Zeleny Reinhard, Harbeck Stefan, Schimmel Heinz
European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Retieseweg 111, B-2440 Geel, Belgium.
J Chromatogr A. 2009 Jan 9;1216(2):249-56. doi: 10.1016/j.chroma.2008.11.061. Epub 2008 Nov 28.
A liquid chromatography-electrospray ionisation tandem mass spectrometry method for the simultaneous detection and quantitation of 5-nitroimidazole veterinary drugs in lyophilised pork meat, the chosen format of a candidate certified reference material, has been developed and validated. Six analytes have been included in the scope of validation, i.e. dimetridazole (DMZ), metronidazole (MNZ), ronidazole (RNZ), hydroxymetronidazole (MNZOH), hydroxyipronidazole (IPZOH), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI). The analytes were extracted from the sample with ethyl acetate, chromatographically separated on a C(18) column, and finally identified and quantified by tandem mass spectrometry in the multiple reaction monitoring mode (MRM) using matrix-matched calibration and (2)H(3)-labelled analogues of the analytes (except for MNZOH, where [(2)H(3)]MNZ was used). The method was validated in accordance with Commission Decision 2002/657/EC, by determining selectivity, linearity, matrix effect, apparent recovery, repeatability and intermediate precision, decision limits and detection capabilities, robustness of sample preparation method, and stability of extracts. Recovery at 1 microg/kg level was at 100% (estimates in the range of 101-107%) for all analytes, repeatabilities and intermediate precisions at this level were in the range of 4-12% and 2-9%, respectively. Linearity of calibration curves in the working range 0.5-10 microg/kg was confirmed, with r values typically >0.99. Decision limits (CCalpha) and detection capabilities (CCbeta) according to ISO 11843-2 (calibration curve approach) were 0.29-0.44 and 0.36-0.54 microg/kg, respectively. The method reliably identifies and quantifies the selected nitroimidazoles in the reconstituted pork meat in the low and sub-microg/kg range and will be applied in an interlaboratory comparison for determining the mass fraction of the selected nitroimidazoles in the candidate reference material currently developed at IRMM.
已开发并验证了一种液相色谱 - 电喷雾电离串联质谱法，用于同时检测和定量冻干猪肉中5 - 硝基咪唑类兽药，这是候选标准物质的选定形式。验证范围包括六种分析物，即二甲硝唑（DMZ）、甲硝唑（MNZ）、罗硝唑（RNZ）、羟基甲硝唑（MNZOH）、羟基异丙硝唑（IPZOH）和2 - 羟甲基 - 1 - 甲基 - 5 - 硝基咪唑（HMMNI）。分析物用乙酸乙酯从样品中提取，在C(18)柱上进行色谱分离，最后通过串联质谱在多反应监测模式（MRM）下使用基质匹配校准和分析物的(2)H(3)标记类似物（MNZOH除外，其使用[(2)H(3)]MNZ）进行鉴定和定量。该方法根据委员会第2002/657/EC号决定进行验证，通过测定选择性、线性、基质效应、表观回收率、重复性和中间精密度、决策限和检测能力、样品制备方法的稳健性以及提取物的稳定性。在1 μg/kg水平下，所有分析物的回收率均为100%（估计范围为101 - 107%），此水平下的重复性和中间精密度分别在4 - 12%和2 - 9%范围内。确认了在0.5 - 10 μg/kg工作范围内校准曲线的线性，r值通常>0.99。根据ISO 11843 - 2（校准曲线法）的决策限（CCalpha）和检测能力（CCbeta）分别为0.29 - 0.44和0.36 - 0.54 μg/kg。该方法能够可靠地鉴定和定量重构猪肉中低至亚μg/kg范围内的选定硝基咪唑类，并将应用于实验室间比对，以确定IRMM目前正在开发的候选参考物质中选定硝基咪唑类的质量分数。
