開發線上樣品前處理技術結合液相層析離子阱質譜儀之非類固醇消炎藥與離子型抗球蟲藥殘留檢驗方法

Development of online sample pretreatment technique coupled liquid chromatography-ion trap mass spectrometry for analysis of non-steroidal anti-inflammatory drug and ionophore coccidiostat residues

張凱鈞(Kai-Chun Chang)

指導教授 ： 鄭建業

中原大學/理學院/化學研究所/博士(2017年)

https://doi.org/10.6840/cycu201700318

摘要

本研究主要針對動物用藥殘留發展樣品前處理之液相層析質譜儀分析方法。研究主題分為二部分，第一部份研究為利用單一陰離子交換管柱發展創新的線上洗沖溶劑轉換技術，進行線上萃取、分離及分析豬隻血清中9種非類固醇消炎藥物。經由改變移動相之pH值，使藥物因不同pKa造成不同解離度，再加上不同分子間π–π作用力與偶極–偶極作用力的差異，造成與靜相之陰離子交換作用力差異性，得以成功的分離9種非類固醇消炎藥(卡布洛芬、雙氯芬酸、氟尼辛、布洛芬、酮洛芬、甲氯芬那酸、甲芬那酸、尼氟滅酸及托芬那酸)。分析的偵測極限值範圍為0.2–2.5 ng/mL，樣品之同一天內與不同天之分析相對標準偏差值範圍分別為2.1–14.5%及2.8–6.5%，準確度為87.2–99.8%，顯示優越的分析靈敏度、準確度與精密度。真實豬隻血清樣品分析結果為豬隻在注射含氟尼辛與托芬那酸針劑1小時後，氟尼辛與托芬那酸可達到最高濃度，其濃度分別為3480 ± 36 ng/mL 與 431 ± 13 ng/mL，於24小時後氟尼辛與托芬那酸於血清樣品的濃度皆降至20 ng/mL以下，均低於法規最高殘留容許量。
研究主題第二部分為發展陽離子交換固相微萃取管柱線上聯結逆相層析管柱形成二維液相層析質譜儀分析系統，分離分析雞蛋中5種離子型抗球蟲藥。第一維使用陽離子交換管柱是因離子型抗球蟲藥容易結合鈉離子而帶有正電荷能與靜態相進行陽離子交換作用，得於線上予以萃取。第二維則以C18逆相層析管柱進行分離分析5種藥物(拉薩羅、馬杜拉黴素、孟寧素、那寧素及沙利黴素)，其質譜儀的偵測極限值範圍為0.15–0.19 ng/mL，樣品之同一天內與不同天之分析相對標準偏差值分別為3.2–9.7%及3.0–4.9%，準確度為90.0–99.6%，顯示優越的分析靈敏度、準確度與精密度。真實雞蛋樣品為雞隻以馬杜拉黴素投藥一週及停藥一週期間所採集之雞蛋，於停藥第2天後可測試到最高濃度100.1 ng/mL，於停藥第7天後仍可測得濃度為12.2 ng/mL殘留。超出法規中不得檢出之規定。

關鍵字

非類固醇消炎藥 ； 離子型抗球蟲藥 ； 豬隻血清 ； 雞蛋 ； 離子交換層析 ； 固相微萃取 ； 二維液相層析 ； 液相層析離子阱質譜儀

並列摘要

This project focuses on two major research topics which concern the development of sample preparation method for veterinary drug residues with liquid chromatography–mass spectrometry. One of the topics was a novel online eluent switching for one single anion exchange column to perform the online extraction, separation and analysis of nine non–steroidal anti–inflammatory drugs in pig serum. The successful extraction and separation of nine non–steroidal anti–inflammatory drugs(carprofen, diclofenac, flunixin, ibuprofen, ketoprofen, meclofenamic acid, mefenamic acid, niflumic acid, and tolfenamic acid)were through their different degree of dissociation by their different pKa values at different pHs plus different intermolecular π–π interactions and dipole–dipole forces with the anion–exchanger on the stationary phase. The limit of detection of the analysis was in the range of 0.2–2.5 ng/mL. The intraday and interday precision were in the range of 2.1–14.5% and 2.8–6.5%, respectively. The analysis accuracy were in the range of 87.2–99.8%. These data show excellent analytical sensitivity, accuracy, and precision. Real pig serum sample analysis showed a greatest concentraction of 3480 ± 36 ng/mL and 431 ± 13 ng/mL for flunixin and tolfenamic acid, respectively, after one hour injection of one flunixin and tolfenamic acid dosage. Both residues were reduced to about 20 ng/mL at the time of 24 h later after injection and were less than the regulated maximum allowed residue level.
The second topic was the development of a two dimensional chromatography–mass spectrometric system by coupling a cation–exchange solid phase microextraction column to a reversed–phase liquid chromatography column for extracting, separating, and analyzing five ionophore coccidiostats in eggs. The first chromatographic dimension performs the online solid–phase extraction by the cation–exchange mechanism between sodium bearing coccidiostats cations and the cation–exchamger stationary phase. The second chromatographic dimension was for the separation of five ionophore coccidiostats(lasalocid, maduramicin, monensin, narasin, and salinomycin)by the C18 reversed–phase column. The limit of detection of the mass spectrometry was in the range of 0.15–0.19 ng/mL. The intraday and interday precision were in the range of 3.2–9.7% and 3.0–4.9%, respectively. The analysis accuracy were in the range of 90.0–99.6%. These data show excellent analytical sensitivity, accuracy, and precision. The real egg samples were collected from hens fed either with or without maduramicin containing feed for one week. The results indicated a greatest maduramicin concentration of 100.1 ng/mL at the second day of drug withdrawal. After seven days of drug withdrawal, the maduramicin residue concentration was 12.2 ng/mL which disobeys the regulated maximum allowed residue level of not detection.

並列關鍵字

non–steroidal anti–inflammatory drugs ； ionophore coccidiostat ； pig serum ； egg ； ion–exchange chromatography ； solid phase microextraction ； two dimensional chromatography ； liquid chromatography–ion trap mass spectrometry

參考文獻

85. 張凱鈞，線上基質去除技術結合液相層析串聯質譜儀檢測雞肉中馬杜拉黴素，中原大學化學系碩士論文，中華民國九十九年七月。

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