Simultaneous Analysis of Free and Sulfated Steroids by Liquid Chromatography/Mass Spectrometry with Selective Mass Spectrometric Scan Modes and Polarity Switching

Abstract

Sulfated steroids can act as a latent form of active free steroids, coexisting with them in biological specimens. To evaluate the metabolic significance of free and sulfated steroid species, a simultaneous analysis of eight free steroids [cholesterol, pregnenolone, 17α-hydroxypregnenolone, progesterone, 17α-hydroxyprogesterone, dehydroepiandrosterone (DHEA), testosterone, and 17β-estradiol] and four biologically relevant sulfated steroids was developed and validated, using selected-ion and multiple-reaction monitoring modes coupled to polarity-switching liquid chromatography/mass spectrometry (LC/MS). All steroids were separated on a reversed-phase phenyl column (50 mm × 2 mm, 3 μm) at a flow rate of 0.5 mL/min. The limits of quantification ranged from 0.1 to 50 ng/mL at extraction recoveries of 94.1-105.5%, while the precision and accuracy were 2.5-9.3% and 92.4-105.9%, respectively. Quantitative results obtained for samples from obese girls showed that the serum levels of DHEA sulfate were significantly increased (P = 0.004), along with the metabolic ratio representing DHEA sulfotransferase (P < 0.02). The developed novel LC/MS method can quantitatively profile both free and sulfated steroids in a single analytical run.

Sulfate-based lipids: Analysis of healthy human fluids and cell extracts

“…In fact, targeted MS-based approaches have been widely used for the SL detection but also due to their ability for structural characterization by tandem mass spectrometry (MS/MS) experiments. Despite the many studies conducted on the identification of SL in plasma (Lee et al, 2016;Mirzaian et al, 2015b), saliva (Slomiany et al, 1988(Slomiany et al, , 1983(Slomiany et al, , 1978, urine (Yao et al, 2016), tear fluid (Lam et al, 2014), gastric fluid (Iwamori et al, 2000), seminal plasma (Sion et al, 2001), lipoproteins (Reis et al, 2013;Serna et al, 2015), red blood cells (Muskiet et al, 1983;Przybylska et al, 1995), blood platelets (Blache et al, 1995), and tissues brain (Moyano et al, 2013), kidney (Tadano-Aritomi et al, 2001), eye lenses (Seng et al, 2014), sperm cells (Fujimoto et al, 2000;Kongmanas et al, 2010;Zhang et al, 2005), hair (Cho et al, 2010;Drosche et al, 1994;Serizawa et al, 1990) and nails (Serizawa et al, 1990) and quantification by multi-reaction monitoring (MRM) (Fong et al, 2013;Kongmanas et al, 2010;Moyano et al, 2013;Sánchez-Guijo et al, 2015;Yao et al, 2016), these were mostly focused on specific steroid sulfates in individual fluids and cells (Esquivel et al, 2018;Fong et al, 2013;Lam et al, 2014;Przybylska et al, 1995;Sánchez-Guijo et al, 2015;Sion et al, 2001;Wang et al, 2018) leaving a whole panel of hig...…”

“…Sulfolipids are a heterogenous class of lipids containing sulfur atom in its structure (Farooqui and Horrocks, 1985;Goldberg, 1961) present in its oxidised state as sulfate-based lipids (SL) and sulfonate-base lipids, or in its reduced state as thioether or thiol ester compounds linked to acyl chains with amino acids. SL appear to be ubiquitous to the whole body and dispersed across fluids (Lam et al, 2014;Larsson et al, 1996;Lee et al, 2016;Muskiet et al, 1983;Sion et al, 2001;Yao et al, 2016) and tissues (Cho et al, 2010;Fujimoto et al, 2000;Moyano et al, 2014;Seng et al, 2014;Serizawa et al, 1990). SL are catalysed by a supergene family of enzymes called sulfotransferases (SULTs) with the transfer of a sulfuryl group (SO3-) to an acceptor hydroxyl group resulting in an increase of water solubility and decrease of biological activity and at the cellular level, SL appear to be gathered in lipid-raft domains (Weerachatyanukul et al, 2007) at the surface of cells (Honke, 2018) contributing to membrane´s electronegative surface charge at physiological pH and thus involved in cell-cell communication processes (Farooqui and Horrocks, 1985;Honke, 2018;Strott and Higashi, 2003) in inflammation (Hu et al, 2007), immunity (Avila et al, 1996;Merten et al, 2001) and infection (Suzuki et al, 2003).…”

“…Despite their occurrence in apparently most fluids and tissues, research on SL is scarce and mostly focused on steroid sulfates (Cho et al, 2010;Eberlin et al, 2010;Esquivel et al, 2018;Lafaye et al, 2004;Lam et al, 2014;Lee et al, 2016;Przybylska et al, 1995;Sánchez-Guijo et al, 2015;Sion et al, 2001;Wang et al, 2018;Yao et al, 2016) due to the relevance of steroid sulfates in sports and anti-doping areas, while SL bearing a glycerol-backbone and a sphingosine-backbone (Scheme 1) remain less studied (Mirzaian et al, 2015a;Slomiany et al, 1978). Due to the SL structural diversity, analysis of SL in complex biological samples is not straightforward limiting the exploitation of SL diagnostic and prognostic value.…”

SULFATION PATHWAYS: Insights into steroid sulfation and desulfation pathways

“…Nevertheless, all these assays require separate runs for the conjugated and free steroids. An integrated method for quantifying free and sulfated steroids in a single LC-MS/ MS run was recently described (Lee et al 2016). It used both SIM and MRM modes as well as polarity switching and was capable of detecting eight free steroids and four sulfated ones.…”

Serum Steroid Ratio Profiles in Prostate Cancer: A New Diagnostic Tool Toward a Personalized Medicine Approach

“…In addition to quantification of steroids by LC-MS/MS, other investigations show that the clinical information correlated with the disease is not only contained in the absolute steroid quantitation, but rather in their ratios ( 33 , 34 ). This reflects their synthesis starting from the precursor cholesterol and the enzymes involved (Figure S1 in Supplementary Material).…”