開發分散液液微萃取法與3D列印封閉式微紙基管柱結合液相層析質譜儀偵測廢水中之濫用藥物

Abstract

本研究旨開發超音波震盪輔助分散液液微萃取與3D列印微流道類管柱裝置與中能量分析廢水中之濫用藥物，以改善現有的紙噴灑游離與萃取方法等分析方法。濫用藥物為近年來所被國際中所重視的健康議題，且越趨逐漸嚴重，影響至個人身心健康、社會經濟與治安犯罪等問題。本研究開發: (1)透過中能量超音波震盪輔助分散液液微萃取法，將檢測68種藥物，即包含興奮劑、新興活性精神物質、鴉片類、止痛藥和苯二氮平類與其代謝物。液液微萃取法其中之乳化作用為實驗重點，透過搖晃、漩渦以及低中高頻之超音波震盪進行。結果顯示透過渦流與中頻超音波震盪時，萃取藥物之80-120%回收率為最多高達33種，而以利進行接下來的於液相層析質譜儀檢測濫用藥物。此方法的線性範圍介於0.001至2 ng mL-1，偵測極限介於0.001至0.1 ng mL-1，展現良好的靈敏性且適用於複雜基質中進行藥物的萃取與偵測藥物的可行性。(2)為了改善過去傳統紙基上的缺陷，因而在微小紙基上建立精準的3D紙基微管柱，並與液相層析質譜系統整合。此方法得以延長分析時間、 減少樣品的擴散、增進紙基層析分離與避免溶劑揮發等能力。此設計之紙基經製造後，提升紙基的強度，避免紙基變形，進而能提供長達90分鐘的分析時間；所創造的微流道結構可以減少樣品的擴散。另外，為了能使紙基微管柱與一般之液相層析管柱具有類似的分離層析效果，而創造出類管柱之構造，獲得良好的分離層析波峰以及避免溶劑揮發。隨後優化噴灑溶劑與流速，達成更佳的質譜訊號。偵測藥物包含24種親水性藥物與1種內標準品，其藥物種類具有新興精神活性物質、興奮劑類、鴉片類、止痛藥和苯二氮平類與其代謝物。將1μL的混合標準品滴在紙基上，並透過噴灑溶劑的帶動，即能在紙基微管柱上層析分離。紙基類管柱所偵測的藥物於溶劑中偵測極限於0.01至1 ng mL-1，顯現具有優秀的靈敏性，而也展現具有良好的線性範圍，介於0.01至100 ng mL-1。此方法偵測真實廢水檢體中的藥物，透過液相層析質譜儀已檢測出甲基安非他命、可待因、麻黃鹼、偽麻黃鹼與嗎啡，將廢水不經樣品前處理直接滴在紙基上，經分析後即可鑑別出目標物。此研究所開發紙基類管柱具有優秀的層析分離效果，為具有潛力可適用於不同基質之樣品。

In this study, liquid chromatography-mass spectrometry (LC-MS) was carried out and combined with a 3D-printed microfluidic device, like a paper column, and dispersive liquid-liquid extraction (DLLME) assisted with medium-frequency sonication for detecting drug abuse in wastewater, improving both paper spray ionization-mass spectrometry (PSI-MS) and the method of extraction. Drug abuse has been referred to as an increasingly important global health topic in recent years. It has caused issues related to individuals’ physical and mental health, the economy, society, security, etc. The objective: (1) DLLME assisted with medium-frequency sonication was optimized for detecting 68 drugs in wastewater, including new psychoactive substances (NPS), stimulants, opioids, painkillers, benzodiazepines, and their metabolites. The main focus of DLLME is emulsification, achieved through shaking, vortexing, and low/medium/high-frequency sonication. The results showed that when vortexing and medium-frequency sonication were conducted, the extraction efficiency of drugs was high, reaching 80-120% recovery for up to 33 classifications, followed by LC-MS for drug detection. This method showed a linear range between 0.001 and 2 ng mL⁻¹, with limits of detection ranging from 0.001 to 0.1 ng mL⁻¹, demonstrating good sensitivity and showing its feasibility for the extraction and detection of drugs in complex matrices. (2) To improve the limitations of conventional paper-based microfluidics, precise 3D-printed micro paper columns (3DP-μPC) were constructed on a miniaturized substrate and integrated with mass spectrometry. This approach enables the extension of analysis time, decreases solvent diffusion, enhances the chromatography of the paper substrate, and avoids volatilization of the solvent. The design of the paper substrate was fabricated to increase the strength of the μPC and to avoid its deformation, further providing up to 90 minutes of analysis time. The microchannel structure on the μPC was created to reduce sample diffusion. Moreover, the column-like structure was designed to obtain chromatographic peaks and to prevent solvent volatilization, giving the μPC a chromatographic separation effect comparable to general liquid chromatography columns. A total of 24 hydrophilic drugs and one internal standard were included for detection. These drugs were classified as new psychoactive substances (NPS), stimulants, opioids, painkillers, benzodiazepines, and their metabolites. A 1 μL standard mixture was dumped onto the μPC and was assisted by solvent elution and driving, to make the chromatographic separation of the 25 selected compounds on it. The LODs of the detected drugs using the μPC ranged from 0.5 to 1 ng mL⁻¹, revealing excellent sensitivity. It also demonstrated a good linear range, ranging from 0.05 to 100 ng mL⁻¹. Drugs in the wastewater were detected using the μPC, including methamphetamine, codeine, ephedrine, pseudoephedrine, and morphine. The sample was dumped directly onto it without sample preparation, and the target analytes were identified after analysis. This study demonstrated the excellent result of chromatographic separtation, its promising technique will be expected on different matrices.