CN106950303A - The assay method of benzene homologues in biological specimen blood - Google Patents
The assay method of benzene homologues in biological specimen blood Download PDFInfo
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Abstract
The present invention is a kind of assay method of benzene homologues in biological specimen blood, i.e., the method for determining 6 kinds of benzene homologues contents in blood simultaneously by purge and trap/gas chromatography/mass spectrometry technology.The present invention is optimized to purge and trap experiment condition, 6 kinds of benzene homologues in blood is enriched with and is parsed, and then carries out qualitative and quantitative analysis with gas chromatography/mass spectrometry and with Internal standard selection characteristic ion.The inventive method is easy to operate, analytical cycle is short, separating degree is good, and the degree of accuracy and precision can meet the requirement of analysis test, be suitable for testing and analyzing while benzene homologues in biological specimen blood.
Description
Technical field
The present invention relates to chemical analysis detection technique field, the measure side of benzene homologues in specifically a kind of biological specimen blood
Method.
Background technology
Benzene homologues are the important source material and solvent of the industries such as chemical industry, medicine, agricultural chemicals, process hides.Because benzene homologues are with stronger
Volatility, be widely present in the environment, because its route of exposure is wide, it has also become a kind of important Occupational Hazard Factors.Due to benzene
It is thing to the very harmful of health, such as causes damage and teratogenesis carcinogenic to hemopoietic system.Therefore how to sudden and violent in benzene homologues
Dew content carries out the focus and urgent problem to be solved that effective measure is the research of current environment medical domain.
At present, the detection method for benzene homologues in air, in water is a lot, and each method is ripe by public institute
Know, compared with water, air etc., blood constituent and physicochemical property are extremely complex, the method for detecting benzene homologues in water and air
It is not particularly suited for the detection to blood.The report on the detection technique of benzene homologues in biological specimen blood is there are no at present.
The content of the invention
The present invention provides a kind of assay method of benzene homologues in biological specimen blood, and at least up to separating degree is good, the degree of accuracy
The requirement of analysis test can be met with precision.
The assay method of benzene homologues in the biological specimen blood provided for above technical problem, the present invention, using purging
Trapping/gas-chromatography/Isotopic Internal Standard MS determines the content of benzene homologues in blood, the assay method specifically include as
Lower step：
The preparation of mixed mark solution：The pure material of benzene homologues is weighed, mixed mark solution is prepared with methanol；
The preparation of Isotopic Internal Standard solution：The isotope species of benzene homologues are weighed, Isotopic Internal Standard solution is prepared with methanol；
The preparation of standard serial solution：Mixed mark solution is taken, the standard series with 5 grades of concentrations above gradients is made into methanol molten
Liquid；Take the standard serial solution of graded concentrations to be added in corresponding sample injection bottle respectively, and sequentially added in each sample injection bottle
Isotopic Internal Standard solution, blank blood and defoamer, finally use pure water constant volume；
The detection of standard serial solution and the drafting of standard curve：The standard serial solution of graded concentrations is passed through into purge and trap instrument
It is measured with gas chromatograph-mass spectrometer, using concentration as abscissa, with benzene homologues in standard serial solution and Isotopic Internal Standard solution
The ratio between peak area of benzene homologues is ordinate, draws the standard curve of each material, and calculating obtains calibration curve equation；
The measure of blood sample：Blood sampling is added in sample injection bottle, sequentially adds Isotopic Internal Standard solution and defoamer, finally fixed with pure water
Hold, be then measured, obtained in blood sample in benzene homologues and Isotopic Internal Standard solution by purge and trap instrument and gas chromatograph-mass spectrometer
The ratio between peak area of benzene homologues, the ratio between peak area is substituted into corresponding calibration curve equation respectively, obtains benzene homologues in blood sample
Content.
The present invention uses P＆T/GC/MS GC-MS Internal standards, and benzene homologues content in biological sample blood is entered
The accurate qualitative and quantitative analysis of row, provides scientific basis, testing result separating degree is good for exposure detection method in human body benzene homologues,
The degree of accuracy and precision can meet the requirement of analysis test.The present invention has used Internal standard.It is quantitative in the prior art
The multiplex external standard method of method or normalization method, but normalization method needs to know the correction factor of each component, is unsuitable for trace impurity
Assay；The concentration comparable of component in the concentration and sample of external standard method requirement reference substance solution, and the accuracy of this method
Influenceed by sample introduction repeatability and experiment condition stability.Internal standard method is to select the pure material not contained in sample as internal standard compound
Add in testing sample solution, ratio is done with component to be measured and interior target peak area, the method for determining constituent content to be measured.Internal standard
Method advantage：In the non-overloading scope of chromatographic column, quantitative result is unrelated with sample size repeatability；As long as measured matter and internal standard compound
Appearance, and can be kept completely separate and can quantify, it is unrelated with other components；It is avoided that the component to be measured tested in each step processing operation is lost
It is quantitative inaccurate that mistake is caused.Internal standard method shortcoming：Internal standard compound is not easy to obtain.The requirement of internal standard compound：Internal standard compound is free of in raw sample
Some components, otherwise can make overlap of peaks and can not accurately measure the peak area of internal standard compound；Internal standard compound has identical with testing compound
Or similar physicochemical property, their retention time should be close, but can be kept completely separate (separating degree >=1.5) each other；Internal standard compound will
Ask and do not disturb, do not react with sample component；Internal standard compound must be the pure material that purity meets the requirements.The present invention is replaced with isotope
Thing replaces the C or H of testing compound with isotope, makes internal standard substance and the existing similar reason of testing compound as internal standard
Changing property can be kept completely separate each other again.The chromatographic behavior classes such as the retention time and abundance of ions of Isotopic Internal Standard and testing compound
Seemingly.
Further, the parameter setting of purge and trap instrument：Using 99. 999% helium as purge gass, purge time is 11
Min, purge flow rate is 40mL/min；Resolution temperature is 250 DEG C, and parsing flow is 300 mL/min, and the parsing time is 2 min；
Baking temperature is 280 DEG C, and baking flow is 200 mL/min, and baking time is 2 min.About 35%-70% blood test results
Mistake comes from before blood examination.To obtain reliable detection data, there is provided true to disease prevention and control center and environmental administration
Accurate experimental data, it is necessary to the interference during strict control blood sampling, blood preseration and detection etc. are a series of.Benzene in blood
It is that thing belongs to the poisonous and harmful organic pollution of trace, it is necessary to be carried out using high bioaccumulation efficiency and highly sensitive analysis and testing technology
Detection.Therefore, the present invention uses purge and trap technology（P&T）The volatile benzenes come in efficiently concentrating biological specimen blood
（Benzene, toluene, ethylbenzene ,/paraxylene and ortho-xylene）, the detection limit of analysis method can be not only reduced, and be not required to organic
Solvent and extraction matrix, can be prevented effectively from the secondary dirt of the generation such as interaction of some compositions in organic reagent and blood sample
Dye.And the present invention is directed to the characteristic of blood, and conditional parameter of purge and trap etc. is targetedly optimized, such as passed through
The influence of analysis purging temperature and purge time, desorption temperature and desorption time to purge and trap efficiency, it is final to determine to be adapted to ginseng
Number.
Further, the parameter setting of gas chromatograph-mass spectrometer：Chromatographic column is DB-624 capillary chromatographic columns, and chromatographic column specification is
30m×0.25mm×1.4μm；Column temperature is 40 DEG C of 3 min of holding of initial temperature, is raised to 150 DEG C with 10 DEG C/min, keeps 0.5
Min, then 210 DEG C are raised to 15 DEG C/min, keep 4 min；Split sampling, split ratio is 20：1；Injector temperature is 250 DEG C；
Carrier gas is 99. 999% helium；Constant current mode, carrier gas flux is 1.2 mL/min；Ion source category is EI, ion source temperature
For 250 DEG C；Transmission line temperature is 280 DEG C；Electron bombardment energy is 70 eV.Gas chromatography-mass spectrography（GC/MS）Can be to trace
Unknown compound carry out qualitative and quantitative analysis, method sensitivity is high, wide using scope, is most important at present, using most
One of analysis and testing technology, has been widely used in VOCs in analysis water, air.Isotopic Internal Standard compound and target compound
Chemical property it is almost just the same, so bioaccumulation efficiency, the extent of damage in test process and matrix influence etc. complete one
Cause, can be used to the measurement error of correction analytical method.
Further, step 1）, prepare the mixed mark solution that each single mark concentration is 100 μ g/mL.
Further, step 2）, prepare the Isotopic Internal Standard solution that each single mark concentration is 200ng/mL.
Further, the set-up procedure of blood sample is included by venipuncture blood collection sample in heparin tube, after sampling
Blood sample in vacuum test tube is poured into sample bottle, and it is stored refrigerated in 4 DEG C.
In summary, one kind purge and trap/gas chromatography/mass spectrometry technology provided by the present invention determines life simultaneously
The method of benzene homologues content in thing sample blood, is optimized to purge and trap experiment condition, makes the benzene in biological specimen blood
Be thing enrichment and parse, then with gas chromatography/mass spectrometry and with Internal standard selection characteristic ion carry out it is qualitative with
Quantitative analysis.By optimizing purge and trap parameter, the inventive method realize to benzene in biological sample blood, toluene, ethylbenzene,
Between/paraxylene, 6 kinds of benzene homologues of ortho-xylene while enrichment, parsing and Accurate Determining.The inventive method detection is limited to
0.002 ~ 0.011 μ g/L, the range of linearity is 0.043 ~ 10.99 μ g/L, and low, high concentration recovery of standard addition scope is respectively
72.78%~81.03%、82.27%~109.09%.Repeat 6 analyses to same sample, RSD scopes are 3.47% ~ 7.36%.
This method is easy to operate, analytical cycle is short, separating degree is good, and the degree of accuracy and precision can meet the requirement of analysis test, is adapted to use
Tested and analyzed in biological specimen blood while 6 kinds of benzene homologues.
Brief description of the drawings
Fig. 1 is each chromatographic peak area change trend under different purging flow velocitys.
Fig. 2 is each chromatographic peak area change trend at a temperature of different purgings.
Each chromatographic peak area change trend when Fig. 3 is different resolution temperatures.
Fig. 4 is each chromatographic peak area change trend under the different parsing times.
Fig. 5 is the selection ion spectrogram for the mixed mark solution that concentration is 1.563 μ g/L.
Embodiment
Benzene homologues contains in present invention use purge and trap/gas-chromatography/Isotopic Internal Standard MS measure blood
Amount, in a kind of typical embodiment, the assay method specifically includes following steps：
The preparation of mixed mark solution：The pure material of benzene homologues is weighed, mixed mark solution is prepared with methanol；
The preparation of Isotopic Internal Standard solution：The isotope species of benzene homologues are weighed, Isotopic Internal Standard solution is prepared with methanol；
The preparation of standard serial solution：Mixed mark solution is taken, the standard series with 5 grades of concentrations above gradients is made into methanol molten
Liquid；Take the standard serial solution of graded concentrations to be added in corresponding sample injection bottle respectively, and sequentially added in each sample injection bottle
Isotopic Internal Standard solution, blank blood and defoamer, finally use pure water constant volume；
The detection of standard serial solution and the drafting of standard curve：The standard serial solution of graded concentrations is passed through into purge and trap instrument
It is measured with gas chromatograph-mass spectrometer, using concentration as abscissa, with benzene homologues in standard serial solution and Isotopic Internal Standard solution
The ratio between peak area of benzene homologues is ordinate, draws the standard curve of each material, and calculating obtains calibration curve equation；
The measure of blood sample：Blood sampling is added in sample injection bottle, sequentially adds Isotopic Internal Standard solution and defoamer, finally fixed with pure water
Hold, be then measured, obtained in blood sample in benzene homologues and Isotopic Internal Standard solution by purge and trap instrument and gas chromatograph-mass spectrometer
The ratio between peak area of benzene homologues, the ratio between peak area is substituted into corresponding calibration curve equation respectively, obtains benzene homologues in blood sample
Content.
In a kind of relative specific embodiment, comprise the following steps：
1）First, High Purity Nitrogen gas hood is opened, ventilate 15 min, operating environment is full of nitrogen.
2）The preparation of mixed mark solution：The pure material of benzene homologues is weighed, it is 100 preferably to be prepared with methanol and obtain each single mark concentration
μ g/mL mixed mark solution.
Specially：A, it is accurate weigh benzene 0.0281g, it is toluene 0.0344g, ethylbenzene 0.0248g, meta-xylene 0.0283g, right
The g of dimethylbenzene 0.0283, the g of ortho-xylene 0.0277, with methanol constant volume to graduation mark, obtain benzene in 10.0mL volumetric flasks
2.81mg/mL, toluene 3.44mg/mL, ethylbenzene 2.48mg/mL, meta-xylene 2.83mg/mL, paraxylene 2.83mg/mL, neighbour
Dimethylbenzene 2.77mg/mL standard liquids；B, the μ L of each standard liquid benzene 356 for accurately taking above-mentioned configuration, the μ L of toluene 291, ethylbenzene 403
μ L, the μ L of meta-xylene 354, the μ L of paraxylene 354, the μ L of ortho-xylene 361 are in 10.0mL volumetric flasks, with methanol constant volume to quarter
Line is spent, the mixed mark solution that each single mark concentration is 100.0 μ g/mL is obtained；C, it is distributed into 1 mL brown ampoule bottles, is placed in after sealing
In refrigerator, 4 DEG C stored refrigerated.
3）The preparation of Isotopic Internal Standard solution：The isotope species of benzene homologues are weighed, is preferably prepared with methanol and obtains each list
Mark the Isotopic Internal Standard solution that concentration is 200ng/mL.
Specially：A, accurately weigh benzene（D5）0.0421g, toluene（D5）0.0435g, ethylbenzene（D10）0.0451g ,/it is right
Dimethylbenzene（D10）0.0452g, ortho-xylene（D4）0.0357g, with methanol constant volume to graduation mark, is obtained in 10.0mL volumetric flasks
To isotope list mark storing solution；B, the benzene for accurately taking above-mentioned preparation（D5）2.375 mL, toluene（D5）2.299mL, ethylbenzene（D10）
2.217 mL ,/paraxylene（D10）2.212mL, ortho-xylene（D4）2.801mL is fixed with methanol in 50.0mL volumetric flasks
Hold to graduation mark, obtain the mixed mark stock solution of isotope that concentration is 200 μ g/mL；C, it is distributed into 1 mL brown ampoule bottles, seals
After be placed in refrigerator, 4 DEG C are stored refrigerated；D, the mixed mark stock solution 1.0mL of above-mentioned isotope is taken, with methanol dilution to 10.00mL, obtained
Interstitial fluid in being the mixed mark of 20.0 μ g/mL isotopes to concentration；E, interstitial fluid 0.1mL in the mixed mark of above-mentioned isotope is taken, with methanol dilution extremely
10.00mL, obtains the Isotopic Internal Standard solution that concentration is 200.0ng/mL；F, it is distributed into 10 mL Solvent Brown bottles, seals
After be placed in refrigerator, 4 DEG C are stored refrigerated.
4）The parameter setting of determining instrument：In continuous mode using to instrument have U.S. Thermo Fisher
The TQU03947 gas chromatograph-mass spectrometers of Scientific companies；The purge and trap instrument of Tekmar companies of the U.S., purge and trap instrument are automatic
Injector；The DB-624 quartz capillary columns of Agilent companies of the U.S.；25 mL core formula scavenging conduits；In 40 mL
It is lined with the brown sample injection bottle of poly tetrafluoroethylene；The 7 mL glass evacuated heparin tube for including liquaemin and sodium fluoride；5 mL palm fibres
Color glass sample bottle.
The parameter setting of purge and trap instrument：Using 99. 999% helium as purge gass, purge time is 11 min, purging stream
Measure as 40mL/min；Resolution temperature is 250 DEG C, and parsing flow is 300 mL/min, and the parsing time is 2 min；Baking temperature is
280 DEG C, baking flow is 200 mL/min, and baking time is 2 min.
The determination of a, purge time and purging flow velocity
Purge time is purging with the product of purging flow velocity with the important parameter that purging flow velocity is purge and trap technology, purge time
Gas cumulative volume.Purge gass cumulative volume is bigger, then purges efficiency higher.But purge gass cumulative volume is excessive, cold-trap can will be captured in
In analyzed component blow off or dispel, reduction purging efficiency and result reappearance.For blood characteristics, and in order to save
Purging flow velocity is optimized as purge time by analysis time, raising operating efficiency, the present invention 11 min of selection.Choose
34th, 36,38,40,42 mL/min are time gradient, take the mixed standard solution of same concentration to be analyzed, relatively more different purging streams
The change of each chromatographic peak area under speed.
Six kinds of Benzene series Concentrations are each chromatographic peak area under 200 ng/L, difference purging flow velocity and become in mixed standard solution
Change trend is shown in Fig. 1.It can be seen that, in the range of 36 ~ 44mL/min, with the increase of purging flow velocity, chromatographic peak area gradually increases；When
When purging flow velocity for 40mL/min, chromatographic peak area reaches maximum；Purge flow velocity to be more than after 40mL/min, chromatographic peak area goes out
Decline is showed.For blood characteristics, present invention selection purging flow velocity is 40 mL/min.
B, the determination for purging temperature
Rise purging temperature can improve purging efficiency, and shorten purge time.Especially when purging highly-water-soluble compound, blow
Sweep influence of the temperature to purging efficiency bigger.It is unfavorable if but purging temperature is too high, the vapor purged out can be caused excessive
In absorption of the component to be measured in cold-trap.And high-moisture can cause the separative efficiency of middle polarity gas chromatographic column to decline, and damage
Its service life of evil.For VOC in water, it is conventional temperature typically to choose 20 ~ 50 DEG C；And blood mesostroma is complicated, more than 40 DEG C
Temperature can cause partially protein to deform, or even blocking pipeline.Therefore, the present invention choose 20,25,30,35,40 DEG C be temperature
Gradient is spent, takes the mixed standard solution of same concentration to be analyzed, the change of each chromatographic peak area at a temperature of relatively more different purgings.
Each chromatographic peak area becomes at a temperature of six kinds of Benzene series Concentrations are 200 ng/L, difference purging in mixed standard solution
Change trend is shown in Fig. 2.It can be seen that, chromatographic peak area increases with the rise of purging temperature.But in view of the characteristic of blood, the present invention
Selection purging temperature is 40 DEG C.
C, resolution temperature determination
Resolution temperature is another key parameter of purge and trap technology.After purging is completed, resolver heats rapidly trap tube,
The compound of collecting trap adsorption is spun off, and gassy system is blown into carrier gas.Resolution temperature is higher, trap tube heating
Time is about short, and parsing is more complete, i.e. purging efficiency is higher, and high resolution temperature advantageously forms sharp symmetrical chromatographic peak.
But resolution temperature is too high to reduce the life-span of collecting trap and adsorbent.For blood characteristics, the present invention chooses 220,230,240,
250th, 260 DEG C are thermograde, take the mixed standard solution of same concentration to be analyzed, each chromatographic peak when comparing different resolution temperatures
The change of area.
Six kinds of Benzene series Concentrations are 200 ng/L in mixed standard solution, and each chromatographic peak area becomes during different resolution temperatures
Change trend is shown in Fig. 3.It can be seen that, in 210 ~ 250 DEG C of temperature ranges, with the increase of resolution temperature, chromatographic peak area gradually increases；
Chromatographic peak area tends to be steady after 250 DEG C.For blood characteristics and in order to protect collecting trap and adsorbent, present invention selection
Resolution temperature is 250 DEG C.
D, the determination for parsing the time
The parsing time is longer, and parsing is more complete, and purging efficiency is higher.But the long parsing time can reduce adsorbent and use the longevity
Life, and cause chromatographic peak to trail.For blood characteristics, we choose 1.0,1.5,2.0,2.5,3.0 min for time gradient, taken
Mixed standard solution with concentration is analyzed, the change of each chromatographic peak area under the relatively more different parsing times.
Six kinds of Benzene series Concentrations are 200 ng/L in mixed standard solution, and each chromatographic peak area becomes under the different parsing times
Change trend is shown in Fig. 4.It can be seen that, in 1.0 ~ 2.0 min temperature ranges, with the growth of parsing time, chromatographic peak area gradually increases
Greatly；Chromatographic peak area tends to be steady after 2.0 min.For blood characteristics and in order to protect adsorbent, present invention selection solution
The analysis time is 2.0 min.
E, the determination for cleaning Flue curing parameter
Clean cycle is set to 1 time in general water.Complicated component in blood is thin containing a large amount of blood clots, cotton-shaped protein and blood
Born of the same parents etc..In order to farthest reduce the loss of volatile materials, pre-treatment step of the invention is simple, so each in blood
Plant transfer conduit and each magnetic valve that material easily blocks purge and trap instrument.Found in experiment, after setting clean cycle as 3 times,
Instrument clogging is significantly reduced.
If the concentration of tested component is higher, instrument background value can be caused higher.So, should set higher baking temperature and
Longer baking time, to reduce the volatile materials remained in instrument.Present invention selection baking temperature is 280 DEG C, baking
Time is 2 min.
The parameter setting of gas chromatograph-mass spectrometer：Chromatographic column be DB-624 capillary chromatographic columns, chromatographic column specification be 30m ×
0.25mm×1.4μm；Column temperature is 40 DEG C of 3 min of holding of initial temperature, is raised to 150 DEG C with 10 DEG C/min, keeps 0.5 min, then
210 DEG C are raised to 15 DEG C/min, 4 min are kept；Split sampling, split ratio is 20：1；Injector temperature is 250 DEG C；Carrier gas is
99. 999% helium；Constant current mode, carrier gas flux is 1.2 mL/min；Ion source category is EI, and ion source temperature is 250
℃；Transmission line temperature is 280 DEG C；Electron bombardment energy is 70 eV.The retention time of every kind of compound is true by full scan pattern
Fixed, mass spectra peak vertex value is determined by SIM patterns, and selection ion is shown in Table 1.
5）The preparation of standard serial solution：Mixed mark solution is taken, the standard system with 5 grades of concentrations above gradients is made into methanol
Row solution；The standard serial solution of graded concentrations is taken to be added in corresponding sample injection bottle respectively, and in each sample injection bottle successively
Isotopic Internal Standard solution, blank blood and defoamer are added, pure water constant volume is finally used；When determining VOC in water, the standard system of preparation
Row solution is typically not added with matrix, but is directly prepared with water；The standard serial solution that the present invention is prepared has added blank blood, so that
Eliminate matrix effect.
Specially：A, by it is mixed mark solution and Isotopic Internal Standard solution recover to room temperature；B, in 15 sample introduction bottles respectively
0.40mL methanol is added, 0.40mL, 100.0 μ g/mL mixed mark solution is taken（A）To first bottle, mix, obtaining concentration is
50.0 μ g/mL mixed mark solution（B）；Similarly, the mixed mark solution of 0.40mL is taken（B）Concentration is obtained for 25.0 μ g/ to second bottle
ML mixed mark solution（C）..., by that analogy, " multiple proportions " dilutes step by step successively, obtained before this concentration for 2500,1250,625,
313rd, 156,78.1,39.1,19.5,9.77,4.89,0ng/mL standard serial solution（Note：Increase a blank）, Ran Houji
It is continuous that with methanol, " multiple proportions " dilutes step by step successively, finally obtain concentration gradient for 12.5,6.25,3.13,1.56,0.78,0.39,
0.20th, 0.10,0.05,0.025,0 ng/mL standard serial solution；C, every grade of 200 μ L standard serial solutions are taken to add
40mL sample injection bottles, add 5mL blank blood, then sequentially add 200 μ L Isotopic Internal Standards solution, a drop defoamer（About
0.039g）With 20 mL pure water, sealed sample bottle（Note：The polytetrafluoroethylene (PTFE) of pad is face-down）, shake 20 times up and down, then will enter
Pure water, sealed sample bottle are filled it up with sample bottle.
6）The detection of standard serial solution and the drafting of standard curve：The standard serial solution of graded concentrations is passed through into purging
Trapping apparatus and gas chromatograph-mass spectrometer are measured, using concentration as abscissa, with benzene homologues in standard serial solution and Isotopic Internal Standard
The ratio between peak area of benzene homologues is ordinate in solution, draws the standard curve of each material, and calculating obtains calibration curve equation；
A, separating resulting
Concentration for 1.563 μ g/L mixed mark solution selection ion spectrogram as shown in figure 5, in figure, 1- benzene；2- toluene；3- ethylbenzene；
Between 4-/paraxylene；5- ortho-xylenes, as can be seen from the figure 5 kinds of benzene homologues obtained good separation（Between/paraxylene
Do not separate）.
B, the range of linearity and detection limit
According to step 5）It is described, 6 kinds of concentration standard serial solutions are prepared, parallel determination is carried out according to above-mentioned experiment condition.With dense
It is abscissa to spend C (μ g/L), with peak area ratio (Y) for ordinate, draws the standard curve of each material.With noise
Than the detection limit that response measures each compound.It the results are shown in Table 2.
C, the rate of recovery and precision
Blank blood sample is taken to make high concentration respectively（1.563 μg/L）And low concentration（0.195 μg/L）The blank of two concentration levels
Recovery of standard addition, each concentration does six Duplicate Samples and determines precision.It the results are shown in Table 3.
RSD meets the requirement less than 20%；The rate of recovery meets the requirement 70% ~ 130%.Standard sample is analyzed,
The measurement range of each compound is within allowed band, it is seen that the monitoring analysis that this method can meet 6 kinds of benzene homologues of measure will
Ask.
7）The preparation of blood sample：By venipuncture blood collection sample in vacuum test tube, it is necessary to assure heparin tube is adopted
It is full, blood sample in vacuum test tube is poured into sample bottle after sampling, and it is stored refrigerated in 4 DEG C；
Specially：By venipuncture blood collection sample in 7 mL vacuum test tubes, gently overturn and mix 20 times, make blood sampling
Liquaemin/sodium fluoride of lenticular fully dissolves in pipe, so as to reduce blood coagulation to greatest extent.By in vacuum test tube after sampling
Sample is gently poured into 5 mL brown screw socket sample bottles, is completely filled with sample bottle, with the bottle with white Teflon pad
Lid is tightened.Sample is stored refrigerated in 4 DEG C, in sampling interior completion analysis in 10 weeks.
8）The measure of blood sample：Blood sampling is added in sample injection bottle, is sequentially added Isotopic Internal Standard solution and defoamer, is finally used
Pure water constant volume, is then measured by purge and trap instrument and gas chromatograph-mass spectrometer, obtains benzene homologues and Isotopic Internal Standard in blood sample
The ratio between peak area of benzene homologues in solution, the ratio between peak area is substituted into corresponding calibration curve equation respectively, obtained in blood sample
The content of benzene homologues.
Specially：5 mL blood samples are taken to add 40 mL sample injection bottles, while 200 μ L Isotopic Internal Standards solution of addition and a drop disappear
Shaken 20 times above and below infusion, plus 20 mL pure water, fill it up with pure water, sealed sample bottle.1min is shaken in ultrasonic oscillator, on
Machine is determined.
The concentration formula of benzene homologues is in blood：In Ci=Cis × 40/5, formula：Ci is tested component in actual sample
Concentration, μ g/L；Cis is the concentration of the counted tested component of standard curve, μ g/L.
The benzene homologues composition in specific blood sample is measured using the above-mentioned assay method further illustrated below.
Using this method, to the benzene series in the biological specimen blood of certain people of contaminated areas 300 and certain regional 200 people of cleaning control
Thing content is determined.The content range of each benzene homologues is shown in Table in the tested blood sample in emphasis contaminated areas and cleaning control area
4.As can be seen from the table, each Benzene series Concentrations in emphasis contaminated areas blood sample, which are relatively cleaned, compares regional higher.
Because this method sensitivity is high, specificity is good, easy to operate, therefore available for point of high-volume biological specimen blood sample
Analysis（Particularly exposure level content is measured in counterweight point pollution Area Inhabitants benzene homologues）.
The prevention and control for setting up relevant disease caused by environmental pollution of this method have important directive significance.
Embodiment 1：
The mL of blood 5 of emphasis contaminated areas resident Mr. Wang is taken, 40 mL sample bottles are added as stated above, 200 μ L are sequentially added
Isotopic Internal Standard solution, a drop defoamer and 20mL pure water.Sealed sample bottle, shakes 20 times up and down.It will be filled it up with again in sample injection bottle
Pure water, sealed sample bottle.1 min is shaken in ultrasonic oscillator, upper machine is determined.Measurement result is shown in Table 5.
It can be seen that six kinds of benzene homologues have detection in Mr. Wang's blood, wherein benzene and toluene concentration are higher in sample, ethylbenzene ,/it is right
Dimethylbenzene and ortho-xylene concentration are relatively low.Pointing out the resident should be noted that prevents from directly or indirectly contacting for a long time with benzene homologues, simultaneously
Serial prophylactico-therapeutic measures should be taken to ensure that it is physically and mentally healthy.
Embodiment 2：
The mL of blood 5 of emphasis contaminated areas resident department is taken, 40 mL sample bottles are added as stated above, 200 μ L are sequentially added
Isotopic Internal Standard solution, a drop defoamer and 20 mL pure water.Sealed sample bottle, shakes 20 times up and down.It will be filled it up with again in sample injection bottle
Pure water, sealed sample bottle.1 min is shaken in ultrasonic oscillator, upper machine is determined.Measurement result is shown in Table 6.
Note：<LOQ represents to be less than detection limit
It can be seen that taking charge of in certain blood has benzene and ortho-xylene detection, wherein the concentration of benzene is higher in sample, and ortho-xylene concentration is relatively low.Carry
Showing that the resident should be noted that prevents from directly or indirectly contacting for a long time with benzene homologues, while serial prophylactico-therapeutic measures should be taken to ensure its body
Heart health.
Embodiment 3：
The mL of blood 5 of emphasis contaminated areas resident Yao is taken, 40 mL sample bottles are added as stated above, 200 μ L are sequentially added
Isotopic Internal Standard solution, a drop defoamer and 20 mL pure water.Sealed sample bottle, shakes 20 times up and down.It will be filled it up with again in sample injection bottle
Pure water, sealed sample bottle.1 min is shaken in ultrasonic oscillator, upper machine is determined.Measurement result is shown in Table 7.
Note：<LOQ represents to be less than detection limit
It can be seen that having benzene and toluene detection in Yao's blood, wherein the concentration of benzene is higher in sample, and toluene concentration is relatively low.Point out the resident
It should be noted that preventing from directly or indirectly contacting for a long time with benzene homologues, while serial prophylactico-therapeutic measures should be taken to ensure that it is physically and mentally healthy.
Embodiment 4：
Take emphasis contaminated areas resident to go into business the mL of blood 5 of certain, 40 mL sample bottles are added as stated above, 200 μ L are sequentially added
Isotopic Internal Standard solution, a drop defoamer and 20 mL pure water.Sealed sample bottle, shakes 20 times up and down.It will be filled it up with again in sample injection bottle
Pure water, sealed sample bottle.1 min is shaken in ultrasonic oscillator, upper machine is determined.Measurement result is shown in Table 8.
Note：<LOQ represents to be less than detection limit
It can be seen that having Low Concentration of Benzene and ethylbenzene detection in certain blood of going into business.Point out the resident should be noted that prevent with benzene homologues it is long-term directly or
Mediate contact.
Embodiment 5：
The mL of blood 5 of emphasis contaminated areas resident Zhao is taken, 40 mL sample bottles are added as stated above, 200 μ L are sequentially added
Isotopic Internal Standard solution, a drop defoamer and 20 mL pure water.Sealed sample bottle, shakes 20 times up and down.It will be filled it up with again in sample injection bottle
Pure water, sealed sample bottle.1 min is shaken in ultrasonic oscillator, upper machine is determined.Measurement result is shown in Table 9.
Note：<LOQ represents to be less than detection limit
It can be seen that have in Zhao's blood benzene, ethylbenzene and/paraxylene detection, wherein the concentration of benzene and ethylbenzene is higher in sample ,/it is right
Xylene concentration is relatively low.Pointing out the resident should be noted that prevents from directly or indirectly contacting for a long time with benzene homologues, while should take and be
Row prophylactico-therapeutic measures ensures that it is physically and mentally healthy.
Claims (6)
1. the assay method of benzene homologues in a kind of biological specimen blood, it is characterised in that：Using purge and trap/gas-chromatography/same
The plain internal standard MS in position determines the content of benzene homologues in blood, and the assay method specifically includes following steps：
The preparation of mixed mark solution：The pure material of benzene homologues is weighed, mixed mark solution is prepared with methanol；
The preparation of Isotopic Internal Standard solution：The isotope species of benzene homologues are weighed, Isotopic Internal Standard solution is prepared with methanol；
The preparation of standard serial solution：Mixed mark solution is taken, the standard series with 5 grades of concentrations above gradients is made into methanol molten
Liquid；Take the standard serial solution of graded concentrations to be added in corresponding sample injection bottle respectively, and sequentially added in each sample injection bottle
Isotopic Internal Standard solution, blank blood and defoamer, finally use pure water constant volume；
The detection of standard serial solution and the drafting of standard curve：The standard serial solution of graded concentrations is passed through into purge and trap instrument
It is measured with gas chromatograph-mass spectrometer, using concentration as abscissa, with benzene homologues in standard serial solution and Isotopic Internal Standard solution
The ratio between peak area of benzene homologues is ordinate, draws the standard curve of each material, and calculating obtains calibration curve equation；
The measure of blood sample：Blood sampling is added in sample injection bottle, sequentially adds Isotopic Internal Standard solution and defoamer, finally fixed with pure water
Hold, be then measured, obtained in blood sample in benzene homologues and Isotopic Internal Standard solution by purge and trap instrument and gas chromatograph-mass spectrometer
The ratio between peak area of benzene homologues, the ratio between peak area is substituted into corresponding calibration curve equation respectively, obtains benzene homologues in blood sample
Content.
2. the assay method of benzene homologues in biological specimen blood according to claim 1, it is characterised in that：Purge and trap instrument
Parameter setting：Using 99. 999% helium as purge gass, purge time is 11 min, and purge flow rate is 40mL/min；Parsing
Temperature is 250 DEG C, and parsing flow is 300 mL/min, and the parsing time is 2 min；Baking temperature is 280 DEG C, and baking flow is
200 mL/min, baking time is 2 min.
3. the assay method of benzene homologues in biological specimen blood according to claim 2, it is characterised in that：Gas chromatograph-mass spectrometer
Parameter setting：Chromatographic column is DB-624 capillary chromatographic columns, and chromatographic column specification is 30m × 0.25mm × 1.4 μm；Column temperature is
40 DEG C of holding 3min of beginning temperature, 150 DEG C are raised to 10 DEG C/min, keep 0.5 min, then be raised to 210 DEG C, holding with 15 DEG C/min
4min；Split sampling, split ratio is 20：1；Injector temperature is 250 DEG C；Carrier gas is 99.999% helium；Constant current mode, is carried
Throughput is 1.2 mL/min；Ion source category is EI, and ion source temperature is 250 DEG C；Transmission line temperature is 280 DEG C；Electronics bangs
Energy is hit for 70 eV.
4. the assay method of benzene homologues in biological specimen blood according to claim 3, it is characterised in that：Prepare each single mark
Concentration is 100 μ g/mL mixed mark solution.
5. the assay method of benzene homologues in biological specimen blood according to claim 4, it is characterised in that：Prepare each single mark
Concentration is 200ng/mL Isotopic Internal Standard solution.
6. the assay method of benzene homologues in the biological specimen blood according to any one in claim 1-5, its feature exists
In：The set-up procedure of blood sample is included by venipuncture blood collection sample in heparin tube, by vacuum test tube after sampling
Blood sample is poured into sample bottle, and stored refrigerated in 4 DEG C.
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| CN115047114B (en) * | 2022-07-13 | 2024-06-04 | 自然资源部第三海洋研究所 | Method for detecting stable isotope composition of benzene series monomer carbon with low trace concentration in water body |
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