Bohne Victoria J Berdikova, Hove Helge, Hamre Kristin
National Institute of Nutrition and Seafood Research, Safe Seafood, P.O. Box 2029 Nordnes, 5817 Bergen, Norway.
J AOAC Int. 2007 Mar-Apr;90(2):587-97.
A method for simultaneous quantitative determination of ethoxyquin (EQ) and its major metabolite in Atlantic salmon tissues, ethoxyquin dimer (EQ dimer), has been developed. The separation was achieved on tandem coupled phenyl-hexyl and C18 columns by 2-phase gradient elution with acetonitrile-ascorbic acid-acetic acid-diethyl amine organized in a 23.5 min sequence. Compounds were extracted with hexane from samples saponified in ethanol-NaOH and protected from air- and light-mediated oxidation by addition of saturated ethylenediaminetetraacetic acid, ascorbic acid, and pyrogallol. The identity of peaks was confirmed by spiking samples with standards verified by proton nuclear magnetic resonance spectrometry, mass spectrometry, and high-performance liquid chromatography. The detection limit (at 358/433 nm) of matrix-spiked EQ was 0.02 and 0.06 microg/L for EQ dimer, with 0.5 g sample weighed and resuspension in 0.5 mL hexane. Linearity was in the range of 0.2-175 microg/L for EQ and 0.3-5100 microg/L for EQ dimer. Two more ubiquitous compounds were identified as de-ethylated EQ and quinone imine. Totally, 14 peaks sharing spectral properties of EQ were separated in a single run, including a major peak present in all muscle samples, termed unknown metabolite of EQ (UMEQ). The concentrations of EQ, EQ dimer, and de-ethylated EQ, as well as concentrations of UMEQ (in arbitrary units), in the muscle were correlated to the amount of EQ fed to the salmon, thus indicating their possible metabolic origin. The pattern of 14 peaks in the muscle showed high specificity and could be used to discriminate between wild salmon and salmon fed EQ-supplemented feed. This method will be a useful tool for studying EQ metabolism and kinetics, and for the routine surveillance of residual levels of dietary EQ in farmed Atlantic salmon.
已开发出一种同时定量测定大西洋鲑鱼组织中乙氧喹（EQ）及其主要代谢产物乙氧喹二聚体（EQ二聚体）的方法。通过在串联的苯基己基柱和C18柱上进行分离，采用乙腈 - 抗坏血酸 - 乙酸 - 二乙胺进行两相梯度洗脱，洗脱顺序为23.5分钟。用己烷从在乙醇 - 氢氧化钠中皂化的样品中提取化合物，并通过添加饱和乙二胺四乙酸、抗坏血酸和焦性没食子酸来防止空气和光介导的氧化。通过向样品中添加经质子核磁共振光谱、质谱和高效液相色谱验证的标准品来确认峰的身份。对于基质加标后的EQ，在358/433nm处的检测限为0.02μg/L，对于EQ二聚体为0.06μg/L，称取0.5g样品并重新悬浮于0.5mL己烷中。EQ的线性范围为0.2 - 175μg/L，EQ二聚体的线性范围为0.3 - 5100μg/L。另外两种普遍存在的化合物被鉴定为脱乙基EQ和醌亚胺。总共在一次运行中分离出14个具有EQ光谱特性的峰，包括所有肌肉样品中存在的一个主要峰，称为EQ的未知代谢物（UMEQ）。肌肉中EQ、EQ二聚体和脱乙基EQ的浓度以及UME（以任意单位计）的浓度与投喂给鲑鱼的EQ量相关，因此表明它们可能的代谢来源。肌肉中14个峰的模式显示出高特异性，可用于区分野生鲑鱼和投喂添加EQ饲料的鲑鱼。该方法将成为研究EQ代谢和动力学以及常规监测养殖大西洋鲑鱼日粮中EQ残留水平的有用工具。
