RIKILT-Institute of Food Safety, P.O. Box 230, 6700 AE Wageningen, The Netherlands.
Anal Chim Acta. 2010 Apr 1;664(1):77-88. doi: 10.1016/j.aca.2010.01.065. Epub 2010 Feb 6.
Biological tests can be used to screen samples for large groups of compounds having a particular effect, but it is often difficult to identify a specific compound when a positive effect is observed. The identification of an unknown compound is a challenge for analytical chemistry in environmental analysis, food analysis, as well as in clinical and forensic toxicology. In this study bioassay-guided fractionation, ultra high performance liquid chromatography combined with time-of-flight mass spectrometry (UHPLC/TOFMS) and accurate mass database searching was tested to detect and identify unknown androgens. Herbal mixtures and sport supplements were tested using an androgen bioassay and modifications in sample preparations were carried out in order to activate inactive pro-androgens, androgen esters and conjugated androgens to enable their detection in the bioassay. Two of the four herbal mixtures tested positive and bioassay-guided fractionation followed by UHPLC/TOFMS of positive fractions resulted in the identification of nortestosterone phenylpropionate, testosterone cyclohexanecarboxylate and methyltestosterone. Three of the four sport supplements reacted toxic in the bioassay or gave inconclusive results and were further investigated using UHPLC/TOFMS in combination with data processing software and an accurate mass database having approximately 40,000 entries. This accurate mass database was derived from the PubChem database on the internet and coupled to the TOFMS software. This resulted in the tentative identification of several androgens, including methylboldenone, testosterone and the androgen esters methyltestosterone propionate or testosterone isobutyrate, testosterone buciclate and methylenetestosterone acetate. The study showed that bioassay-guided fractionation in combination with UHPLC/TOFMS analysis is a useful procedure to detect, isolate and identify unknown androgens in suspected samples. As an alternative, the use of data processing software in combination with an accurate mass database and coupled on-line with the TOFMS instrument software enabled the identification of androgens and androgen esters in the chromatogram even without bioassay-guided fractionation.
生物测试可用于筛选具有特定作用的大量化合物的样品，但当观察到阳性效应时，通常难以确定特定的化合物。在环境分析、食品分析以及临床和法医毒理学中，分析化学面临的挑战是鉴定未知化合物。在这项研究中，采用生物测定指导的分步分离法、超高效液相色谱与飞行时间质谱联用（UHPLC/TOFMS）和精确质量数据库搜索，检测并鉴定未知雄激素。使用雄激素生物测定法测试了草药混合物和运动补品，并对样品制备进行了修改，以使活性前雄激素、雄激素酯和共轭雄激素失活，以便在生物测定中检测到它们。测试的四种草药混合物中的两种呈阳性，阳性馏分的生物测定指导分步分离后，再进行 UHPLC/TOFMS 分析，结果鉴定出了苯丙酸诺龙、环已烷羧酸睾酮和甲睾酮。四种运动补品中的三种在生物测定中反应有毒或结果不确定，因此使用 UHPLC/TOFMS 与数据处理软件结合，并使用包含约 40,000 项的精确质量数据库进一步进行了调查。这个精确质量数据库是从互联网上的 PubChem 数据库中提取的，并与 TOFMS 软件耦合。这导致了对几种雄激素的暂定鉴定，包括甲基勃地酮、睾酮和雄激素酯甲睾酮丙酸酯或睾酮异丁酸酯、睾酮布西酯和甲睾酮醋酸酯。该研究表明，生物测定指导的分步分离与 UHPLC/TOFMS 分析相结合，是一种有用的程序，可以检测、分离和鉴定可疑样品中的未知雄激素。作为替代方案，使用数据处理软件与精确质量数据库结合，并与在线 TOFMS 仪器软件耦合，即使没有生物测定指导的分步分离，也能在色谱图中鉴定出雄激素和雄激素酯。
