Johanna Lindner (Presenter)
Institute of Laboratory Medicine, LMU Munich
Authorship: Johanna Lindner (1), Michael Vogeser (1), Stefanie H. Grimm (1)
(1) Institute of Laboratory Medicine, Hospital of the Ludwig Maximilians Universität Munich, Germany
| Short Abstract We developed an UHPLC-MS/MS assay that combines a simple sample preparation with a powerful MS method quantifying a broad steroid panel (cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 17-OH-progesterone, 11-deoxycorticosterone, progesterone, aldosterone, testosterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate) in human serum. After a manual protein precipitation step, the eluates were directly injected into the UHPLC-MS system. Stable isotope-labelled counterparts of the targeted analytes were employed as internal standards. For detection the mass spectrometer operated in the ESI positive mode. Chromatographic separation of all isobaric compounds was achieved employing a Kinetex Biphenyl column. In combination with a mobile phase consisting of 0.2 mM ammonium fluoride and methanol we were able to establish a selective and sensitive method. |
Long Abstract
Background: Steroid hormones and their corticoid precursors are important biomarkers to diagnose and monitor a broad spectrum of endocrine disorders. For a better understanding of these disorders, the assessment of steroid profiles is an important field in clinical research. Therefore, we developed an UHPLC-MS/MS assay that combines a simple and effective sample preparation with a powerful MS method quantifying a broad steroid panel (cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 17-OH-progesterone, 11-deoxycorticosterone, progesterone, aldosterone, testosterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate).
Methods: After a manual protein precipitation step using zinc trifluoroacetate in methanol as a novel approach, the eluates were directly injected into the UHPLC-MS system. For chromatographic separation we investigated different stationary and mobile phases. In order to enable a reliable quantification the respective stable isotope-labelled counterparts of the targeted analytes were employed as internal standards. For detection we used a Xevo TQ-S mass spectrometer operating in the ESI positive mode. Multiple reaction monitoring was performed for all analytes.
Results: Excellent chromatographic separation of all isobaric compounds (corticosterone ↔ 11-deoxycortisol ↔ 21-deoxycortisol and 17-OH-progesterone ↔ 11-deoxycorticosterone) was achieved employing a Kinetex Biphenyl column (150 x 2.1 mm, 1.7 µm). In combination with an innovative mobile phase consisting of 0.2 mM ammonium fluoride and methanol we were able to establish a highly selective and sensitive UHPLC-MS/MS method for the quantification of twelve endogenous steroids in human serum.
Conclusions: We could demonstrate that Biphenyl columns are a powerful tool for comprehensive, MS based steroid assays including various isobaric substances.
References & Acknowledgements:
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