Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain.
Anal Bioanal Chem. 2012 Feb;402(5):1879-88. doi: 10.1007/s00216-011-5611-1. Epub 2012 Jan 13.
A novel and fast routine method for the simultaneous determination and confirmation of clenbuterol in bovine and human urine samples by gas chromatography electron ionization mass spectrometry (GC-EI-MS) has been developed. The method employs isotope dilution mass spectrometry (IDMS) and is based on a combination of minimal labeling (a single (13)C label in the molecule) and isotope pattern deconvolution (IPD). This new methodology does not require the construction of a methodological calibration graph, and was compared with the classical IDMS procedure employed in clenbuterol analysis based on the use of a deuterated compound as internal standard (d(9)-clenbuterol) and a calibration curve. The sample preparation consists of simple extraction with dichloromethane, which was dried and derivatized with chloro(chloromethyl)dimethylsilane, generating a cyclic dimethylsilamorpholine (DMS) derivative suitable for GC(EI)MS detection and identification. This compound produces five intense ions in the electron ionization source, which allow the presence of clenbuterol to be confirmed in just one analysis, as demanded by European Union directives. The accuracy of the method was studied by performing recovery experiments at different concentration levels (from 0.3 to 5 ng g(-1)) in 5 mL bovine urine samples using two labeled compounds: an in-house-synthesized (13)C(1)-clenbuterol and a commercially available d(9)-clenbuterol. The detection limit of the method in human urine was 0.050 ng g(-1) with a sample volume of 10 mL, and is thus suitable for antidoping control purposes. Finally, the (13)C(1)-clenbuterol standard was employed for the determination of clenbuterol in two reference materials, BCR-503 and BCR-504 (lyophilized bovine urine). The concentrations obtained were in agreement with the certified values, with a reproducibility of below 1% RSD.
一种新颖、快速的方法，用于通过气相色谱电子电离质谱（GC-EI-MS）同时测定和确证牛和人尿样中的克伦特罗。该方法采用同位素稀释质谱（IDMS），基于最小标记（分子中单个（13)C 标记）和同位素模式反卷积（IPD）的组合。这种新方法不需要构建方法校准曲线，并与基于使用氘代化合物作为内标（d(9)-克伦特罗）和校准曲线的克伦特罗分析中使用的经典 IDMS 程序进行了比较。样品制备包括用二氯甲烷简单萃取，萃取物干燥并用氯（氯甲基）二甲基硅烷衍生化，生成适合 GC(EI)MS 检测和鉴定的环状二甲基硅烷吗啉（DMS）衍生物。该化合物在电子电离源中产生五个强烈的离子，允许仅通过一次分析即可确认克伦特罗的存在，这是欧盟指令所要求的。通过在 5 毫升牛尿样品中在不同浓度水平（0.3 至 5 ng g(-1))下进行回收实验，使用两种标记化合物：一种内部合成的（13)C(1)-克伦特罗和一种市售的 d(9)-克伦特罗，研究了该方法的准确性。该方法在人尿中的检测限为 0.050 ng g(-1)，样品体积为 10 毫升，因此适合于反兴奋剂控制目的。最后，（13)C(1)-克伦特罗标准用于两种参考物质 BCR-503 和 BCR-504（冻干牛尿）中克伦特罗的测定。所获得的浓度与认证值一致，重复性低于 1%RSD。
