Determination of neurosteroids in human cerebrospinal fluid in the 21st century: A review.

Determination of steroid hormones synthesized by the human body plays an important role in various fields of endocrinology. Neurosteroids (NS) are steroids that are synthesized in the central (CNS) or peripheral nervous system (PNS), which is not only a source but also a target for neurosteroids. They are discussed as possible biomarkers in various cognitive disorders and research interest in this topic raises continuously. Nevertheless, knowledge on functions and metabolism is still limited, although the concept of neurosteroids was already introduced in the 1980s. Until today, the analysis of neurosteroids is truly challenging. The only accessible matrix for investigations of brain metabolism in living human beings is cerebrospinal fluid (CSF), which therefore becomes a very interesting specimen for analysis. However, neurosteroid concentrations are expected to be very low and the available amount of cerebrospinal fluid is limited. Further, high structural similarities of endogenous neurosteroids challenges analysis. Therefore, comprehensive methods, highly selective and sensitive for a large range of concentrations for different steroids in one aliquot are required and under continuous development. Although research has been increasingly intensified, still only few data are available on reference levels of neurosteroids in human cerebrospinal fluid. In this review, published literature of the last twenty years, as a period with relatively contemporary analytical methods, was systematically investigated. Considerations on human cerebrospinal fluid, different analytical approaches, and available data on levels of in analogy to periphery conceivable occurring neurosteroids, including (pro-) gestagens, androgens, corticoids, estrogens, and steroid conjugates, and their interpretation are intensively discussed.

Splitless hyphenation of SFC with MS by APCI, APPI, and ESI exemplified by steroids as model compounds.

A systematic evaluation of splitless hyphenation of supercritical fluid chromatography (SFC) with mass spectrometry (MS) was performed using different techniques for ambient pressure ionization. Interfaces commonly known from HPLC-MS/MS, i.e. electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo ionization (APPI), were tested for their suitability in SFC-MS/MS. A triple quadrupole MS was used for data evaluation in a targeted multi-analyte design using endogenous steroids as model compounds. Individual optimization of the ionization parameters was performed in multi-dimensional design for best support of ionization in all three techniques. A post-column make-up was used to avoid analyte precipitation in the transfer capillary but also to support ionization independently from mobile phase composition. Buffer choice and concentration as well as temperature were found crucial in ESI and APCI. Best results for the multi-analyte method were obtained in both techniques using ammonium fluoride as make-up buffer. Instead of buffer solutions different organic solvents were used as dopants in APPI to support ionization. The mobile phase constituent isopropanol was already found to support ionization in APPI, however, for many analytes the addition of toluene resulted in superior results in terms of intensity. Comparing the optimized methods in terms of limit of detection (LOD), limit of quantification (LOQ), and sensitivity (slope of calibration curve) ESI was the best choice for the multiple analyte design. Only a few analytes resulted in a different optimum ionization, if focused on separately. In terms of linear dynamic range, APCI and APPI proved superior to ESI, where calibration over the whole range of concentrations (from LOD up to 5000 pg ∗ µL-1) required quadratic regression.

Methods in endogenous steroid profiling - A comparison of gas chromatography mass spectrometry (GC-MS) with supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS).

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.