Witting Michael, Rudloff Hans-Christian, Thondamal Manjunatha, Aguilaniu Hugo, Schmitt-Kopplin Philippe
Research Unit Analytical BioGeoChemistry, Department of Environmental Sciences, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.
Research Unit Analytical BioGeoChemistry, Department of Environmental Sciences, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Jan 26;978-979:118-21. doi: 10.1016/j.jchromb.2014.12.005. Epub 2014 Dec 12.
Separation of isomeric molecular species, e.g. double bond position isomers, is a challenging task for liquid chromatography. The two steroid hormones Δ4- and Δ7-dafachronic acid (DA) represent such an isomeric pair. DAs are 3-ketosteroids found in the nematode Caenorhabditis elegans and generated from cholesterol. Δ4- and Δ7-DA have important biological activities and are produced by two different biological pathways in C. elegans. Here we have described a fast separation method for these two isomers using a 1.3 μm core-shell particle in less than 10 min together with a simple MeOH extraction. Using this method we were able to independently quantify Δ4- and Δ7-DA in C. elegans independently from each other and limits of detection of about 5 ng/ml for each isomer were achieved with a good day-to-day reproducibility. As proof-of-principle the method has been applied to the quantification of DAs in worms fed ad libitum or under bacterial deprivation.
分离同分异构分子物种，例如双键位置异构体，对于液相色谱来说是一项具有挑战性的任务。两种甾体激素Δ4-和Δ7-脱皮甾酸（DA）就代表了这样一对异构体。DA是在秀丽隐杆线虫中发现的3-酮甾体，由胆固醇生成。Δ4-和Δ7-DA具有重要的生物活性，并且在秀丽隐杆线虫中通过两种不同的生物途径产生。在此，我们描述了一种快速分离这两种异构体的方法，该方法使用1.3μm核壳颗粒，在不到10分钟的时间内完成分离，并结合简单的甲醇萃取。使用该方法，我们能够相互独立地定量秀丽隐杆线虫中的Δ4-和Δ7-DA，并且每种异构体的检测限约为5 ng/ml，且具有良好的日常重现性。作为原理验证，该方法已应用于自由摄食或细菌剥夺条件下蠕虫中DA的定量分析。
