JPH07294507A - Analysis of microcystines - Google Patents
Analysis of microcystinesInfo
- Publication number
- JPH07294507A JPH07294507A JP9126494A JP9126494A JPH07294507A JP H07294507 A JPH07294507 A JP H07294507A JP 9126494 A JP9126494 A JP 9126494A JP 9126494 A JP9126494 A JP 9126494A JP H07294507 A JPH07294507 A JP H07294507A
- Authority
- JP
- Japan
- Prior art keywords
- column
- microcystines
- liquid
- microcystins
- amino acids
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、アオコ（藍藻類が水中
で異常発生したもの）および湖沼水、河川水などに含ま
れる毒性成分のミクロシスチン類の分析法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for analyzing microcystins, which are toxic components contained in water-bloom (abnormal generation of cyanobacteria in water), lake water, river water and the like.
【０００２】[0002]
【従来の技術】富栄養化の進んだ湖沼では藍藻類が発生
し、水面にアオコと呼ばれる水の華を形成するが、この
中には毒素（例えばミクロシスチン類）を生産するもの
がある。湖沼の水は上水道、家畜の飲み水、養魚用とし
て利用されているため、毒素生産の有無を管理するのは
水質管理上重要になってくる。2. Description of the Related Art In a eutrophic lake, blue-green algae are generated to form a flower of water called a blue-green algae on the surface of the water. Since the water in lakes is used for water supply, drinking water for livestock, and for fish farming, it is important to control the presence or absence of toxin production in terms of water quality control.
【０００３】そのため、従来よりアオコ中に含まれる毒
素のミクロシスチン類の分析が試みられており、その一
般的な手法としては、ミクロシスチン類を液体クロマト
グラフ用分離カラムにより分離し、ミクロシスチン類そ
のものがもつＵＶ吸収を紫外分光光度検出器により検出
する方法が知られている。Therefore, it has been attempted to analyze microcystins, which are toxins contained in water-bloom, and the general method is to separate the microcystins by a separation column for liquid chromatograph. A method is known in which the UV absorption of itself is detected by an ultraviolet spectrophotometric detector.
【０００４】[0004]
【発明が解決しようとする課題】しかしながら、従来の
手法では、ミクロシスチン類が持つＵＶ吸収の極大波長
の約２４０ｎｍ域には、ミクロシスチン以外にも多くの
有機物がＵＶ吸収を持つため、検出の選択性に乏しく、
実試料を分析したとき他の夾雑成分による妨害を受けや
すかった。However, according to the conventional method, since many organic substances other than microcystin have UV absorption in the maximum wavelength range of about 240 nm of UV absorption of microcystins, the detection of Poor selectivity,
When real samples were analyzed, they were susceptible to interference by other contaminants.
【０００５】そこで、本発明は、ミクロシスチン類の選
択性に優れた新規な分析法を提供することを目的とす
る。[0005] Therefore, an object of the present invention is to provide a novel assay method having excellent selectivity for microcystins.
【０００６】[0006]
【課題を解決するための手段】本件発明者は、鋭意検討
した結果、液体クロマトグラフ用分離カラムにより分離
されたミクロシスチン類を加水分解し、アミノ酸に変化
させた後、該アミノ酸を蛍光物質に変換すれば、ミクロ
シスチン類を選択的に検出できることを見出だし、本発
明をなすに至った。Means for Solving the Problems The inventors of the present invention have conducted extensive studies and as a result, hydrolyzed microcystins separated by a separation column for a liquid chromatograph to convert them into amino acids, and then converted the amino acids into fluorescent substances. It was found that the microcystins can be selectively detected by conversion, and the present invention has been completed.
【０００７】すなわち、本発明は、アオコ中に含まれる
毒素のミクロシスチン類を液体クロマトグラフ用カラム
によって分離する工程と、該分離液を水酸化ナトリウム
水溶液と混合・加熱し、ミクロシスチン類をアミノ酸に
変換させる工程と、該アミノ酸を蛍光誘導体化試薬と反
応させて、発蛍光物質に誘導体化させる工程と、誘導体
化された発蛍光物質を検出する工程とからなるミクロシ
スチン類の分析法である。That is, according to the present invention, a step of separating microcystins, which are toxins contained in water-bloom, by a column for liquid chromatography, and mixing and heating the separated liquid with an aqueous sodium hydroxide solution, the microcystins are separated by Is a method for analyzing microcystins, which comprises a step of converting the amino acid into a fluorescent derivatizing reagent by reacting the amino acid with a fluorescent derivatizing reagent, and a step of detecting the derivatized fluorescent substance. .
【０００８】ここで、液体クロマトグラフ用カラムとし
ては、例えばオクタデシル基を感応基とするＯＤＳカラ
ムを用いることができるが、これには限定されず、ミク
ロシスチン類を分離できるものならば何でも良い。ま
た、移動相としては、例えばリン酸緩衝液、メタノール
を用いることができる。Here, as the liquid chromatographic column, for example, an ODS column having an octadecyl group as a sensitive group can be used, but the column is not limited to this and any column capable of separating microcystins may be used. As the mobile phase, for example, a phosphate buffer solution or methanol can be used.
【０００９】分離されたミクロシスチン類と混合させる
水酸化ナトリウム水溶液の濃度は、特に限定されない
が、０．１Ｍ前後の範囲が好ましい。また、加熱温度
は、ミクロシスチン類の加水分解という見地からは温度
をできる限り上げるのが好ましいが、１７０℃を越える
と移動相内で気泡が発生するので、１６０℃前後の範囲
が好ましい。The concentration of the aqueous sodium hydroxide solution to be mixed with the separated microcystins is not particularly limited, but is preferably in the range of about 0.1M. Further, the heating temperature is preferably as high as possible from the viewpoint of hydrolysis of microcystins, but when it exceeds 170 ° C., bubbles are generated in the mobile phase, so that the heating temperature is preferably around 160 ° C.
【００１０】蛍光誘導体化試薬としては、例えば、Ｏ−
フタルアルデヒド（ＯＰＡ）を用いることができ、ＯＰ
ＡはＮ−アセチル−Ｌ−システインとアルカリ性下で反
応させる。ＯＰＡの濃度は、通常のアミノ酸分析で使用
される範囲のものをそのまま用いることができる。Examples of fluorescent derivatization reagents include O-
Phthalaldehyde (OPA) can be used, OP
A is reacted with N-acetyl-L-cysteine under alkaline conditions. Regarding the concentration of OPA, the concentration within the range used in ordinary amino acid analysis can be used as it is.
【００１１】誘導体化された発蛍光物質は、公知の蛍光
検出器で検出される。The derivatized fluorescent substance is detected by a known fluorescence detector.
【００１２】[0012]
【作用】本発明では、液体クロマトグラフ用カラムから
の分離液中に含まれるミクロシスチン類は、水酸化ナト
リウム水溶液と混合・加熱することにより加水分解さ
れ、アミノ酸を生成する。生成したアミノ酸は蛍光誘導
体化試薬と反応して発蛍光物質に変化するので、蛍光検
出器で検出する。In the present invention, the microcystins contained in the separated liquid from the liquid chromatograph column are hydrolyzed by mixing and heating with an aqueous solution of sodium hydroxide to produce an amino acid. Since the produced amino acid reacts with the fluorescent derivatization reagent and changes into a fluorescent substance, it is detected by a fluorescence detector.
【００１３】[0013]
【実施例】本発明の方法を実施する装置の概略図を図１
に示す。図中、１は移動相溜、２は移動相送液ポンプ、
３は試料注入器、４は液体クロマトグラフ用カラム、５
は水酸化ナトリウム水溶液溜、６は水酸化ナトリウム水
溶液送液ポンプ、７はカラム４からの分離液と水酸化ナ
トリウム水溶液とを混合する混合コイル、８は混合コイ
ル７を収容するオーブン、９は冷却水槽、１０はＯＰＡ
試薬槽、１１はＯＰＡ試薬送液ポンプ、１２はＯＰＡ試
薬と分離液を反応させる反応コイル、１３は液体クロマ
トグラフ用カラム４と反応コイル１２を収容するカラム
オーブン、１４は蛍光検出器、１５はデータ処理器、１
６は廃液溜を各々示す。1 is a schematic diagram of an apparatus for carrying out the method of the present invention.
Shown in. In the figure, 1 is a mobile phase reservoir, 2 is a mobile phase liquid delivery pump,
3 is a sample injector, 4 is a liquid chromatograph column, 5
Is a sodium hydroxide aqueous solution reservoir, 6 is a sodium hydroxide aqueous solution feed pump, 7 is a mixing coil for mixing the separated liquid from the column 4 and the sodium hydroxide aqueous solution, 8 is an oven containing the mixing coil 7, and 9 is cooling Water tank, 10 is OPA
A reagent tank, 11 is an OPA reagent feed pump, 12 is a reaction coil for reacting the OPA reagent with the separation liquid, 13 is a column oven containing the liquid chromatography column 4 and the reaction coil 12, 14 is a fluorescence detector, and 15 is a fluorescence detector. Data processor, 1
6 shows the waste liquid reservoirs, respectively.
【００１４】かかる装置において、移動相送液ポンプ２
から供給された移動相は、試料注入器３を経由して液体
クロマトグラフ用カラム４に送られる。移動相の流れが
定常状態になった後、試料を試料注入器３より注入し、
注入された試料中のミクロシスチン類は、カラム４を通
過する過程で試料中の共存物質から分離される。In such a device, the mobile phase liquid feed pump 2
The mobile phase supplied from is sent to the liquid chromatograph column 4 via the sample injector 3. After the mobile phase flow has reached a steady state, a sample is injected from the sample injector 3,
Microcystins in the injected sample are separated from coexisting substances in the sample in the process of passing through the column 4.
【００１５】カラム４から出た分離液は、送液ポンプ６
から供給された水酸化ナトリウム水溶液と混合され、そ
の混合管はオーブン８で例えば１６０℃に加熱される。
この工程でミクロシスチン類は加水分解され、アミノ酸
へと変化する。その後、分離液は一旦冷却水槽９により
室温付近まで冷却される。The separated liquid discharged from the column 4 is fed by a liquid feed pump 6
Is mixed with the sodium hydroxide aqueous solution supplied from the above, and the mixing tube is heated in the oven 8 to 160 ° C., for example.
In this process, microcystins are hydrolyzed and converted into amino acids. Then, the separated liquid is once cooled to near room temperature by the cooling water tank 9.
【００１６】次の段階で、分離液は送液ポンプ１１から
供給されたＯＰＡ試薬と反応コイル１２で反応させら
れ、その反応コイル１２はカラムオーブン１３内で４５
℃に温調される。この工程で、すでにアミノ酸に変化し
たミクロシスチン類はＯＰＡ試薬と反応して発蛍光物質
に変化する。In the next step, the separated liquid is reacted with the OPA reagent supplied from the liquid feed pump 11 in the reaction coil 12, and the reaction coil 12 in the column oven 13
The temperature is adjusted to ℃. In this step, the microcystins that have already been converted to amino acids react with the OPA reagent and change to fluorescent substances.
【００１７】最後の段階で、発蛍光物質に変化した分離
液は蛍光検出器１４に導かれ、検出される。このとき検
出された蛍光強度は電気信号に変換されてデータ処理器
１５へ送られ、クロマトグラムとして記録される。な
お、検出後の発蛍光物質は、廃液溜１６に捨てられる。At the final stage, the separated liquid converted into the fluorescent substance is guided to the fluorescence detector 14 and detected. The fluorescence intensity detected at this time is converted into an electric signal, sent to the data processor 15, and recorded as a chromatogram. The fluorescent substance after detection is discarded in the waste liquid reservoir 16.
【００１８】以上の装置を用いて、次の分析条件により
アオコ中のミクロシスチン類を分析した。 ＜分析条件＞ カラム４ ：ＳＴＲ ＯＤＳ−II（４．６ｍｍＩ．Ｄ．
−１５０ｍｍｌ） 移 動 相：５０ｍＭりん酸（ナトリウム）緩衝溶液
（ｐＨ３．０）／メタノール＝９／１１ 移動相流量：０．８ｍｌ／ｍｉｎ オーブン４ ：４５℃ ＮａＯＨ ：０．１Ｍ ＮａＯＨ流量：０．２ｍｌ／ｍｉｎ 混合コイル７：０．８ｍｍＩ．Ｄ．−８ｍｍｌ オーブン８ ：１６０℃ ＯＰＡ試薬 ：Ａ液とＢ液の混合液 Ａ液：Ｎ−アセチル−Ｌ−システイン５００ｍｇを含む
０．０５Ｍほう酸（ナトリウム）緩衝溶液（pH 9.2）５
００ｍｌ Ｂ液：Ｏ−フタルアルデヒド４００ｍｇを含むエタノー
ル溶液７ｍｌ ＯＰＡ流量 ：０．２ｍｌ／ｍｉｎ 反応コイル１２：０．５ｍｍＩ．Ｄ．−１ｍｍｌ 検 出 器 ：蛍光検出器 Ｅｘ＝３４５ｎｍ，Ｅｍ＝
４５０ｎｍUsing the above apparatus, microcystins in water-bloom were analyzed under the following analytical conditions. <Analysis conditions> Column 4: STR ODS-II (4.6 mm ID.
-150 mml) Mobile phase: 50 mM phosphate (sodium) buffer solution (pH 3.0) / methanol = 9/11 Mobile phase flow rate: 0.8 ml / min Oven 4: 45 ° C NaOH: 0.1 M NaOH flow rate: 0. 2 ml / min mixing coil 7: 0.8 mmI. D. -8 mL Oven 8: 160 ° C. OPA Reagent: Mixture of Solution A and Solution B Solution A: 0.05 M borate (sodium) buffer solution (pH 9.2) containing 500 mg of N-acetyl-L-cysteine 5
00 ml B solution: ethanol solution containing 400 mg of O-phthalaldehyde 7 ml OPA flow rate: 0.2 ml / min Reaction coil 12: 0.5 mm I.D. D. -1 mlm Detector: Fluorescence detector Ex = 345 nm, Em =
450 nm
【００１９】＜分析結果＞上述の条件で分析した結果を
図２に示す。図中、ＰＲがミクロシスチンＰＲ、ＹＲが
ミクロシスチンＹＲ、ＬＲがミクロシスチンＬＲを示
す。図より明らかなように、本発明によれば夾雑物質の
影響を受けずに、ミクロシスチン類を選択的に検出でき
ることがわかる。<Analysis Results> The results of analysis under the above conditions are shown in FIG. In the figure, PR indicates microcystin PR, YR indicates microcystin YR, and LR indicates microcystin LR. As is clear from the figure, according to the present invention, microcystins can be selectively detected without being affected by contaminants.
【００２０】[0020]
【発明の効果】本発明によれば、試料中に含まれるミク
ロシスチン類以外の有機物は、蛍光物質を生成しないの
で、ミクロシスチン類のみを選択的に高感度に検出でき
る。According to the present invention, since organic substances other than microcystins contained in a sample do not generate fluorescent substances, only microcystins can be selectively detected with high sensitivity.
【図１】本発明の方法を実施するための装置の概略図1 is a schematic diagram of an apparatus for performing the method of the present invention.
【図２】本発明の方法によりアオコ中のミクロシスチン
類を分析した分析結果を示す図FIG. 2 is a diagram showing the results of analysis of microcystins in water-bloom by the method of the present invention.
１：移動相溜 ３：試料注入器 ４：液体クロマトグラフ用カラム ５：水酸化ナトリウ
ム水溶液溜 ７：混合コイル ９：冷却水槽 １０：ＯＰＡ試薬槽 １４：蛍光検出器1: Mobile Phase Reservoir 3: Sample Injector 4: Liquid Chromatographic Column 5: Sodium Hydroxide Aqueous Solution Reservoir 7: Mixing Coil 9: Cooling Water Tank 10: OPA Reagent Tank 14: Fluorescence Detector
Claims (1)
スチン類を液体クロマトグラフ用カラムによって分離す
る工程と、該分離液を水酸化ナトリウム水溶液と混合・
加熱し、ミクロシスチン類をアミノ酸に変化させる工程
と、該アミノ酸を蛍光誘導体化試薬と反応させて、発蛍
光物質に誘導体化させる工程と、誘導体化された発蛍光
物質を検出する工程とからなるミクロシスチン類の分析
法。1. A step of separating microcystins, which are toxins contained in water-bloom, etc., by a column for liquid chromatography, and mixing the separated liquid with an aqueous sodium hydroxide solution.
The method comprises the steps of heating and converting microcystins into amino acids, reacting the amino acids with a fluorescent derivatization reagent to derivatize the fluorescent substance, and detecting the derivatized fluorescent substance. Analysis method for microcystins.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9126494A JPH07294507A (en) | 1994-04-28 | 1994-04-28 | Analysis of microcystines |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9126494A JPH07294507A (en) | 1994-04-28 | 1994-04-28 | Analysis of microcystines |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07294507A true JPH07294507A (en) | 1995-11-10 |
Family
ID=14021572
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9126494A Pending JPH07294507A (en) | 1994-04-28 | 1994-04-28 | Analysis of microcystines |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07294507A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003079365A (en) * | 2001-09-07 | 2003-03-18 | Kanegafuchi Chem Ind Co Ltd | Method for detecting environmentally harmful chemical substance in high sensitivity |
| KR100381718B1 (en) * | 2000-06-26 | 2003-04-26 | 한국생명공학연구원 | The colorimetric quantification of microcystin using protein phosphatase |
| CN103163001A (en) * | 2013-03-27 | 2013-06-19 | 云南省环境监测中心站 | Method for extracting and purifying microcystic toxins LR and RR by taking cyanobacterial bloom in Dian Lake as raw material |
| CN110186886A (en) * | 2019-05-29 | 2019-08-30 | 扬州大学 | The inversion method of Microcystins in Water MC-LR concentration |
| CN112525873A (en) * | 2020-11-12 | 2021-03-19 | 滨州医学院 | Fluorescent chemical sensor for detecting microcystin-LR and preparation method thereof |
-
1994
- 1994-04-28 JP JP9126494A patent/JPH07294507A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100381718B1 (en) * | 2000-06-26 | 2003-04-26 | 한국생명공학연구원 | The colorimetric quantification of microcystin using protein phosphatase |
| JP2003079365A (en) * | 2001-09-07 | 2003-03-18 | Kanegafuchi Chem Ind Co Ltd | Method for detecting environmentally harmful chemical substance in high sensitivity |
| CN103163001A (en) * | 2013-03-27 | 2013-06-19 | 云南省环境监测中心站 | Method for extracting and purifying microcystic toxins LR and RR by taking cyanobacterial bloom in Dian Lake as raw material |
| CN110186886A (en) * | 2019-05-29 | 2019-08-30 | 扬州大学 | The inversion method of Microcystins in Water MC-LR concentration |
| CN110186886B (en) * | 2019-05-29 | 2022-02-11 | 扬州大学 | Inversion method of microcystin MC-LR concentration in water body |
| CN112525873A (en) * | 2020-11-12 | 2021-03-19 | 滨州医学院 | Fluorescent chemical sensor for detecting microcystin-LR and preparation method thereof |
| CN112525873B (en) * | 2020-11-12 | 2023-03-24 | 滨州医学院 | Fluorescent chemical sensor for detecting microcystin-LR and preparation method thereof |
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