JP2004053531A

JP2004053531A - Quantification method of terminal amino acid type organophosphorus herbicide

Info

Publication number: JP2004053531A
Application number: JP2002214417A
Authority: JP
Inventors: Toshishige Suzuki; 鈴木　敏重; Pacheco Tanaka Alfred; アルフレド・パチェコ・タナカ; Yukiko Takahashi; 高橋　由紀子
Original assignee: National Institute of Advanced Industrial Science and Technology AIST
Current assignee: National Institute of Advanced Industrial Science and Technology AIST
Priority date: 2002-07-23
Filing date: 2002-07-23
Publication date: 2004-02-19
Anticipated expiration: 2022-07-23
Also published as: JP3656106B2

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of quantifying a terminal amino acid type organophosphorus herbicide in a sample highly sensitively, quickly, accurately by simple operation. <P>SOLUTION: The sample including the terminal amino acid type organophosphorus herbicide is added to an aqueous solution of a metal complex of a complexing fluorescence reagent having an anthracene structure part and a polyamine structure part, and an aqueous solution having weakly acidic or neutral pH (preferably pH5-7) is prepared, and fluorescence is generated by excitation light. The concentration of the terminal amino acid type organophosphorus herbicide in the sample is determined based on the increase degree of the fluorescence intensity, or determined by collating the fluorescence intensity with a calibration curve showing the relation between the terminal amino acid type organophosphorus herbicide concentration and the fluorescence intensity. <P>COPYRIGHT: (C)2004,JPO

Description

【０００１】
【発明の属する技術分野】
本発明は、末端アミノ酸型有機リン系除草剤を、高感度で迅速に定量する方法に関するものである。
【０００２】
【従来の技術】
末端アミノ酸型有機リン系除草剤は、水溶性で、比較的毒性が少なく、生物分解性がよいため、多用されている。自然界や農作物における末端アミノ酸型有機リン系除草剤の残留量をモニターするため、高感度で簡便な定量法が求められている。末端アミノ酸型有機リン系除草剤の定量法としては、揮発性化合物へ誘導体化した後、ＧＣやＧＣ−ＭＳを用いる方法が知られているが（Ｃ．Ｄ．Ｓｔａｌｉｋａｓ、Ｃ．Ｎ．Ｋｏｎｉｄａｒｉ、Ｊ．Ｃｈｒｏｍａｔｏｇｒａｐｈｙ　Ａ、９０７巻、１頁、２００２年）、この方法は煩雑な誘導体化反応と特殊な装置を必要とするし、また、イオンクロマトグラフィー法は、末端アミノ酸型有機リン系除草剤を成分分離し、定量する方法として研究されているが、通常用いられる電気伝導度による検出法では感度の点で十分満足しうるものではない。
【０００３】
比色法や蛍光法は高感度で簡便な定量法ではあるが、末端アミノ酸型有機リン系除草剤はそれ自身発色団を持たず、また発色や蛍光を与えるためのラベル化も困難である。モリン（２′，３，４′，５，７‐ペンタヒドロキシフラボン）のＡｌ（ＩＩＩ）錯体の示す蛍光の該除草剤による消光現象を利用する方法が知られているが（Ｍ．Ｌｏｖｄａｈｌ、Ｊ．Ｃｈｒｏｍａｔｏｇｒａｐｈｙ、６０２巻、１９７頁、１９９２年）、該除草剤の濃度増大に従って蛍光が減少するため、蛍光が微少になると測定誤差を生じやすくなるなど精度等の点で問題がある。
【０００４】
【発明が解決しようとする課題】
本発明は、このような事情の下、試料中の末端アミノ酸型有機リン系除草剤を、簡便な操作で、高感度かつ迅速に、しかも精度よく定量する方法を提供することを課題とする。
【０００５】
【課題を解決するための手段】
本発明者らは、試料中の末端アミノ酸型有機リン系除草剤の定量について鋭意研究を重ねた結果、水溶液中、錯形成性蛍光試薬、中でもＮ‐（９‐アントリルメチル）‐ポリアミンの示す蛍光強度が金属イオンとりわけＣｕ（ＩＩ）イオンでの錯体形成により減少すること、またこの形成された錯体に末端アミノ酸型有機リン系除草剤を加えると再び蛍光強度が増大することを見出し、これらの知見に基づいて本発明をなすに至った。
【０００６】
すなわち、本発明は、
（１）アントラセン構造部とポリアミン構造部とを有する錯形成性蛍光試薬の金属錯体の水溶液に、末端アミノ酸型有機リン系除草剤を含む試料を加え、かつｐＨを弱酸性ないし中性とした水溶液を調製したのち、励起光により蛍光を発生させ、該蛍光強度の増大度に基づいて該試料中の末端アミノ酸型有機リン系除草剤の濃度を求めることを特徴とする末端アミノ酸型有機リン系除草剤の定量方法、及び
（２）アントラセン構造部とポリアミン構造部とを有する錯形成性蛍光試薬の金属錯体の水溶液に、末端アミノ酸型有機リン系除草剤を含む試料を加え、かつｐＨを弱酸性ないし中性とした水溶液を調製したのち、励起光により蛍光を発生させ、該蛍光強度を、末端アミノ酸型有機リン系除草剤濃度と蛍光強度との関係を示す検量線と照合して、該試料中の末端アミノ酸型有機リン系除草剤の濃度を求めることを特徴とする末端アミノ酸型有機リン系除草剤の定量方法、
を提供するものである。
【０００７】
【発明の実施の形態】
本発明の好ましい態様としては、以下のとおりのものが挙げられる。
（３）前記のｐＨを弱酸性ないし中性とした水溶液が、ｐＨを５〜７とした水溶液である前記（１）又は（２）記載の方法。
（４）錯形成性蛍光試薬が、Ｎ‐（アントリルメチル）‐ポリアミンである前記（１）、（２）又は（３）記載の方法。
（５）Ｎ‐（アントリルメチル）‐ポリアミンがＮ‐（９‐アントリルメチル）‐２‐ピリジルメチルアミンである前記（４）記載の方法。
（６）前記金属錯体を形成する金属イオンが、銅（ＩＩ）イオン、ニッケル（ＩＩ）イオン及び水銀（ＩＩ）イオンの中から選ばれた少なくとも１種である前記（１）ないし（５）のいずれかに記載の方法。
（７）末端アミノ酸型有機リン系除草剤が、Ｎ‐（ホスホノメチル）グリシン、４‐〔ヒドロキシ（メチル）ホスフィノ〕‐ＤＬ‐ホモアラニン、ＤＬ‐ホモアラニン‐４‐イル（メチル）ホスフィン酸、Ｌ‐２‐アミノ‐４‐〔（ヒドロキシ）（メチル）＝ホスフィノイル〕ブチリル‐Ｌ‐アラニル‐Ｌ‐アラニン、４‐〔ヒドロキシ（メチル）ホスフィノ〕‐Ｌ‐ホモアラニル‐Ｌ‐アラニル‐Ｌ‐アラニン又はそれらの塩である前記（１）ないし（６）のいずれかに記載の方法。
【０００８】
本発明で用いる錯形成性蛍光試薬は、光エネルギーを受容するアントラセン構造部分と、金属イオンを捕捉するポリアミン構造部分とを有するものであって、ポリアミン構造部分については、金属イオン、好ましくはＣｕ（ＩＩ）イオン、Ｎｉ（ＩＩ）イオン及びＨｇ（ＩＩ）イオンの中から選ばれた少なくとも１種、中でもＣｕ（ＩＩ）イオンと安定な錯体を形成するものであれば特に制限されないが、エチレンジアミン、ジエチレントリアミン、２‐アミノメチルピリジン構造のものが好ましい。このような錯形成性蛍光試薬としては、例えばアントラセン構造部分におけるアントラセン環を形成する炭素原子と、ポリアミン構造部分における窒素原子とが直接結合しているかあるいはアルキレン基を介して結合しているものなどが挙げられ、このようなものとしては、例えばＮ‐（アントリルメチル）‐ポリアミン、Ｎ‐（アントリルメチル）‐アミノアルコール、Ｎ‐（アントリルメチル）‐アザサイクリックアミンなどが挙げられ、中でもＮ‐（９‐アントリルメチル）‐ポリアミン、例えばＮ‐（９‐アントリルメチル）‐２‐ピリジルメチルアミン、Ｎ‐（９‐アントリルメチル）‐ジエチレントリアミンが好ましい。
【０００９】
これらの錯形成性蛍光試薬は、水溶液中、ｐＨが弱酸性ないし中性、好ましくは５〜７で強い蛍光を示すが、金属イオン、とりわけＣｕ（ＩＩ）、Ｎｉ（ＩＩ）、Ｈｇ（ＩＩ）イオンと錯体を形成すると消光が起こる。この錯体水溶液に、末端アミノ酸型有機リン系除草剤を加えると蛍光が回復し、この除草剤の添加量に比例して蛍光強度は増大する。このような現象は、末端アミノ酸型有機リン系除草剤が、Ｃｕ（ＩＩ）イオンなどの金属イオンと結合することで錯体から該金属イオンを奪い取り、その結果除草剤の添加量に応じて錯形成性蛍光試薬の遊離が進行し、再び蛍光を発するようになるという特有の挙動に基づくものである。
【００１０】
末端アミノ酸型有機リン系除草剤は、有機リン構造部とアミノ酸末端構造部とを有する除草剤であればよく、アミノ酸末端構造部にはペプチド構造をもつものも包含され、このようなものの例としては、Ｎ‐（ホスホノメチル）グリシン、４−〔ヒドロキシ（メチル）ホスフィノ〕‐ＤＬ‐ホモアラニン、ＤＬ‐ホモアラニン‐４‐イル（メチル）ホスフィン酸、Ｌ‐２‐アミノ‐４‐〔（ヒドロキシ）（メチル）＝ホスフィノイル〕ブチリル‐Ｌ‐アラニル‐Ｌ‐アラニン、４‐〔ヒドロキシ（メチル）ホスフィノ〕‐Ｌ‐ホモアラニル‐Ｌ‐アラニル‐Ｌ‐アラニン又はそれらの塩（例えばアルカリ金属塩、アンモニウム塩、イソプロピルアミン塩やエタノールアミン塩等の有機アミン塩など）が挙げられ、市販品としてはグリホセート、ビアラホス（Ｌ‐２‐アミノ‐４‐〔（ヒドロキシ）（メチル）＝ホスフィノイル〕ブチリル‐Ｌ‐アラニル‐Ｌ‐アラニンナトリウム塩）、及びグルホシネート（アンモニウム‐ＤＬ‐ホモアラニン‐４‐イル（メチル）ホスフィネート）などが挙げられる。
次に、この蛍光発現反応の一例のスキームを示す。
【００１１】
【化１】

【００１２】
この反応スキームに示されるように、蛍光の消光された、Ｎ‐（９‐アントリルメチル）‐２‐ピリジルメチルアミンのＣｕ（ＩＩ）錯体水溶液に対し、Ｎ‐（ホスホノメチル）グリシンが加えられると、これがＣｕ（ＩＩ）イオンと結合するように作用し、錯形成性蛍光試薬のＮ‐（９‐アントリルメチル）‐２‐ピリジルメチルアミンが遊離され、それにより蛍光が発現される。
【００１３】
定量に際して用いられる錯形成性蛍光試薬の濃度は、５×１０^−５〜１０^−７Ｍ（Ｍ＝モル／リットル）の範囲で用いられる。また、Ｃｕ（ＩＩ）イオンなどの金属イオンは、錯形成性蛍光試薬の半当量になるように水溶液として添加される。生成した金属錯体の水溶液に、末端アミノ酸型有機リン系除草剤の試料水溶液を加え、ｐＨを調整した後にその蛍光強度を測定する。ｐＨ条件は弱酸性ないし中性、好ましくはｐＨ５〜７、中でもｐＨ６〜７の範囲であるのが蛍光変化がシャープとなるので好ましい。ｐＨの調整にあたっては、ＰＩＰＥＳ〔ピペラジン‐１，４‐ビス（２‐エタンスルホン酸）〕緩衝液が好ましく用いられる。
【００１４】
蛍光を発生させるための励起光の波長は、３５０〜４３０ｎｍの範囲で選ばれるが、３６８ｎｍとするのが蛍光強度を最大化しうるので好ましい。
【００１５】
本発明方法では、前記したように、蛍光試薬の金属錯体を含む水溶液に、末端アミノ酸型有機リン系除草剤を含む試料を加え、かつｐＨを弱酸性ないし中性に調整したのち、励起光により蛍光を発生させ、該蛍光強度の増大度に基づいて該試料中の末端アミノ酸型有機リン系除草剤の濃度が求められる。
【００１６】
本発明方法は、測定系中にアニオンが比較的多量に混在してもそれによる影響はほとんどなく、例えば末端アミノ酸型有機リン系除草剤の濃度が０．１８ｐｐｍ（１×１０^−５Ｍ）程度の場合、これに対し、塩化物イオン、硫酸イオン、リン酸イオン、硝酸イオンが１×１０^−３Ｍ程度共存しても妨害を受けずに末端アミノ酸型有機リン系除草剤を定量することができる。一方、Ｆｅ（ＩＩＩ）、Ａｌ（ＩＩＩ）、Ｃｕ（ＩＩ）、Ｚｎ（ＩＩ）、Ｎｉ（ＩＩ）、Ｃａ（ＩＩ）等の金属イオンの共存は、末端アミノ酸型有機リン系除草剤の定量を妨害するが、キレート樹脂により金属イオンをあらかじめ除去することにより防ぐことができる。この際に用いられるキレート樹脂としては、イミノ二酢酸、ＥＤＴＡ、ジエチレントリアミン‐Ｎ，Ｎ，Ｎ′，Ｎ′‐テトラ酢酸等のアミノポリカルボン酸の構造をもつ金属捕捉基を有するものが好ましい。
【００１７】
【発明の効果】
本発明方法によれば、水中の微量末端アミノ酸型有機リン系除草剤を室温で迅速に蛍光光度定量することができる。本発明方法は、蛍光強度の変化がシャープであり、また可視部に蛍光を持つため、ハンディＵＶランプ等の紫外線灯を用いた目視による判定が可能であること、試薬ならびにその金属錯体が水溶性であることから、末端アミノ酸型有機リン系除草剤を含む、河川水、湖沼水、地下水などの環境試料中や、農作物中の末端アミノ酸型有機リン系除草剤のモニターやイオンクロマト分離後の迅速定量に対し、きわめて実用価値が高いと言える。
【００１８】
【実施例】
次に実施例によって本発明をさらに詳細に説明するが、本発明はこれらの例によって何ら限定されるものではない。
なお、Ｍはモル／リットル濃度を示す。
【００１９】
実施例１
５０ｍｌメスフラスコに９×１０^−５ＭのＮ‐（９‐アントリルメチル）‐２‐ピリジルメチルアミン水溶液を５ｍｌ、４．５×１０^−５Ｍの銅（ＩＩ）イオンを供与する硫酸銅水溶液を５ｍｌ、１ＭのＰＩＰＥＳ緩衝液１ｍｌを加えたものを用意し、これに直接純水を加えて全量を５０ｍｌとしたコントラスト試料と、また、これにＮ‐（ホスホノメチル）グリシンを含む検液を後述の各所定量加え、さらに純水を加えて全量を５０ｍｌとした各実施試料とを調製した。これら試料を２、３分室温で放置した後に、蛍光光度計にて励起波長３６８ｎｍとした際の蛍光スペクトルを得た。添加したＮ‐（ホスホノメチル）グリシンの濃度が高くなるに従い、蛍光波長４１９ｎｍにおける蛍光強度が増大することが認められた。蛍光スペクトルの変化を図１に示す。図１において、横軸は励起波長３６８ｎｍの際の蛍光波長を、縦軸は相対蛍光強度を表す。Ｎ‐（９‐アントリルメチル）‐２‐ピリジルメチルアミンおよびＣｕ（ＩＩ）イオンの濃度はそれぞれ９．０×１０^−６Ｍ、４．５×１０^−６Ｍ、反応溶液のｐＨは６．８である。スペクトルは下から上へ順に、コントラスト試料、Ｎ‐（ホスホノメチル）グリシン濃度（ｐｐｂ）で、４３、８５、１２８、１７０、２１２、２５４、２９５、３３６、３７８の検液をそれぞれ添加した各実施試料についてのものである。
【００２０】
実施例２
実施例１における図１の各最大ピークの蛍光強度とＮ‐（ホスホノメチル）グリシン濃度の対数値とを関数として図２に示す検量線を作成した。全量２５ｍｌの測定検液に対して、１７０ｐｐｂ〜１７ｐｐｍの範囲のＮ‐（ホスホノメチル）グリシンの定量が可能であることが分る。図２の検量線において、横軸はＮ‐（ホスホノメチル）グリシン濃度（×１０^−５Ｍ）の対数値、縦軸は相対蛍光強度を表わす。測定における励起波長は３６８ｎｍであり、蛍光検出波長は４１９ｎｍである。Ｎ‐（９‐アントリルメチル）‐２‐ピリジルメチルアミンおよびＣｕ（ＩＩ）イオンの濃度はそれぞれ２×１０^−５Ｍ、１×１０^−５Ｍ、反応溶液のｐＨは６．８である。
【００２１】
実施例３
Ｎ‐（９‐アントリルメチル）‐２‐ピリジルメチルアミンの蛍光強度とｐＨの関係をＣｕ（ＩＩ）イオンの存在下、非存在下で調べた。３×１０^−６ＭのＮ‐（９‐アントリルメチル）‐２‐ピリジルメチルアミン水溶液１００μｌ、１．５×１０^−６Ｍの硫酸銅水溶液１００μｌを加え、ｐＨを調整後に、イオン交換水を用いて全量２５ｍｌとした。各々の溶液の蛍光光度及びｐＨを測定した。４１９ｎｍの蛍光の強度とｐＨの関係を図３にグラフで示す。図３において、横軸は溶液のｐＨ、縦軸は相対蛍光強度を表す。●はＣｕ（ＩＩ）イオン非存在下での、また、○はＣｕ（ＩＩ）イオン存在下での蛍光強度変化を、△はその蛍光強度の差をそれぞれ示す。
【００２２】
実施例４
Ｎ‐（ホスホノメチル）グリシン（１×１０^−５Ｍ）に対しそれぞれ２０倍当量の銅、亜鉛、ニッケル及び鉄を含む、ｐＨ６．８に調整した全量５０ｍｌの水溶液からなる模擬検液を、ジエチレントリアミン‐Ｎ，Ｎ，Ｎ′，Ｎ′‐テトラ酢酸構造をもつ金属捕捉基を有するキレート樹脂２ｇを充填したカラムに通液する。通液後の溶液とカラム洗液をあわせ、さらに水を加えて全量を１００ｍｌとする。この中から５ｍｌを取り、これにＮ‐（９‐アントリルメチル）‐２‐ピリジルメチルアミン（１×１０^−４Ｍ）０．９ｍｌ、銅（ＩＩ）イオン（１×１０^−４Ｍ）０．４５ｍｌ、ＰＩＰＥＳ緩衝液（０．１Ｍ、ｐＨ６．８）１ｍｌを加え、さらに水で全量を１０ｍｌとし、４１９ｎｍの蛍光の強度を３回測定し、得られた蛍光強度を各検量線にあてはめ、模擬検液中のＮ‐（ホスホノメチル）グリシンの濃度を求めた。その結果を、３回の測定の平均値ならびに真値とともに表１に示す。
【００２３】
【表１】

【図面の簡単な説明】
【図１】実施例１において、Ｎ‐（ホスホノメチル）グリシンの添加濃度による蛍光スペクトルの変化を示すグラフ。
【図２】実施例２において作成した検量線を示すグラフ。
【図３】実施例３における蛍光強度とｐＨの関係を示すグラフ。[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to a method for rapidly and quantifying a terminal amino acid type organophosphorus herbicide with high sensitivity.
[0002]
[Prior art]
Terminal amino acid type organophosphorus herbicides are widely used because they are water-soluble, have relatively low toxicity, and have good biodegradability. In order to monitor the residual amount of the terminal amino acid type organophosphorus herbicide in nature and crops, a highly sensitive and simple quantitative method is required. As a method for quantifying the terminal amino acid type organophosphorus herbicide, a method using GC or GC-MS after derivatization to a volatile compound is known (CD Stalikas, CN Konidari, J. Chromatography A, Vol. 907, p. 1, 2002), this method requires a complicated derivatization reaction and a special apparatus, and the ion chromatography method uses a terminal amino acid type organophosphorus herbicide. Although it has been studied as a method for separating and quantifying components, the detection method using electric conductivity that is usually used is not sufficiently satisfactory in terms of sensitivity.
[0003]
The colorimetric method and the fluorescent method are highly sensitive and simple quantitative methods, but the terminal amino acid type organophosphorus herbicide does not have a chromophore itself, and it is difficult to label it to give color and fluorescence. A method is known which utilizes the quenching phenomenon by the herbicide of the fluorescence exhibited by the Al (III) complex of morin (2 ', 3,4', 5,7-pentahydroxyflavone) (M. Lovdahl, J Chromatography, vol. 602, p. 197, 1992), the fluorescence decreases with an increase in the concentration of the herbicide.
[0004]
[Problems to be solved by the invention]
Under such circumstances, an object of the present invention is to provide a method for quantifying a terminal amino acid-type organophosphorus herbicide in a sample with a simple operation, with high sensitivity, promptly and accurately.
[0005]
[Means for Solving the Problems]
The present inventors have conducted intensive studies on the quantification of terminal amino acid-type organophosphorus herbicides in a sample. As a result, the present invention shows that complex-forming fluorescent reagents, especially N- (9-anthrylmethyl) -polyamine, are present in aqueous solution. They found that the fluorescence intensity was reduced by complex formation with metal ions, especially Cu (II) ions, and that the addition of a terminal amino acid type organophosphorus herbicide to the formed complex increased the fluorescence intensity again. The present invention has been made based on the findings.
[0006]
That is, the present invention
(1) An aqueous solution in which a sample containing a terminal amino acid-type organophosphorus herbicide is added to an aqueous solution of a metal complex of a complex-forming fluorescent reagent having an anthracene structure portion and a polyamine structure portion, and the pH is weakly acidic or neutral. And then generating fluorescence by excitation light, and determining the concentration of the terminal amino acid type organophosphorus herbicide in the sample based on the degree of increase in the fluorescence intensity. (2) adding a sample containing a terminal amino acid-type organophosphorus herbicide to an aqueous solution of a metal complex of a complexing fluorescent reagent having an anthracene structure part and a polyamine structure part, and weakening the pH After preparing a neutral or aqueous solution, fluorescence is generated by excitation light, and the fluorescence intensity is compared with a calibration curve showing the relationship between the concentration of the terminal amino acid type organophosphorus herbicide and the fluorescence intensity. To method of quantifying terminal amino acid type organophosphorus herbicides and obtains the concentration of terminal amino acid type organophosphorus herbicide in the sample,
Is provided.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
Preferred embodiments of the present invention include the following.
(3) The method according to (1) or (2), wherein the aqueous solution whose pH is weakly acidic to neutral is an aqueous solution whose pH is 5 to 7.
(4) The method according to (1), (2) or (3), wherein the complexing fluorescent reagent is N- (anthrylmethyl) -polyamine.
(5) The method according to the above (4), wherein the N- (anthrylmethyl) -polyamine is N- (9-anthrylmethyl) -2-pyridylmethylamine.
(6) The metal ion forming the metal complex is at least one selected from the group consisting of copper (II) ion, nickel (II) ion and mercury (II) ion. The method according to any of the above.
(7) The terminal amino acid type organophosphorus herbicide is N- (phosphonomethyl) glycine, 4- [hydroxy (methyl) phosphino] -DL-homoalanine, DL-homoalanin-4-yl (methyl) phosphinic acid, L-2 -Amino-4-[(hydroxy) (methyl) = phosphinoyl] butyryl-L-alanyl-L-alanine, 4- [hydroxy (methyl) phosphino] -L-homoalanyl-L-alanyl-L-alanine or salts thereof The method according to any one of the above (1) to (6), wherein
[0008]
The complexing fluorescent reagent used in the present invention has an anthracene structure portion that receives light energy and a polyamine structure portion that captures a metal ion. For the polyamine structure portion, a metal ion, preferably Cu ( II) ions, Ni (II) ions and Hg (II) ions, and are not particularly limited as long as they form a stable complex with Cu (II) ions. Those having a 2,2-aminomethylpyridine structure are preferred. Examples of such complex-forming fluorescent reagents include those in which a carbon atom forming an anthracene ring in an anthracene structure portion and a nitrogen atom in a polyamine structure portion are directly bonded or bonded via an alkylene group. Such as N- (anthrylmethyl) -polyamine, N- (anthrylmethyl) -amino alcohol, N- (anthrylmethyl) -azacyclic amine, etc. Among them, N- (9-anthrylmethyl) -polyamine such as N- (9-anthrylmethyl) -2-pyridylmethylamine and N- (9-anthrylmethyl) -diethylenetriamine are preferred.
[0009]
These complexing fluorescent reagents show strong fluorescence at pH from weakly acidic to neutral, preferably from 5 to 7 in aqueous solution, but show metal ions, especially Cu (II), Ni (II) and Hg (II). Quenching occurs when a complex is formed with the ion. When an amino acid-terminated organophosphorus herbicide is added to the aqueous complex solution, the fluorescence recovers, and the fluorescence intensity increases in proportion to the amount of the herbicide added. Such a phenomenon occurs because the terminal amino acid-type organophosphorus herbicide binds to a metal ion such as Cu (II) ion to deprive the complex of the metal ion and, as a result, complex formation in accordance with the amount of the added herbicide. This is based on a specific behavior in which the release of the sexual fluorescent reagent proceeds and emits fluorescence again.
[0010]
The terminal amino acid type organophosphorus herbicide may be any herbicide having an organophosphorus structure portion and an amino acid terminal structure portion, and the amino acid terminal structure portion also includes those having a peptide structure. Represents N- (phosphonomethyl) glycine, 4- [hydroxy (methyl) phosphino] -DL-homoalanine, DL-homoalanin-4-yl (methyl) phosphinic acid, L-2-amino-4-[(hydroxy) (methyl ) = Phosphinoyl] butyryl-L-alanyl-L-alanine, 4- [hydroxy (methyl) phosphino] -L-homoalanyl-L-alanyl-L-alanine or salts thereof (eg alkali metal salts, ammonium salts, isopropylamine Salt and organic amine salts such as ethanolamine salt), and commercially available glyphosate Vialaphos (L-2-amino-4-[(hydroxy) (methyl) = phosphinoyl] butyryl-L-alanyl-L-alanine sodium salt) and glufosinate (ammonium-DL-homoalanine-4-yl (methyl) phosphinate) And the like.
Next, a scheme of an example of the fluorescence expression reaction is shown.
[0011]
Embedded image

[0012]
As shown in this reaction scheme, when N- (phosphonomethyl) glycine is added to a fluorescence-quenched aqueous solution of a Cu (II) complex of N- (9-anthrylmethyl) -2-pyridylmethylamine. This acts to bind to Cu (II) ions and liberates the complexing fluorescent reagent N- (9-anthrylmethyl) -2-pyridylmethylamine, thereby developing fluorescence.
[0013]
The concentration of the complexing fluorescent reagent used in the quantification is in the range of 5 × 10 ⁻⁵ to 10 ⁻⁷ M (M = mol / liter). Further, a metal ion such as Cu (II) ion is added as an aqueous solution so as to be a half equivalent of the complex-forming fluorescent reagent. A sample aqueous solution of a terminal amino acid-type organophosphorus herbicide is added to the generated aqueous solution of the metal complex, the pH is adjusted, and the fluorescence intensity is measured. The pH condition is preferably weakly acidic to neutral, preferably in the range of pH 5 to 7, and particularly preferably in the range of pH 6 to 7, since the fluorescence change becomes sharp. In adjusting the pH, a PIPES [piperazine-1,4-bis (2-ethanesulfonic acid)] buffer is preferably used.
[0014]
The wavelength of the excitation light for generating fluorescence is selected in the range of 350 to 430 nm, but is preferably 368 nm because the fluorescence intensity can be maximized.
[0015]
In the method of the present invention, as described above, a sample containing a terminal amino acid type organophosphorus herbicide is added to an aqueous solution containing a metal complex of a fluorescent reagent, and the pH is adjusted to be weakly acidic to neutral, and then the mixture is irradiated with excitation light. Fluorescence is generated, and the concentration of the terminal amino acid type organophosphorus herbicide in the sample is determined based on the degree of increase in the fluorescence intensity.
[0016]
According to the method of the present invention, even if a relatively large amount of anions is mixed in the measurement system, there is almost no effect due to the mixture. For example, the concentration of the terminal amino acid type organophosphorus herbicide is about 0.18 ppm (1 × 10 ⁻⁵ M). In contrast, in the case of the above, even if chloride ion, sulfate ion, phosphate ion, and nitrate ion coexist in an amount of about 1 × 10 ⁻³ M, the terminal amino acid type organophosphorus herbicide can be quantified without interference. it can. On the other hand, the coexistence of metal ions such as Fe (III), Al (III), Cu (II), Zn (II), Ni (II), and Ca (II) allows the quantitative determination of terminal amino acid type organophosphorus herbicides. The interference can be prevented by previously removing metal ions with a chelating resin. As the chelate resin used at this time, a resin having a metal capturing group having an aminopolycarboxylic acid structure such as iminodiacetic acid, EDTA, diethylenetriamine-N, N, N ', N'-tetraacetic acid is preferable.
[0017]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to the method of this invention, the amount of terminal amino acid type organophosphorus herbicides in water can be rapidly and fluorometrically determined at room temperature. The method of the present invention has a sharp change in fluorescence intensity and has fluorescence in the visible region, so that visual judgment using an ultraviolet lamp such as a handy UV lamp is possible, and the reagent and its metal complex are soluble in water. Therefore, monitoring of terminal amino acid-type organophosphorus herbicides in agricultural samples, such as river water, lake water, groundwater, etc., including terminal amino acid-type organophosphorus herbicides, and rapid separation after ion chromatography separation It can be said that the practical value is extremely high for quantitative determination.
[0018]
【Example】
Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
In addition, M shows a mol / liter concentration.
[0019]
Example 1
5 ml of 9 × 10 ⁻⁵ M aqueous solution of N- (9-anthrylmethyl) -2-pyridylmethylamine in a 50 ml volumetric flask, aqueous solution of copper sulfate donating 4.5 × 10 ⁻⁵ M copper (II) ions Was prepared by adding 1 ml of a 1 M PIPES buffer solution, and a pure sample was directly added with pure water to make the total volume 50 ml. A test solution containing N- (phosphonomethyl) glycine was also described later. , And each of the working samples was adjusted to a total volume of 50 ml by adding pure water. After allowing these samples to stand at room temperature for a few minutes, a fluorescence spectrum at an excitation wavelength of 368 nm was obtained with a fluorometer. It was recognized that the fluorescence intensity at a fluorescence wavelength of 419 nm increased as the concentration of the added N- (phosphonomethyl) glycine increased. FIG. 1 shows the change in the fluorescence spectrum. In FIG. 1, the horizontal axis represents the fluorescence wavelength at the excitation wavelength of 368 nm, and the vertical axis represents the relative fluorescence intensity. The concentrations of N- (9-anthrylmethyl) -2-pyridylmethylamine and Cu (II) ions are 9.0 × 10 ⁻⁶ M and 4.5 × 10 ⁻⁶ M, respectively, and the pH of the reaction solution is 6. 8 The spectra are, in order from bottom to top, a contrast sample, and each working sample to which N- (phosphonomethyl) glycine concentration (ppb) of 43, 85, 128, 170, 212, 254, 295, 336, 378 was added. Is about.
[0020]
Example 2
A calibration curve shown in FIG. 2 was prepared as a function of the fluorescence intensity of each maximum peak in FIG. 1 and the logarithmic value of N- (phosphonomethyl) glycine concentration in Example 1. It can be seen that the amount of N- (phosphonomethyl) glycine in the range of 170 ppb to 17 ppm can be determined for a total test solution of 25 ml. In the calibration curve of FIG. 2, the horizontal axis represents the logarithmic value of the N- (phosphonomethyl) glycine concentration (× 10 ⁻⁵ M), and the vertical axis represents the relative fluorescence intensity. The excitation wavelength in the measurement is 368 nm, and the fluorescence detection wavelength is 419 nm. The concentrations of N- (9-anthrylmethyl) -2-pyridylmethylamine and Cu (II) ions are 2 × 10 ⁻⁵ M and 1 × 10 ⁻⁵ M, respectively, and the pH of the reaction solution is 6.8.
[0021]
Example 3
The relationship between the fluorescence intensity and the pH of N- (9-anthrylmethyl) -2-pyridylmethylamine was examined in the presence and absence of Cu (II) ion. 100 μl of a 3 × 10 ⁻⁶ M aqueous solution of N- (9-anthrylmethyl) -2-pyridylmethylamine and 100 μl of a 1.5 × 10 ⁻⁶ M aqueous copper sulfate solution were added, and after adjusting the pH, ion-exchanged water was added. The total volume was 25 ml. The fluorescence intensity and pH of each solution were measured. FIG. 3 is a graph showing the relationship between the fluorescence intensity at 419 nm and the pH. In FIG. 3, the horizontal axis represents the pH of the solution, and the vertical axis represents the relative fluorescence intensity. ● indicates the change in fluorescence intensity in the absence of Cu (II) ions, ○ indicates the change in fluorescence intensity in the presence of Cu (II) ions, and △ indicates the difference in the fluorescence intensity.
[0022]
Example 4
A simulated test solution consisting of 50 ml of an aqueous solution adjusted to pH 6.8 and containing 20 equivalents of copper, zinc, nickel and iron with respect to N- (phosphonomethyl) glycine (1 × 10 ⁻⁵ M) was prepared by diethylenetriamine- The solution is passed through a column filled with 2 g of a chelating resin having a metal capturing group having an N, N, N ', N'-tetraacetic acid structure. The solution after the passage and the column washing solution are combined, and water is further added to make a total volume of 100 ml. 5 ml was taken from this, and 0.9 ml of N- (9-anthrylmethyl) -2-pyridylmethylamine (1 × 10 ⁻⁴ M) and copper (II) ion (1 × 10 ⁻⁴ M) were added thereto. .45 ml and 1 ml of PIPES buffer (0.1 M, pH 6.8) were added, and the total amount was further made up to 10 ml with water. The intensity of fluorescence at 419 nm was measured three times, and the obtained fluorescence intensity was applied to each calibration curve. The concentration of N- (phosphonomethyl) glycine in the simulated test solution was determined. The results are shown in Table 1 together with the average value and the true value of the three measurements.
[0023]
[Table 1]

[Brief description of the drawings]
FIG. 1 is a graph showing a change in a fluorescence spectrum according to the concentration of N- (phosphonomethyl) glycine added in Example 1.
FIG. 2 is a graph showing a calibration curve created in Example 2.
FIG. 3 is a graph showing the relationship between fluorescence intensity and pH in Example 3.

Claims

A sample containing a terminal amino acid-type organophosphorus herbicide was added to an aqueous solution of a metal complex of a complexing fluorescent reagent having an anthracene structure portion and a polyamine structure portion, and an aqueous solution having a weakly acidic to neutral pH was prepared. Thereafter, fluorescence is generated by the excitation light, and the concentration of the terminal amino acid type organophosphorus herbicide in the sample is determined based on the degree of increase in the fluorescence intensity. Method.

A sample containing a terminal amino acid-type organophosphorus herbicide was added to an aqueous solution of a metal complex of a complexing fluorescent reagent having an anthracene structure portion and a polyamine structure portion, and an aqueous solution having a weakly acidic to neutral pH was prepared. Thereafter, fluorescence is generated by the excitation light, and the fluorescence intensity is compared with a calibration curve showing the relationship between the concentration of the terminal amino acid type organophosphorus herbicide and the fluorescence intensity, and the terminal amino acid type organophosphorus herbicide in the sample is determined. A method for quantifying a terminal amino acid type organophosphorus herbicide, comprising determining the concentration of the herbicide.

The method according to claim 1 or 2, wherein the aqueous solution whose pH is weakly acidic to neutral is an aqueous solution whose pH is 5 to 7.

4. The method according to claim 1, wherein the complexing fluorescent reagent is N- (anthrylmethyl) -polyamine.

The method according to claim 4, wherein the N- (anthrylmethyl) -polyamine is N- (9-anthrylmethyl) -2-pyridylmethylamine.

The method according to any one of claims 1 to 5, wherein the metal ions forming the metal complex are at least one selected from copper (II) ions, nickel (II) ions, and mercury (II) ions. .

The terminal amino acid type organophosphorus herbicide is N- (phosphonomethyl) glycine, 4- [hydroxy (methyl) phosphino] -DL-homoalanine, DL-homoalanin-4-yl (methyl) phosphinic acid, L-2-amino- 4-[(hydroxy) (methyl) = phosphinoyl] butyryl-L-alanyl-L-alanine, 4- [hydroxy (methyl) phosphino] -L-homoalanyl-L-alanyl-L-alanine or a salt thereof Item 7. The method according to any one of Items 1 to 6.