CN103592399A - Method capable of simultaneously measuring organochlorine pesticide concentration and synthetic musk concentration in human serum - Google Patents
Method capable of simultaneously measuring organochlorine pesticide concentration and synthetic musk concentration in human serum Download PDFInfo
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Abstract
The invention relates to a method capable of simultaneously measuring organochlorine pesticide concentration and synthetic musk concentration in human serum. According to the method, the aim of measuring organochlorine pesticide and synthetic musk in the serum is realized through a chemical pretreatment method of liquid-liquid extraction, gel permeation chromatography and silica gel alumina column, in combination with a detection technology of gas chromatography-electron capture detection (GC-ECD) and gas chromatography-mass spectrometry (GC-MS). The method provided by the invention belongs to the technical field of physico-chemical analysis measurement of organic chemicals. In the method provided by the invention, the serum pretreatment process comprises organic solvent liquid-liquid extraction, gel permeation chromatographic column degreasing, silica gel alumina column purification, separation and concentrating to constant volume, finally, GC-ECD and GC-MS are used for detection, detection spectrograms of organochlorine pesticide and synthetic musk in serum sample are respectively obtained, and concentration values of organochlorine pesticide and synthetic musk in the serum are calculated through a special formula. The method provided by the invention is a steady, relatively high-sensitivity and high-precision effective measurement method.
Description
Technical field
The present invention relates to a kind of stable and repeatability method of organo-chlorine pesticide and synthetic musk concentration in human body serum simultaneously preferably, the detection technique that the process for chemically pretreating by liquid-liquid extraction-gel permeation chromatographic column-silica gel alumina column detects (GC-ECD) and gas chromatography-mass spectrography (GC-MS) in conjunction with gas chromatography electron capture reaches the object of measuring organo-chlorine pesticide and musk ambrette in serum.The invention belongs to the physicochemical analysis determination techniques field of organic chemicals.
Background technology
Organic chlorine agriculture chemicals (OCPs) belongs to first batch of positively controlled 12 kinds of persistence organic pollutants of the Convention of Stockholm > > agreement of international convention < < persistence organic pollutant, organo-chlorine pesticide has stronger hydrophobic lipophilicity and environment refractory organics, and can be with Air Flow long-distance migration, by food chain enrichment, be passed in the mankind's body in the highest trophic level, cause health hazard.DDT and HCH have been proved has endocrine disrupting and potential carcinogenic impact.China was once the production of organic chlorine agriculture chemicals and used big country, 20% of the total consumption in the use amount Wei40 Wan Dun，Zhan world of past DDT.Although Yi China forbids successively since the eighties, because the pollution situation of its environmental persistence ，Qi China is still comparatively serious, in soil, water and various food and human sample, still can detect the DDTs of high level etc.Organo-chlorine pesticide is easily enriched in the rich fatty tissue of human body, comprises that subcutaneous fat, breast milk and serum are medium.Ubiquity organo-chlorine pesticide in human serum, level of pollution is about hundreds of to thousands of ng/g fat weights.It is the main path that human body is exposed to organo-chlorine pesticide that meals are taken in.So far in early days, many countries and regions utilize the pollution tendencies of OCPs in the evaluation of OCPs contents level in human serum environment to 20 century 70s, and the forbidding situation of evaluating OCPs.UNEP (United Nations Environment Program) also repeatedly generally investigates the OCPs content in the serum sample of various places, countries in the world.American-European countries all in succession starts correlative study project the OCPs in human body is exposed and monitored.Yet China's research is in this respect started late, early stage research focuses mostly on the content analysis of in river wastewater and bed mud OCPs, after the nineties, just there are successively OCPs content and distribution in the domestic megapolitan's sample body of bibliographical information, but the detection method that lacks unified standard, therefore the human body of domestic OCPs exposes to study and much all lacks comparability, has lacked for a long time about the assessment to domestic background crowd's exposure and health effect since OCPs forbidding.Studies show that, persistence OCPs is mainly exposed and is entered human body by diet, and OCPs concentration also can be along with food chain amplifies step by step, and the health hazard that the mankind in food chain top are subject to is maximum, therefore need to adhere to for a long time the human body of OCPs to expose and monitor.Research method, aspect sample pretreatment, often use freeze-drying, soxhlet extraction, gel permeation chromatography.The determination and analysis of the most of OCPs of academia depends on vapor-phase chromatography, common are gas chromatography-electron capture detection method (GC-ECD), gas chromatography-flame ion detection method (GC-FID), GC-MS(gas chromatography-mass spectrography) (GC-MS) and gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS) etc.Because vapor-phase chromatography has been a kind of quite perfect, ripe instrumental method now, its detectability, precision, accuracy and analysis efficiency have all obtained the extensive approval of academia, therefore this thinking of content that will overturn GC method analysis OCPs is substantially impossible within the quite a long time, unless instrument analysis technology has again new breakthrough.For analytical work person, more energy may concentrate on the optimization of GC parameter, the improvement of sample pretreatment, reagent such as select at the aspect, and the supplementing to improve and also need to drop into suitable manpower and materials of database.
Musk ambrette is that a kind of polarity is less, has the compound of stronger lipophilic hydrophobic nature, can be along food chain transmission.As adding fragrant class material, they are widely used in cosmetics and skincare product, articles for washing and food, tobacco and medicine.Musk ambrette mainly comprises Jiale muskiness, tonalid, ketone musk and musk xylene etc.As a class novel environmental pollutant, musk ambrette is subject to the extensive concern of international community, because immoderate production before the mankind and use cause a large amount of musk ambrette materials in use along with sanitary sewage enters each water body environment, and finally in each medium of food chain entered environment, up to now, researchist has all found the existence of musk ambrette in the fat of superficial water, sediment, atmosphere, dust, fish, birds, marine mammal and human body, breast milk.There are some researches show, synthetic musk can induce other toxicants that its toxicity is strengthened, some Moschus has environmental hormone effects and genetoxic effect, therefore even there is report to point out that nitro musk comprises that dimethylbenzene and ketone musk are all potential endocrine disruptors, from the nineties, start production and the use that Japan and European Union have all stopped nitro musk in succession.China is the main production and application base of world's essence and flavoring agent and nursing materials.Along with domestic crowd's growth in the living standard, to adding the use of fragrant class commodity, increase rapidly, especially some coastal developed regions approach European countries' level especially.But the potential health hazard for musk ambrette does not also cause enough attention, for the interpolation of nitro and polycyclic musk, use and all there is no restriction.The main route of exposure of human body of musk ambrette has been proved to be skin osmotic absorption, is secondly diet and dust.Therefore human body is the main exposure colony of musk ambrette, is necessary further to probe into the Study on mechanism such as distribution and damage of Moschus in human body.The distribution research of current domestic Moschus in environment concentrates on the distribution research in water body environment mostly, distribution characteristics such as Moschus in bed mud in river and in sewage treatment plant's Inlet and outlet water and excess sludge, only a 2-3 piece of writing literature research distribution of Moschus that (comprises breast milk and blood) in human body, limitation due to method and sample collection, the existence of musk ambrette in human serum of both at home and abroad also having only had so far several pieces of bibliographical informations, but the material detecting and sample recall rate are all very low, detection method needs further improve and optimize.
Organo-chlorine pesticide and musk ambrette are held respectively the representative species of type organic contaminant and the novel organic contaminant of environment as tradition, for them, detect in human sample and not only can supplement the basic data that domestic human body organic contaminant detects, simultaneously also for the dissimilar organic contaminant of comprehensive evaluation provides and more optimized and more rational background data the health hazard of human body.At present also there is no one piece of bibliographical information method and data of organo-chlorine pesticide and musk ambrette in human body serum simultaneously, therefore, the present invention has positive promotion meaning for level of pollution and comprehensive evaluation two pollutants of organo-chlorine pesticide in researching human body and musk ambrette to the health effect of human body.
Summary of the invention
The object of this invention is to provide a kind of can stablizing and the good simultaneously method of organo-chlorine pesticide and synthetic musk concentration in human body serum of repeatability.
The present invention is a kind of measures the method for organo-chlorine pesticide and synthetic musk concentration in human serum simultaneously, it is characterized in that having following process and step:
A, liquid-liquid extraction and constant weight fat
Measure 8ml blood serum sample and put into separating funnel, add respectively 0.005 μ g recovery indicant TCMX+PCB209, D3-AHTN and D15-MX, add subsequently the potassium oxalate of 8ml 8% and 10ml ethanol fully to shake up, then add the ether vibration of 5ml; Described mixed liquor is carried out to liquid-liquid extraction: first add 20ml normal hexane, fully vibration, stratification carries out separatory after liquid level layering, gets supernatant liquor and pours in heart bottle, stand-by; Lower floor's turbid solution of collecting is poured in separating funnel again, added 10ml normal hexane, fully vibration, stratification, gets supernatant liquor and pours in former stand-by heart bottle; Lower floor's turbid solution, by above-mentioned same method re-extract three times step by step, discards lower floor's turbid solution afterwards; By and and be collected in clarified solution in former heart bottle and be placed on Rotary Evaporators and revolve steaming, revolve and steam to volume 2～3ml; Then be transferred in the brown vial of weighing in advance, with the soft nitrogen of nitrogen, blow near dry, be put in constant weight 2-3 days in exsiccator, the fat of constant weight is weighed, record fat weight;
B, upper gel permeation chromatographic column (GPC post) degrease
The fat of above-mentioned constant weight is dissolved with the mixed liquor of a small amount of normal hexane and methylene chloride, and both volume ratios are normal hexane: methylene chloride=1: 1; Then cross gel permeation chromatographic column (GPC post); This column packing is Bio-Beads S-X3 gel, its effect is to make sample pass through GPC post and separation fat wherein, leacheate is the normal hexane of 185ml and the mixed liquor of methylene chloride (v/v=1:1), consumption socket joint is got the leacheate of front 65ml, discards, and then with heart bottle graft, gets remaining 120ml leacheate, by this leacheate, be placed on Rotary Evaporators and revolve steaming, revolve and steam to 2ml, stand-by;
C, silica gel alumina composite post processing excessively
Above-mentioned stand-by 2ml solution is crossed to silica gel aluminium oxide pillar to be processed, silica gel and the aluminium oxide (silica gel: aluminium oxide is 4cm:2cm) of certain proportion height are mainly housed in this post, and its effect is mainly remove the impurity in solution and organo-chlorine pesticide and musk ambrette are collected in different drip washing components; First add 5ml normal hexane to carry out drip washing, discard leacheate, then add 20ml 7:3 normal hexane: methylene chloride mixed solution, leacheate is collected in heart bottle, is considered as a component; Use again afterwards 15ml 1:5 normal hexane: methylene chloride mixed liquor carries out drip washing, in heart bottle, collect equally, be considered as two components, two component leacheates are placed in respectively on Rotary Evaporators and revolve steaming, revolve and steam to 2ml, solution is transferred in brown vial, with the soft nitrogen of nitrogen, blow to dry, stand-by;
D, instrument detect
In a component brown vial, add the pentachloronitrobenzene of 3 μ l 1ppm as interior mark, and 47 μ l isooctane, enter gas chromatography, with electron capture detector, carry out the detection of organo-chlorine pesticide class material; In two component brown vial, add 5 μ l 2ppm hexamethylbenzenes as interior mark, and the isooctane of 45 μ l, enter gas chromatograph-mass spectrometer (GCMS) (GC-MS) and carry out the detection of musk ambrette class material;
The chromatogram testing conditions of organo-chlorine pesticide is as follows: HP-5 (30m * 0.25mm i.d., 0.25 μ m) capillary column, and carrier gas is helium, constant current, post flow is 1.0ml/min, without split sampling, sample size is 1 μ l; 230 ℃ of injector temperatures, 110 ° of C of post beginning temperature, 1.5 ° of C/min rise to 170 ° of C, retain 5 min, and 2 ° of C/min rise to 226 ° of C, and 40 ° of C/min rise to 280 ° of C, retain 10 min, and 300 ° of C of rear operation retain 5min.Detector temperature is made as 315 ° of C.
The chromatograph condition of musk ambrette and mass spectrometer condition are as follows respectively: chromatographic condition HP-5 (30m * 0.25mm i.d., 0.25 μ m) capillary column; Carrier gas is helium, constant current, and post flow is 1.0ml/min, without split sampling, sample size is 1 μ l; 280 ℃ of injector temperatures, 80 ° of C of post beginning temperature, 8 ° of C/min rise to 160 ° of C, retain 1 min, 4 ° of C/min rise to 175 ° of C, retain 2 min, and 3 ° of C/min rise to 184 ° of C, retain 1 min, 20 ° of C/min rise to 280 ° of C, and 300 ° of C of rear operation retain 5min; Mass spectrum condition: adopt electron ionization (EI) mode, ion source temperature is made as 250 ° of C, 280 ° of C of interface temperature; Selectivity ion fractional scanning (SIM).The mark scanning ion of each material is: Jiale muskiness (HHCB), m/z 243,258 and 213; Tonalid (AHTN), m/z 243,258 and 159; Musk xylene (MX), m/z 282 and 297; Ketone musk (MK), m/z 279 and 294; Jiale muskiness-lactone (HHCB-lactone), m/z 257 and 272; AHTN-
d 3, m/z 246; MX-
d 15 , m/z 188 and 297;
E, concentration are calculated
A, drawing standard curve
With the mixed sample of organo-chlorine pesticide, configure the standard solution of finite concentration gradient; Target substance in the mixed mark of organo-chlorine pesticide comprises hexachloro-benzene (HCB), four kinds of isomerss of benzene hexachloride (α-, β-, γ-and δ-HCH) and DDT with and metabolic product (comprise 2,4 '-DDT, 4,4 '-DDT, 2,4 '-DDD, 4,4 '-DDD, 2,4 '-DDE and 4,4 '-DDE); Using TCMX and PCB-209 as recovery indicant, be configured to concentration and be respectively 10 μ g/L, 20 μ g/L, 40 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L, 200 μ g/L, the standard specimen solution of eight variable concentrations points of 400 μ g/L, and add 60 μ g/L pentachloronitrobenzenes (PCNB) for internal standard compound matter; Above-mentioned standard solution is entered to gas chromatography, with electron capture detector, detect, record the response of each concentration point; To obtaining the peak area (S of target compound after spectrogram integration
heading), and the peak area (S of internal standard compound matter
in mark), by both ratio, i.e. relative response R=S
heading/ S
in mark, and using R as ordinate, standard specimen concentration C is horizontal ordinate, forces initial point, the typical curve of render target material draws the graticule slope a of a few class target substances; The mixed mark of musk ambrette comprises Jiale muskiness (HHCB), tonalid (AHTN), celestolide (ADBI), phantolid (AHMI), special Lars (ATII), musk xylene (MX), ketone musk (MK) and Jiale muskiness-lactone (HHCB-lactone); Take D3-AHTN and D15-MX as recovery indicant, be configured to concentration and be respectively 20 μ g/L, 40 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L, 200 μ g/L, 300 μ g/L, the standard specimen solution of eight concentration point of 400 μ g/L, and the hexamethylbenzene (HMB) that adds 200 μ g/L is internal standard compound matter, enter gas chromatograph-mass spectrometer (GCMS) (GC-MS) to detect; The same organo-chlorine pesticide of graticule method for drafting of musk ambrette;
B, concentration are calculated
Adopt internal standard method to calculate; According to retention time and other, such as qualitative standards such as characteristic ions, the target compound detecting in actual blood serum sample is carried out qualitatively, afterwards target compound is gone out to peak and carry out peak area integration, i.e. S
order, internally mark compound also integration, i.e. S simultaneously
in, S
orderwith S
inratio be relative response R, then according to the slope a of the target compound typical curve of above-mentioned correspondence, can draw the upper machine concentration value C of target compound in sample
upper machine; Specific formula for calculation is as follows:
C
upper machine=R/a
In formula: C
upper machine---machine concentration on sample (unit: μ g/L),
R---relative response, R=S
order/ S
in,
The slope of standard curve of a---target compound;
In order to remove the background interference in experiment, according to above-mentioned formula, first calculate the concentration value C of dummy
blank, by C
upper machinededuct C
blankvalue, then be multiplied by the constant volume of sample, finally divided by the fat weight after serum extraction constant weight, the result of gained is the concentration of organo-chlorine pesticide and musk ambrette in serum, that is: C
sample concentration=(C
upper machine-C
blank) * constant volume (mL)/fat weight (g);
Dummy replaces aforementioned serum to carry out the preprocessing process of above-mentioned four steps with pure water.
The inventive method is in conjunction with detecting organo-chlorine pesticide in breast milk and blood in all kinds of documents, polychlorinated biphenyl, the disposal route of PBDE and musk ambrette etc., in the stable and repeated while human body serum preferably of energy, the concentration of organo-chlorine pesticide and musk ambrette, is a kind of method of better suited high accuracy.
Accompanying drawing explanation
Fig. 1. be the collection of illustrative plates of organo-chlorine pesticide standard specimen in the present invention.
Fig. 2 (a). be the collection of illustrative plates of musk ambrette in the present invention (MX) standard specimen.
Fig. 2 (b). be the collection of illustrative plates of musk ambrette in the present invention (HHCB, AHTN etc.) standard specimen.
Fig. 3. be the collection of illustrative plates of organo-chlorine pesticide material in mesostroma mark-on of the present invention.
Fig. 4 (a). be the collection of illustrative plates of musk ambrette (MX) material in mesostroma mark-on of the present invention.
Fig. 4 (b). be the collection of illustrative plates of musk ambrette (HHCB, AHTN etc.) material in mesostroma mark-on of the present invention.
Fig. 4 (c). be the collection of illustrative plates of musk ambrette (HHCB-lactone) material in mesostroma mark-on of the present invention.
Fig. 5. be the collection of illustrative plates of the organo-chlorine pesticide that detects in human serum sample.
Fig. 6 (a). be the collection of illustrative plates of the musk ambrette (MX) that detects in human serum sample.
Fig. 6 (b). be the collection of illustrative plates of the musk ambrette (HHCB, AHTN etc.) that detects in human serum sample.
Fig. 6 (c). be the collection of illustrative plates of the musk ambrette (HHCB-lactone) that detects in human serum sample.
Fig. 7. be the canonical plotting of HHCB in experimental example.
Embodiment
After now specific embodiments of the invention being described in.
Embodiment:
In the present embodiment, process and the step of assay method are as follows:
A, liquid-liquid extraction and constant weight fat
Measure 8ml blood serum sample and put into separating funnel, add respectively 0.005 μ g recovery indicant TCMX+PCB209, D3-AHTN and D15-MX, add subsequently potassium oxalate and the 10ml ethanol of 8ml 8% fully to shake up, add again the ether vibration of 5ml, described mixed liquor is carried out to liquid-liquid extraction: first add 20ml normal hexane, fully vibration, stratification, after liquid level layering, carry out separatory, get supernatant liquor and pour in heart bottle, stand-by; Lower floor's turbid solution of collecting is poured in separating funnel again, added 10ml normal hexane, fully vibration, stratification, gets supernatant liquor and pours in former stand-by heart bottle; Lower floor's turbid solution, by above-mentioned same method re-extract three times step by step, discards lower floor's turbid solution afterwards; By and and be collected in clarified solution in former heart bottle and be placed on Rotary Evaporators and revolve steaming, revolve and steam to volume 2～3ml; Then be transferred in the brown vial of weighing in advance, with the soft nitrogen of nitrogen, blow near dry, be put in constant weight 2-3 days in exsiccator, the fat of constant weight is weighed, record fat weight;
B, upper gel permeation chromatographic column (GPC post) degrease
The fat of above-mentioned constant weight is dissolved with the mixed liquor of a small amount of normal hexane and methylene chloride, and both volume ratios are normal hexane: methylene chloride=1: 1; Then cross gel permeation chromatographic column (GPC post); This column packing is Bio-Beads S-X3 gel, its effect is to make sample pass through GPC post and separation fat wherein, leacheate is normal hexane and the methylene chloride (v/v=1: mixed liquor 1) of 185ml, consumption socket joint is got the leacheate of front 65ml, discards, and then with heart bottle graft, gets remaining 120ml leacheate, by this leacheate, be placed on Rotary Evaporators and revolve steaming, revolve and steam to 2ml, stand-by;
C, silica gel alumina composite post processing excessively
Above-mentioned stand-by 2ml solution is crossed to silica gel aluminium oxide pillar to be processed, silica gel and the aluminium oxide (silica gel: aluminium oxide is 4cm:2cm) of certain proportion height are mainly housed in this post, and its effect is mainly remove the impurity in solution and organo-chlorine pesticide and musk ambrette are collected in different drip washing components; First add 5ml normal hexane to carry out drip washing, discard leacheate, then add 20ml 7:3 normal hexane: methylene chloride mixed solution, leacheate is collected in heart bottle, is considered as a component; Use again afterwards 15ml 1:5 normal hexane: methylene chloride mixed liquor carries out drip washing, in heart bottle, collect equally, be considered as two components, two component leacheates are placed in respectively on Rotary Evaporators and revolve steaming, revolve and steam to 2ml, solution is transferred in brown vial, with the soft nitrogen of nitrogen, blow to dry, stand-by;
D, instrument detect
In a component brown vial, add the pentachloronitrobenzene of 3 μ l 1ppm as interior mark, and 47 μ l isooctane, enter gas chromatography, with electron capture detector, carry out the detection of organo-chlorine pesticide class material; In two component brown vial, add 5 μ l 2ppm hexamethylbenzenes as interior mark, and the isooctane of 45 μ l, enter gas chromatograph-mass spectrometer (GCMS) (GC-MS) and carry out the detection of musk ambrette class material;
The chromatogram testing conditions of organo-chlorine pesticide is as follows: HP-5 (30m * 0.25mm i.d., 0.25 μ m) capillary column, and carrier gas is helium, constant current, post flow is 1.0ml/min, without split sampling, sample size is 1 μ l; 230 ℃ of injector temperatures, 110 ° of C of post beginning temperature, 1.5 ° of C/min rise to 170 ° of C, retain 5 min, and 2 ° of C/min rise to 226 ° of C, and 40 ° of C/min rise to 280 ° of C, retain 10 min, and 300 ° of C of rear operation retain 5min; Detector temperature is made as 315 ° of C.
The chromatograph condition of musk ambrette and mass spectrometer condition are as follows respectively: chromatographic condition HP-5 (30m * 0.25mm i.d., 0.25 μ m) capillary column; Carrier gas is helium, constant current, and post flow is 1.0ml/min, without split sampling, sample size is 1 μ l; 280 ℃ of injector temperatures, 80 ° of C of post beginning temperature, 8 ° of C/min rise to 160 ° of C, retain 1 min, 4 ° of C/min rise to 175 ° of C, retain 2 min, and 3 ° of C/min rise to 184 ° of C, retain 1 min, 20 ° of C/min rise to 280 ° of C, and 300 ° of C of rear operation retain 5min; Mass spectrum condition: adopt electron ionization (EI) mode, ion source temperature is made as 250 ° of C, 280 ° of C of interface temperature; Selectivity ion fractional scanning (SIM).The mark scanning ion of each material is: Jiale muskiness (HHCB), m/z 243,258 and 213; Tonalid (AHTN), m/z 243,258 and 159; Musk xylene (MX), m/z 282 and 297; Ketone musk (MK), m/z 279 and 294; Jiale muskiness-lactone (HHCB-lactone), m/z 257 and 272; AHTN-
d 3 , m/z 246; MX-
d 15 , m/z 188 and 297;
E, concentration are calculated
A, drawing standard curve
With the mixed sample of organo-chlorine pesticide, configure the standard solution of finite concentration gradient; Target substance in the mixed mark of organo-chlorine pesticide comprises hexachloro-benzene (HCB), four kinds of isomerss of benzene hexachloride (α-, β-, γ-and δ-HCH) and DDT with and metabolic product (comprise 2,4 '-DDT, 4,4 '-DDT, 2,4 '-DDD, 4,4 '-DDD, 2,4 '-DDE and 4,4 '-DDE); Using TCMX and PCB-209 as recovery indicant, be configured to concentration and be respectively 10 μ g/L, 20 μ g/L, 40 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L, 200 μ g/L, the standard specimen solution of eight variable concentrations points of 400 μ g/L, and add 60 μ g/L pentachloronitrobenzenes (PCNB) for internal standard compound matter.Above-mentioned standard solution is entered to gas chromatography, with electron capture detector, detect, record the response of each concentration point; To obtaining the peak area (S of target compound after spectrogram integration
heading), and the peak area (S of internal standard compound matter
in mark), by both ratio, i.e. relative response R=S
heading/ S
in mark, and using R as ordinate, standard specimen concentration C is horizontal ordinate, forces initial point, the typical curve of render target material draws the graticule slope a of a few class target substances.The mixed mark of musk ambrette comprises Jiale muskiness (HHCB), tonalid (AHTN), celestolide (ADBI), phantolid (AHMI), special Lars (ATII), musk xylene (MX), ketone musk (MK) and Jiale muskiness-lactone (HHCB-lactone); Take D3-AHTN and D15-MX as recovery indicant, be configured to concentration and be respectively 20 μ g/L, 40 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L, 200 μ g/L, 300 μ g/L, the standard specimen solution of eight concentration point of 400 μ g/L, and the hexamethylbenzene (HMB) that adds 200 μ g/L is internal standard compound matter, enters gas chromatograph-mass spectrometer (GCMS) (GC-MS) and detect.The same organo-chlorine pesticide of graticule method for drafting of musk ambrette;
B, concentration are calculated
Adopt internal standard method to calculate; According to retention time and other, such as qualitative standards such as characteristic ions, the target compound detecting in actual blood serum sample is carried out qualitatively, afterwards target compound is gone out to peak and carry out peak area integration, i.e. S
order, internally mark compound also integration, i.e. S simultaneously
in, S
orderwith S
inratio be relative response R, then according to the slope a of the target compound typical curve of above-mentioned correspondence, can draw the upper machine concentration value C of target compound in sample
upper machine, specific formula for calculation is as follows:
C
upper machine=R/a
In formula: C
upper machine---machine concentration on sample (unit: μ g/L),
R---relative response, R=S
order/ S
in,
The slope of standard curve of a---target compound,
In order to remove the background interference in experiment, according to above-mentioned formula, first calculate the concentration value C of dummy
blank, by C
upper machinededuct C
blankvalue, then be multiplied by the constant volume of sample, finally divided by the fat weight after serum extraction constant weight, the result of gained is the concentration of organo-chlorine pesticide and musk ambrette in serum, that is:
C
sample concentration=(C
upper machine-C
blank) * constant volume (mL)/fat weight (g)
Dummy replaces aforementioned serum to carry out the preprocessing process of above-mentioned four steps with pure water.
In the present invention about the collection of illustrative plates of organo-chlorine pesticide and musk ambrette standard specimen referring to Fig. 1 and Fig. 2 (a) and (b).In the chromatogram of Fig. 1, enumerate whole tested organo-chlorine pesticide standard specimens, comprised hexachloro-benzene (HCB), four kinds of isomerss of benzene hexachloride (α-, β-, γ-and δ-HCH) and DDT with and metabolic product (comprise 2,4 '-DDT, 4,4 '-DDT, 2,4 '-DDD, 4,4 '-DDD, 2,4 '-DDE and 4,4 '-DDE).Whole tested musk ambrette standard specimens in the chromatogram of Fig. 2 (a) and (b), have been enumerated, comprise Jiale muskiness (HHCB), tonalid (AHTN), celestolide (ADBI), phantolid (AHMI), special Lars (ATII), musk xylene (MX), ketone musk (MK) and Jiale muskiness-lactone (HHCB-lactone).
In the present invention about the collection of illustrative plates of organo-chlorine pesticide in matrix mark-on and musk ambrette material referring to Fig. 3 and Fig. 4 (a) and (b), (c).These two groups of chromatograms are in matrix calf serum, to have added artificially the standard sample of organo-chlorine pesticide and musk ambrette, and after the pretreatment process of aforementioned four steps the chromatogram of gained.PCNB in Fig. 3 is interior mark, and TCMX and PCB-209 are recovery indicant.HMB in Fig. 4 (a) and (b), (c) is interior mark, D
3-AHTN is recovery indicant.
In the present invention about the collection of illustrative plates of organo-chlorine pesticide in human serum and musk ambrette referring to Fig. 5 and Fig. 6 (a) and (b), (c).Blood serum sample is the actual serum sample of human body that hospital gathers, and after the pre-service of aforementioned four steps, obtains chromatogram.
In the present invention, the canonical plotting of the HHCB illustrating for example of embodiment is referring to Fig. 7.The equation of HHCB typical curve is y=0.0028x, and wherein y is instrument response peak area value, and x is compound concentration value.
The mensuration of organo-chlorine pesticide and musk ambrette in human serum sample
According to aforementioned computing formula, 10 blood serum samples are detected, what obtain the results are shown in Table 1 and table 2:
Note: <LOD-represent that this material, cannot be quantitative under method detectability.
According to the U.S. EPA regulation recovery, belong to normal tolerance interval between 80%-120%, the organo-chlorine pesticide in the present invention in human serum and the recovery of musk ambrette all, within the scope of this, illustrate that this experimental technique is reliable.
Concentration calculated example:
In serum sample 2, the instrument response peak area of the internal standard compound matter HMB of musk ambrette is S
in=898539, HHCB is S
order=77737, its relative response R=S
order/ S
in=77737/898539=0.0865, according to typical curve y=0.0028x, obtains concentration:
C
upper machine=x=R/a=0.0865/0.0028=32.04 μ g/L
In the blank that corresponding this sample is same batch, the instrument response peak area of interior mark HMB is S
in sky=806713, HHCB is S
empty order=19428, its relative response R=S
order/ S
in=19428/806713=0.0241, according to typical curve y=0.0028x, obtains concentration:
C
blank=x=R/a=0.0241/0.0028=8.92 μ g/L
Sample all constant volume to 0.05 mL, then fat weight normalization per sample, obtain actual sample concentration (unit: ng/g fat weight), that is:
C
sample concentration=(C
upper machine-C
blank) * constant volume (mL)/fat weight (g)=(32.04-8.92) * 0.05/0.0362=31.9 ng/g fat weight.
Remaining target compound calculates its concentration value in the same manner.
Claims (1)
1. a method of simultaneously measuring organo-chlorine pesticide and synthetic musk concentration in human serum, is characterized in that having following process and step:
A, liquid-liquid extraction and constant weight fat: measure 8ml blood serum sample and put into separating funnel, add respectively 0.005 μ g recovery indicant TCMX+PCB209, D3-AHTN and D15-MX, add subsequently potassium oxalate and the 10ml ethanol of 8ml 8% fully to shake up, then add the ether vibration of 5ml; Described mixed liquor is carried out to liquid-liquid extraction: first add 20ml normal hexane, fully vibration, stratification carries out separatory after liquid level layering, gets supernatant liquor and pours in heart bottle, stand-by; Lower floor's turbid solution of collecting is poured in separating funnel again, added 10ml normal hexane, fully vibration, stratification, gets supernatant liquor and pours in former stand-by heart bottle; Lower floor's turbid solution, by above-mentioned same method re-extract three times step by step, discards lower floor's turbid solution afterwards; By and and be collected in clarified solution in former heart bottle and be placed on Rotary Evaporators and revolve steaming, revolve and steam to volume 2～3ml; Then be transferred in the brown vial of weighing in advance, with the soft nitrogen of nitrogen, blow near dry, be put in constant weight 2-3 days in exsiccator, the fat of constant weight is weighed, record fat weight;
B, upper gel permeation chromatographic column (GPC post) degrease: the fat of above-mentioned constant weight is dissolved with the mixed liquor of a small amount of normal hexane and methylene chloride, and both volume ratios are normal hexane: methylene chloride=1: 1; Then cross gel permeation chromatographic column (GPC post); This column packing is Bio-Beads S-X3 gel, its effect is to make sample pass through GPC post and separation fat wherein, leacheate is normal hexane and the methylene chloride (v/v=1: mixed liquor 1) of 185ml, consumption socket joint is got the leacheate of front 65ml, discards, and then with heart bottle graft, gets remaining 120ml leacheate, by this leacheate, be placed on Rotary Evaporators and revolve steaming, revolve and steam to 2ml, stand-by;
C, silica gel alumina composite post processing excessively: above-mentioned stand-by 2ml solution is crossed to silica gel aluminium oxide pillar and process, silica gel and the aluminium oxide (silica gel: aluminium oxide is 4cm:2cm) of certain proportion height are mainly housed in this post, and its effect is mainly remove the impurity in solution and organo-chlorine pesticide and musk ambrette are collected in different drip washing components; First add 5ml normal hexane to carry out drip washing, discard leacheate, then add 20ml 7:3 normal hexane: methylene chloride mixed solution, leacheate is collected in heart bottle, is considered as a component; Use again afterwards 15ml 1:5 normal hexane: methylene chloride mixed liquor carries out drip washing, in heart bottle, collect equally, be considered as two components, two component leacheates are placed in respectively on Rotary Evaporators and revolve steaming, revolve and steam to 2ml, solution is transferred in brown vial, with the soft nitrogen of nitrogen, blow to dry, stand-by;
D, instrument detect: in a component brown vial, add the pentachloronitrobenzene of 3 μ l 1ppm as interior mark, and 47 μ l isooctane, enter gas chromatography, with electron capture detector, carry out the detection of organo-chlorine pesticide class material; In two component brown vial, add 5 μ l 2ppm hexamethylbenzenes as interior mark, and the isooctane of 45 μ l, enter gas chromatograph-mass spectrometer (GCMS) (GC-MS) and carry out the detection of musk ambrette class material;
The chromatogram testing conditions of organo-chlorine pesticide is as follows: HP-5 (30m * 0.25mm i.d., 0.25 μ m) capillary column, and carrier gas is helium, constant current, post flow is 1.0ml/min, without split sampling, sample size is 1 μ l; 230 ℃ of injector temperatures, 110 ° of C of post beginning temperature, 1.5 ° of C/min rise to 170 ° of C, retain 5 min, and 2 ° of C/min rise to 226 ° of C, and 40 ° of C/min rise to 280 ° of C, retain 10 min, and 300 ° of C of rear operation retain 5min.Detector temperature is made as 315 ° of C;
The chromatograph condition of musk ambrette and mass spectrometer condition are as follows respectively: chromatographic condition HP-5 (30m * 0.25mmi.d., 0.25 μ m) capillary column; Carrier gas is helium, constant current, and post flow is 1.0ml/min, without split sampling, sample size is 1 μ l; 280 ℃ of injector temperatures, 80 ° of C of post beginning temperature, 8 ° of C/min rise to 160 ° of C, retain 1 min, 4 ° of C/min rise to 175 ° of C, retain 2 min, and 3 ° of C/min rise to 184 ° of C, retain 1 min, 20 ° of C/min rise to 280 ° of C, and 300 ° of C of rear operation retain 5min; Mass spectrum condition: adopt electron ionization (EI) mode, ion source temperature is made as 250 ° of C, 280 ° of C of interface temperature; Selectivity ion fractional scanning (SIM); The mark scanning ion of each material is: Jiale muskiness (HHCB), m/z 243,258 and 213; Tonalid (AHTN), m/z 243,258 and 159; Musk xylene (MX), m/z 282 and 297; Ketone musk (MK), m/z 279 and 294; Jiale muskiness-lactone (HHCB-lactone), m/z 257 and 272; AHTN-
d 3 , m/z 246; MX-
d 15 , m/z 188 and 297;
E, concentration are calculated:
A, drawing standard curve: the standard solution that configures finite concentration gradient with the mixed sample of organo-chlorine pesticide; Target substance in the mixed mark of organo-chlorine pesticide comprises hexachloro-benzene (HCB), four kinds of isomerss of benzene hexachloride (α-, β-, γ-and δ-HCH) and DDT with and metabolic product (comprise 2,4 '-DDT, 4,4 '-DDT, 2,4 '-DDD, 4,4 '-DDD, 2,4 '-DDE and 4,4 '-DDE); Using TCMX and PCB-209 as recovery indicant, be configured to concentration and be respectively 10 μ g/L, 20 μ g/L, 40 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L, 200 μ g/L, the standard specimen solution of eight variable concentrations points of 400 μ g/L, and add 60 μ g/L pentachloronitrobenzenes (PCNB) for internal standard compound matter; Above-mentioned standard solution is entered to gas chromatography, with electron capture detector, detect, record the response of each concentration point; To obtaining the peak area (S of target compound after spectrogram integration
heading), and the peak area (S of internal standard compound matter
in mark), by both ratio, i.e. relative response R=S
heading/ S
in mark, and using R as ordinate, standard specimen concentration C is horizontal ordinate, forces initial point, the typical curve of render target material draws the graticule slope a of a few class target substances; The mixed mark of musk ambrette comprises Jiale muskiness (HHCB), tonalid (AHTN), celestolide (ADBI), phantolid (AHMI), special Lars (ATII), musk xylene (MX), ketone musk (MK) and Jiale muskiness-lactone (HHCB-lactone); Take D3-AHTN and D15-MX as recovery indicant, be configured to concentration and be respectively 20 μ g/L, 40 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L, 200 μ g/L, 300 μ g/L, the standard specimen solution of eight concentration point of 400 μ g/L, and the hexamethylbenzene (HMB) that adds 200 μ g/L is internal standard compound matter, enter gas chromatograph-mass spectrometer (GCMS) (GC-MS) to detect; The same organo-chlorine pesticide of graticule method for drafting of musk ambrette;
B, concentration are calculated: adopt internal standard method to calculate; According to retention time and other, such as qualitative standards such as characteristic ions, the target compound detecting in actual blood serum sample is carried out qualitatively, afterwards target compound is gone out to peak and carry out peak area integration, i.e. S
order, internally mark compound also integration, i.e. S simultaneously
in, S
orderwith S
inratio be relative response R, then according to the slope a of the target compound typical curve of above-mentioned correspondence, can draw the upper machine concentration value C of target compound in sample
upper machine; Specific formula for calculation is as follows:
C
upper machine=R/a
In formula: C
upper machine---machine concentration on sample (unit: μ g/L),
R---relative response, R=S
order/ S
in,
The slope of standard curve of a---target compound;
In order to remove the background interference in experiment, according to above-mentioned formula, first calculate the concentration value C of dummy
blank, by C
upper machinededuct C
blankvalue, then be multiplied by the constant volume of sample, finally, divided by the fat weight after serum extraction constant weight, the result of gained is the concentration of organo-chlorine pesticide in serum (OCPs) and musk ambrette (musks), i.e. C
sample concentration=(C
upper machine-C
blank) * constant volume (mL)/fat weight (g); Dummy replaces aforementioned serum to carry out the preprocessing process of above-mentioned four steps with pure water.
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| CN104165953A (en) * | 2014-08-15 | 2014-11-26 | 余新威 | Extraction and purification method for analyzing OCPs in marine shellfish tissue |
| CN105467053A (en) * | 2015-12-04 | 2016-04-06 | 中华人民共和国东莞出入境检验检疫局 | Rapid detection method for artificial musk residues in daily chemical product |
| CN105699527A (en) * | 2016-02-02 | 2016-06-22 | 环境保护部华南环境科学研究所 | Detection method of content of brominated flame retardant and phosphorus-based flame retardant in blood serum |
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