Freie Universität Berlin, Institute of Pharmacy, Königin-Luise-Str. 2+4, 14195 Berlin, Germany.
Agilent Technologies GmbH, Hewlett-Packard-Str. 8, 76337 Waldbronn, Germany.
J Chromatogr A. 2018 Jun 15;1554:101-116. doi: 10.1016/j.chroma.2018.04.035. Epub 2018 Apr 17.
In various fields of endocrinology, the determination of steroid hormones synthesised by the human body plays an important role. Research on central neurosteroids has been intensified within the last years, as they are discussed as biomarkers for various cognitive disorders. Their concentrations in cerebrospinal fluid (CSF) are considered to be regulated independently from peripheral fluids. For that reason, the challenging matrix CSF becomes a very interesting specimen for analysis. Concentrations are expected to be very low and available amount of CSF is limited. Thus, a comprehensive method for very sensitive quantification of a set of analytes as large as possible in one analytical aliquot is desired. However, high structural similarities of the selected panel of 51 steroids and steroid sulfates, including numerous isomers, challenges achievement of chromatographic selectivity. Since decades the analysis of endogenous steroids in various body fluids is mainly performed by gas chromatography (GC) coupled to (tandem) mass spectrometry (MS(/MS)). Due to the structure of the steroids of interest, derivatisation is performed to meet the analytical requirements for GC-MS(/MS). Most of the laboratories use a two-step derivatisation in multi-analyte assays that was already published in the 1980s. However, for some steroids this elaborate procedure yields multiple isomeric derivatives. Thus, some laboratories utilize (ultra) high performance liquid chromatography ((U)HPLC)-MS/MS as alternative but, even UHPLC is not able to separate some of the isomeric pairs. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to GC and (U)HPLC may help to overcome these issues. Within this project the two most promising methods for endogenous steroid profiling were investigated and compared: the "gold standard" GC-MS and the orthogonal separation technique SFC-MS/MS. Different derivatisation procedures for gas chromatographic detection were explored and the formation of multiple derivatives described and confirmed. Taken together, none of the investigated derivatisation procedures provided acceptable results for further method development to meet the requirements of this project. SFC with its unique selectivity was able to overcome these issues and to distinguish all selected steroids, including (pro-)gestagens, androgens, corticoids, estrogens, and steroid sulfates with appropriate selectivity. Valued especially in the separation of enantiomeric analytes, SFC has shown its potential as alternative to GC. The successful separation of 51 steroids and steroid sulfates on different columns is presented to demonstrate the potential of SFC in endogenous steroid profiling.
在内分泌学的各个领域，测定人体合成的类固醇激素都起着重要的作用。近年来，人们对中枢神经甾体的研究也日益加强，因为它们被认为是各种认知障碍的生物标志物。它们在脑脊液（CSF）中的浓度被认为与外周液无关。正因为如此，具有挑战性的基质 CSF 成为了一个非常有趣的分析样本。其浓度预计非常低，并且 CSF 的可用量有限。因此，需要一种全面的方法来尽可能灵敏地定量分析一个分析物组。然而，所选 51 种类固醇和类固醇硫酸盐（包括许多异构体）的分析物组的高度结构相似性，给色谱选择性的实现带来了挑战。几十年来，各种体液中内源性类固醇的分析主要通过气相色谱（GC）与（串联）质谱（MS(/MS)）联用进行。由于感兴趣的类固醇的结构，需要进行衍生化以满足 GC-MS(/MS) 的分析要求。大多数实验室在多分析物测定中使用两步衍生化，这一方法早在 20 世纪 80 年代就已经发表。然而，对于某些类固醇，这种复杂的程序会产生多种异构体衍生物。因此，一些实验室利用（超）高效液相色谱（UHPLC）-MS/MS 作为替代方法，但即使是 UHPLC 也无法分离某些异构体对。超临界流体色谱（SFC）作为与 GC 和（UHPLC）正交的分离技术，可能有助于克服这些问题。在本项目中，研究并比较了两种最有前途的内源性类固醇分析方法：“金标准”GC-MS 和正交分离技术 SFC-MS/MS。探索了不同的衍生化程序以进行气相色谱检测，并描述和确认了多种衍生物的形成。总的来说，没有一种研究的衍生化程序能提供可接受的结果，以满足本项目的要求进一步开发方法。SFC 具有独特的选择性，能够克服这些问题并区分所有选定的类固醇，包括孕激素、雄激素、皮质类固醇、雌激素和类固醇硫酸盐，具有适当的选择性。SFC 在分离对映异构体分析物方面特别有价值，它已经显示出作为 GC 的替代方法的潜力。不同的 SFC 柱上 51 种类固醇和类固醇硫酸盐的成功分离表明了 SFC 在内源性类固醇分析中的潜力。
