The dynamics of a serum steroid profile after stimulation with intravenous ACTH.

BACKGROUND: Stimulation with intravenous adrenocorticotropic hormone (ACTH) is a widely used diagnostic procedure to characterize the adrenocortical function. Currently, the response of serum cortisol, mainly quantified by immunoassays, is the only established read-out of this test. By using liquid chromatography coupled with mass spectrometry (LC-MS/MS) simultaneous determination of several steroids that respond to ACTH stimulation is now possible. The aim of this study was to further characterize the typical effect of exogenous ACTH (250 mg) on a LC-MS/MS-serum steroid profile. METHODS: A set of 36 paired samples (pre-/post-IV-ACTH) was investigated (age range 22-58, 26 female and 10 male individuals). Serum steroid profiling was performed using a LC-MS/MS method covering cortisol, cortisone, corticosterone, 11-deoxycortisol, 17-OH-progesterone and 11-deoxycorticosterone. RESULTS: The concentrations of all measured steroids increased after stimulation with ACTH, except for cortisone. Serum corticosterone, 11-deoxycorticosterone and 11-deoxycortisol showed markedly more pronounced relative increases compared to cortisol. The strongest response was observed for corticosterone (15-fold median relative increase, compared to 1.4-fold median increase of cortisol). CONCLUSION: Serum steroid profiling using LC-MS/MS after stimulation with IV ACTH demonstrates highly dynamic response patterns. Further studies should address in particular serum corticosterone as a potential novel marker of biochemical stress response.

Ferromagnetic particles as a rapid and robust sample preparation for the absolute quantification of seven eicosanoids in human plasma by UHPLC-MS/MS.

We used ferromagnetic particles as a novel technique to deproteinize plasma samples prior to quantitative UHPLC-MS/MS analysis of seven eicosanoids [thromboxane B2 (TXB2), prostaglandin E2 (PGE2), PGD2, 5-hydroxyeicosatetraenoic acid (5-HETE), 11-HETE, 12-HETE, arachidonic acid (AA)]. A combination of ferromagnetic particle enhanced deproteination and subsequent on-line solid phase extraction (on-line SPE) realized quick and convenient semi-automated sample preparation-in contrast to widely used manual SPE techniques which are rather laborious and therefore impede the investigation of AA metabolism in larger patient cohorts. Method evaluation was performed according to a protocol based on the EMA guideline for bioanalytical method validation, modified for endogenous compounds. Calibrators were prepared in ethanol. The calibration curves were found to be linear in a range of 0.1-80ngmL(-1) (TXB2, PGE2, PGD2), 0.05-40ngmL(-1) (5-HETE, 11-HETE), 0.5-400ngmL(-1) (12-HETE) and 25-9800ngmL(-1) (AA). Regarding all analytes and all quality controls, the resulting precision data (inter-assay 2.6 %-15.5 %; intra-assay 2.5 %-15.1 %, expressed as variation coefficient) as well as the accuracy results (inter-assay 93.3 %-125 %; intra-assay 91.7 %-114 %) were adequate. Further experiments addressing matrix effect, recovery and robustness, yielded also very satisfying results. As a proof of principle, the newly developed LC-MS/MS assay was employed to determine the capacity of AA metabolite release after whole blood stimulation in healthy blood donors. For this purpose, whole blood specimens of 5 healthy blood donors were analyzed at baseline and after a lipopolysaccharide (LPS) induced blood cell activation. In several baseline samples some eicosanoids levels were below the Lower Limit of Quantification. However, in the stimulated samples all chosen eicosanoids (except PGD2) could be quantified. These results, in context with those obtained in validation, demonstrate the applicability of ferromagnetic particles for the sample preparation for eicosanoids in human plasma. Thus, we conclude that ferromagnetic particle enhanced deproteination is a promising novel tool for sample preparation in LC-MS/MS, which is of particular interest for automation in clinical mass spectrometry, e.g. in order to further address eicosanoid analysis in larger patient cohorts.

Isotope Inversion Experiment evaluating the suitability of calibration in surrogate matrix for quantification via LC-MS/MS-Exemplary application for a steroid multi-method.

For quotable quantitative analysis of endogenous analytes in complex biological samples by isotope dilution LC-MS/MS, the creation of appropriate calibrators is a challenge, since analyte-free authentic material is in general not available. Thus, surrogate matrices are often used to prepare calibrators and controls. However, currently employed validation protocols do not include specific experiments to verify the suitability of a surrogate matrix calibration for quantification of authentic matrix samples. The aim of the study was the development of a novel validation experiment to test whether surrogate matrix based calibrators enable correct quantification of authentic matrix samples. The key element of the novel validation experiment is the inversion of nonlabelled analytes and their stable isotope labelled (SIL) counterparts in respect to their functions, i.e. SIL compound is the analyte and nonlabelled substance is employed as internal standard. As a consequence, both surrogate and authentic matrix are analyte-free regarding SIL analytes, which allows a comparison of both matrices. We called this approach Isotope Inversion Experiment. As figure of merit we defined the accuracy of inverse quality controls in authentic matrix quantified by means of a surrogate matrix calibration curve. As a proof-of-concept application a LC-MS/MS assay addressing six corticosteroids (cortisol, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, and 17-OH-progesterone) was chosen. The integration of the Isotope Inversion Experiment in the validation protocol for the steroid assay was successfully realized. The accuracy results of the inverse quality controls were all in all very satisfying. As a consequence the suitability of a surrogate matrix calibration for quantification of the targeted steroids in human serum as authentic matrix could be successfully demonstrated. The Isotope Inversion Experiment fills a gap in the validation process for LC-MS/MS assays quantifying endogenous analytes. We consider it a valuable and convenient tool to evaluate the correct quantification of authentic matrix samples based on a calibration curve in surrogate matrix.