CN103048398A - Method for determining microcystin MC-LR in water body - Google Patents
Method for determining microcystin MC-LR in water body Download PDFInfo
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Abstract
本发明公开了一种水体中微囊藻毒素MC-LR的测定方法，属于环保技术领域。所述的水体中微囊藻毒素MC-LR的测定方法即利用GF/C玻璃纤维滤纸过滤待测水样中的藻细胞，用C18硅胶为填料的固相萃取柱SPE柱分离纯化过滤后的待测水样，并用高效液相色谱进行检测，然后通过预先获得的不同浓度的微囊藻毒素MC-LR标准品所对应的出峰面积的标准曲线求得待测水样中微囊藻毒素MC-LR的含量。本发明的一种水体中微囊藻毒素MC-LR的测定方法具有步骤简单、操作方便、洗脱液的用量及对环境的毒性都有所降低，对操作人员更安全，检测成本低等优点，最低检测限为0.002mg/L。
The invention discloses a method for measuring microcystin MC-LR in water, belonging to the technical field of environmental protection. The determination method of microcystin MC-LR in the water body is to use GF/C glass fiber filter paper to filter the algae cells in the water sample to be tested, and use C18 silica gel as a filler for solid phase extraction column SPE column to separate and purify the algal cells The water sample to be tested is detected by high-performance liquid chromatography, and then the microcystin in the water sample to be tested is obtained by the standard curve of the peak area corresponding to the microcystin MC-LR standard of different concentrations obtained in advance. MC-LR content. The method for measuring microcystin MC-LR in water of the present invention has the advantages of simple steps, convenient operation, reduced eluent consumption and environmental toxicity, safer for operators, and low detection cost. , the lowest detection limit is 0.002mg/L.
Description
Technical field
The present invention relates to the assay method of a kind of Microcystins in Water MC-LR, belong to environmental technology field.
Background technology
Along with the large-scale outburst that continues of global fresh water bloom, the secondary metabolite Microcystin that discharges in the blue-green algae death process has become the natural Toxic that a class extensively exists in the water body.Microcystin is seven peptide monocyclic compounds, up to the present, has found 80 kinds of different Microcystin hypotypes.Wherein take variable amino acid as leucine the microcapsule phycotoxin MC-LR of (L) and arginine (R) for the most common, toxicity is the strongest, at present known toxicity is only second to dioxin.The environmental behaviours such as home to return to of the distribution of microcapsule phycotoxin MC-LR in environment, migration, accumulation have been subject to efforts at environmental protection person's special concern.
Can be divided into biological toxicology method, chemical analysis, immunodetection to the detection method of Microcystins in Water MC-LR at present.
1, biological test method: take mouse filled with and feed or lumbar injection is identified the toxicity of algae toxin.Its advantage is easy to be directly perceived, can judge comparatively roughly whether extract is toxic.Have simple to operate, visual result, the advantage such as quick, but need to consume more toxin, sensitivity and selectivity are not high, can't accurate quantitative analysis, can not distinguish the isomeride type of toxin.And the standing charges of mouse are high, workload is large.
2, immunodetection: utilize toxin to bring out immune response and produce antibody, utilize antibody that the specific recognition of antigen is come various toxin are detected.Mainly comprise enzyme-linked immunosorbent assay, phosphoprotein phosphatase inhibition analysis method etc.Immunodetection is simple, analysis speed is fast, sensitivity is higher, but can not play good discriminating effect by contratoxin, false positive reaction occurs sometimes.
3, chemical analysis: comprise high performance liquid chromatography (HPLC), thin-layer chromatography (TLC) high performance liquid chromatography and mass spectrum (HPLC-MS), gas chromatography and the methods such as mass spectrum (GC-MS) coupling, Capillary Electrophoresis (CE).
GC analytic approach in the chemical analysis is quick, accurate, sensitive, trace, but can only measure the total amount of algae toxin, and with high content of technology, complicated operation, and instrument and equipment is expensive, can not generally adopt.
TLC method degree of separation is limited, and is also difficult to its practical application.The CE method is short detection time, and degree of separation is higher, and sample size is little, but will be inferior to HPLC aspect the sensitivity of monitoring.
The HPLC method accurately, sensitive, favorable reproducibility, and can analyze simultaneously different algae toxin isomeride, operate relatively simple, instrument and equipment moderate cost, but need to be by algae toxin standard model in qualification process.Comparatively speaking, HPLC is a kind of analysis means of commonly using.
Because biological test and immune detection all need biological specimen, and have above-mentioned uncertainty in the mensuration process, therefore in environmental monitoring, use not general.
The HPLC method is fast because of its analysis speed, method favorable reproducibility and use more general.But in sample pretreatment, still there is following problem: adopt resin as enriching column during (1) sample pretreatment, volume is larger, and elution step is many, consumes organic solvent many, and organic solvent relates to the relatively large solvents of toxicity such as phenixin, acetone, and is larger to operating personnel's relative risk; (2) the C18 silicagel column that adopts conventional specification is to the example enrichment purifying, and because its size is larger, required filler and solvent are more, exist waste solvent and filler to utilize inadequate phenomenon, and the filler recovery utilization factor is lower, and is disposable often, has greater environmental impacts.
Summary of the invention
The objective of the invention is to provide for technical matterss such as existing consumption of organic solvent are large in the assay method that solves above-mentioned Microcystins in Water MC-LR, toxicity is large, step is various a kind of simple, fast, accurately, the assay method of the Microcystins in Water MC-LR of environmental protection.
Technical scheme of the present invention
The assay method of a kind of Microcystins in Water MC-LR, namely utilize the GF/C glass fiber filter paper to filter frustule in the water sample to be measured, be water sample to be measured after the solid-phase extraction column SPE column separating purification of filler filters with C18 silica gel, and measure it with the high efficiency liquid phase Reversed-phase Chromatographic System and go out peak area, then obtain the content of microcapsule phycotoxin MC-LR in the water sample to be measured by the corresponding typical curve that goes out peak area of the microcapsule phycotoxin MC-LR standard items of the variable concentrations that obtains in advance.
The assay method of above-mentioned a kind of Microcystins in Water MC-LR specifically comprises the steps:
(1), the pre-service of water sample
Be that the GF/C glass fiber filter paper of 1.2 μ m is filtered and removed other suspended particulate substances in frustule and the water sample with 5-10mL water sample to be measured through specification, the water sample to be measured of collecting after filtering is stand-by;
(2), the extraction of the activation of solid-phase extraction column SPE post and microcapsule phycotoxin MC-LR
Filler in the solid-phase extraction column SPE post is C18 silica gel, and the filler of each sample column is 500 mg, and solid-phase extraction column SPE post activates with 5mL methyl alcohol first, and regulates with the Milli-Q water of 10mL;
The specification SPE post of described solid-phase extraction column SPE post is solid phase extraction column, column jecket capacity 6mL;
With the water sample to be measured after the filtration of step (1), with the SPE column extracting after the above-mentioned activation, water sample to be measured after the filtration advances the SPE extraction column with the speed of 10mL/min, keep separator, other chaff interferences pass through adsorbent, the separator of gained is with the washed with methanol of 5mL20% 1 time, and the chaff interference that makes previous reservation optionally drip washing falls;
Separator after 20% washed with methanol is with the pure methanol-eluted fractions of 3mL, and eluent is MC-LR extract in the water sample to be measured, and the control temperature is 45 ℃ blows to doing with nitrogen, is dissolved in 200 μ L Chromatographic Pure Methanols, in-20 ℃ of preservations;
(3), the drafting of microcapsule phycotoxin MC-LR typical curve
Get the MC-LR standard model, be made into the MC-LR standard items methanol solution of variable concentrations with chromatogram alcohol methyl alcohol, measure appearance time corresponding to its variable concentrations MC-LR standard items methanol solution at the peak area of 6min with the HPLC Reversed-phase Chromatographic System, then take concentration as horizontal ordinate, take peak area as ordinate, draw that the microcapsule phycotoxin MC-LR standard items of variable concentrations are corresponding to go out peak area, obtain the corresponding typical curve that goes out peak area of the microcapsule phycotoxin MC-LR standard items of variable concentrations;
(4), the high-efficient liquid phase chromatogram HPLC of MC-LR is measured in the water sample
Get in the water sample to be measured that step (2) handles well the MC-LR extract with the Chromatographic Pure Methanol constant volume to 1mL, obtain MC-LR extract methanol solution in the water sample to be measured, measure with the HPLC Reversed-phase Chromatographic System, then obtain its corresponding MC-LR concentration value according to the typical curve of step (3) gained, be the MC-LR concentration value of the concentrated 5-10 of water sample to be measured after doubly;
Be measured value should be actual value in the water sample to be measured 5-10 doubly because sampling 5-10mL, after filtration, extraction concentrates, dries up, finally be settled to 1mL and detect.
The assay method of above-mentioned a kind of Microcystins in Water MC-LR, lowest detection is limited to 0.002mg/L.
Technique effect of the present invention
The assay method of a kind of microcapsule phycotoxin MC-LR of the present invention, owing to adopting the SPE pillar of 6mL specification that water sample is carried out pre-service, required water sample to be measured only is 5-10mL, and employing methyl alcohol is the drip washing solvent, so that comparing all with classic method, the consumption of eluent and toxicity decreases, safer to operating personnel.Further, because the minimizing of required water sample amount to be measured, thereby relative minimizing discharging of waste liquid amount, filler is fully utilized, and is more friendly to environment, and testing cost is low.
Further, the assay method of a kind of microcapsule phycotoxin MC-LR of the present invention, owing to adopting SPE pillar-HPLC coupling technique, it is the analytical approach of collection sample pretreatment and analyzing and testing one, therefore, have that step is simple, easy to operate, analysis speed is fast, solvent consumption is few, the method favorable reproducibility, be easy to the advantages such as robotization.
Further, the assay method of a kind of microcapsule phycotoxin MC-LR of the present invention, in the operating process, water sample to be measured at first can filter removal with suspended particulate substance and frustule after the GF/C glass fiber filter paper is filtered, after advancing C18 silica gel and being the SPE post of filler, can see obviously that light yellow material is adsorbed on the filler, wherein may comprise some pigments interferes things, after with 20% washed with methanol, and with pure methyl alcohol desorption, the concentrated laggard HPLC Reversed-phase Chromatographic System of testing sample is detected evaluation, peak shape is very clear, and on detectability, can well detect the content of MC-LR, lowest detection is limited to 0.002mg/L.
Description of drawings
The corresponding canonical plotting that goes out peak area of the microcapsule phycotoxin MC-LR standard items of Figure 1A, variable concentrations, wherein 0.002≤x≤0.2mg/L;
The corresponding canonical plotting that goes out peak area of the microcapsule phycotoxin MC-LR standard items of Figure 1B, variable concentrations, wherein x 〉=0.2mg/L;
Fig. 2, embodiment 1 contain the HPLC figure that takes microcapsule phycotoxin MC-LR assay in the tap water water sample out of that MC-LR is 1mg/L;
The HPLC of microcapsule phycotoxin MC-LR assay figure in Fig. 3, the embodiment 2 man-made lake Xiayang lake water water samples.
Embodiment
Also by reference to the accompanying drawings the present invention is further set forth below by specific embodiment, but do not limit the present invention.
Filler C18 silica gel in the used SPE post of the present invention, the fertile magnificent filter paper in Hangzhou company limited produces;
The used solid-phase extraction column SPE post of the present invention is solid phase extraction column, column jecket capacity 6mL, and the fertile magnificent filter paper in Hangzhou company limited produces;
The GF/C glass fiber filter paper of the 1.2 μ m that the present invention is used, the Shanghai fast science equipment company limited that rubs;
The MC-LR standard items molecular formula C that the present invention is used
49H
74N
10O
12, molecular weight 995.2, her Jim Press Science and Technology Ltd. produces HPLC purity 〉=95%;
The methyl alcohol that the present invention is used, chromatographic grade.
The assay method of a kind of Microcystins in Water MC-LR specifically comprises the steps:
(1), the pre-service of water sample
Take from the water water sample and add the MC-LR standard model and obtain tap water water sample to be measured, and make that the MC-LR theoretical value is 1mg/L in the tap water water sample to be measured.
Getting 5mL tap water water sample to be measured, is the GF/C glass fiber filter paper filtration of 1.2 μ m through specification, and the tap water water sample to be measured of collecting after filtering is stand-by;
(2), the extraction of the activation of solid-phase extraction column SPE post and microcapsule phycotoxin MC-LR
Filler in the SPE post is C18 silica gel, and the SPE filler of each sample column is 500mg, and the SPE post activates with 5mL methyl alcohol first, and regulates with the Milli-Q water of 10mL;
With the tap water water sample to be measured after the filtration of step (1), with the SPE column extracting after the above-mentioned activation, tap water water sample to be measured after the filtration advances the SPE extraction column with the speed of 10mL/min, keep separator, other chaff interferences pass through adsorbent, the separator of gained is with the washed with methanol of 5mL20% 1 time, and the chaff interference that makes previous reservation optionally drip washing falls;
Separator after the washed with methanol pure methyl alcohol desorption of 3mL, residue is MC-LR extract in the tap water water sample to be measured, and 45 ℃ of nitrogen blow to doing, and are dissolved in 200 μ L Chromatographic Pure Methanols, in-20 ℃ of preservations;
(3), the drafting of microcapsule phycotoxin MC-LR typical curve
Be respectively successively the MC-LR standard items methanol solution of 0.002mg/L, 0.01mg/L, 0.02mg/L, 0.05mg/L, 0.1mg/L, 0.2mg/L, 0.5mg/L, 1mg/L, 2mg/L, 5mg/L, 10mg/L with the Chromatographic Pure Methanol compound concentration, the MC-LR standard items methanol solution that adopts the HPLC Reversed-phase Chromatographic System to measure variable concentrations is the peak area that at 6min place at appearance time;
Measuring used Reversed-phase Chromatographic System model is LC-2000, and Japanese JASCO company produces, and used chromatographic column is the special post Hypersil ODS 25 μ m of Dalian Erie, and column size is 4.6 * 250 mm;
Condition determination: mobile phase is methyl alcohol: the volume ratio of water is 60/40, and to transfer pH with trifluoroacetic acid be 3.2, and flow velocity is 0.8mL/min, and column temperature remains on 25 ℃, and ultraviolet detects wavelength 238nm, and sample size is 10 μ L;
Take the concentration of MC-LR standard items methanol solution as horizontal ordinate, take peak area as ordinate, the drawing standard curve finally obtains typical curve such as Figure 1A, shown in Figure 1B, can draw from Figure 1A, Figure 1B:
Y=8.15x+0.8313, R
2=0.9266, wherein y is peak area, and x is MC-LR standard items methanol solutions, 0.002≤x≤0.2mg/L;
Y=0.1975x+2.434, R
2=9843, wherein y is peak area, and x is MC-LR standard items methanol solutions, x 〉=0.2mg/L;
(4), the mensuration of high-efficient liquid phase chromatogram HPLC
Get in the tap water water sample to be measured that step (2) handles well the MC-LR extract with the Chromatographic Pure Methanol constant volume to 1mL, obtain MC-LR extract methanol solution in the water sample to be measured, with measuring with the described HPLC Reversed-phase Chromatographic System of step (3), the same step of condition determination (3), measurement result as shown in Figure 2, from Fig. 2, obtain appearance time at the peak area of 6min, then obtaining MC-LR concentration according to the typical curve of step (3) gained is 4.6mg/L, because tap water water sample to be measured is sampled as 5mL in the testing process, after filtration, extraction is concentrated, dry up, finally being settled to 1mL detects, therefore the MC-LR concentration of practical measurement is 5 times of tap water water sample to be measured, and namely MC-LR concentration is 0.92mg/L in the actual tap water water sample to be measured.
Determine the concentration 0.92mg/L of the microcapsule phycotoxin MC-LR in the tap water water sample to be measured by the assay method of above-mentioned Microcystins in Water MC-LR, the actual addition in itself and the step (1) is that concentration is that 1mg/L differs 8%.The assay method that shows thus a kind of Microcystins in Water MC-LR of the present invention is reliable, accuracy is high.
The assay method of a kind of Microcystins in Water MC-LR specifically comprises the steps:
(1), the pre-service of water sample
Getting man-made lake Xiayang lake water water sample 5mL is the GF/C glass fiber filter paper filtration of 1.2 μ m through specification, and the man-made lake to be measured Xiayang lake water water sample of collecting after filtering is stand-by;
(2), solid-phase extraction column SPE(Solid-Phase Extraction) activation of post and the extraction of microcapsule phycotoxin MC-LR
Filler in the SPE post is C18 silica gel, and the filler of each sample column is 500mg, and the SPE post activates with 5mL methyl alcohol first, and regulates with the Milli-Q water of 10mL;
With the man-made lake to be measured Xiayang lake water water sample after the filtration of step (1), with the SPE column extracting after the above-mentioned activation, man-made lake Xiayang lake water water sample to be measured after the filtration advances the SPE extraction column with the speed of 10mL/min, keep separator, other chaff interferences pass through adsorbent, the separator of gained is with the washed with methanol of 5mL20% 1 time, and the chaff interference that makes previous reservation optionally drip washing falls;
Separator after the washed with methanol pure methyl alcohol desorption of 3mL, residue is MC-LR extract in the lake water water sample of man-made lake Xiayang, and 45 ℃ of nitrogen blow to doing, and are dissolved in 200 μ L Chromatographic Pure Methanols, in-20 ℃ of preservations;
(3), the drafting of microcapsule phycotoxin MC-LR typical curve
Step (3) with embodiment 1;
(4), the mensuration of high-efficient liquid phase chromatogram HPLC
Get in the man-made lake to be measured Xiayang lake water water sample that step (2) handles well the MC-LR extract with the Chromatographic Pure Methanol constant volume to 1mL, obtain MC-LR extract methanol solution in the lake water water sample of man-made lake to be measured Xiayang, measure with the HPLC Reversed-phase Chromatographic System, the same step of condition determination (3), the result as shown in Figure 3, from Fig. 3, obtain appearance time at the peak area of 6min, then the concentration of obtaining MC-LR according to the typical curve of step (3) gained is 2.3mg/L, because man-made lake Xiayang lake water water sample is sampled as 5mL in the testing process, after filtration, extraction is concentrated, dry up, finally being settled to 1mL detects, therefore the MC-LR concentration of practical measurement is 5 times of man-made lake to be measured Xiayang lake water water sample, and namely the concentration of MC-LR is 0.46mg/L in the actual man-made lake to be measured Xiayang lake water water sample.
Foregoing only is the basic explanation of the present invention under conceiving, and according to any equivalent transformation that technical scheme of the present invention is done, all should belong to protection scope of the present invention.
Claims (5)
1. the assay method of a Microcystins in Water MC-LR, it is characterized in that utilizing the GF/C glass fiber filter paper to filter frustule in the water sample to be measured, be water sample to be measured after the solid-phase extraction column SPE column separating purification of filler filters with C18 silica gel, and measure it with the high efficiency liquid phase Reversed-phase Chromatographic System and go out peak area, then obtain the content of microcapsule phycotoxin MC-LR in the water sample to be measured by the corresponding typical curve that goes out peak area of the microcapsule phycotoxin MC-LR standard items of the variable concentrations that obtains in advance.
2. the assay method of a kind of Microcystins in Water MC-LR as claimed in claim 1, the specification that it is characterized in that described GF/C glass fiber filter paper is 1.2 μ m.
3. the assay method of a kind of Microcystins in Water MC-LR as claimed in claim 1 is characterized in that described SPE post is solid phase extraction column, column jecket capacity 6mL.
4. such as the assay method of claim 1,2 or 3 described a kind of Microcystins in Water MC-LR, it is characterized in that specifically comprising the steps:
(1), the pre-service of water sample
5-10mL water sample to be measured is filtered through the GF/C glass fiber filter paper, and the water sample of collecting after filtering is stand-by;
(2), the extraction of the activation of solid-phase extraction column SPE post and microcapsule phycotoxin MC-LR
Solid-phase extraction column SPE column packing is C18 silica gel, and the filler of each sample column is 500mg, and solid-phase extraction column SPE post activates with 5mL methyl alcohol first, and regulates with the Milli-Q water of 10mL;
With the water sample to be measured after the filtration of step (1), with the SPE column extracting after the above-mentioned activation, the water sample to be measured after the filtration advances the SPE extraction column with the speed of 10mL/min, keeps separator, other chaff interferences pass through adsorbent, the washed with methanol of the separator usefulness 5mL20% of gained 1 time;
Separator after 20% washed with methanol is with the pure methanol-eluted fractions of 3mL, and eluent is MC-LR extract in the water sample to be measured, and the control temperature is 45 ℃ blows to doing with nitrogen, is dissolved in 200 μ L Chromatographic Pure Methanols, in-20 ℃ of preservations;
(3), the drafting of microcapsule phycotoxin MC-LR typical curve
Get the MC-LR standard model, be made into the MC-LR standard items methanol solution of variable concentrations with Chromatographic Pure Methanol, measure appearance time corresponding to its variable concentrations MC-LR standard items methanol solution at the peak area of 6min with the HPLC Reversed-phase Chromatographic System, then take concentration as horizontal ordinate, take peak area as ordinate, draw that the microcapsule phycotoxin MC-LR standard items of variable concentrations are corresponding to go out peak area, obtain the corresponding typical curve that goes out peak area of the microcapsule phycotoxin MC-LR standard items of variable concentrations;
(4), the high-efficient liquid phase chromatogram HPLC of MC-LR is measured in the water sample
Get in the water sample to be measured that step (2) handles well the MC-LR extract with the Chromatographic Pure Methanol constant volume to 1mL, obtain MC-LR extract methanol solution in the water sample to be measured, measure with the HPLC Reversed-phase Chromatographic System, then obtain its corresponding MC-LR concentration value according to the corresponding typical curve that goes out peak area of the microcapsule phycotoxin MC-LR standard items of the variable concentrations of step (3) gained, be the MC-LR concentration value of the concentrated 5-10 of water sample to be measured after doubly.
5. the assay method of a kind of Microcystins in Water MC-LR as claimed in claim 4, it is characterized in that described HPLC Reversed-phase Chromatographic System model is LC-2000, Japan JASCO company produces, used chromatographic column is the special post Hypersil ODS 25 μ m of Dalian Erie, and column size is 4.6 * 250mm;
Condition determination: mobile phase is methyl alcohol: the volume ratio of water is 60/40, and to transfer pH with trifluoroacetic acid be 3.2, and flow velocity is 0.8mL/min, and column temperature remains on 25 ℃, and ultraviolet detects wavelength 238nm, and sample size is 10 μ L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103543219A (en) * | 2013-09-17 | 2014-01-29 | 北京市水产科学研究所 | Method for extracting cyanobacteria toxin from urban eutrophic cyanobacterial bloom water |
| CN103940651A (en) * | 2014-04-17 | 2014-07-23 | 中国环境科学研究院 | Pretreatment method for measuring intracellular microcystin in natural water |
| CN109444306A (en) * | 2018-12-24 | 2019-03-08 | 赵文晋 | A kind of method that efficient liquid phase fluorescence chromatography detects microcapsule algae toxin in water |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1546991A (en) * | 2003-12-05 | 2004-11-17 | 浙江大学 | Determination method of trace microcystin in water |
| CN1687114A (en) * | 2005-03-24 | 2005-10-26 | 浙江大学医学院 | Method for purifying microcysin LR and RR synchronously |
| CN101285824A (en) * | 2008-04-22 | 2008-10-15 | 济南市供排水监测中心 | Blue algae polluted water microcystic toxins checking method |
| CN102435703A (en) * | 2011-11-25 | 2012-05-02 | 中国环境科学研究院 | Method for simultaneously detecting multiple microcystins in water |
-
2012
- 2012-12-05 CN CN2012105130869A patent/CN103048398A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1546991A (en) * | 2003-12-05 | 2004-11-17 | 浙江大学 | Determination method of trace microcystin in water |
| CN1687114A (en) * | 2005-03-24 | 2005-10-26 | 浙江大学医学院 | Method for purifying microcysin LR and RR synchronously |
| CN101285824A (en) * | 2008-04-22 | 2008-10-15 | 济南市供排水监测中心 | Blue algae polluted water microcystic toxins checking method |
| CN102435703A (en) * | 2011-11-25 | 2012-05-02 | 中国环境科学研究院 | Method for simultaneously detecting multiple microcystins in water |
Non-Patent Citations (7)
| Title |
|---|
| P.P. SHEN ET AL.: "Analysis of microcystins in cyanobacteria blooms and surface water samples from Meiliang Bay, Taihu Lake, China", 《ENVIRONMENT INTERNATIONAL》, vol. 29, no. 5, 31 August 2003 (2003-08-31) * |
| ROLAND W. MOOLLAN ET AL.: "Some comments on the determination of microcystin toxins in waters by high-performance liquid chromatography", 《ANALYST》, vol. 121, 29 February 1996 (1996-02-29) * |
| 乔素兰: "高效液相色谱法检测水中微囊藻毒素的实验条件优化", 《环境监测管理与技术》, vol. 19, no. 6, 31 December 2007 (2007-12-31) * |
| 庄一廷: "固相萃取-高效液相色谱法测定某水库型水源地微囊藻毒素", 《福建分析测试》, vol. 17, no. 3, 25 July 2008 (2008-07-25) * |
| 林瑶 等: "固相萃取-高效液相色谱法测定水中痕量微囊藻毒素", 《中国卫生检验杂志》, vol. 20, no. 9, 30 September 2010 (2010-09-30) * |
| 陈海燕 等: "高效液相色谱法同时测定水中三种微囊藻毒素", 《中国卫生检验杂志》, vol. 21, no. 2, 28 February 2011 (2011-02-28) * |
| 马颖: "固相萃取-高效液相色谱法检测饮用水中痕量微囊藻毒素", 《净水技术》, vol. 30, no. 6, 28 December 2011 (2011-12-28) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103543219A (en) * | 2013-09-17 | 2014-01-29 | 北京市水产科学研究所 | Method for extracting cyanobacteria toxin from urban eutrophic cyanobacterial bloom water |
| CN103940651A (en) * | 2014-04-17 | 2014-07-23 | 中国环境科学研究院 | Pretreatment method for measuring intracellular microcystin in natural water |
| CN109444306A (en) * | 2018-12-24 | 2019-03-08 | 赵文晋 | A kind of method that efficient liquid phase fluorescence chromatography detects microcapsule algae toxin in water |
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