CN102636604A - Quantitative detecting method for estrogen in milk - Google Patents
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Abstract
The invention provides a quantitative detecting method for estrogen in milk. The quantitative detecting method includes steps of 1) performing derivatization pretreatment for a milk sample; and 2) measuring the content of the estrogen in the milk sample by the aid of online solid-phase extraction and two-dimensional liquid chromatogram tandem mass spectrum analysis. Owning to sample derivatization measurement, detection sensitivity is greatly improved. A two-dimensional liquid chromatogram tandem mass spectrum system is used for detecting, accordingly, online automatic purification of the sample is realized, and the detection efficiency of the sample is greatly improved. In addition, the analysis method is fine in recovery rate, high in automation degree and simple and easy in operation, and can fast and synchronously analyze the content of various types of trace estrogen in the sample.
Description
Technical field
The present invention relates to the Food Inspection field, specifically, relate to estrogenic quantitative detecting method in the milk.
Background technology
Over past ten years, estrogen receives extensive concern as typical incretion interferent.Type estrogen incretion interferent (EDCs) is one type and can combines with ERs, disturbs normal hormone secretion in the body, causes the reproduction cell quality to descend, and the compound that three causes serious harms such as (carcinogenic, teratogenesis, mutagenesis).Estrogen can cause human endocrine unbalance, and especially the low dosage long term exposure has produced specific crowd (like fetus, ewborn infant and prepuberal children) and seriously influenced, and has developed into social concern like sex premature, obesity etc.Estrogenic another big harm is exactly its carcinogenesis.External epidemiology survey shows that the incidences of disease such as nearly decades of carcinoma of testis, the cancer of the uterus are ascendant trend year by year, suffers from the cancer number and increases the increase significant correlation with animal products such as the change esp meat of eating patterns, egg, milk.In view of the material impact of estrogen in the animal derived food, be necessary estrogen content in the milk is monitored, with easy exposed population groups' such as protection infant health to human health.Set up estrogen high-sensitivity detecting method in the milk, for the background survey of estrogen content in the food with carry out risk assessment work, significant.
Realize estrogenic science risk assessment, high-sensitivity detecting method is crucial.The enough sensitivity of steroid hormone detection method in european union directive 2003/74/EC [42] the regulation animal product can be distinguished hormone that normal physiological level and abuse and misuse the cause phenomenon that exceeds standard.But often in ppt (ng/L) level, general chemical detection method is difficult to reach estrogen in the animal product.Utilize chromatogram and mass spectrometric hyphenated technique possibility to be provided for detecting the trace hormonal readiness.But because estrin structure is stable and nonpolar characteristic, being difficult to ionization or Ionization Efficiency low is that it detects difficult point.In addition, estrogenic liquid chromatography-tandem mass spectrometry detects all need use the negative ion detecting pattern, and sensitivity is very low.Even the LC-MSMS of uses advanced (API5000) detects estrogen, sensitivity also can only reach 0.1 μ g/kg, is difficult to satisfy the requirement of estrogen trace detection in the animal product.Improving detection sensitivity is the key that estrogen detects in the milk.Utilize derivative reagent to measure the estrogen in the milk, can improve detection sensitivity greatly, but, import metallic ions such as sodium, potassium because derivatization causes extra matrix interference, serious to mass detector harm.Therefore set up sample extraction method efficiently, effectively removing matrix interference becomes problem demanding prompt solution.
Summary of the invention
The objective of the invention is to milk sample matrix complicated; Estrogen content is low under the normal condition; Detection method requires problems such as high; The analytical technology of a kind of high sensitivity, high accurancy and precision, online rapid extraction sample is provided, realizes quantitative rapid automatized mensuration trace estrogen in the milk sample.
In order to realize the object of the invention, estrogenic quantitative detecting method in a kind of milk of the present invention may further comprise the steps: 1) milk sample is carried out the derivatization pre-service; 2) analyze through online SPE-two-dimensional liquid chromatography tandem mass spectrum, measure estrogenic content in the milk sample.Wherein said estrogen is one or more in oestrone, alpha-estradiol, beta estradiol and the estriol.
Preferably, derive described in the step 1) and turn to the dansyl Cl derivatization.Concrete operations are:
Get the 3mL milk sample, add mark spiral mixing in 3 kinds of deuterium-labelled estrogen; Sample adds 30% trichloroacetic acid 100 μ L sedimentation albumen; Add ethyl acetate and normal hexane solvent 8mL that equal-volume mixes behind the mixing, 10min is extracted in concussion, 10000 leave heart 10min after, the separation upper solution dries up with nitrogen; Repeat to extract sample 2 times, the residue that nitrogen is dried up is with the dissolving of 1mL acetonitrile, hatches spiral mixing 30s behind the 5min ,-20 ℃ of freezing 30min for 50 ℃; Sample leaves heart 5min in 10000 then; Upper solution is transferred to evaporate to dryness in another test tube; Add 150 μ L sodium bicarbonate buffer liquid spirals sample dissolution again, add 150 μ L dansyl Cl acetone soln mixings then, 50 ℃ of derivatization 5min; With 0.22 μ m filtering with microporous membrane sample, supply next step to detect in the sample introduction bottle of packing into and use.
Online SPE-two-dimensional liquid chromatography tandem mass spectrum analysis comprises: with appearance on the sample in the sample introduction bottle, carry out online SPE and the analysis of two-dimensional liquid chromatography tandem mass spectrum.Online SPE two-dimensional liquid chromatography tandem mass spectrum system building is as shown in Figure 1.Online SPE two-dimensional liquid chromatography tandem mass spectrum (online SPE LC-MS/MS) has comprised double-colored spectrum pump, interchange valve and on-line preconcentration post.Wherein pump A is used for the sample in-line purification, and pump B is used for sample chromatogram post compartment analysis.Sample uses the post switching technology to get into analytical column then and measures at first through enriching column wash-out impurity and keep target compound, realizes that the semi-automation of sample is extracted.
I. drawing standard curve, matrix is added inner mark method ration:
Take by weighing 8 parts of blank milk samples; Every part of 3mL; Add the interior mark of 3 kinds of deuterium-labelled estrogen of same concentrations respectively; Interior target final concentration is 50ng/L, adds the estrogen standard items of variable concentrations successively, makes its final concentration be respectively 0ng/L, 2.0ng/L, 5.0ng/L, 10.0ng/L, 20.0ng/L, 40.0ng/L, 80.0ng/L and 100ng/L; Appearance is carried out online SPE-two-dimensional liquid chromatography tandem mass spectrum analysis on the sample; The ratio of tested component peaks area and internal standard compound peak area is ordinate in the sample to add, and is horizontal ordinate drawing standard curve with the ratio of tested concentration of component in the standard solution and internal standard compound concentration;
II. estrogenic quantitative in the milk sample:
With appearance on the pretreated milk sample of step 1), carry out online SPE-two-dimensional liquid chromatography tandem mass spectrum analysis, with typical curve sample is carried out quantitatively, the response of determinand all should be in the range of linearity of Instrument measuring in the sample solution.
Sample purification, elution requirement (comprising flow velocity and gradient) that said SPE is adopted are seen table 1.
The elution requirement that table 1 SPE is adopted
| Step | T.T. (min) | Flow velocity (μ L/min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 0.00 | 500 | 45.0 | 55.0 |
| 1 | 2.50 | 500 | 45.0 | 55.0 |
| 2 | 2.60 | 500 | 1.0 | 99.0 |
| 3 | 4.00 | 500 | 1.0 | 99.0 |
| 4 | 4.20 | 100 | 45.0 | 55.0 |
[0016]?
| 5 | 13.00 | 100 | 45.0 | 55.0 |
Mobile phase A wherein: 0.1% formic acid water; Mobile phase B: 0.1% formic acid methyl alcohol.
Preferably, the sample purification post that uses of SPE is strata-X on-line SPEcartridge (Phenomenex company).
Said liquid-phase chromatographic analysis condition (gradient elution step and flow velocity) is seen table 2.
Table 2 liquid-phase chromatographic analysis condition (gradient elution step and flow velocity)
| Step | T.T. (min) | Flow velocity (μ L/min) | Moving phase C (%) | Moving phase D (%) |
| 0 | 0.00 | 450 | 50.0 | 50.0 |
| 1 | 2.50 | 450 | 50.0 | 50.0 |
| 2 | 9.50 | 450 | 15.0 | 85.0 |
| 3 | 10.00 | 450 | 5.0 | 95.0 |
| 4 | 11.00 | 450 | 5.0 | 95.0 |
| 5 | 11.50 | 450 | 50.0 | 50.0 |
| 6 | 13.00 | 450 | 50.0 | 50.0 |
Moving phase C:0.1% formic acid water wherein; Moving phase D:0.1% formic acid acetonitrile.
The liquid-phase chromatographic analysis post is Eclipse Plus, Phenyl-Hexyl, 2.1x100mm, 3.5 μ m (Agilent company).
The timetable that wherein valve switches between sample purification and the sample analysis is:
The mass spectrophotometry condition is:
Ion gun: electro-spray ionization source (ESI), the positive ion mode detects; Ion gun voltage :+5500V; Temperature: 500 ℃; Gas 1 (GS1, N in the source
2) pressure 65psi; Gas 2 (GS2, N
2) pressure 55psi; Gas curtain gas (N
2) pressure 15psi; Scan mode is many reactive ion monitorings (MRM); Collision gas (CAD, N
2) pressure 5psi.
Advantage of the present invention is:
(1) the present invention adopts analyte derivative mensuration, has greatly improved detection sensitivity; Adopt two-dimensional liquid chromatography-tandem mass spectrum system to detect, realize the on-line automaticization purification of sample, greatly improved the detection efficiency of sample.
(2) the analytical approach recovery of the present invention is good, automaticity is high, operation is simple, fast the content of multiple trace estrogen in the Synchronization Analysis sample.
Description of drawings
Fig. 1 is online SPE two-dimensional liquid chromatography system simulation figure, and wherein Figure 1A is the sample introduction pattern, and Figure 1B is a mode determination.
Fig. 2 is the chromatogram of 0.05 μ g/L standard solution; Chromatogram is followed successively by: Fig. 2 A is oestrone (retention time RT=7.13min); Fig. 2 B is a mark (retention time RT=7.11min) in the oestrone, and Fig. 2 C is beta estradiol (retention time RT=6.44min) and alpha-estradiol (retention time RT=6.61min), and Fig. 2 D is a mark (retention time RT=6.41min) in the beta estradiol; Fig. 2 E is estriol (retention time RT=4.65min), and Fig. 2 F is a mark (retention time RT=4.64min) in the estriol.
Fig. 3 is oestrone (E1), alpha-estradiol (α-E2), beta estradiol (β-E2), four kinds of estrogenic typical curves of estriol.Horizontal ordinate is estrogen concentrations (ng/L), and ordinate is e surge area and internal standard compound peak area ratio.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment, the raw materials used commercial goods that is.
The pre-service of embodiment 1 test sample
Get the mixed uniformly milk sample of 3mL, add mark in 3 kinds of deuterium-labeled estrogen, the spiral mixing.Interior mark ultimate density is 50ppt.Then, in sample, add 30% trichloroacetic acid 100 μ L, mixing.Add 8mL ethyl acetate and normal hexane (1: 1) solvent, after 10min was extracted in concussion, 10000 left heart 10min.Shift supernatant, nitrogen dries up under 50 ℃ of water bath condition.Repeat to extract twice.The residue that nitrogen is dried up dissolves with the 1mL acetonitrile, hatches 5min for 50 ℃, spiral mixing 30s, and room temperature leaves standstill 5min, places-20 ℃ of freezing 30min.Sample 10000 leaves heart 5min under 4 ℃ of conditions, supernatant is transferred in another test tube, and nitrogen dries up under 50 ℃ of water bath condition.(adding 150 μ L concentration then is 1mg/mL dansyl Cl acetone soln mixing for 100mM, pH=10.5) spiral dissolved residue with 150 μ L sodium bicarbonate buffer liquid.Place 60 ℃ of water-bath derivatization 5min fast.The quick centrifugal 1min of sample, with the organic filtering with microporous membrane of 0.22 μ m, to be measured in the sample introduction bottle of packing into.
The condition that online SPE-two-dimensional liquid chromatography tandem mass spectrum is selected: 2 Agilent 1200SL liquid chromatography binary pump, G1367C type automatic sampler, G1316B type column oven is joined the Valco six-way valve.Mass spectrometer is that API 5000 is furnished with electro-spray ionization source (ESI) and Analyst 1.4.2 data processing software,
Sample purification is with the online solid phase extraction column of strata-X (20mm * 2.0mm * 20 μ m is available from Phenomenex company).Sample analysis is with Eclipse Plus benzene hexyl column (2.1mm * 100mm * 3.5 μ m is available from Agilent company).Ion gun: electro-spray ionization source (ESI), the positive ion mode detects; Ion gun voltage :+5500V; Temperature: 500 ℃; Gas 1 (GS1, N in the source
2) pressure 65psi; Gas 2 (GS2, N
2) pressure 55psi; Gas curtain gas (N
2) pressure 15psi; Scan mode is many reactive ion monitorings (MRM); Collision gas (CAD, N
2) pressure 5psi.
The liquid chromatography-tandem mass spectrometry coupling is 0.5ng/L to four kinds of estrogenic detection lower limits.
The drafting of embodiment 3 typical curves
Add internal standard method with matrix and carry out quantitative measurement.The drafting of internal standard method typical curve comprises: prepare blank milk sample, take by weighing 8 parts, every part of 3mL.Add the interior mark of same concentrations respectively, interior target final concentration is 50ng/L.Add the estrogen standard items of variable concentrations successively, make the standard point ultimate density of typical curve be respectively 0ng/L, 2.0ng/L, 5.0ng/L, 10.0ng/L, 20.0ng/L, 40.0ng/L, 80.0ng/L and 100ng/L.Above-mentioned matrix is added standard by extracting and liquid chromatography tandom mass spectrometry determination with the sample same procedure; Ratio with tested component peaks area and internal standard compound peak area in the interpolation sample is ordinate; The ratio of tested concentration of component and internal standard compound concentration is horizontal ordinate drawing standard working curve in the standard solution; With standard working curve sample is carried out quantitatively, the response of determinand all should be in the range of linearity of Instrument measuring in the sample solution (Fig. 2 A-Fig. 2 F, Fig. 3).
The mensuration of embodiment 4 sample recovery rates
Prepare a milk sample, take by weighing 7 parts, every part of 3mL.Label 0-6.No. 0 sample is made as blank, and 1-2 number, each sample adds the 10ng/L standard items; 3-4 number, each sample adds the 20ng/L standard items; 5-6 number, each sample adds the 100ng/L standard items.Prepare the standard solution of same concentrations in addition, carry out the sample recovery rate experiment of basic, normal, high three concentration.Add the peak area of determinand in the sample through standard and deduct the peak area of determinand in the blank sample and the peak area ratio calculation sample recovery of standard solution, the result sees table 3.
Table 3 adds the recovery experimental result
Embodiment 5 utilizes online SPE-two-dimensional liquid chromatography tandem mass spectrum to detect estrogenic content in the milk
Gather milk sample to be measured, take back the laboratory and carry out Treatment Analysis.Each sample is established 2 replications.Get the 3mL sample, mark back mixing in adding.Add 8mL ethyl acetate and normal hexane (1: 1) solvent, 10min is extracted in concussion.Centrifugal 10min under 10000 commentaries on classics collects supernatant in another test tube, and nitrogen dries up under 50 ℃ of water bath condition.Repeat to extract sample 2 times.The residue that nitrogen is dried up dissolves with the 1mL acetonitrile, hatches 5min for 50 ℃, spiral mixing 30s.Room temperature leaves standstill 5min, places-20 ℃ of freezing 30min.Sample 10000 leaves heart 5min under 4 ℃ of conditions, supernatant is transferred in another test tube, and nitrogen dries up under 50 ℃ of water bath condition.(adding 150 μ L concentration then is 1mg/mL dansyl Cl acetone soln mixing for 100mM, pH=10.5) spiral dissolved residue with 150 μ L sodium bicarbonate buffer liquid.Place 60 ℃ of water-bath derivatization 5min fast.The quick centrifugal 1min of sample, with the organic filtering with microporous membrane of 0.22 μ m, to be measured in the sample introduction bottle of packing into.
Utilize online SPE-two-dimensional liquid chromatography tandem mass spectrum to measure the estrogen concentrations of above-mentioned testing sample.The condition that online SPE-two-dimensional liquid chromatography tandem mass spectrum is selected: API5000 type series connection level Four bar mass spectrometer (Applied Biosystem company, the U.S.).Be furnished with electro-spray ionization source (ESI) and Analyst 1.4.2 data processing software.The instrument condition of liquid chromatography part: 2 Agilent 1200SL liquid chromatography binary pump, G1367C type automatic sampler, G1316B type column oven is joined the Valco six-way valve.Fig. 1 is seen in the installation of two-dimensional liquid chromatography.Sample purification is with the online solid phase extraction column of strata-X (20mm * 2.0mm * 20 μ m), Phenomenex company.Sample analysis is with Eclipse Plus benzene hexyl column (2.1mm * 100mm * 3.5 μ m), Agilent company.Ion gun: electro-spray ionization source (ESI), the positive ion mode detects; Ion gun voltage :+5500V; Temperature: 500 ℃; Gas 1 (GS1, N in the source
2) pressure 65psi; Gas 2 (GS2, N
2) pressure 55psi; Gas curtain gas (N
2) pressure 15psi; Scan mode is many reactive ion monitorings (MRM); Collision gas (CAD, N
2) pressure 5psi.Liquid chromatography moving phase is:
(1) sample in-line purification mobile phase A: 0.1% formic acid water; Mobile phase B: 0.1% formic acid methyl alcohol.The gradient elution step is 0-2.5min, 55%B, flow velocity 500 μ l/min; 2.5-2.6min, 99%B, flow velocity 500 μ l/min; 2.6-4.0min, 99%B, flow velocity 500 μ l/min; 4.0-4.2min, 55%B, flow velocity 100 μ l/min; 4.2-13.0min, 55%B, flow velocity 100 μ l/min.
(2) sample chromatogram post separated flow phase C:0.1% formic acid water; Moving phase D:0.1% formic acid acetonitrile.Concrete gradient elution step and flow velocity are 0-2.5min, 50%B; 2.5-9.5min, rise to 85%B; 9.5-10.0 minute, rise to 95%B; 10-11min keeps 95%B; 11-11.5min, getting back to 50%B, 11.5-13min keeps 50%B.Whole chromatographic separation process flow velocity is 450 μ l/min.
Sample purification and analysis valve switching schedule are: 0min, and valve position is changed to a left side, 1.0min, valve position switches to the right side.2.5min valve position switches Hui Zuowei.
Testing result is as shown in table 4.
Four kinds of estrogenic content (pg/mL) in table 4 milk sample
Annotate: N.D. representes not detect.
Can know that from table 4 result the detection method that the present invention sets up can high sensitivity, detect the trace estrogen in the milk sample high accurancy and precision.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (8)
1. estrogenic quantitative detecting method in the milk is characterized in that, may further comprise the steps:
1) milk sample is carried out the derivatization pre-service;
2) analyze through online SPE-two-dimensional liquid chromatography tandem mass spectrum, measure estrogenic content in the milk sample.
2. method according to claim 1 is characterized in that, said estrogen is one or more in oestrone, alpha-estradiol, beta estradiol and the estriol.
3. method according to claim 1 is characterized in that, derives described in the step 1) to turn to the dansyl Cl derivatization.
4. method according to claim 3 is characterized in that, dansyl Cl derivatization concrete grammar is:
Get the 3mL milk sample, add mark spiral mixing in 3 kinds of deuterium-labelled estrogen; Sample adds 30% trichloroacetic acid 100 μ L sedimentation albumen; Add ethyl acetate and normal hexane solvent 8mL that equal-volume mixes behind the mixing, 10min is extracted in concussion, 10000 leave heart 10min after, the separation upper solution dries up with nitrogen; Repeat to extract sample 2 times, the residue that nitrogen is dried up is with the dissolving of 1mL acetonitrile, hatches spiral mixing 30s behind the 5min ,-20 ℃ of freezing 30min for 50 ℃; Sample leaves heart 5min in 10000 then; Upper solution is transferred to evaporate to dryness in another test tube; Add 150 μ L sodium bicarbonate buffer liquid spirals sample dissolution again, add 150 μ L dansyl Cl acetone soln mixings then, 50 ℃ of derivatization 5min; With 0.22 μ m filtering with microporous membrane sample, supply next step to detect and use.
5. method according to claim 4 is characterized in that step 2) concrete operations are:
I. drawing standard curve:
Take by weighing 8 parts of blank milk samples; Every part of 3mL; Add the interior mark of 3 kinds of deuterium-labelled estrogen of same concentrations respectively; Interior target final concentration is 50ng/L, adds the estrogen standard items of variable concentrations successively, makes its final concentration be respectively 0ng/L, 2.0ng/L, 5.0ng/L, 10.0ng/L, 20.0ng/L, 40.0ng/L, 80.0ng/L and 100ng/L; Appearance is carried out online SPE-two-dimensional liquid chromatography tandem mass spectrum analysis on the sample; The ratio of tested component peaks area and internal standard compound peak area is ordinate in the sample to add, and is horizontal ordinate drawing standard curve with the ratio of tested concentration of component in the standard solution and internal standard compound concentration;
II. estrogenic quantitative in the milk sample:
With appearance on the pretreated milk sample of step 1), carry out online SPE-two-dimensional liquid chromatography tandem mass spectrum analysis, with typical curve sample is carried out quantitatively, the response of determinand all should be in the range of linearity of Instrument measuring in the sample solution.
6. method according to claim 5 is characterized in that, the elution requirement that SPE is adopted is:
Mobile phase A wherein: 0.1% formic acid water; Mobile phase B: 0.1% formic acid methyl alcohol.
7. according to claim 5 or 6 described methods, it is characterized in that the liquid-phase chromatographic analysis condition is:
Moving phase C:0.1% formic acid water wherein; Moving phase D:0.1% formic acid acetonitrile.
8. according to claim 5 or 6 described methods, it is characterized in that the mass spectrophotometry condition is: ion gun: electro-spray ionization source (ESI), the positive ion mode detects; Ion gun voltage :+5500V; Temperature: 500 ℃; Gas 1 (GS1, N in the source
2) pressure 65psi; Gas 2 (GS2, N
2) pressure 55psi; Gas curtain gas (N
2) pressure 15psi; Scan mode is many reactive ion monitorings (MRM); Collision gas (CAD, N
2) pressure 5psi.
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| CN105334282B (en) * | 2015-10-27 | 2017-05-03 | 哈尔滨工业大学 | Co-detecting method for environmental estrogens in surface water body |
| CN105842379A (en) * | 2016-06-02 | 2016-08-10 | 云南省农业科学院质量标准与检测技术研究所 | Method for measuring phenolic estrogen by means of derivation |
| CN109187827A (en) * | 2018-08-14 | 2019-01-11 | 成都益康谱科技有限公司 | Estrogen and its metabolite online test method in human urine |
| CN109187827B (en) * | 2018-08-14 | 2021-06-18 | 成都益康谱科技有限公司 | On-line detection method for estrogen and metabolite thereof in human urine |
| CN109374765A (en) * | 2018-10-31 | 2019-02-22 | 中国科学院遗传与发育生物学研究所 | A method of endogenous witchweed lactone in full-automatic online SPE-LC-MS/MS Quantitative Analysis of Plant Samples |
| CN117805304A (en) * | 2024-02-29 | 2024-04-02 | 中国环境科学研究院 | Method and kit for detecting hormone in blood sample |
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