CN1715907A - The gas chromatography analysis method of micro ethanol in the blood of human body - Google Patents
The gas chromatography analysis method of micro ethanol in the blood of human body Download PDFInfo
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- CN1715907A CN1715907A CN 200410062970 CN200410062970A CN1715907A CN 1715907 A CN1715907 A CN 1715907A CN 200410062970 CN200410062970 CN 200410062970 CN 200410062970 A CN200410062970 A CN 200410062970A CN 1715907 A CN1715907 A CN 1715907A
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Abstract
The invention discloses the gas chromatography analysis method of micro ethanol in a kind of blood of human body.This method is selected chromatographic grade divinylbenzene porous polymer stationary phase, perhaps selects to support white diatomite stationary phase and two kinds of stationary phase of chromatographic grade divinylbenzene porous polymer stationary phase of 5% methyl polysiloxane, the preparative chromatography post; The control chromatographic condition is a column temperature: 150 ℃; Temperature of vaporization chamber: 150 ℃; Fid detector temperature: 160 ℃; Carrier gas flux: 22ml/min; Press before the post: 0.14MPa; Hydrogen: 42ml/min; Air: 270ml/min; Input mode: column sample injection; Sample size: 0.5-5 μ l; Micro ethanol in the blood of human body is carried out gas chromatographic analysis.The present invention is easy and simple to handle, and the accuracy height does not need blood sample is carried out special processing, can finish from sampling, pre-service, sample feeding and analytical procedure in several minutes.
Description
Technical field
The present invention relates to the gas chromatography analysis method of micro ethanol in a kind of blood of human body.
Background technology
Driving when intoxicated is one of important hidden danger of urban traffic safety, so the accurate detection by quantitative of ethanol (alcohol) seems extremely important in the blood of human body.Ethanol enters blood by digestion and spreads all over each tissue of whole body, the effect of human body mainly is paralysis and suppresses central nervous system, and the power and the ethanol in blood content of effect is closely related.Because the complex process of diet and metabolism also can detect micro ethanol sometimes in ordinary person's blood of not drinking.Therefore American-European many countries have all formulated statutory standards to driver's ethanol in blood concentration minimum, generally at 50-80mg/100ml.China's current law is forbidded strictly to drive when intoxicated, all discovery drivers have alcohol reaction, all can be imposed punishment by driving after drinking, yet, if there is not the ethanol in blood concentration data then to lack the penalty foundation, often controversial, therefore set up ethanol in blood quick and precisely and the analytical approach that is fit to Chinese instrument and equipment situation have practical significance.
Whether the check driver drinks two class methods is arranged at present, the first kind is that ethanol content in (or breathe out) gas is blown in check, mainly contain following several method: 1) vehicular alcohol concentration controller, just connect the circuit start vehicle when ethanol content is qualified in confirming driver's expiration; 2) utilize chemical reaction to make ethanol and developer effect (as trivalent chromium), make the pocket tester of ethanol expiration detector tube colour developing, determine roughly concentration range of ethanol according to different the comparing of colour developing with standard color comparison card with the hexavalent chrome reduction au bleu of yellow; 3) adopt semiconductor (majority is a tin ash) to measure the alcohol tester of alcohol as gas sensor; 4) utilize fuel cell to make the portable detector of sensor.Shortcoming such as wherein preceding two kinds of method ubiquity measured value instabilities, poor reliability, measuring error are big, can only be qualitative can not be quantitative, testing result is prone to false positive and can not generally uses; The gas sensor of portable semiconductor alcohol tester is subject to other gas and disturbs, especially very responsive to humidity, be difficult to get rid of influence, and its relatively short output fading period and relative less linear segments, make that its measurement accuracy is poor, linear and stability can not reach requirement fully, and various countries also cancel in succession detecting with this equipment and drive when intoxicated in recent years; Make the alcohol content tester of sensor with fuel cell, because it has favorable linearity and very high precision, adopted by countries in the world as metering and measuring equipment, right this imported product costs an arm and a leg, also need frequent calibration, calibration cycle is one to six months, reaches higher precision for making the result, must guarantee each sampling volume unanimity, and because the characteristic of fuel cell, output data has the decline phenomenon when continuous coverage.Above method all is the ethanol content that detects in blowing, and calculates the blood determining alcohol by ethanol content in blowing again.Calculate the blood determining alcohol by the ethanol content in blowing, be subjected to factors such as temperature, humidity, blow gas pressure and air-blowing quantity and people's individual difference, in vivo the influence of digestion time length after particularly drinking, accuracy and stability are not high, as suspicious alcohol user's Preliminary inspection, its conclusion does not possess legal effect only.Second class is a vapor-phase chromatography, can measure the concentration of ethanol in blood, is method comparatively accurately, can be divided into the head space method again, solvent extraction, protein precipitation method, dilution method etc. by the disposal route of its blood sample.Wherein head space method report is maximum, and this method is not introduced interfering material, but the quantitative more head space automation degree of equipment that depends on of accuracy, often quantitatively accuracy is relatively poor for simple machine.Solvent extraction equipment is simple, but will introduce solvent, easily causes the difficulty on interference and the chromatographic resolution, also easily causes the ethanol loss when concentrated.The protein precipitation method is that the blood supernatant after the centrifuging is added reagent again, and with precipitation albumen wherein, purpose is the protection chromatographic column, and method is easier, but the reagent of introducing also may cause interference or pollute chromatographic system.Dilution method has reduced the pollution of blood sample to sampling system and chromatographic column because of having diluted blood, but can reduce the sensitivity of method simultaneously.
Summary of the invention
Purpose of the present invention is exactly in order to solve the deficiencies in the prior art part, and the gas chromatography analysis method of micro ethanol in a kind of blood of human body is provided.
The present invention realizes by following measure:
Thinking of the present invention is that the requirement analytical approach is easy and simple to handle, and the accuracy height does not need blood sample is carried out special processing, can finish from sampling, pre-service, sample feeding and analytical procedure in several minutes.
The gas chromatography analysis method of micro ethanol in a kind of blood of human body is characterized in that extracting human body blood sample 0.5 microlitre～2 milliliters in pin that contains anti-coagulants or container; Select chromatographic grade divinylbenzene porous polymer stationary phase, perhaps select to support white diatomite stationary phase and two kinds of stationary phase of chromatographic grade divinylbenzene porous polymer stationary phase of 5% methyl polysiloxane, the preparative chromatography post; The control chromatographic condition is a column temperature: 150 ℃; Temperature of vaporization chamber: 150 ℃; Fid detector temperature: 160 ℃; Carrier gas flux: 22ml/min; Press before the post: 0.14MPa; Hydrogen: 42ml/min; Air: 270ml/min; Input mode: column sample injection; Sample size: 0.5-5 μ l; Micro ethanol in the blood of human body is carried out gas chromatographic analysis.
Preparation process that the above " supports the white diatomite stationary phase of 5% methyl polysiloxane " for the methyl polysiloxane of 5 parts of weight is dissolved in 100 parts of isopyknic methylene chloride of weight white diatomite carrier in, again 100 parts of weight granularities, 80～140 purpose white diatomite carriers are immersed solution, treating that solvent volatilizees naturally is coated on the white diatomite carrier methyl polysiloxane equably, makes the white diatomite stationary phase that supports 5% methyl polysiloxane.
0.6～2 meter of the total column length of the said chromatographic column of the present invention, 1～4 millimeter of external diameter φ.
The stationary phase of preparative chromatography post of the present invention can single selection chromatographic grade divinylbenzene porous polymer stationary phase, is filled into preparative chromatography post in glass or the stainless-steel tube; Similarly also can select to support the white diatomite stationary phase and the chromatographic grade divinylbenzene porous polymer stationary phase preparative chromatography post of 5% methyl polysiloxane, wherein support 8～15 centimetres of the white diatomite stationary phase packed column head part of 5% methyl polysiloxane, remainder fill color spectrum level divinylbenzene porous polymer stationary phase.
The used carrier gas of the present invention is selected from N
2, H
2, He, any one gas among the Ar.
Blood anticoagulant of the present invention is selected from medical commodity heparin or liquor sodii citratis.
For further specifying the present invention, now carry out more detailed description:
The present invention relates to two parts of chromatography instrument system and blood sample analysis method.The chromatography instrument system is made of the gas chromatograph that chromatographic column is housed and data processor or chromatographic work station; The blood sample analysis method comprises that blood specimen collection is handled and sample is injected analytic system analyze two steps.
One. the chromatography instrument system
Chromatography instrument of the present invention system is equipped with the mentioned chromatographic column of the present invention for being equipped with the routine or the micro gas chromatograph of packed column injection port and flame ionization ditector (FID) or photoionization detector (PID) in the chromatogram stove.Chromatographic data is by data processor or chromatographic work station computing.
1) chromatographic column preparation: with the methyl polysiloxane of 5 parts of weight be dissolved in 100 parts of isopyknic methylene chloride of weight white diatomite carrier in, again 100 parts of weight granularities, 80～140 purpose white diatomite carriers are immersed solution, treating that solvent volatilizees naturally is coated on the white diatomite carrier methyl polysiloxane equably, makes the white diatomite stationary phase that supports 5% methyl polysiloxane.
Single selection chromatographic grade divinylbenzene porous polymer stationary phase is filled in glass or the stainless-steel tube and is prepared into chromatographic column; White diatomite stationary phase and the chromatographic grade divinylbenzene porous polymer stationary phase that similarly also can select to support 5% methyl polysiloxane are prepared into chromatographic column, support 8～15 centimetres of the white diatomite stationary phase packed column head part of 5% methyl polysiloxane, remainder fill color spectrum level divinylbenzene porous polymer stationary phase.
0.6～2 meter of the total column length of chromatographic column, 1～4 millimeter of external diameter φ.
Two kinds of column performance unanimities, the white diatomite stationary phase that supports 5% methyl polysiloxane by replacing post head branch can prolong the chromatographic column specially life-span.
2) chromatographic column is aging: logical N in chromatographic column
2, H
2, He, any one gas among the Ar, flow 10～50ml/ minute, temperature programme: 50 ℃ kept 5 minutes, and were warming up to 100 ℃ (keeping 5 minutes) with 10 ℃/min, be warming up to 150 ℃ (keeping 5 minutes) with 5 ℃/min, be warming up to 250 ℃ (keeping 60～120 minutes) with 2 ℃/min again.
3) chromatographic condition: column temperature: 150 ℃ (constant temperature); Temperature of vaporization chamber: 150 ℃; Fid detector temperature: 160 ℃; Carrier gas (N
2, H
2, He, any one gas among the Ar) and flow: 22ml/min; Press before the post: 0.14MPa; Hydrogen: 42ml/min; Air: 270ml/min; Input mode: column sample injection; Sample size: 0.5-5 μ l.
Two. the blood sample analysis method
1) gets people's finger tip blood or ear-lobe blood and can carry out the single express-analysis, repeatedly analyze, and need keep sample, then need vein to take a blood sample 1～2 milliliter in order to rechecking as need.Division is as follows:
A. take out/arrange conventional blood anticoagulant agent solution (as heparin, liquor sodii citratis etc.) for several times with the microsyringe of 1～5 microlitre, make the inner surface of needle tubing that one deck anti-coagulants as thin as a wafer be arranged; Get 0.5～5 microlitre in people's finger tip blood or the ear-lobe blood with 1～5 microlitre microsyringe, directly inject off-the-shelf gas chromatograph injection port and analyze, behind the sample introduction microsyringe continue to take out/arrange blood anticoagulant solution for several times, for future use.
B. extract 1～2 milliliter of people's venous samples can and inject anticoagulant tube, place hydro-extractor under 3500-6000 rev/min of rotating speed, to make the blood sample layering in centrifugal 2-7 minute; Absorption gained supernatant 0.5-5 microlitre is injected in the off-the-shelf gas chromatograph injection port to be analyzed.Blood sample after the described processing should be measured as early as possible, in order to avoid ethanol volatilization and have an effect and cause the concentration change influence accurately to be measured with enzyme or microorganism in the blood sample is preserved as true the need, can be positioned in 4 ℃ of refrigerators and preserve two days, in order to rechecking.
2) the ethanol appearance time is between 0.7～0.8 minute, quantivative approach is an external standard method, and is qualitative with the ethanol retention time, quantitative with peak area or peak height, typical curve is set up in ethanol standard water liquor analysis with preparation, utilizes data processor or chromatographic work station deal with data output report.
The present invention compares with existing square technology, has following substantive distinguishing features:
1) the present invention need not blood is carried out albumen precipitation, need not with an organic solvent blood sample to be extracted etc., has reduced a large amount of sample pretreatment work.
2) the present invention does not have specific (special) requirements to instrument and equipment, possess the routine or the micro gas chromatograph that dispose packed column injection port and flame ionization ditector (FID) or photoionization detector (PID), and routine data treatment facility, one root chromatogram column, one Daepori leads to hydro-extractor, and can carry out fast detecting work behind several 1～5 major equipments such as microlitre microsyringe.
3) the present invention only needs finger tip or one of ear-lobe blood can finish an express-analysis; When extracting the venous blood of 1～2ml, after repeatedly doing the sample analysis, also can carry out short-term and preserve, so that recheck when having objection to sample.
4) range of linearity of the present invention surpasses 10
3, linearly dependent coefficient R is 0.9999, recovery scope is that the relative standard deviation RSD that analyzes for 98～105%, 6 times is less than 2.6.
5) the present invention measures the lowest detectable limit of ethanol in blood content and is: 1mg/100ml.
6) the present invention is easy fast, gets final product the analyzing and testing work of ethanol content in the perfect aspect inner blood in several minutes.
Equally, the present invention also is applicable to the fast measuring of ethanol in the animal blood, also is applicable to the fast measuring of ethanol in the body fluid such as people and animals urine, also is applicable to simultaneously the mensuration of other alcohols in blood and the body fluid such as methyl alcohol.
Description of drawings
Fig. 1 is a micro ethanol fast measuring chromatogram in the blood of human body among the embodiment 1.
Embodiment
Embodiment 1
1) preparation of chromatographic column
Glass or stainless steel chromatogram post blank pipe are cleaned and dries up with nitrogen, after clogging a little glass or quartz fibre, post tail one end connects vacuum pump, connect glass funnel at column cap one end, chromatographic grade divinylbenzene porous polymer stationary phase is poured in the glass funnel, when bleeding, the post tail knocks column jecket with suitable dynamics, the chromatographic column blank pipe of in batches stationary phase being packed into installs that the first end of rear pillar is clogged a little glass or the quartz fibre shutoff is prepared into chromatographic column specially.1 meter of chromatogram column length, 3 millimeters of external diameter φ.
2) chromatographic column is aging:
The chromatographic column of having loaded is installed in the gas chromatography stove case, column cap one termination vaporizer, post tail one end does not connect, it is 10～50ml/ minute that the post tail records carrier gas flux, temperature programme: 50 ℃ kept 5 minutes, be warming up to 100 ℃ (keeping 5 minutes) with 10 ℃/min, be warming up to 150 ℃ (keeping 5 minutes), be warming up to 250 ℃ (keeping 60～120 minutes) with 2 ℃/min again with 5 ℃/min.
3) blood sample analysis
3 alcohol users having been carried out the actual analysis of ethanol in blood content measures.
Adopt preparation to have the gas chromatograph of packed column injection port and fid detector to analyze; Carry out data acquisition and processing (DAP) with chromatographic work station; With the aqueous solution that contains chromatographically pure ethanol as external standard liquor analysis set up the external standard typical curve, concentration of alcohol is respectively 1000,500,100,10,1mg/100ml, the operating conditions during analysis is: column temperature: 150 ℃ (constant temperature); Temperature of vaporization chamber: 150 ℃; Fid detector temperature: 160 ℃; Carrier gas N
2Flow: 22ml/min; Press before the post: 0.14MPa; H
2: 42ml/min; Air r:270ml/min; Input mode: column sample injection; Sample size: 1 μ l.
Under the aforesaid operations condition, from rare to dense, accurately draw the above-mentioned standard solution of 1 μ l successively respectively with microsyringe, peak area logarithm value and concentration logarithm value with ethanol are drawn the double-log typical curve, this curve has good linear at 1mg/100ml in the 1000mg/100ml scope, linear equation is: y=1.0226x+2.6892, linearly dependent coefficient R=0.9999.Add a certain amount of ethanol and mixing in the serum that does not contain ethanol, measure the repeatability of ethanol retention time and concentration 6 times, the relative standard deviation RSD of this method is less than 2.6% as can be known.Add certain density ethanol standard solution at blank blood sample and the medium volume of blood sample that contains a certain amount of ethanol, with the recovery of sample introduction after one times of the serum dilution with method of testing, obtaining this method average recovery rate is 98-105%.
The young male that 3 about 55kg of body weight are arranged, drink respectively Luzhou Old Cellar liquor each about 20,20 and 30ml, place the pipe that is added with anti-coagulants at 30,40 and 50 minutes posterior vein blood drawing 2ml respectively, under 4000 rev/mins of rotating speeds centrifugal 5 minutes, draw supernatant 1 μ l sample introduction with microsyringe, analyze by the aforesaid operations condition, ethanol goes out the peak about 0.75 minute, substantially noiseless, the gained spectrogram contains ethanol 10.3,2.49 and 6.12mg/100ml respectively as shown in Figure 1 in 3 human bloods.
Embodiment 2
1) chromatographic column preparation
Glass or stainless steel chromatogram post blank pipe are cleaned and dries up with nitrogen, after clogging a little glass or quartz fibre, post tail one end connects vacuum pump, connect little glass funnel at column cap one end, chromatographic grade divinylbenzene porous polymer stationary phase is poured in the glass funnel, when bleeding, the post tail knocks column jecket with suitable dynamics, the chromatographic column blank pipe of in batches stationary phase being packed into, the post head branch is left a blank 10 centimetres and is filled the white diatomite stationary phase support 5% methyl polysiloxane with quadrat method, filling a little glass or quartz fibre separated between two kinds of stationary phase, installs the first end of rear pillar and clogs the chromatographic column that a little glass or quartz fibre shutoff are prepared into pre-column.1.5 meters of chromatogram column lengths, 1 millimeter of external diameter φ.
2) chromatographic column is aging:
With embodiment 1.
3) blood sample analysis
With embodiment 1.
Claims (5)
1, the gas chromatography analysis method of micro ethanol in a kind of blood of human body is characterized in that extracting human body blood sample 0.5 microlitre～2 milliliters in pin that contains anti-coagulants or container; Select chromatographic grade divinylbenzene porous polymer stationary phase, perhaps select to support white diatomite stationary phase and two kinds of stationary phase of chromatographic grade divinylbenzene porous polymer stationary phase of 5% methyl polysiloxane, the preparative chromatography post; The control chromatographic condition is a column temperature: 150 ℃; Temperature of vaporization chamber: 150 ℃; Fid detector temperature: 160 ℃; Carrier gas flux: 22ml/min; Press before the post: 0.14MPa; Hydrogen: 42ml/min; Air: 270ml/min; Input mode: column sample injection; Sample size: 0.5-5 μ l; Micro ethanol in the blood of human body is carried out gas chromatographic analysis.
2,, it is characterized in that 0.6～2 meter of the total column length of chromatographic column, 1～4 millimeter of external diameter φ as the said method of claim 1.
3,, it is characterized in that supporting 8～15 centimetres of the white diatomite stationary phase packed column head part of 5% methyl polysiloxane, remainder fill color spectrum level divinylbenzene porous polymer stationary phase as claim 1 or 2 said methods.
4,, it is characterized in that carrier gas is selected from N as the said method of claim 1
2, H
2, He, any one gas among the Ar.
5,, it is characterized in that blood anticoagulant is selected from medical commodity heparin or liquor sodii citratis as the said method of claim 1.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100561217C (en) * | 2006-06-29 | 2009-11-18 | 上海神开石油化工装备股份有限公司 | A kind of oil gas proximate analysis method that only needs air and hydrogen |
| CN102841167A (en) * | 2011-06-20 | 2012-12-26 | 苏州赛分科技有限公司 | Liquid chromatographic column |
| CN102998402A (en) * | 2011-09-15 | 2013-03-27 | 上海安谱科学仪器有限公司 | Special gas chromatography column for analysis of benzene compounds in environment |
| CN106568848A (en) * | 2015-10-08 | 2017-04-19 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting ethanol in living lactobacillus beverage |
| CN107110843A (en) * | 2014-12-19 | 2017-08-29 | 萨默费尼根有限公司 | The method of the blood divided for cellulation |
| CN110887908A (en) * | 2019-12-05 | 2020-03-17 | 中维安全检测认证集团有限公司 | Method for detecting ethylene in gas by gas chromatography |
| CN114235987A (en) * | 2021-11-25 | 2022-03-25 | 广东烟草广州市有限公司 | Method and device for identifying authenticity of electronic cigarette based on headspace-gas chromatography |
-
2004
- 2004-07-02 CN CN 200410062970 patent/CN1715907A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100561217C (en) * | 2006-06-29 | 2009-11-18 | 上海神开石油化工装备股份有限公司 | A kind of oil gas proximate analysis method that only needs air and hydrogen |
| CN102841167A (en) * | 2011-06-20 | 2012-12-26 | 苏州赛分科技有限公司 | Liquid chromatographic column |
| CN102998402A (en) * | 2011-09-15 | 2013-03-27 | 上海安谱科学仪器有限公司 | Special gas chromatography column for analysis of benzene compounds in environment |
| CN107110843A (en) * | 2014-12-19 | 2017-08-29 | 萨默费尼根有限公司 | The method of the blood divided for cellulation |
| CN107110843B (en) * | 2014-12-19 | 2019-03-08 | 萨默费尼根有限公司 | The method of blood for cellulation division |
| CN106568848A (en) * | 2015-10-08 | 2017-04-19 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting ethanol in living lactobacillus beverage |
| CN110887908A (en) * | 2019-12-05 | 2020-03-17 | 中维安全检测认证集团有限公司 | Method for detecting ethylene in gas by gas chromatography |
| CN114235987A (en) * | 2021-11-25 | 2022-03-25 | 广东烟草广州市有限公司 | Method and device for identifying authenticity of electronic cigarette based on headspace-gas chromatography |
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