Parallel targeted and non-targeted quantitative analysis of steroids in human serum and peritoneal fluid by liquid chromatography high-resolution mass spectrometry

Abstract

We developed and validated a liquid chromatography high-resolution mass spectrometry method for the absolute quantification of 51 steroids for clinical analysis of human serum and, for the first time, peritoneal fluid. Data acquisition was performed in both targeted and untargeted mode simultaneously, thus allowing the accurate and precise quantification of the main components of the classical steroid pathways (17 steroids) as well as the analysis of 34 additional non-classical steroids. For targeted analysis, validation was performed according to FDA guidelines, resulting, among other parameters, in accuracy < 13% RSD and precision < 10% relative error, for both inter- and intra-day validation runs. By establishing steroid-specific response factors, the calibration curves of the targeted analytes can be extended to untargeted analytes. This approach opens novel possibilities for the post hoc analysis of clinical samples as the data can be examined for virtually any steroid even after data acquisition, enabling facile absolute quantification once a standard becomes available. We demonstrate the applicability of the approach to evaluate the differences in steroid content between peripheral serum and peritoneal fluid across the menstrual cycle phases, as well as the effect of the synthetic gestagen dienogest on the steroid metabolome. Graphical abstract

No extra-adrenal aldosterone production in various human cell lines

“…For the LC-MS analysis, 500 µL cell aliquots were spiked with 38 µL internal standard mix and steroids subsequently extracted using solid-phase extraction on an OasisPrime HLB 96-well plate according to the protocol previously published ( Andrieu et al 2022 ). The LC-MS system consists of a Vanquish UHPLC (equipped with an ACQUITY UPLC HSS T3 Column, 100 Å, 1.8 µm, 1 mm × 100 mm; Waters, Switzerland) coupled to a Q Exactive Orbitrap Plus (both from ThermoFisher Scientific).…”

Adrenal Abcg1 Controls Cholesterol Flux and Steroidogenesis

“…This terminal procedure was chosen because it allowed the collection of paired blood and adrenal tissue samples while causing a significantly lower stress response in mice compared to other euthanasia methods ( 35 ). Twenty-five µL of serum was used for further liquid chromatography-mass spectrometry (LC-MS) analysis using an established in house LC-MS method ( 36 ). Briefly, samples were collected and stored at −20 °C.…”

Exploring the Potential of Sulfur Moieties in Compounds Inhibiting Steroidogenesis

“…Steroid profiling using mass spectrometry: Steroid profiling in NCI H295R cells was performed using a liquid chromatography high-resolution mass spectrometry (LC-HRMS) method as previously described and validated with 1 µM of the unlabeled substrate added to the cell culture [ 28 , 29 , 30 ]. Steroids from 500 µL cell media, plus 38 µL of a mixture of internal standards (at 3.8 nM each), were extracted using solid-phase extraction with an OasisPrime HLB 96-well plate.…”