Steroid analysis

Analysis of steroids by tandem mass spectrometry coupled with gas chromatography

Specialized in the analysis of neurosteroids, this platform makes it possible to characterize the entire steroidome, from tissues of the central and peripheral nervous system as well as biological fluids of animal or human origin.

Activities

The analytical chemistry technological platform makes it possible to characterize a complete steroidome from tissues and biological fluids thanks to the cutting-edge technology of quadrupole tandem mass spectrometry coupled with very high resolution gas chromatography (GC-MS/ MS). It is today the most sensitive reference technology (up to the femtomole), and precise for determining a steroid map allowing the analysis of up to 80 steroids, including progestogens, androgens, estrogens, gluco- and mineralocorticoids , steroid sulfates, oxysterols, vitamin D and all their precursors and metabolites. The specificity of this technology makes it possible to unambiguously characterize steroids with very similar structures such as position isomers or stereoisomers.

The combination of a sample processing protocol including extraction, solid phase extraction (SPE) and high performance liquid chromatography (HPLC) steps, with the actual analysis by GC-MS/MS, allows to analyze steroids with very different physicochemical properties in any type of tissues (a few mg) such as cerebral sub-regions, adrenal glands, testicles, liver, on whole organisms (C. Elegans, zebrafish…) as well than any type of biological fluids (25-50 µl) such as plasma, saliva, cerebrospinal fluid and urine, with very high reliability. The introduction of steroids labeled with stable isotopes (13C- or 2H) as internal standards gives access to very precise quantification over the entire range of steroids.

The expertise of our group is recognized nationally and internationally and our platform is at the center of many collaborations, whether in fundamental or clinical research. We carry out analyzes as part of the unit’s research projects, with national (INSERM, CNRS, INRAE) and international external academic partners. We also have collaborations for clinical studies (Kremlin-Bicêtre University Hospital, Tenon Hospital) and provide services for national and international industrial partners. Our platform is unique and internationally recognized in the fields of endocrinology, neuroendocrinology and neurosciences, and is at the origin of major discoveries on various pathologies, whether in experimental animal models (mouse, rat, etc.) or humans.

The analytical platform is made up of 2 laboratories. A laboratory is dedicated to the different stages of sample preparation, for the extraction, fractionation and purification of biological extracts. The second laboratory is dedicated to the chemical derivation of steroids and analysis by mass spectrometry.

Equipements

The sample preparation laboratory is equipped with an ultracentrifuge, a sonication bath, a water bath heated to 60°C and surmounted by an evaporation system under compressed air for the extraction from tissues and biological fluids, and 3 chemical fume cupboards. It is also composed of a Solid Phase Extraction (SPE) system which allows, by selective adsorption of steroids according to their physicochemical properties on a hydrophobic solid phase, to purify the biological extracts by eliminating the majority of the more hydrophilic compounds and more hydrophobic while isolating the more hydrophilic steroids (sulfate steroids) from unconjugated steroids. The SPE device makes it possible to process 24 samples simultaneously and to carry out the filtration of the biological extracts. These are then again purified and fractionated by High Performance Liquid Chromatography (HPLC) coupled to a fraction collector. This step allows isolation of the more apolar ketone steroids from the more polar hydroxylated steroids because the subsequent step of chemical derivation is specific to the chemical structure of the steroids.

Le laboratoire analytique est climatisé et à humidité contrôlée. Il est doté d’un poste de dérivation chimique avec un bac à température contrôlé surmonté d’un système d’évaporation sous azote. Cette étape indispensable permet de réaliser des réactions d’acylation des stéroïdes hydroxylés afin d’augmenter la sensibilité et la sélectivité analytique.

The platform is composed of 3 mass spectrometers

– a quadripolar type mass spectrometer coupled to a gas phase chromatograph (GC-MS) TRACE 1300 – ISQ (ThermoFisher) which allows the development of the identification of steroids and the analysis of steroid sulfates.

– two tandem quadrupole mass spectrometers coupled to a gas chromatograph (GC-MS/MS): TRACE 1300 – TSQ8000 (ThermoFisher) and TRACE 1300 – TSQ9000 (ThermoFisher). GC-MS/MS is the reference technique in terms of sensitivity and specificity for identifying and quantifying steroids in purified biological extracts.

Team

Resp. Opérationel. Philippe LIERE (ingénieur de recherche, Philippe.Liere@inserm.fr)

Antoine PIANOS (ingénieur d’étude)

Maria Sanchez Garcia (assistante ingénieure, CDD)

Resp. Scientifique. Michael Schumacher (DR1, Michael.Schumacher@inserm.fr)

Principales publications

– Liere P, Liu GJ, Pianos A, Middleton RJ, Banati RB, Akwa Y. 2023. The Comprehensive Steroidome in Complete TSPO/PBR Knockout Mice under Basal Conditions. International J. Molecular Sciences. 24, 2474.

– Fernandez N, Petit A, Pianos A, Haddad L, Schumacher M, Liere P, Guennoun R. 2023. Aging Is Associated With Lower Neuroactive Steroids and Worsened Outcomes Following Cerebral Ischemia in Male Mice. Endocrinology.164, 1–24.

– Goudet G, Douet C, Pianos A, Haddad L, , Reigner F, Deleuze S, Liere P. 2022. Saliva and plasma steroidome in mare during reproductive stages: A GC-MS/MS study. Frontiers in Animal Science. 3, 10.3389/fanim.1055179.hal-03841907.

– Vacher CM, O’Reilly JJ, Salzbank J, Lacaille H, Bakalar D, Sebaoui-Illoul S, Liere P, Clarkson-Paredes C, Sasaki T, Sathyanesan A, Imamura Kawasawa Y, Popratiloff A, Hashimoto-Torii K, Gallo V, Schumacher M, Penn AA. 2021. Placental neurosteroids shape cerebellar development and social behavior. Nature Neuroscience. 24, 1392-1401.

– Liere P, Cornil CA, de Bournonville MP, Pianos A, Keller M, Schumacher M, Balthazart J. 2019. Steroid profiles in quail brain and serum: Sex and regional differences and effects of castration with steroid replacement. J Neuroendocrinol. Feb;31(2):e12681

– Berkane N, Liere P, Lefevre G, Alfaidy N, Nahed RA, Vincent J, Oudinet JP, Pianos A, Cambourg A, Rozenberg P, Galichon P, Rousseau A, Simon T, Schumacher M, Chabbert-Buffet N, Hertig A. 2018. Abnormal steroidogenesis and aromatase activity in preeclampsia. Placenta. Sep;69:40-49.

– Guennoun R, Fréchou M, Gaignard P, Liere P, Slama A, Schumacher M, Denier C, Mattern C. 2019. Intranasal administration of progesterone: A potential efficient route of delivery for cerebroprotection after acute brain injuries. Neuropharmacology. Feb;145(Pt B):283-291.

– Zhu X, Fréchou M, Liere P, Zhang S, Pianos A, Fernandez N, Denier C, Mattern C, Schumacher M, Guennoun R. 2017. A Role of Endogenous Progesterone in Stroke Cerebroprotection Revealed by the Neural-Specific Deletion of Its Intracellular Receptors. J Neurosci. Nov 8;37(45):10998-11020.

– Liere P, Pianos A, Oudinet JP, Schumacher M, Akwa Y. 2017. Differential effects of the 18-kDa translocator protein (TSPO) ligand etifoxine on steroidogenesis in rat brain, plasma and steroidogenic glands: Pharmacodynamic studies. Psychoneuroendocrinology. 83:122134.

– Gonzalez Deniselle MC, Liere P, Pianos A, Meyer M, Aprahamian F, Cambourg A, Di Giorgio NP, Schumacher M, De Nicola AF, Guennoun R. 2016. Steroid Profiling in Male Wobbler Mouse, a Model of Amyotrophic Lateral Sclerosis. Endocrinology. 157(11):4446-4460.

– Liere P, Schumacher M. 2015. Mass spectrometric analysis of steroids: all that glitters is not gold. Expert Rev Endocrinol Metab 10:463-465.

– Schumacher M, Guennoun R, Mattern C, Oudinet JP, Labombarda F, De Nicola AF, Liere P. 2015. Analytical challenges for measuring steroid responses to stress, neurodegeneration and injury in the central nervous system. Steroids. 103/42-57. Review

– Liu A, Margaill I, Zhang S, Labombarda F, Coqueran B, Delespierre B, Liere P, Marchand-Leroux C, O’Malley BW, Lydon JP, De Nicola AF, Sitruk-Ware R, Mattern C, Plotkine M, Schumacher M, Guennoun R. 2012. Progesterone receptors: a key for neuroprotection in experimental stroke. Endocrinology. 153(8):3747-57.

– Hertig A, Liere P, 2010. New markers in preeclampsia. Clin Chim. Acta, 411, 1591-1595.

– Hertig A, Liere P, Chabbert-Buffet N, Fort J, Pianos A, Eychenne B, Cambourg A, Schumacher M, Berkane N, Lefevre G, Uzan S, Rondeau E, Rozenberg P, Rafestin-Oblin ME. 2010. Steroid profiling in preeclamptic women:evidence for aromatase deficiency. Am. J. Obstet. Gynecol., 203(5): 03(5):477.e1-9.

– Liere P, Pianos A, Eychenne B, Cambourg A, Bodin K, Griffiths W, Schumacher M, Baulieu EE, Sjövall J. 2009. Analysis of pregnenolone and dehydroepiandrosterone in rodent brain: cholesterol autoxidation is the key. J Lipid Res. 50(12):2430-44.

– Schumacher M, Liere P, Akwa Y, Rajkowski K, Griffiths W, Bodin K, Sjövall J, Baulieu EE. 2008. Pregnenolone sulfate in the brain: a controversial neurosteroid. Neurochem Int. 52(4-5):522-40.

– Meffre D, Pianos A, Liere P, Eychenne B, Cambourg A, Schumacher M, Stein DG, Guennoun R. 2007. Steroid profiling in brain and plasma of male and pseudopregnant female rats after traumatic brain injury: analysis by gas chromatography/mass spectrometry. Endocrinology. 148(5):2505-17.

– Broué F, Liere P, Kenyon C, Baulieu EE. 2007. A steroid hormone that extends the lifespan of Caenorhabditis elegans. Aging Cell. 6(1):87-94.

– Liere, P., A. Pianos, B. Eychenne, A. Cambourg, S. Liu, W. Griffiths, M. Schumacher, J. Sjovall and E. E. Baulieu. 2004. Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone and absence of their sulfated counterparts in rodent brain. J Lipid Res., 45, 2287-2302.

– Weill-Engerer, S., J. P. David, V. Sazdovitch, P. Liere, B. Eychenne, A. Pianos, M. Schumacher, A. Delacourte, E. E. Baulieu and Y. Akwa. 2002. Neurosteroid quantification in human brain regions: comparison between Alzheimer’s and nondemented patients. J Clin Endocrinol Metab. 87: 5138-43.

– P. Liere, Y. Akwa, S. Weill-Engerer, B. Eychenne, A. Pianos, P. Robel, J. Sjövall, M. Schumacher and E.E. Baulieu, 2000, Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry, Journal of Chromatography B., 739, 301.