以液相層析質譜儀結合QuEChERS技術建立水產品中殘留三苯基甲烷類染料之研究

Determination of Triphenylmethanees Dye in Seafood by Liquid Chromatography Coupled with Tandem Mass Spectrometry

林建安(Chien-An Lin) ； 廖伶莉(Ling-Li Liao) ； 陳婉玲(Wan-Ling Chen) ； 陳素娥(Su-O Chen)

《化學》 76卷1期 (2018/03) Pp. 103-112

https://doi.org/10.6623/chem.201803_76(1).002

摘要

三苯基甲烷類染料(triphenylmethane dyes, TPMs)是以三苯基甲烷為主體構成之芳香烴化合物，常見的染料有孔雀綠、甲基藍、結晶紫…等，本研究以其中七項染料及兩種代謝物作為主要探討對象。由於他們的抗菌、抗真菌和抗寄生蟲的特性及該類型染料取得容易亦具效果顯著之特性，因此常用於水產養殖業。但TPMs對人體具有毒性(誘變及致癌)，在美國和歐盟皆禁止農、漁業等相關產業對於TPMs的使用。該類型染料具有迅速代謝特性，且在海鮮組織中易形成還原性代謝物。分析物本身具有高不穩定性，因此造成樣品前處理和儀器分析時有困難度。本研究的目的為建立出具快速、簡便亦準確之分析檢驗方法，擬以QuEChERS為前處理方式有效縮短前處理流程，透過使用甲酸銨及氯乙烷為主要萃取溶劑，並使用食鹽、C18等粉劑來達到萃取、淨化之效果。在研究過程中因三苯基甲烷類染料本身的不穩性，需添加適量抗氧化劑來有效減緩標的分析物的氧化反應，以維持標的分析物。挑選TPMs中常見的其中七項染料及其兩種代謝物(Malachite green, Leuco malachite green, Crystal violet, Leuco crystal violet, Methylene blue, Methyl Red, Azure B, Nile Blue及Victoria Blue)為主要研究對象，本研究以液相層析串聯質譜儀為主要分析工具，分別使用5 mM醋酸銨(pH = 4)及0.1%甲酸乙腈溶液為移動相，分析管柱為ZORBAX Eclipse Plus C18, 3.0 × 100 mm 1.8-Micron進行層析分離探討測定，該檢測方法組織檢量線範圍為0.5～100 ng/mL，r均可達到0.995以上，定量極限為0.5 ppb，添加回收部分分別於空白檢體(蝦類、吳郭魚)中添加2.5及5 ppb，其平均回收率介於60%～120%間，變異係數為2.15%～6.90%。本研究適合作為「TPMs類染料」初步篩查檢驗方法。

關鍵字

三苯基甲烷類染料 ； QuEChERS ； 液相層析串聯質譜儀

並列摘要

Triphenylmethane dyes (TPMs) are synthetic dyes used for a wide range in industrial applications. In aquaculture production, many companies used TPMs becaused of antibacterial, anti-fungal, anti-parasitic properties, low cost...etc. properties. But its toxicity to humans (mutagenic & carcinogenic), US and EU have developed a zero tolerance policy to prohibit agriculture, fisheries and other related industries for the use of TPMs. This type of dye has a rapid metabolic property, and forms a reducing metabolite easyily in the aquaculture products tissue. Besides, the difficulty of sample pretreatment and instrumental analysis caused by analyte instability. In order to improve the inspection of aquatic products, it is urgent to establish a quick, simple and accurate test method. In this study, a simple method was developed and validated to determine residues of seven triphenylmethane dyes and their metabolites (Malachite green, Leuco malachite green, Crystal violet, Leuco crystal violet, Methylene blue, Methyl Red, Azure B, Nile Blue and Victoria Blue) in aquaculture products by using LC/MS/MS. A modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) technique was carried out to prepare the sample, in addition, both of the optimal extraction and cleanup conditions were established adopting an experimental design. The validation parameters from the modified method were obtained to ensure complying with the requirements of substances in aquaculture products, which establish by regulatory agencies. The validation results indicated that the Nine TPMs can be identified and quantified in a concentration range between 0.5 and 100 ng/mL, and possessed the recovery between 100% and 97.1% (n = 15). Under reproducible conditions, RSD had precision between 2.15% and 6.9%. Therefore, the modified QuEChERS method has the advantages of simplicity, sensitivity and rapidity and can be used for simultaneous determination of analytes in aquatic products.

並列關鍵字

Triphenylmethane dyes ； QuEChERS ； LC/MS/MS