CN100460865C - Method for measuring lead in blood by using hydride generating atomic spectrum method - Google Patents
Method for measuring lead in blood by using hydride generating atomic spectrum method Download PDFInfo
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Abstract
Present invention relates to method for determining blood specimen lead content by hydride generation-atomic spectrum method. It contains 1, haemoid pretreatment, loading flow in centrifuge tube then adding blood sample and oscillating to mix uniformly, after centrifuging after resting for 2-10 minute to obtain liquid to be measured, 2, establishing standard curve, diluting step by step 1000 milligrams/liter to 10 microgram/litre, determining by hydride generation-atomic spectrum method to obtain lead standard curve, 3, determining sample, determining sample liquid using hydride generation-atomic spectrum method and comparing with standard series quantify. Said method has simple operation, fine stability and reliability, high determination data repetitiveness and accuracy with low cost.
Description
Technical field
The invention provides a kind of acid protein precipitation method and handle blood sample, measure the method for lead content in the blood sample then with hydride generation-atomic spectroscopy.
Background technology
Lead is a kind of famous poisonous element, can cause alimentary canal, and hemopoietic system and nervous system are impaired, and is very big to human body (particularly children) harm.But lead is widely used in fields such as accumulator, gasoline knock-reducer, alloy, radioactivity protection again simultaneously, can't thoroughly eliminate in a short time.This has just caused comparatively serious saturnism, particularly is in the children of growth and development stage, and saturnine ratio is very high, probably endangers the whole population quality of China.Saturnine control comprises primary prevention (stopping using doped fuel, apparatus and paint etc.) and secondary prevention (carrying out the children ' s lead poisoning examination in maternal and child care project and other public health projects) and takes non-pharmaceutical method to treat means prevention children ' s lead poisonings such as saturnism.Carry out secondary prevention, just need the lead content in the human body, for government formulates the relevant laws rules, the control saturnism provides scientific basis.On the other hand, patient is carried out clinical lead content detect, will help to judge the cause of disease, provide strong foundation for formulating correct therapeutic scheme.
Current detection human body lead content mainly relies on the detection to blood lead, and main method has zinc protoporphyrin method, differetial-potential leaching, graphite oven atomic absorption, hydride generation-atomic spectroscopy etc.Wherein preceding two kinds of method detection limits are higher, and accuracy of measurement is relatively poor, seldom use now; Graphite oven atomic absorption is the standard method that current blood lead is measured, but its instrument cost is higher, and operation is comparatively complicated, is difficult to be generalized to grass-roots unit in the such developing country of China.Hydride generation-atomic spectroscopy is used for blood lead and measures practice has preferably been arranged in recent years, this method can really accomplish trace, accurately, cheap, measure blood lead easily, so be highly susceptible to promoting.
Current, adopt hydride generation-atomic spectroscopy to survey blood lead and still have some defectives, mainly concentrate on two aspects: require sample is cleared up when at first being hydride generation-atomic spectroscopy survey blood lead, digestion process length consuming time and easily pollution, acid also can't be caught up with totally; On the other hand, the acidity to reaction when hydride takes place to survey blood lead requires very harshness, and available acidity scope has deviation will cause measurement result significantly on the low side less than 0.5% (percent by volume) slightly.
Summary of the invention
The purpose of this invention is to provide a kind of hydride generation-atomic spectroscopy and measure the method for blood lead content.
The purpose of the method for the invention is to be achieved through the following technical solutions:
A kind of method with hydride generation-atomic spectroscopy measurement blood lead content, step are divided into the pre-treatment of first step blood sample, the foundation and the 3rd step sample determination of the second step typical curve.The pre-treatment of described first step blood sample is every current-carrying that adds 2.9 milliliters～2.95 milliliters in the centrifuge tube after the cleaning, add 50～100 microlitre blood samples then and vibrate and mix, left standstill again 2～10 minutes, under 3000～12000 rev/mins of rotating speeds centrifugal 2～15 minutes then, obtain the sample liquid to be measured of clear; The foundation of the second step typical curve, with the plumbous standard reserving solution stepwise dilution of the 1000 mg/litre standard solution that is the variable concentrations of 0～10 micrograms per litre, measure the typical curve that obtains lead with hydride generation-atomic spectroscopy, this process comprises that described current-carrying carries standard solution and reacts with reductive agent to the reaction block place, generate the hydride of the lead of gaseous state, the alanate of gaseous state is delivered to atomization in the atomizer by carrier gas then, record corresponding spectral signal with atomic spectroscopy, just can obtain plumbous typical curve with concentration of standard solution and spectral signal mapping; The 3rd step sample determination, use hydride generation-atomic spectroscopy working sample liquid to be measured, this process comprises that current-carrying carries sample liquid to be measured and reacts with reductive agent to the reaction block place, generate the hydride of the lead of gaseous state, the alanate of gaseous state is delivered to atomization in the atomizer by carrier gas then, record corresponding spectral signal with atomic spectroscopy, more quantitative with typical curve.
Wherein, the component of described reductive agent is potassium borohydride or sodium borohydride, the potassium ferricyanide of 10 grams per liters-30 grams per liter and the weak acid of 5 grams per liters-20 grams per liter of the NaOH of 10 grams per liters-30 grams per liter or potassium hydroxide, 5 grams per liters-30 grams per liter;
Described weak acid is tartrate, oxalic acid or boric acid.
Described current-carrying is that percent by volume is 1.0%～4.5% inorganic strong acid solution.
Described inorganic acid is hydrochloric acid, nitric acid, perchloric acid or sulfuric acid.
The present invention has used a kind of new simple and convenient blood sample disposal route, and by experiment repeatedly, obtain a kind of novel reagent proportioning, enlarged the available acidity scope of measuring, make that the data of measuring are more reliable and more stable, the consumptive material of experiment also is dirt cheap, and method is accurate, quick, convenient, is very suitable for promoting at different medical unit.
The sampling Graphite Furnace Atomic Absorption method of the present invention and standard is relatively, and is simple to operate, fast and also accuracy high, the repeatability of determination data and collimation are good; Main is the low price of instrument, and method of operating simply is convenient to grasp, and the consumptive material of experiment is common chemical reagent, and is with low cost, is very suitable for promoting in the medical institutions of basic unit.
This method is compared with traditional hydride generation-atomic spectrum method, has the following advantages:
1. blood sample pre-treating method of the present invention has avoided traditional hydride generation-atomic spectrum sounding lead method to carry out long-time complicated digestion process to blood sample, has reduced contamination of heavy, and has avoided catching up with sour incomplete problem.
2. improved the reagent that hydride reacts, the available acidity range expansion to 1.5% (percent by volume) that hydride is reacted has guaranteed the stability and the reliability of measuring process.
3. method of the present invention can add current-carrying adds a cover preservation in the centrifuge tube, be easy to carry and preserve, and can very easily blood sample be adopted in the centrifuge tube that adds current-carrying when taking blood sample, adds a cover and avoids contaminated.Use hydro-extractor capacious, once can more than 100 sample of centrifugal treating, can greatly improve the speed of measurement.
4. the experiment consumptive material that method of the present invention is used all is very common, cheap, greatly reduces financial cost.
Embodiment
Method with hydride generation-atomic spectroscopy measurement blood lead content of the present invention comprises: the pre-treatment of first step blood sample, the foundation of the second step typical curve, the 3rd step sample determination.
The pre-treatment of first step blood sample, it is every current-carrying that adds 2.9 milliliters～2.95 milliliters in the centrifuge tube after the cleaning, add 50～100 microlitre blood samples then and vibrate and mix, left standstill again 2～10 minutes, under 3000～12000 rev/mins of rotating speeds centrifugal 2～15 minutes then, obtain the sample liquid to be measured of clear; The foundation of the second step typical curve, a plurality of standard solution with the plumbous standard reserving solution stepwise dilution of the 1000 mg/litre variable concentrations that is 0～10 micrograms per litre, general desirable 3-6 points, measure the typical curve that obtains lead with hydride generation-atomic spectroscopy, this process comprises that current-carrying carries standard solution and reacts with reductive agent to the reaction block place, generate the hydride of the lead of gaseous state, the alanate of gaseous state is delivered to atomization in the atomizer by carrier gas then, record corresponding spectral signal with atomic spectroscopy, just can obtain plumbous typical curve with concentration of standard solution and spectral signal mapping; The 3rd step sample determination, use hydride generation method-atomic spectroscopy working sample liquid to be measured, this process comprises that current-carrying carries sample liquid to be measured and reacts with reductive agent to the reaction block place, generate the hydride of the lead of gaseous state, the alanate of gaseous state is delivered to atomization in the atomizer by carrier gas then, record corresponding spectral signal with atomic spectroscopy, more quantitative with typical curve.
Described vibration can be adopted equipment such as vortex mixer or ultrasonic mixer, and duration of oscillation got final product in about 10-60 seconds.
The method of mensuration blood lead content of the present invention is on prior art scheme basis, study the influence of factors such as acidity, carrier gas flux, shield gas flow amount, reductive agent proportioning, hollow cathode lamp current, thereby obtained the condition that hydride generation method is measured blood lead; And investigated the influence of interfering ion, common bivalent cation in the whole blood: magnesium (Mg
2+), iron (Fe
3+), zinc (Zn
2+), cobalt (Co
2+), cadmium (Cd
2+), nickel (Ni
2+), calcium (Ca
2+), copper (Cu
2-) waiting 8 kinds of ions, their physiological concentration does not have significantly interference to atom fluorimetry is plumbous, so this method has selectivity preferably.This method has been improved original hydride generation reagent, wherein the key component of the reductive agent of second and third step use has: the NaOH or the potassium hydroxide of 10 grams per liters-30 grams per liter, the potassium borohydride or the sodium borohydride of 5 grams per liters-30 grams per liter, the potassium ferricyanide of 10 grams per liters-30 grams per liter and adding 5 grams per liters-20 grams per liter weak acid.
Described weak acid can be tartrate, oxalic acid or boric acid etc.
Described current-carrying is that percent by volume is 1.0%～4.5% inorganic strong acid solution, example hydrochloric acid, nitric acid, perchloric acid or sulfuric acid etc.
Venous blood that the actual blood sample that uses among the present invention be to extract or tip blood or adopt the venous blood of heparin or ethylenediamine tetraacetic acid (EDTA) anti-freezing, wherein (must not above 3 months) back measurement can directly be measured or be placed in the refrigerator and preserved a period of time to the venous blood of anti-freezing.
Be the accuracy of the method for inspection, the accepted standard material is from the freeze-drying ox blood GBW09139 and the GBW09140 of CDC, and their calibration value is respectively 96 ± 20ppb and 248 ± 35ppb.
Embodiment 1
The pre-treatment of first step blood sample, in the centrifuge tube after the cleaning every to add 2.9 milliliters percent by volume be that 3.5% nitric acid is current-carrying, add 100 microlitre blood samples then, be equivalent to dilute 30 times, get 4 centrifuge tubes after the cleaning in addition, every adds 3 milliliters percent by volume is 3.5% nitric acid, as reagent blank, with above-mentioned all centrifuge tubes all on vortex mixer vibration mixed 30 seconds, left standstill again 10 minutes, and descended centrifugal finishing centrifugal 10 minutes at 3000 rev/mins then, obtain the sample liquid to be measured of clear, be placed on the specimen holder centrifuge tube taking-up to be measured;
The foundation of the second step typical curve, the plumbous standard solution (GBW08611) that with percent by volume is 3.5% nitric acid stepwise dilution 1000 mg/litre is to 10 micrograms per litre, get five points of 0,2,4,8,10 micrograms per litre then, with 30 grams per liter NaOH+10 grams per liter potassium borohydrides+20 grams per liter potassium ferricyanides+10 grams per liter boric acid is reductive agent, and percent by volume is 3.5% nitric acid (HNO
3) be current-carrying, (see patent No. CN01274858.7 with sequential injection hydride generation-atomic fluorescence spectrometer, denomination of invention " the sequential injection sampling device that is used for atomic fluorescence spectrometer ") measures the typical curve that can obtain lead, the condition enactment of instrument sees Table 1, and the condition enactment of sequential injection sees Table 2.
The 3rd step sample determination adopts sequential injection hydride generation-atomic fluorescence spectrometer, uses and identical conditioned measurement sample liquid to be measured of second step, obtains the lead content in the blood sample.
Introduce the experimental data of one group of sample below, in experiment, adopt the hydride Generation-Atomic Fluorescence Spectrometry to measure the venous samples can of 2 parts of standard lyophilization ox blood samples and 14 parts of anticoagulant heparins, every part of 2 in blood sample work is parallel.Measured the venous samples can of 14 parts of anticoagulant heparins simultaneously with graphite oven atomic absorption, the measurement result that obtains is shown in table 3.
As shown in Table 3, two groups of blood lead values of the GBW09139 that this method records, GBW09140 all drop in the range of uncertainty of mark thing, this method are described accurately and reliably.In addition, as shown in Table 4, the blood lead measurement result of sampling Graphite Furnace Atomic Absorption and two kinds of methods of hydride generation-atomic fluorescence is through paired t-test, obtain t=-1.84, P=0.088〉0.05, in 95% fiducial interval range, so these two kinds of methods measure blood lead contents and do not have significant difference, and this has also proved the reliability of method provided by the invention.
Table 1. hydride generation-atomic fluorescence sounding lead instrument condition
| Total current (mA) | 40～80 | Carrier gas (mL/min) | 300～800 |
| Auxilliary cathode current (mA) | 15～40 | Shielding gas (mL/min) | 600～1000 |
| Negative high voltage (V) | 250～300 | Integral time (s) | 5～12 |
| Body of heater height (mm) | 8～12 | Sampling delay (s) | 1～3 |
| Sample size (mL) | 0.8 | Reading mode | Peak area |
The sequential injection condition of table 2. hydride generation-atomic fluorescence sounding lead
Table 3. sampling Graphite Furnace Atomic Absorption and the contrast of hydride generation-atomic fluorescence blood lead experimental result
| Sample number | Sampling Graphite Furnace Atomic Absorption (micrograms per litre) | Atomic fluorescence (micrograms per litre) |
| GBW09139 (96 ± 20 micrograms per litre) | \ | 93±6(S.D.) |
| GBW09140 (248 ± 35 micrograms per litre) | \ | 255±4(S.D.) |
| Blood sample 1 | 52.8 | 51.4 |
| Blood sample 2 | 56.5 | 59.1 |
| Blood sample 3 | 41.6 | 41.1 |
| Blood sample 4 | 37.2 | 42.2 |
| Blood sample 5 | 38.6 | 56.7 |
| Blood sample 6 | 66.5 | 54.5 |
| Blood sample 7 | 36.3 | 40.1 |
| Blood sample 8 | 54.7 | 67.7 |
| Blood sample 9 | 95.5 | 95.4 |
| Blood sample 10 | 46.0 | 53.4 |
| Blood sample 11 | 61.5 | 59.1 |
| Blood sample 12 | 49.8 | 55.4 |
| Blood sample 13 | 28.6 | 36.4 |
| Bent sample 14 | 139.4 | 142.1 |
Table 4. paired t-test result
| Pairing | Mean value | Standard deviation | Standard error mean value | 95% fiducial interval | The t value | P |
| Sampling Graphite Furnace Atomic Absorption-hydride generation-atomic fluorescence | -3.54 | 7.20 | 1.92 | -7.70<t<0.61 | -1.84 | 0.088 |
Embodiment 2:
The pre-treatment of first step blood sample, in the centrifuge tube after the cleaning every to add 2.95 milliliters percent by volume be that 4.5% hydrochloric acid is current-carrying, add 50 microlitre blood samples then, be equivalent to dilute 60 times, get 4 centrifuge tubes after the cleaning in addition, every adds 3 milliliters percent by volume is 4.5% hydrochloric acid, as reagent blank, with above-mentioned all centrifuge tubes all on ultrasonator vibration mixed 60 seconds, left standstill again 2 minutes, and descended centrifugal finishing centrifugal 5 minutes at 10000 rev/mins then, obtain the sample liquid to be measured of clear, be placed on the specimen holder centrifuge tube taking-up to be measured;
The foundation of the second step typical curve, the plumbous standard reserving solution (GBW08611) that with percent by volume is 4.5% hydrochloric acid stepwise dilution 1000 mg/litre is to 10 micrograms per litre, get five points of 0,2,4,8,10 micrograms per litre then, with 30 grams per liter potassium hydroxide+30 grams per liter sodium borohydrides+30 grams per liter potassium ferricyanides+5 grams per liter oxalic acid is reductive agent, percent by volume is that 4.5% hydrochloric acid is current-carrying, can obtain plumbous typical curve with hydride generation-atomic absorption spectrometry, the condition enactment of instrument sees Table 5.
The 3rd step sample determination adopts hydride generation-Atomic Absorption Spectrometer, uses and identical conditioned measurement sample liquid to be measured of second step, obtains the lead content in the blood sample.
Two groups of blood lead values of the GBW09139 that the present embodiment method records, GBW09140 are respectively 91 and 243, all drop in the range of uncertainty of mark thing, this method are described accurately and reliably.
Table 5. hydride generation-atomic absorption instrument condition
| Wavelength | 283.3nm | Spectrum can be with | 0.4nm |
| Lamp current | 3.0mA | Flow rate of carrier gas | 160ml/min |
| The quartz ampoule heating voltage | 150V |
Embodiment 3
The pre-treatment of first step blood sample, in the centrifuge tube after the cleaning every to add 2.95 milliliters percent by volume be that 2% perchloric acid is current-carrying, add 50 microlitre blood samples then, be equivalent to dilute 60 times, get 4 centrifuge tubes after the cleaning in addition, every adds 3 milliliters percent by volume is 2% perchloric acid, as reagent blank, with above-mentioned all centrifuge tubes all on ultrasonator vibration mixed 50 seconds, left standstill again 4 minutes, and descended centrifugal finishing centrifugal 7 minutes at 8000 rev/mins then, obtain the sample liquid to be measured of clear, be placed on the specimen holder centrifuge tube taking-up to be measured;
The foundation of the second step typical curve, the plumbous standard reserving solution (GBW08611) that with percent by volume is 2% perchloric acid stepwise dilution 1000 mg/litre is to 10 micrograms per litre, get five points of 0,2,4,8,10 micrograms per litre then, with 15 grams per liter potassium hydroxide+5 grams per liter sodium borohydrides+10 grams per liter potassium ferricyanides+20 grams per liter tartrate is reductive agent, percent by volume is that 2% perchloric acid is current-carrying, measure the typical curve that can obtain lead with hydride generation-atomic fluorescence spectrometer, the condition enactment of instrument sees Table 6.
The 3rd step sample determination adopts hydride generation-atomic fluorescence spectrometer, uses and identical conditioned measurement sample liquid to be measured of second step, obtains the lead content in the blood sample.
Two groups of blood lead values of the GBW09139 that the present embodiment method records, GBW09140 are respectively 99 and 263, all drop in the range of uncertainty of mark thing, this method are described accurately and reliably.
Table 6. hydride generation-atomic fluorescence sounding lead instrument condition
| Total current (mA) | 40～80 | Carrier gas (mL/min) | 300～800 |
| Auxilliary cathode current (mA) | 15～40 | Shielding gas (mL/min) | 600～1000 |
| Negative high voltage (V) | 250～300 | Integral time (s) | 5～12 |
| Body of heater height (mm) | 8～12 | Sampling delay (s) | 1～3 |
| Sample size (mL) | 1.2 | Reading mode | Peak area |
Embodiment 4
The pre-treatment of first step blood sample, in the centrifuge tube after the cleaning every to add 2.95 milliliters percent by volume be that 1% sulfuric acid is current-carrying, add 50 microlitre blood samples then, be equivalent to dilute 60 times, get 4 centrifuge tubes after the cleaning in addition, every adds 3 milliliters percent by volume is 1% sulfuric acid, as reagent blank, with above-mentioned all centrifuge tubes all on vortex mixer vibration mixed 50 seconds, left standstill again 4 minutes, and descended centrifugal finishing centrifugal 2 minutes at 12000 rev/mins then, obtain the sample liquid to be measured of clear, be placed on the specimen holder centrifuge tube taking-up to be measured;
The foundation of the second step typical curve, the plumbous standard reserving solution (GBW08611) that with percent by volume is 1% sulfuric acid stepwise dilution 1000 mg/litre is to 10 micrograms per litre, get five points of 0,2,4,8,10 micrograms per litre then, with 10 grams per liter potassium hydroxide+20 grams per liter sodium borohydrides+15 grams per liter potassium ferricyanides+5 grams per liter citric acids is reductive agent, percent by volume is that 1% sulfuric acid is current-carrying, measure the typical curve that can obtain lead with hydride generation-inductive coupling plasma emission spectrograph, the condition enactment of instrument sees Table 7.
The 3rd step sample determination adopts hydride generation-inductive coupling plasma emission spectrograph, uses and identical conditioned measurement sample liquid to be measured of second step, obtains the lead content in the blood sample.
Two groups of blood lead values of the GBW09139 that present embodiment records, GBW09140 are respectively 89 and 261, all drop in the range of uncertainty of mark thing, this method are described accurately and reliably.
Table 7. hydride generation-inductive coupling plasma emission spectrograph condition
| Power | 1.2kW | Wavelength | 220.353nm |
| Height of observation | 11mm | Nebulizer gas pressure | 360kPa |
| The cold gas flow velocity | 14L/min |
Claims (3)
1. method of measuring blood lead content with hydride generation-atomic spectroscopy, step is divided into the pre-treatment of first step blood sample, the foundation and the 3rd step sample determination of the second step typical curve, it is characterized in that: the pre-treatment of described first step blood sample is every current-carrying that adds 2.9 milliliters～2.95 milliliters in the centrifuge tube after the cleaning, add 50～100 microlitre blood samples then and vibrate and mix, left standstill again 2～10 minutes, under 3000～12000 rev/mins of rotating speeds centrifugal 2～15 minutes then, obtain the sample liquid to be measured of clear; The foundation of the second step typical curve, plumbous standard inventory solution stepwise dilution with 1000 mg/litre is the standard solution of the variable concentrations of 0～10 micrograms per litre, measure the typical curve that obtains lead with hydride generation-atomic spectroscopy, this process comprises that described current-carrying carries standard solution and reacts with reductive agent to the reaction block place, generate the hydride of the lead of gaseous state, the alanate of gaseous state is delivered to atomization in the atomizer by carrier gas then, record corresponding spectral signal with atomic spectroscopy, just can obtain plumbous typical curve with concentration of standard solution and spectral signal mapping; The 3rd step sample determination, use hydride generation-atomic spectroscopy working sample liquid to be measured, this process comprises that current-carrying carries sample liquid to be measured and reacts with reductive agent to the reaction block place, generate the hydride of the lead of gaseous state, the alanate of gaseous state is delivered to atomization in the atomizer by carrier gas then, record corresponding spectral signal with atomic spectroscopy, more quantitative with typical curve, wherein, the component of described reductive agent is the NaOH or the potassium hydroxide of 10 grams per liters-30 grams per liter, the potassium borohydride or the sodium borohydride of 5 grams per liters-30 grams per liter, the weak acid of the potassium ferricyanide of 10 grams per liters-30 grams per liter and 5 grams per liters-20 grams per liter, described current-carrying are that percent by volume is 1.0%～4.5% inorganic strong acid solution.
2. the method for measurement blood lead content according to claim 1 is characterized in that: described weak acid is tartrate, oxalic acid or boric acid.
3. the method for measurement blood lead content according to claim 1 and 2 is characterized in that: described inorganic acid is hydrochloric acid, nitric acid, perchloric acid or sulfuric acid.
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| CN101344486B (en) * | 2007-07-10 | 2010-09-22 | 中国船舶重工集团公司第七二五研究所 | Hydride-atomic fluorescence optical spectrum rapid measuring method for micro-trace amount of tellurium in hull steel |
| CN102353662B (en) * | 2011-07-05 | 2012-12-05 | 浙江出入境检验检疫局检验检疫技术中心 | Detection method for determining migration quantity of trace lead, cadmium, arsenic and antimony in food contact material by sequential injection-HG-AFS method |
| CN114486776A (en) * | 2021-12-31 | 2022-05-13 | 合肥国轩高科动力能源有限公司 | Method for testing mercury content in graphite negative electrode material of lithium ion battery |
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