CN105572238B - A kind of method of estrogen in detection livestock waste - Google Patents
A kind of method of estrogen in detection livestock waste Download PDFInfo
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- CN105572238B CN105572238B CN201410550857.0A CN201410550857A CN105572238B CN 105572238 B CN105572238 B CN 105572238B CN 201410550857 A CN201410550857 A CN 201410550857A CN 105572238 B CN105572238 B CN 105572238B
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Abstract
The invention discloses a kind of method for detecting estrogen in livestock waste, this method includes sample extraction, sample purification, sample elution and sample determination step, the estrogen in cultivation solid waste is mainly effectively extracted using methylene chloride/methanol mixed solution as solvent, the rate of recovery is more than 80%；A large amount of oil substances in sample are effectively removed using silica gel pillar；Analyzed with HPLC/FL, method is simple, universal, and fluorescence detector selectivity is high, strong antijamming capability, and a variety of estrogen separation are obvious, and peak shape is symmetrical, and concentration peak area linear is on speaking terms.
Description
Technical field
The invention belongs to environmental analysis technical field, and in particular to a kind of method for detecting estrogen in livestock waste.
Background technology
The third-largest global great environmental that Environmental Hormone Pollution is considered as after depletion of the ozone layer, greenhouse effects is asked
Topic.Environmental hormone is defined as to the normal behaviour of biology and reproduction, the synthesis for developing related normal hormonal, storage, secretion, body
The processes such as interior conveying, combination and removing produce the material of blanketing.Environmental hormone is considered as to cause mankind spermatozoon quantity
Reduce, teratospermia, the principal element of female infertility phenomenon, also and into human immunity systemic-function decline, endocrine is different
Often, malignant tumour increases, nervous system occur the diseases such as obstacle, newborn teratogenesis, hypoplasia rise year by year it is closely related.Together
When, environmental hormone can influence the internal system, immune system and nervous system of wild animal, so as to be caused to the ecosystem
Threaten.Environmental Hormone Pollution is closely related with mankind's activity.Sanitary sewage and breeding waste are considered as the master of environmental hormone
Want source.Hormone of greatest concern is mainly estrogen at present, including estradiol, ethinyloestradiol, oestrone, bisphenol-A etc..Sanitary sewage
Discharge with breeding waste is their main paths into environment.
With the understanding endangered environmental hormone, it is extremely urgent to establish efficient, reliable analysis method.Environmental Hormone Pollution
The characteristics of be：First, it is widely distributed, it there may be in various media, including water body, soil, biological sample；Second, it is dense
Spend it is low, it is generally horizontal in ng/L, but the characteristics of due to hormone itself, even in such low concentration, can also produce significant life
Thing effect.At present, the analysis of estrogen mainly detects (HPLC/MS/MS) technology by high performance liquid chromatography-tandem mass, general
The sensitivity of logical liquid chromatogram-ultraviolet detection (HPLC/DAD) is difficult to meet to require.Relative to the extensive of common HPLC/DAD
Using HPLC/MS/MS belongs to high-end precision instrument, and cost is high, and the requirement to operating personnel is also high, and general analysis laboratory is not
Possess.In addition, HPLC/MS/MS analyses have very high requirement to sample pre-treatments.For water sample, first have to by solid phase
(SPE) enrichment is extracted, then to carry out the purified treatment of complexity, can just enter HPLC/MS/MS analyses.The purpose of purification is to the greatest extent
The various impurity in sample may be removed, otherwise severe jamming can be produced to mass spectral analysis, influence result accuracy.In a word, ring
The quantitative analysis complex steps of environment hormone, take, very high to personnel and instrument requirements, financial cost is also very high.
The analysis method of environmental hormone relative maturity in water.The discharge of breeding waste and returning to the field are hormones in environment
One of main source.Relative to water sample, the composition of breeding waste wants the more of complexity, especially containing a certain amount of grease.It is female
Hormone hydrophily is not strong, very low with water extraction recovery, it is therefore necessary to which with organic solvent, but which kind of solvent is suitable, at present also not
It is clear.In addition, organic solvent during hormone is extracted, simultaneously can extract the grease in sample.Grease enters nothing
All it is fatal by high performance liquid chromatography separation below, or mass spectral analysis.A variety of factors above, it is discarded for cultivating
Hormone substance in thing, there is presently no reliable extraction, analysis method.
The content of the invention
It is an object of the invention to provide one kind cultivation solid waste, (such as chicken farm, pig farm include excrement, fine hair, food
Thing residue etc.) in estradiol, ethinyloestradiol, oestrone, the quantitative analysis method of the estrogen such as bisphenol-A, this method can effectively overcome
The interference of various impurity in sample, while relative inexpensiveness, it is adapted to the analysis of batch samples.
The purpose of the present invention is achieved in the following ways：
A kind of method of estrogen in detection livestock waste, it is characterised in that this method comprises the following steps：
(1) sample extraction：Plant solid waste 3-10g is taken, using volume ratio as 2:1-4:1 dichloromethane and methanol
Mixed solution is solvent, is extracted under ultrasonic wave added, combining extraction liquid, centrifugation, takes supernatant, blowing solvent with nitrogen is allowed to wave
Hair, until surplus solution 1-2mL；
(2) sample purification：Silica gel 2-5g is taken, is activated with n-hexane, it is 1 to load blade diameter length ratio：It is quiet in 10-15 glass column
Put, treat that solid sedimentation is complete, discharge n-hexane, flush its liquid level and silicagel column, purification pillar is prepared；Glass column footpath is high
Than being preferably 1：10；
The sample n-hexane dissolution that step (1) is obtained, then purification pillar is uploaded to, cleaned with n-hexane, Ran Houyong
Nitrogen dries up silicagel column；
(3) sample elution：Eluted with acetonitrile, collect eluent, be concentrated into 1-2ml；
(4) sample determines：Quantitative analysis, the excitation wave of fluorescence detector are carried out with high performance liquid chromatography-fluorescence device
Long and launch wavelength is respectively 280nm and 310nm.
The product ratio preferably 2 of the mixed solution body of dichloromethane and methanol in this method:1.
Extraction conditions described in above method step (1) is extraction 2-3 times, extracts 5-15 minutes every time.
Silica gel particle diameter used in above method step (2) was 80-100 mesh sieves.The preferred diameter 1cm of described glass column,
Column length 10-15cm.
Sample 10-20mL n-hexane dissolutions, then upload to purification pillar, use again in addition in above method step (2)
30mL n-hexanes clean, and are then dried up silicagel column with nitrogen.The dosage of n-hexane used is 10-20ml, and grease is as non-
Polar substances, it is easy to washed off by n-hexane, and estrogen has certain polarity, is retained substantially by silica gel absorption.
Acetonitrile content used in above method step (3) is 2-5mL, can will remain estrogen acetonitrile on a silica gel column
Elute.
Comprising the concrete steps that for above method step (2) silica gel n-hexane activation will add n-hexane stirring pasty state in silica gel
Glass column is poured into again.
Because estradiol, ethinyloestradiol, oestrone, bisphenol-A etc. estrogen molecule structure are similar, there is similar fluorescent characteristic,
It can be analyzed with fluorescence detector, above method step (4) high performance liquid chromatography-fluorescence device uses when being analyzed
Chromatographic column is C18Reversed-phase column, mobile phase are first alcohol and water, and flow velocity 1mL/min, using gradient elution, methanol initial concentration is
30%, 50% was increased in 15 minutes and is kept for 10 minutes, then continued to rise to 100% in 10 minutes again, and keep 5
Minute.
The present invention is to remove oily matter therein using the main purpose of sample purification, to avoid to follow-up instrument point
Analysis has an impact, and is committed step.Purification is completed on purification pillar.It is preferably glass material to purify pillar, and blade diameter length ratio is best
For 1：10, there is piston solvent can be controlled to flow out below.Filler is 80-100 mesh silica gel, and silica gel adds n-hexane stirring pasty state
Glass column is poured into, is stood, treats that solid settles, pays attention to that bubble can not be left among silicagel column.Open following piston, discharge just oneself
Alkane, flush its liquid level and silicagel column.
Beneficial effects of the present invention compared with the prior art：
(1) methylene chloride/methanol mixed solvent effectively extracts the estrogen in cultivation solid waste, and the rate of recovery is 80%
More than.
(2) present invention can effectively remove a large amount of oil substances in sample using silica gel pillar.
(3) analyzed with HPLC/FL, with respect to HPLC/MS/MS, instrument is relatively cheap, universal, low to analysis personnel requirement.
Fluorescence detector selectivity is high, and antijamming capability is stronger than mass detector, high sensitivity, and a variety of estrogen separation are obvious, peak shape
Symmetrically, concentration-peak area linear relationship is good.
(4) this method to the rate of recovery of estrogen in cultivation solid waste more than 80%, to estradiol, ethinyloestradiol,
Oestrone, the test limit of bisphenol-A are respectively 3.2,1.8,5.4,2.8 μ g/kg, the inventive method specificity is strong, the degree of accuracy, precision
Degree, reappearance are preferable, are adapted to the content detection of estrogen in solid waste, are the easy-to-use methods being worthy to be popularized.
Brief description of the drawings
Fig. 1 is oestrone, estradiol, the chromatographic fractionation figure of ethinyloestradiol in chicken farm solid waste (being added with estrogen standard specimen).
Fig. 2 is the standard curve of estradiol, oestrone and ethinyloestradiol, and oestrone, estradiol, the retention time of ethinyloestradiol are respectively
22.3rd, 26.5 and 28.8min, 3 components reach baseline separation, have well linear in the range of 1 μ g/kg-1mg/kg
Relation.
The content of estradiol in the solid waste of Fig. 3 chicken farms.
Fig. 4 is the chromatogram that estrogen is detected in the waste of chicken farm.
Embodiment
The present invention is further described below by way of specific embodiment：
Embodiment 1
The detection of estrogen in the waste of chicken farm.
Sample is derived from Nanjing Suburb chicken farm.Often come out of steamer a collection of broiler chicken (cycle is 6 weeks) in the chicken farm, just clears up once
Cage includes the solid waste of excrement, fine hair, swill etc., and waste is concentrated and banks up storage.After some batches are accumulated,
Waste centrally disposes again.So, produced by the solid waste in chicken farm is actually some batch broiler chicken, its at the middle and upper levels be firm
Just stack fresh, and the time of lower floor is more remote.3 samples have been taken altogether, and 3,2, No. 1 are taken respectively from from surface 0.3,0.6
With the sample (calculating that pilling up time is respectively 12 weeks, 24 weeks and 36 weeks or so) of 0.9 meter of depths.The more sample of lower floor, during placement
Between it is more long.Analysis result is shown in Fig. 3, it is found that the estrogen in the solid waste of chicken farm is mainly estradiol, with the increase of depth,
The content of estrogen gradually reduces, and shows that estradiol is degraded.
5g will be respectively weighed after 3 sample dryings, made respectively with the methylene chloride/methanol mixed solution of 10mL volume ratios 2/1
Extracted 3 times for solvent supersonic, 10 minutes every time, combining extraction liquid, centrifugation, take supernatant, be placed in 40 DEG C of water-baths, promoted with nitrogen
Enter solvent volatilization, to remaining 1mL or so stopping.
Silica gel purification pillar is prepared, using the silica gel that 2g particle diameters are 100 mesh, is activated with n-hexane.1ml concentrating samples are used
Silica gel pillar is poured into after 10mL n-hexane dissolutions, then pillar is eluted with 30mL n-hexanes, is finally dried up with nitrogen.It is dried
Silicagel column is eluted with 2mL acetonitriles, is collected leacheate, is concentrated to 1mL.Sample is analyzed with Hitachi LC20TA types HPLC/FL.Color
Spectrum post is C18Reversed-phase column (250 × 4mm, 5 μm, Supelco), mobile phase are first alcohol and water, flow velocity 1mL/min, gradient elution, first
Alcohol initial concentration is 30%, and 50% was increased in 15 minutes and is kept for 10 minutes, then continued to raise in next 10 minutes
To 100%, and kept for 5 minutes.Detector excitation wavelength and launch wavelength are respectively 280nm and 310nm.
Testing result shows, ethinyloestradiol is not detected in the waste of chicken farm, detects micro oestrone, but containing a large amount of
Estradiol, see Fig. 4.Estradiol in 0.3 meter from surface in waste at concentrations up to 720 μ g/kg, but concentration is with sample depth
It is remarkably decreased, 335 and 201 μ g/kg is being dropped to from surface 0.6 and 0.9 meter of depths concentration.Because the discarded object of bottom banks up the time
It is longer, upper strata it is more fresh, this explanation estradiol there occurs natural decomposition.
Claims (7)
- A kind of 1. method for detecting estrogen in livestock waste, it is characterised in that described estrogen is estradiol, and ethinyloestradiol is female Ketone, bisphenol-A；This method comprises the following steps：(1) sample extraction：Plant solid waste 3-10g is taken, using volume ratio as 2:1-4:The mixing of 1 dichloromethane and methanol Solution is solvent, is extracted under ultrasonic wave added, combining extraction liquid, centrifugation, takes supernatant, blowing solvent with nitrogen is allowed to volatilize, directly To surplus solution 1-2mL；(2) sample purification：Silica gel 2-5g is taken, is activated with n-hexane, it is 1 to load blade diameter length ratio：In 10-15 glass column, stand, treat Solid sedimentation is complete, discharges n-hexane, flushes its liquid level and silicagel column, purification pillar is prepared；The sample n-hexane dissolution that step (1) is obtained, then purification pillar is uploaded to, cleaned with n-hexane, then use nitrogen Silicagel column is dried up；(3) sample elution：Eluted with acetonitrile, collect eluent, be concentrated into 1-2ml；(4) sample determines：Carry out quantitative analysis with high performance liquid chromatography-fluorescence device, the excitation wavelength of fluorescence detector and Launch wavelength is respectively 280nm and 310nm；High performance liquid chromatography-fluorescence device uses chromatographic column as C when being analyzed18Instead Xiang Zhu, mobile phase are first alcohol and water, and flow velocity 1mL/min, using gradient elution, methanol initial concentration is 30%, in 15 minutes It is increased to 50% and is kept for 10 minutes, then continued to rise to 100% in 10 minutes again, and kept for 5 minutes.
- 2. the method for estrogen in detection livestock waste according to claim 1, it is characterised in that in this method step (1) The product of the mixed solution body of dichloromethane and methanol is than being 2:1.
- 3. the method for estrogen in detection livestock waste according to claim 1, it is characterised in that in this method step (1) Described extraction conditions is extraction 2-3 times, extracts 5-15 minutes every time.
- 4. the method for estrogen in detection livestock waste according to claim 1, it is characterised in that the silicon used in step (2) Micelle footpath was 80-100 mesh sieves.
- 5. the method for estrogen in detection livestock waste according to claim 1, it is characterised in that the second used in step (3) Nitrile dosage is 2-5mL.
- 6. the method for estrogen in detection livestock waste according to claim 1, it is characterised in that step (2) silica gel is with just Hexane activation comprises the concrete steps that n-hexane stirring pasty state will be added in silica gel pours into glass column again.
- 7. the method for estrogen in detection livestock waste according to claim 1, it is characterised in that the glass in step (2) The blade diameter length ratio of post is 1：10.
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| CN102735768A (en) * | 2012-06-06 | 2012-10-17 | 北京师范大学 | Process for jointly detecting estrogens and their associations in livestock and poultry excrements |
| CN103364238A (en) * | 2012-04-10 | 2013-10-23 | 中国科学院大连化学物理研究所 | Selective separation of PCNs (polychlorinated naphthalenes congeners), HBCDss (hexabromocyclododecanes) and TBBPA (tetrabromobisphenol A) in complex samples |
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| US20090134325A1 (en) * | 2007-11-27 | 2009-05-28 | Goldman Mildred M | Methods for detecting estradiol by mass spectrometry |
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| CN103364238A (en) * | 2012-04-10 | 2013-10-23 | 中国科学院大连化学物理研究所 | Selective separation of PCNs (polychlorinated naphthalenes congeners), HBCDss (hexabromocyclododecanes) and TBBPA (tetrabromobisphenol A) in complex samples |
| CN102735768A (en) * | 2012-06-06 | 2012-10-17 | 北京师范大学 | Process for jointly detecting estrogens and their associations in livestock and poultry excrements |
| CN103901140A (en) * | 2014-04-28 | 2014-07-02 | 中国环境科学研究院 | Pretreatment method applied to analysis of tetrabromobisphenol A in biologically poisoned excrement |
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| Tso, Jerry;Dutta, Sudarshan;Inamdar, Shreeram.Simultaneous Analysis of Free and Conjugated Estrogens, Sulfonamides, and Tetracyclines in Runoff Water and Soils Using Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry.《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》.2011,第59卷(第6期), * |
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Effective date of registration: 20200415 Address after: Luyuan 214200 Yixing Road, Wuxi city of Jiangsu Province, No. 501 6C Co-patentee after: NANJING INSTITUTE OF ENVIRONMENTAL SCIENCES, MEE Patentee after: WUXI HUANCHUANG ENVIRONMENTAL TECHNOLOGY DEVELOPMENT Co.,Ltd. Co-patentee after: NANJING AGRICULTURAL University Address before: Luyuan 214200 Yixing Road, Wuxi city of Jiangsu Province, No. 501 6C Patentee before: WUXI HUANCHUANG ENVIRONMENTAL TECHNOLOGY DEVELOPMENT Co.,Ltd. |