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Highlights • Steroid analysis using LC/ESI-MS/MS is reliable and sensitive. • ESI efficiency of estrogens is limited. • Chemical derivatization of steroids improves their detectability. • Simultaneous quantification of E2, E1, cortisol and cortisone was achieved., The presence of estrogens, androgens and glucocorticoids as well as their receptors and steroid converting enzymes in adipose tissue has been established. Their contribution to diseases such as obesity, diabetes and hormone-dependent cancers is an active area of research. Our objective was to develop a LC–MS/MS method to quantify bioactive estrogens and glucocorticoids simultaneously in human adipose tissue. Estrogens and glucocorticoids were extracted from adipose tissue samples using solid-phase extraction. Estrogens were derivatized using 1-(2,4-dinitro-5-fluorophenyl)-4-methylpiperazine (PPZ) and methyl iodide to generate a permanently charged molecule (MPPZ). Steroids were separated and quantified by LC–MS/MS. The limit of quantitation for the steroids was between 15 and 100 pg per sample. Accuracy and precision were acceptable (

Estrogens regulate many diverse biological processes in health and disease. They circulate at a wide range of concentrations in females generating several active metabolites (hydroxy and methoxyestrogens). The metabolites are assumed to be present in much lower levels and are thought to contribute to diseases such as pulmonary arterial hypertension (PAH). Estrogen metabolites are challenging to quantify in plasma and currently available immunoassays are non-specific. Here we have developed and validated a novel assay to simultaneously quantify parent estrogens and their metabolites by mass spectrometry (MS). Estrogens were extracted from human plasma using solid phase extraction and derivatized using 1-(5-fluoro-2, 4-dinitrophenyl)-4-methylpiperazine (PPZ) before quaternization by methylation (“MPPZ”). MPPZ derivatives were separated and quantified by liquid chromatography tandem MS (LC-MS/MS) in positive electrospray ionization mode, using a QTrap 6500 + coupled to a Shimadzu Nexera X2. Separation was achieved using an ACE Excel 2 C18-PFP column (2 μm, 2.1 mm × 150 mm). The limits of quantification (LOQ) were 0.43–2.17 pg on column with a linear range from 2 or 10 - 2000 pg mL-1. Intra and inter-day precision and accuracy were acceptable (, Graphical abstract Image 1, Highlights • LC-MS/MS method for simultaneous quantification of estrogens and their bioactive metabolites in human plasma. • High recoveries and reduced matrix effects using MCX-SPE® extraction cartridges followed by MPPZ derivatization. • Generation of stable derivatives allowing quantification of estrogens in low endogenous levels.

Estrogens circulate at concentrations less than 20 pg/mL in men and postmenopausal women, presenting analytical challenges. Quantitation by immunoassay is unreliable at these low concentrations. Liquid chromatography tandem mass spectrometry (LC–MS/MS) offers greater specificity and sometimes greater sensitivity, but ionization of estrogens is inefficient. Introduction of charged moieties may enhance ionization, but many such derivatives of estrogens generate non-specific product ions originating from the “reagent” group. Therefore an approach generating derivatives with product ions specific to individual estrogens was sought. Estrogens were extracted from human plasma and serum using solid phase extraction and derivatized using 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS). Electrospray in positive mode with multiple reaction monitoring using a QTrap 5500 mass spectrometer was used to quantify “FMP” derivatives of estrogens, following LC separation. Transitions for the FMP derivatives of estrone (E1) and estradiol (E2) were compound specific (m/z 362→238 and m/z 364→128, respectively). The limits of detection and quantitation were 0.2 pg on-column and the method was linear from 1–400 pg/sample. Measures of intra- and inter-assay variability, precision and accuracy were acceptable (, Graphical abstract fx1, Highlights • Quantitative analysis of low amounts of estrone and estradiol in plasma and serum. • Quantitation across physiological range in men and pre- and post-menopausal women. • Methylpyridinium ether derivatives improve analytical specificity and sensitivity.