JP6815032B2 - How to detect drugs - Google Patents
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- 229940079593 drug Drugs 0.000 title claims description 31
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Description
本発明は、薬物の検出方法に関し、特に、検出対象となる薬物を他の物質と区別し、誤検出を防ぐことが可能な、薬物の検出方法に関する。 The present invention relates to a method for detecting a drug, and more particularly to a method for detecting a drug, which can distinguish a drug to be detected from other substances and prevent erroneous detection.
近年、レクリエーション薬としての薬物乱用は、薬物依存につながる可能性があるとして、世界中で深刻な社会問題として認識されている。特にアンフェタミン系の薬物は覚醒及び食欲不振作用を誘発する強力な中枢神経系刺激剤であることが知られている（非特許文献１−３）。 In recent years, substance abuse as a recreational drug has been recognized as a serious social problem worldwide as it can lead to drug addiction. In particular, amphetamine-based drugs are known to be potent central nervous system stimulants that induce arousal and anorexia (Non-Patent Documents 1-3).
メタンフェタミン（以下、「ＭＡ」という。）は、日本における乱用薬物として最も一般的に使用されているもので、違法な所持、使用等により毎年１万人以上が逮捕されているため、薬物問題における我が国における最重要課題とされている（非特許文献４）。 Methamphetamine (hereinafter referred to as "MA") is the most commonly used drug of abuse in Japan, and more than 10,000 people are arrested every year for illegal possession and use, so it is a drug problem. It is regarded as the most important issue in Japan (Non-Patent Document 4).
尿、血液、唾液などの生体試料を用いたＭＡの定量は、ＭＡの接種による乱用を直接確認できるため有効な手段として普及している。ＭＡの定量を行う方法としては、ガスクロマトグラフィー、液体クロマトグラフィーおよびキャピラリー電気泳動クロマトグラフィー等と組み合わせた質量分析法が高感度検出のために使用されている（非特許文献１、５）。 Quantification of MA using biological samples such as urine, blood, and saliva has become widespread as an effective means because abuse due to inoculation of MA can be directly confirmed. As a method for quantifying MA, mass spectrometry combined with gas chromatography, liquid chromatography, capillary electrophoresis chromatography and the like is used for high-sensitivity detection (Non-Patent Documents 1 and 5).
電気化学発光（以下、「ＥＣＬ」という。）は、電極反応によって生じた発光化学種が、後続化学反応によって励起状態となり、発光する現象である（非特許文献６、７）。この方法は、蛍光分光法のような励起光源は不要であるため高感度の分析が可能である上、装置が比較的小型であるため、各種現場におけるその場分析が可能である特長を有する。さらに、この方法は前述した尿や血液などの半透明な生体試料でも検出器の構成を工夫することで分析が行えるという特徴がある。このため、ＥＣＬ法は、生物学的試料中の薬物検出の手段として、注目を集めている（非特許文献７−９）。 Electrochemical chemiluminescence (hereinafter referred to as "ECL") is a phenomenon in which a luminescent chemical species generated by an electrode reaction is excited by a subsequent chemical reaction and emits light (Non-Patent Documents 6 and 7). This method has the advantage that high-sensitivity analysis is possible because it does not require an excitation light source such as fluorescence spectroscopy, and in-situ analysis is possible at various sites because the device is relatively small. Furthermore, this method is characterized in that even translucent biological samples such as urine and blood can be analyzed by devising the configuration of the detector. Therefore, the ECL method has attracted attention as a means for detecting drugs in biological samples (Non-Patent Document 7-9).
前述のとおり、ＥＣＬ法は、高い選択性で生体試料中のＭＡのスクリーニングを行うことを可能とするが、一部の物質においては、ＭＡと同様のECL活性を呈することがあるため、当該物質が試料中に含まれていることにより、MAに対する偽陽性を示す可能性がある。これにより、生体試料中に前記性質を示すような検出妨害物質が含まれる場合には、ＭＡの検出を行う際に悪影響があるという課題があった。 As described above, the ECL method makes it possible to screen MA in a biological sample with high selectivity, but since some substances may exhibit the same ECL activity as MA, the substance concerned. May show false positives for MA due to the inclusion of in the sample. As a result, when the biological sample contains a detection-interfering substance exhibiting the above-mentioned properties, there is a problem that there is an adverse effect when detecting MA.
また、生体試料中に含まれる検出対象となるＭＡを、液−液抽出により有機溶媒相に濃縮することで選択的にMAを抽出する技術を適用した場合にも、有機溶媒相中のMAはECL活性を示さず、そのままの状態では検出できないという課題があった。 In addition, even when the technique of selectively extracting MA by concentrating MA to be detected contained in a biological sample into an organic solvent phase by liquid-liquid extraction is applied, the MA in the organic solvent phase remains. There was a problem that it did not show ECL activity and could not be detected as it was.
そこで、上記課題を解決するため、本発明者らは、鋭意研究を行い、有機溶媒中のMAを検出するためのECL用検出液を簡便な混合溶媒系で達成できることを見出し、本発明を完成させるに至った。すなわち、本発明は、ＭＡと他の物質とを明確に区別可能にする、新規な薬物の検出方法を提供することを目的とする。 Therefore, in order to solve the above problems, the present inventors conducted diligent research and found that a detection solution for ECL for detecting MA in an organic solvent could be achieved with a simple mixed solvent system, and completed the present invention. I came to let you. That is, it is an object of the present invention to provide a novel drug detection method that makes it possible to clearly distinguish MA from other substances.
本発明に係る第一の形態は、アンフェタミン系の薬物を検出する方法であって、
試料に疎水性の有機溶媒を添加する工程と、前記試料から検出対象となる前記薬物を前記有機溶媒に抽出する工程と、前記抽出後の有機溶媒を分取し、両親媒性を有する分散剤を添加する工程と、電気化学発光法により前記有機溶媒と前記分散剤との混合溶液の分析を行う工程と、を備えることを特徴とする薬物の検出方法である。
The first form according to the present invention is a method for detecting an amphetamine-based drug.
A step of adding a hydrophobic organic solvent to the sample, the steps of extracting the drug to be detected from the sample to the organic solvent, was separated and the organic solvent after the extraction, dispersing agent having amphiphilic Is a method for detecting a drug, which comprises a step of adding the organic solvent and a step of analyzing a mixed solution of the organic solvent and the dispersant by an electrochemical luminescence method.
また、本発明に係る第二の形態は、前記有機溶媒は、ヘキサン、酢酸エチル、n-オクタノール、クロロホルムから選択される１または２ 以上の溶媒であることを特徴とする請求項１記載の検出方法である。 The second embodiment according to the present invention, the organic solvent medium is hexane, ethyl acetate, n- octanol, according to claim 1, characterized in that the one or more solvents selected from chloroform This is a detection method.
また、本発明に係る第三の形態は、前記分散剤は、メタノール、エタノール、n-プロパノール、アセトン、アセトニトリルから選択される１または２以上の両親媒性溶媒であることを特徴とする請求項１または２ 記載の検出方法である。 Also, claims a third embodiment according to the present invention, the dispersing agent, characterized methanol, ethanol, n- propanol, acetone, that is one or more amphiphilic solvent selected from acetonitrile 1 or 2 is the detection method.
また、本発明に係る第四の形態は、前記試料と前記有機溶媒の混合溶液に機械的作用を印加する工程と、をさらに備えることを特徴とする、請求項１−３のいずれか１項記載の検出方法である。 A fourth aspect of the present invention further comprises a step of applying a mechanical action to the mixed solution of the sample and the organic solvent, according to any one of claims 1-3. The detection method described.
また、本発明に係る第五の形態は、前記薬物がメタンフェタミンであることを特徴とする、請求項１−４のいずれか１項記載の溶媒である。 The fifth aspect of the present invention is the solvent according to any one of claims 1-4, characterized in that the drug is methamphetamine.
また、本発明に係る第六の形態は、前記有機溶媒と前記分散剤の混合溶媒のｐＨ値を調製する工程と、をさらに備えることを特徴とする、請求項１−５のいずれか１項記載の検出方法である。A sixth aspect of the present invention further comprises a step of adjusting the pH value of the mixed solvent of the organic solvent and the dispersant, according to any one of claims 1-5. The detection method described.
また、本発明に係る第七の形態は、前記機械的作用を印加する工程は遠心分離機による遠心分離工程を含むことを特徴とする請求項４に記載の検出方法である。
The seventh aspect according to the present invention is the detection method according to claim 4 , wherein the step of applying the mechanical action includes a centrifugation step by a centrifuge .
本発明に係る薬物の検出方法によれば、検出対象と同様のＥＣＬ活性を示す物質が含有した試料を用いて薬物の検出を行った場合でも、正確にMAのみを選択的に検出することが可能となる。 According to the drug detection method according to the present invention, even when a drug is detected using a sample containing a substance showing the same ECL activity as the detection target, only MA can be accurately and selectively detected. It will be possible.
以下、本発明に係る薬物の検出方法の実施の形態について説明する。 Hereinafter, embodiments of the drug detection method according to the present invention will be described.
本発明に係る薬物の検出方法は、尿等の試料と有機溶媒とを混合し、検出対象の薬物を有機溶媒に濃縮させた後、分散剤を添加し、これを電気化学発光法で分析することにより、検出対象である薬物を高い選択性で検出することを可能にする方法である。 In the method for detecting a drug according to the present invention, a sample such as urine is mixed with an organic solvent, the drug to be detected is concentrated in the organic solvent, a dispersant is added, and this is analyzed by an electrochemical luminescence method. This is a method that makes it possible to detect a drug to be detected with high selectivity.
本発明に係る薬物の検出方法において、分析に用いる試料の種類は特に限定するものでないが、尿、血液、汗等の液体の生体試料であることが望ましい。また、試料の前処理を行うことにより液体試料として取り扱うことが可能であれば、皮膚、毛髪等の固体の生体試料または錠剤等の試料についても適用することが可能である。 In the method for detecting a drug according to the present invention, the type of sample used for analysis is not particularly limited, but a liquid biological sample such as urine, blood, or sweat is desirable. Further, if it can be handled as a liquid sample by pretreating the sample, it can also be applied to a solid biological sample such as skin and hair or a sample such as a tablet.
本発明に係る薬物の検出方法において、使用する有機溶媒の種類は特に制限されず、疎水性の溶媒であれば、公知の溶媒から任意に適用可能である。本発明で適用可能な有機溶媒の例としては、ヘキサン、酢酸エチル、n-オクタノール、クロロホルムが挙げられ、特に酢酸エチルが好適である。ここで、疎水性とは、水に不溶または難溶な性質をいい、化学的には極性が低いことをいう。 In the method for detecting a drug according to the present invention, the type of organic solvent used is not particularly limited, and any known hydrophobic solvent can be applied as long as it is a hydrophobic solvent. Examples of the organic solvent applicable in the present invention include hexane, ethyl acetate, n-octanol, and chloroform, and ethyl acetate is particularly preferable. Here, hydrophobicity refers to the property of being insoluble or sparingly soluble in water, and chemically has low polarity.
本発明に係る薬物の検出方法において、使用する分散剤の種類は特に制限なく、両親媒性を有する溶媒であれば、適用することが可能である。両親媒性の分散剤の例としては、メタノール、エタノール、n-プロパノール、アセトン、アセトニトリルが挙げられる。 In the method for detecting a drug according to the present invention, the type of dispersant used is not particularly limited, and any solvent having amphipathic properties can be applied. Examples of amphipathic dispersants include methanol, ethanol, n-propanol, acetone and acetonitrile.
本発明において、検出対象となる薬物としては、アンフェタミン系の薬物、特にメタンフェタミンが好適である。生体試料中のメタンフェタミンはアルカリ性の条件にすることにより、水に不溶となる性質を有するため、有機溶媒へ効果的に抽出される。そのため、本発明に係る溶媒を用いて、分離（抽出）を行うことにより、有機溶媒に抽出及び濃縮される。これにより、ビタミンＣ等の生体由来の成分によるECL検出の際の妨害を抑えることができる特長を有する。また、本発明に係る検出方法は、薬物以外の物質、例えば医薬品、農薬等についても適用可能である。 In the present invention, amphetamine-based drugs, particularly methamphetamine, are suitable as the drug to be detected. Methamphetamine in a biological sample has the property of being insoluble in water when it is made alkaline, so it is effectively extracted into an organic solvent. Therefore, by performing separation (extraction) using the solvent according to the present invention, it is extracted and concentrated in an organic solvent. As a result, it has a feature that interference with ECL detection by a biological component such as vitamin C can be suppressed. The detection method according to the present invention is also applicable to substances other than drugs, such as pharmaceuticals and pesticides.
本発明においては、生体試料と有機溶媒の混合溶液に機械的作用を印加しても良い。機械的作用の印加によって、生体試料中の検出対象を効率よく有機溶媒に濃縮することが可能となる。機械的作用を印加する方法は、特に制限するものではないが、水平型及び垂直型振とう器による振とう過程、並びに遠心分離器による遠心分離過程が好適に適用可能である。 In the present invention, a mechanical action may be applied to a mixed solution of a biological sample and an organic solvent. By applying a mechanical action, it becomes possible to efficiently concentrate the detection target in the biological sample in an organic solvent. The method of applying the mechanical action is not particularly limited, but a shaking process using a horizontal type and a vertical type shaker and a centrifugation process using a centrifuge are preferably applicable.
本発明においては、有機溶媒と分散剤の混合溶液のpH調整を行っても良い。pH調整方法は特に制限なく、公知の方法から任意に選択可能である。pH調整方法の例としては、リン酸または水酸化ナトリウム水溶液を添加する方法が挙げられる。本発明に好適な値は、分析方法により異なり、例えば、電気化学発光法を用いた場合には、ｐＨ値は7以上の中性からアルカリ性が望ましい。 In the present invention, the pH of a mixed solution of an organic solvent and a dispersant may be adjusted. The pH adjusting method is not particularly limited and can be arbitrarily selected from known methods. Examples of the pH adjusting method include a method of adding an aqueous solution of phosphoric acid or sodium hydroxide. The value suitable for the present invention differs depending on the analysis method. For example, when the electrochemical luminescence method is used, the pH value is preferably 7 or more, neutral to alkaline.
本発明において、有機溶媒と分散剤との混合溶液を分析する方法については、特に制限はなく、公知の方法から任意に選択することが可能である。分析方法の例としては、ガスクロマトグラフィー、液体クロマトグラフィーおよび質量分析と組み合わせたキャピラリー電気泳動クロマトグラフィー、電気化学発光法等が挙げられ、特に電気化学発光法を用いた方法が好適である。 In the present invention, the method for analyzing the mixed solution of the organic solvent and the dispersant is not particularly limited and can be arbitrarily selected from known methods. Examples of the analysis method include capillary electrophoresis chromatography combined with gas chromatography, liquid chromatography and mass spectrometry, an electrochemical luminescence method, and the like, and a method using an electrochemical luminescence method is particularly preferable.
以下に本発明に係る薬物の検出方法の実施例として、水/酢酸エチル/メタノール混合溶媒を用いて、電気化学測定によりMAの検出を行った例について説明する。 Hereinafter, as an example of the method for detecting a drug according to the present invention, an example in which MA is detected by electrochemical measurement using a mixed solvent of water / ethyl acetate / methanol will be described.
MA塩酸塩（大日本住友製薬社製）を添加した尿5.0 mLを試験管に分取し、0.1 moldm-3の水酸化ナトリウム水溶液0.5 mLを添加してアルカリ性とした。 5.0 mL of urine containing MA hydrochloride (manufactured by Sumitomo Dainippon Pharma) was dispensed into a test tube, and 0.5 mL of 0.1 moldm-3 aqueous sodium hydroxide solution was added to make it alkaline.
この溶液に1.0mLの酢酸エチルを添加し、10分間振とう機で攪拌した。その後、2000 x gの遠心分離機を用いて、10分間遠心及び分離を行い、尿と酢酸エチルとを相分離させることで、尿資料中のMAを、酢酸エチルに抽出させた。 1.0 mL of ethyl acetate was added to this solution, and the mixture was stirred with a shaker for 10 minutes. Then, using a 2000 x g centrifuge, centrifugation and separation were carried out for 10 minutes, and urine and ethyl acetate were phase-separated to extract MA in urine data into ethyl acetate.
抽出後の酢酸エチルを0.10 mL分取し、リン酸緩衝溶液（PBS）でpHを調整した1.0 mmoldm-3のRu(bpy)3 2+水溶液0.50 mL、メタノール0.30 mL、を添加し、混合溶媒を調製した。 0.10 mL of ethyl acetate after extraction was taken, and 0.50 mL of 1.0 mmol dm- 3 Ru (bpy) 3 2+ aqueous solution and 0.30 mL of methanol, whose pH was adjusted with phosphate buffered solution (PBS), were added, and the mixed solvent was added. Was prepared.
前記混合溶媒を用いて、電気化学発光法によるMAの検出を行った。電気化学発光測定は、微小電解ガラスセル中で行い、グラッシーカーボン作用電極、銀/塩化銀参照電極及び白金対極を用いた一般的な3電極方式によって行った。この電気化学発光測定は、電気化学アナライザによって制御した。また、電極表面からの微弱発光であるＥＣＬは、作用電極に向き合う形で光電子倍増管を設置することで検出した。また、全てのＥＣＬ測定は暗室で行われた。 MA was detected by the electrochemical luminescence method using the mixed solvent. The electrochemical emission measurement was performed in a microelectrolytic glass cell, and was performed by a general three-electrode method using a glassy carbon working electrode, a silver / silver chloride reference electrode, and a platinum counter electrode. This electrochemical luminescence measurement was controlled by an electrochemical analyzer. In addition, ECL, which is weak light emission from the electrode surface, was detected by installing a photomultiplier tube facing the working electrode. Also, all ECL measurements were performed in a dark room.
図１（実施例１）に、電気化学発光測定の結果を示す。図から、明瞭なシグナルが検出されていることが認められる。これは、本実施例に係る方法により、MAの検出が正しく行われたことを示している。 FIG. 1 (Example 1) shows the results of electrochemical luminescence measurement. From the figure, it can be confirmed that a clear signal is detected. This indicates that MA was detected correctly by the method according to this example.
生体試料から、液−液抽出により有機溶媒相にMAを濃縮した場合、ＥＣＬ活性を示さず、電気化学発光法による検出ができない。この点を検証するため、濃縮後の酢酸エチルに対して、分散剤を用いず、直接電気化学発光測定を行った。測定に使用した機器、測定方法は、実施例１と同様である。 When MA is concentrated in an organic solvent phase by liquid-liquid extraction from a biological sample, it does not show ECL activity and cannot be detected by the electrochemical luminescence method. In order to verify this point, direct chemiluminescence measurement was performed on the concentrated ethyl acetate without using a dispersant. The equipment and measurement method used for the measurement are the same as those in the first embodiment.
図１（比較例１）に、電気化学発光測定の結果を示す。図からは、シグナルは確認されなかった。このことは、電気化学発光法によるMAの検出には、分散剤を添加した、本発明に係る混合溶媒が有効であることを示している。 FIG. 1 (Comparative Example 1) shows the results of electrochemical luminescence measurement. No signal was confirmed from the figure. This indicates that the mixed solvent according to the present invention to which a dispersant is added is effective for the detection of MA by the electrochemical luminescence method.
電気化学発光法によるMAの検出における、ｐＨの影響を検証するため、リン酸緩衝溶液を用いて、混合溶媒のｐＨ値を変化させた場合のＥＣＬ発光強度を測定した。測定に使用した機器、測定方法は実施例１と同様である。 In order to verify the effect of pH on the detection of MA by the electrochemical luminescence method, the ECL emission intensity when the pH value of the mixed solvent was changed was measured using a phosphate buffer solution. The equipment and measurement method used for the measurement are the same as those in the first embodiment.
図２（実施例２）に、それぞれのｐＨ値において、本発明に係る混合溶媒を用いた電気化学発光測定を行った結果のグラフを示す。図から、ｐＨ7以上の中性−アルカリ性において、大きな電気化学発光強度を達成していることが認められる。また、pH7の中性条件下で、特に大きな発光強度が得られており、これは、本発明に係る検出方法では、よりマイルドな条件でMAの検出を行うことが可能であることが示されている。
FIG. 2 (Example 2) shows a graph of the results of electrochemiluminescence measurement using the mixed solvent according to the present invention at each pH value. From the figure, it can be seen that a large electrochemical luminescence intensity is achieved at neutral-alkali at pH 7 or higher. Further, a particularly large emission intensity was obtained under a neutral condition of pH 7, which indicates that the detection method according to the present invention can detect MA under milder conditions. ing.
Claims (7)
試料に疎水性の有機溶媒を添加する工程と、
前記試料から検出対象となる前記薬物を前記有機溶媒に抽出する工程と、
前記抽出後の有機溶媒を分取し、両親媒性を有する分散剤を添加する工程と、
電気化学発光法により前記有機溶媒と前記分散剤との混合溶液の分析を行う工程と、を備えることを特徴とする、薬物の検出方法。 A method for detecting amphetamine drugs
The process of adding a hydrophobic organic solvent to the sample and
A step of extracting the drug to be detected from the sample to the organic solvent,
The step of separating the organic solvent after extraction and adding an amphipathic dispersant,
Characterized in that it and a step of performing an analysis of the mixed solution of the dispersing agent and the organic solvent by electrochemiluminescence detection method of drug.
The detection method according to claim 4, wherein the step of applying the mechanical action includes a centrifugation step by a centrifuge.
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