Hybridspe

Blood for plasma alkylresorcinol analysis were drawn in a non-fasting state. Plasma alkylresorcinols were measured using normal-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), after extraction from EDTA-plasma using supported liquid extraction (SLE) [28 (link)]. Briefly, 20 µL of 10 ng/mL internal standard (alkylresorcinol C19:0 d4, Reseachem AG, Bergsdorf, Switzerland) was added to 100 µL of plasma, and the sample extracted twice with 800 µL acetone on a SLE plate (HybridSPE, Sigma-Aldrich). Pooled extract was dried and resuspended in 50 µL of heptane:ethanol (95:5 v/v) and 10 µL injected onto an LC-MS/MS system (Shimadzu LCMS 8040, Shimadzu Europa GmbH, Duisburg, Germany). Alkylresorcinol homologues C17:0, C19:0, C21:0, C23:0 and C25:0 were quantified relative to the internal standard. Batch quality control samples had inter- and intra-batch variation < 15%.

All experiments were performed with a method that is capable of quantifying fifteen steroids in a single run and is described in detail elsewhere [6 (link)]. In short, the assay characteristics are here described for aldosterone (ALDO) and 17-hydroxyprogesterone (17P)—the analytes covered by this work. Prior to analysis, 100 µL of a calibrator, QC or serum sample were spiked with 20 µL of an internal standard solution (Chromsystems) and then subjected to protein precipitation by a methanolic zinc sulphate solution followed by phospholipid removal by the means of HybridSPE (Merck/Sigma Aldrich). A 40 µL aliquot of the purified sample was used for analysis with the LC-MS/MS system (Sciex API6500+ triple quad mass spectrometer hyphenated to an Agilent 1290 Infinity II UHPLC). Chromatographic separation was performed on a biphenyl stationary phase (Restek Raptor Biphenyl, 100 mm × 2.1 mm, 2.7 µm, BGB Analytik, Boeckten, Switzerland) by gradient elution (water/methanol with 0.2 mM NH4F as additive), resulting in retention times of 2.8 min (ALDO) and 3.8 min (17P). Analyte detection was performed in the positive ESI mode for both analytes, recording two mass transitions each starting from the respective [M+H]⁺ peaks (ALDO: m/z 361.3 → 315.2/343.2; 17P: m/z 331.3 → 97.0/109.1).