water into the sample injection bottle, and sealing the sample bottle. Shaking in ultrasonic oscillator for 1min, and testing on computer. The results are shown in Table 6.
Note: < LOQ indicates a value lower than the detection limit
It can be seen that benzene and o-xylene are detected in certain blood, wherein the benzene concentration in the sample is higher, and the o-xylene concentration is lower. The resident is prompted to pay attention to prevent the resident from directly or indirectly contacting the benzene series for a long time, and meanwhile, a series of prevention and treatment measures are taken to ensure the physical and psychological health of the resident.
Example 3:
5mL of blood of a resident Yao in a key polluted area is taken, added into a 40mL sample bottle according to the method, and then added with 200 muL of isotope internal standard solution, one drop of antifoaming agent and 20mL of pure water in sequence. The sample vial was sealed and shaken up and down 20 times. And filling pure water into the sample injection bottle, and sealing the sample bottle. Shaking in ultrasonic oscillator for 1min, and testing on computer. The results are shown in Table 7.
Note: < LOQ indicates a value lower than the detection limit
It can be seen that benzene and toluene are detected in some blood of Yao, wherein the benzene concentration is higher and the toluene concentration is lower in the sample. The resident is prompted to pay attention to prevent the resident from directly or indirectly contacting the benzene series for a long time, and meanwhile, a series of prevention and treatment measures are taken to ensure the physical and psychological health of the resident.
Example 4:
5mL of blood of residents Jia in the key polluted area is taken, added into a 40mL sample bottle according to the method, and then added with 200 mu L of isotope internal standard solution, one drop of defoaming agent and 20mL of pure water in sequence. The sample vial was sealed and shaken up and down 20 times. And filling pure water into the sample injection bottle, and sealing the sample bottle. Shaking in ultrasonic oscillator for 1min, and testing on computer. The results are shown in Table 8.
Note: < LOQ indicates a value lower than the detection limit
Low-concentration benzene and ethylbenzene are detected in Jiaozang blood. The resident is prompted to pay attention to prevent direct or indirect contact with the benzene series for a long period of time.
Example 5:
5mL of Zhao-somewhat blood of residents in key polluted areas is taken, added into a 40mL sample bottle according to the method, and then added with 200 mu L of isotope internal standard solution, one drop of antifoaming agent and 20mL of pure water in sequence. The sample vial was sealed and shaken up and down 20 times. And filling pure water into the sample injection bottle, and sealing the sample bottle. Shaking in ultrasonic oscillator for 1min, and testing on computer. The results are shown in Table 9.
Note: < LOQ indicates a value lower than the detection limit
It can be seen that benzene, ethylbenzene and meta/para-xylene are detected in Zhao certain blood, wherein the concentrations of benzene and ethylbenzene in the sample are higher, and the concentrations of meta/para-xylene are lower. The resident is prompted to pay attention to prevent the resident from directly or indirectly contacting the benzene series for a long time, and meanwhile, a series of prevention and treatment measures are taken to ensure the physical and psychological health of the resident.
Claims (1)
1. A method for measuring benzene series in biological sample blood is characterized in that: the method for measuring the content of the benzene series in the blood by adopting a sweeping trapping/gas chromatography/isotope internal standard mass spectrum combined method specifically comprises the following steps:
preparing mixed standard solutions with each single standard concentration of 100 mu g/mL: weighing pure substances of benzene series, and preparing a mixed standard solution by using methanol;
preparing isotope internal standard solutions with the single standard concentration of 200 ng/mL: weighing isotope substances of benzene series, and preparing isotope internal standard solution by using methanol;
preparation of standard series of solutions: taking the mixed standard solution, and preparing a standard series solution with a concentration gradient of more than 5 grades by using methanol; respectively adding standard series solutions with different concentrations into corresponding sample injection bottles, sequentially adding an isotope internal standard solution, blank blood and a defoaming agent into each sample injection bottle, and finally fixing the volume by using pure water;
detection of standard series solution and drawing of standard curve: measuring the standard series solutions with different concentrations by a purging and trapping instrument and a gas chromatograph, drawing a standard curve of each substance by taking the concentration as a horizontal coordinate and taking the ratio of the peak areas of the benzene series in the standard series solutions and the benzene series in the isotope internal standard solutions as a vertical coordinate, and calculating to obtain a standard curve equation;
measurement of blood samples: the preparation process of the blood sample comprises the steps of collecting the blood sample in a blood collection tube through venipuncture, pouring the blood sample in a vacuum blood collection tube into a sample bottle after sampling, and refrigerating and storing at 4 ℃; adding a blood sample into a sample feeding bottle, sequentially adding an isotope internal standard solution and a defoaming agent, finally fixing the volume by pure water, measuring by a purging and trapping instrument and a mass spectrometer to obtain the peak area ratio of the benzene series in the blood sample and the benzene series in the isotope internal standard solution, and respectively substituting the peak area ratio into corresponding standard curve equations to obtain the content of the benzene series in the blood sample;
wherein, the parameter setting of the purging and trapping instrument is as follows: taking 99.999% helium as purge gas, wherein the purge time is 11min, and the purge flow is 40 mL/min; the resolving temperature is 250 ℃, the resolving flow is 300 mL/min, and the resolving time is 2 min; the baking temperature is 280 ℃, the baking flow is 200 mL/min, and the baking time is 2 min; setting parameters of the gas chromatograph-mass spectrometer: the chromatographic column is a DB-624 capillary chromatographic column, and the specification of the chromatographic column is 30m multiplied by 0.25mm multiplied by 1.4 mu m; keeping the column temperature at 40 deg.C for 3min, heating to 150 deg.C at 10 deg.C/min, keeping for 0.5min, heating to 210 deg.C at 15 deg.C/min, and keeping for 4 min; split-flow sample injection, the split-flow ratio is 20: 1; the temperature of a sample inlet is 250 ℃; the carrier gas is 99.999% helium; in the constant-current mode, the carrier gas flow is 1.2 mL/min; the ion source type is EI, and the ion source temperature is 250 ℃; the temperature of the transmission line is 280 ℃; the electron bombardment energy was 70 eV.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710169700.7A CN106950303B (en) | 2017-03-21 | 2017-03-21 | Method for measuring benzene series in biological sample blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710169700.7A CN106950303B (en) | 2017-03-21 | 2017-03-21 | Method for measuring benzene series in biological sample blood |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106950303A CN106950303A (en) | 2017-07-14 |
| CN106950303B true CN106950303B (en) | 2020-01-21 |
Family
ID=59473624
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710169700.7A Expired - Fee Related CN106950303B (en) | 2017-03-21 | 2017-03-21 | Method for measuring benzene series in biological sample blood |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106950303B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110018252A (en) * | 2019-04-10 | 2019-07-16 | 珠海天祥粤澳质量技术服务有限公司 | The detection method of 1,3,5- trichloro-benzenes in water quality |
| CN112043592A (en) * | 2020-08-26 | 2020-12-08 | 山西医科大学 | Liquid passing device and radiopharmaceutical preparation equipment |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101876652B (en) * | 2010-07-02 | 2012-09-05 | 中国烟草总公司郑州烟草研究院 | Method for measuring benzene and benzene series in printing ink |
| CN103675134B (en) * | 2013-12-05 | 2015-05-13 | 上海金玺实验室有限公司 | Method for detecting benzene series pollutants in gas by using internal standard method |
| CN105181831A (en) * | 2015-08-28 | 2015-12-23 | 安徽省三环纸业(集团)有限公司 | Determination method of benzene and benzene series in tipping paper |
| CN105467030B (en) * | 2015-11-23 | 2018-07-13 | 内蒙古蒙牛乳业（集团）股份有限公司 | The method for detecting benzene homologues in dairy products |
-
2017
- 2017-03-21 CN CN201710169700.7A patent/CN106950303B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN106950303A (en) | 2017-07-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110187037B (en) | System and method for measuring content of 57 volatile organic compounds in ambient air | |
| Huang et al. | An improved dual-stage protocol to pre-concentrate mercury from airborne particles for precise isotopic measurement | |
| Perkins et al. | Application of routine analysis procedures to a direct mass spectrometry technique: Selected ion flow tube mass spectrometry (SIFT-MS) | |
| CN110221013A (en) | Simple, Practical Determination Method of VOCs in underground water | |
| CN103675134A (en) | Method for detecting benzene series pollutants in gas by using internal standard method | |
| Jayanty | Evaluation of sampling and analytical methods for monitoring toxic organics in air | |
| CN111337608A (en) | Method for measuring volatile organic compounds in solid pollution source waste gas | |
| CN106950303B (en) | Method for measuring benzene series in biological sample blood | |
| Watson et al. | Air monitoring: New advances in sampling and detection | |
| CN112697909A (en) | Detection method of dioxin compound | |
| Vallecillos et al. | Evaluation of active sampling strategies for the determination of 1, 3-butadiene in air | |
| Vallecillos et al. | Determination of 1, 3-butadiene degradation products in air samples by thermal desorption-gas chromatography-mass spectrometry | |
| CN104914184A (en) | Cold trap capturing-gas chromatography/mass spectrum combined detection method for furan in cigarette mainstream smoke | |
| CN111650299A (en) | High-sensitivity determination method for trace volatile organic compounds in ambient air | |
| CN113358769A (en) | Method for determining benzene series in water | |
| Olynyk et al. | Simultaneous Qualitative and Quantitative Analyses, I. Precision Study of Compounds Amenable to the Inert Gas-Purge-and-Trap Method | |
| Hino et al. | Determination of very volatile organic compounds in environmental water by injection of a large amount of headspace gas into a gas chromatograph | |
| JP2805716B2 (en) | Analysis of impurities in organometallic compounds | |
| CN113866314B (en) | Method for measuring content of nonafluorobutyl ethyl ether in soil and sediment | |
| RU2789634C1 (en) | Method for determining furan and methylfuran in atmospheric air by capillary gas chromatography with a mass selective detector using the low-temperature concentration method | |
| CN105044239B (en) | The cold trap trapping gas chromatography/mass spectrometry detection method of nitromethane in a kind of gaseous phase of main stream smoke of cigarette | |
| RU2308716C1 (en) | Method of determining activity of alkylfluorophosphonate vapors in air | |
| KR102169404B1 (en) | Portable real-time environmental pollutant measurement system | |
| JPH1164316A (en) | Method for analyzing air in clean room for organic gas | |
| Wang et al. | Determination of water at the parts per million level in buta-1, 3-diene and associated solvents by gas chromatography |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211208 Address after: 610000 No. 1106, building 1, No. 1388, middle section of Tianfu Avenue, Chengdu hi tech Zone, China (Sichuan) pilot Free Trade Zone, Chengdu, Sichuan Patentee after: CHENGDU TAIMENG SOFTWARE Co.,Ltd. Address before: 030001 No. 56, Xinjian South Road, Shanxi, Taiyuan Patentee before: SHANXI MEDICAL University |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200121 |