Data for analysis of catechol estrogen metabolites in human plasma by liquid chromatography tandem mass spectrometry

Analysis of catechol estrogens (2 & 4 hydroxy-estrone and estradiol) has proven troublesome by liquid chromatography tandem mass spectrometry due to their low concentrations, short half-lives and temperature-labile nature. Derivatization to methyl piperazine analogues has been reported for a panel of 9 estrogens in, “Derivatization enhances analysis of estrogens and their bioactive metabolites in human plasma by liquid chromatography tandem mass spectrometry” (Denver et al., 2019). Data show alteration of the base catalyst in this method was required to allow detection of catechol estrogens to low levels. Data also highlight the challenges faced in chromatographic separation of isomers and isotopologues, which were partially overcome by employing an extended column length and reduced oven temperature. In addition, data analysis displayed significant matrix effects during quantitation in plasma, following solid-phase extraction, despite efficient recoveries

Derivatization enhances analysis of estrogens and their bioactive metabolites in human plasma by liquid chromatography tandem mass spectrometry

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 (<20% at LOQ and <15% above). These derivatives demonstrated minimal degradation upon short-term storage at 15 °C (<20%) and longer term at −20 °C (<20%). Using this approach, estrone (E1) and estradiol (E2) were detected in plasma (0.5 mL) from healthy women and those with PAH but downstream metabolites 16-hydroxy-E1, 16-hydroxy-E2, 2-methoxy-E1 and 4-methoxy-E1 were only detected in plasma from diseased patients. These findings will next be tested robustly in large patient cohorts. This novel LC-MS/MS analysis of estrogens and their bioactive metabolites, using MPPZ derivatization, opens doors for the simultaneous analysis of a panel of estrogens in human plasma, across the endogenous range of concentrations encountered in health and disease

Estrogen metabolites in a small cohort of patients with idiopathic pulmonary arterial hypertension.

Increased risk and severity of idiopathic pulmonary arterial hypertension (iPAH) is associated with elevated estradiol in men and postmenopausal women. Pulmonary arteries synthesise estradiol via aromatase and metabolise it via CYP1B1 to mitogenic metabolites; SNPs in aromatase and CYP1B1 have been associated with PAH. This suggests that estradiol metabolism could be altered in iPAH. This proof-of-concept study profiles estradiol and several metabolites of estradiol simultaneously in serum from iPAH patients and controls. We show that the estradiol and metabolite profile is altered in iPAH and that 16-hydroxyestrone and 16-hydroxyestradiol accumulate in iPAH patients with 16-hydroxyestrone levels relating to disease severity

A pilot study to examine association of BMI with functional class and six minute walk distance in idiopathic and heritable PAH : possible association with estrogen metabolism

Abstract: The hypothesis that a relationship exists between body mass index (BMI), functional class, and 6 min walk distance (6MWD) in Group 1‐pulmonary arterial hypertension (PAH) was examined. Analysis of data from the UK National Cohort Study for heritable pulmonary arterial/idiopathic PAH suggests increased BMI is a predictor of worse functional class and shorter 6MWD; increased body‐weight in mice and man may be associated with increased estrogen metabolism

Simultaneous quantification of estrogens and glucocorticoids in human adipose tissue by liquid-chromatography-tandem mass spectrometry

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 (<20%). Using this method, estradiol, estrone, cortisone and cortisol were quantified in adipose tissue from women with and without breast cancer. This novel assay of estrogens and glucocorticoids by LC–MS/MS coupled with derivatization allowed simultaneous quantification of a panel of steroids in human adipose tissue across the endogenous range of concentrations encountered in health and disease

Current strategies for quantification of estrogens in clinical research

Estrogens and their bioactive metabolites play key roles in regulating diverse processes in health and disease. In particular, estrogens and estrogenic metabolites have shown both protective and non-protective effects on disease pathobiology, implicating the importance of this steroid pathway in disease diagnostics and monitoring. All estrogens circulate in a wide range of concentrations, which in some patient cohorts can be extremely low. However, elevated levels of estradiol are reported in disease. For example, in pulmonary arterial hypertension (PAH) elevated levels have been reported in men and postmenopausal women. Conventional immunoassay techniques have come under scrutiny, with their selectivity, accuracy and precision coming into question. Analytical methodologies such as gas and liquid chromatography coupled to single and tandem mass spectrometric approaches (GC–MS, GC–MS/MS, LC–MS and LC–MS/MS) have been developed to quantify endogenous estrogens and in some cases their bioactive metabolites in biological fluids such as urine, serum, plasma and saliva. Liquid-liquid or solid-phase extraction approaches are favoured with derivatization remaining a necessity for detection in lower volumes of sample. The limits of quantitation of individual assays vary but are commonly in the range of 0.5–5 pg/mL for estrone and estradiol, with limits for their bioactive metabolites being higher. This review provides an overview of current approaches for measurement of unconjugated estrogens in biological matrices by MS, highlighting the advances in this field and the challenges remaining for routine use in the clinical and research environment

Sex and estrogen metabolism in Pulmonary Arterial Hypertension (PAH)

Pulmonary arterial hypertension (PAH) is a progressive, life-limiting disease, characterised by increased pulmonary vascular resistance and right ventricular hypertrophy leading to right heart failure and death. In PAH, a sexual dimorphism is present with a strong sex bias in disease penetrance (4:1 female to male ratio). Despite being more susceptible to the development of PAH, females display improved right ventricular (RV) function and survival rate in comparison to males. As a result, a sex paradox exists and sex hormones have been implicated in the development and progression of PAH, with numerous studies displaying roles for estrogen, its metabolites and receptors in disease progression. Little is known about the systemic endogenous levels of estrogen and its metabolites, or their relationship to sexual dimorphism in PAH. This is partially due to the fact that current methods to quantify estrogens commonly employ immunoassays, which measure only one metabolite at a time and often over-estimate levels due to cross-reacting antibodies. Therefore, an improved assay to analyse a panel of estrogen metabolites is required to quantify the wide range of endogenous concentrations in females and males. The main aim of this work was to develop, validate and apply a liquid chromatography tandem mass spectrometry (LC-MS/MS) approach to quantify multiple estrogens simultaneously. Estrogens were extracted from serum or plasma by solid phase extraction on Oasis MCX® cartridges followed by derivatisation using 1-(5-fluoro-2, 4-dinitrophenyl)-4-methylpiperazine (MPPZ). Nine derivatives were quantified using a Shimadzu Nexera X2 interfaced with a QTrap 6500+. Recovery was 90–110% and ion suppression minimal (0 - 30%). The limits of quantification were between 2–6 pg/mL with acceptable precision and accuracy (<15%). Estrogen-MPPZ derivatives also demonstrated minimal degradation upon short-term storage at 15°C (auto-sampler) and longer-term at -20°C (<20%). Application of this method to clinical samples from various cohorts of PAH and portopulmonary hypertension (PPHTN) patient samples allowed detection of parent estrogens (estrone (E1), estradiol (E2), and its isomer α-estradiol (17αE2)) alongside specific metabolites (16-hydroxyestrone (16OHE1), 16-hydroxyestradiol (16OHE2), 2-methoxyestrone (2MeOE1), 2-methoxyestradiol (2MeOE2), 4-methoxyestrone (4MeOE1) and 4-methoxyestradiol (4MeOE2)). In summary, sex-dependent differences in estrogen profiles were displayed in comparisons of PAH and non-PAH control samples. In females, E1 and E2 concentrations were reduced in PAH, with an elevation in 16OHE1 and/or 16OHE2, dependent on disease classification. Conversely, in males, E1 and E2 were elevated alongside increased 16OHE1 and, in PPHTN only, 16OHE2. In addition, a proof-of-concept trial of estrogen receptor alpha (ERα) antagonism by fulvestrant administration (intramuscular, 500 mg dose) in five postmenopausal women demonstrated a reduction in 16OHE2 concentrations following 9 weeks. Comparison of estradiol quantification by LC-MS/MS and immunoassays in two studies provided evidence of over-estimation by immunoassay. In the first, a correlation between the two methods was apparent with lower concentrations consistently quantified by LC-MS/MS than by immunoassay. In the second study, elevated estradiol concentrations following fulvestrant treatment were measured by immunoassay but not by LC-MS/MS. This suggests structural similarities between fulvestrant and estradiol causes cross-reactivity during immunoassay detection, a finding stated in a number of research papers. Furthermore, in vitro, 16OHE2 caused proliferation of rat and human pulmonary artery smooth muscle cells (PASMCs). The proliferative phenotype was confined to female rat PASMCs and female human PAH-PASMCs, with no apparent effect in male rats or in female and male control human PASMCs. RNA sequencing also identified potential upstream effects of estradiol and estrogen receptors in female PAH PASMCs. This study is the first to simultaneously quantify estrogen and the bioactive metabolites in PAH patients, providing a sensitive and selective method to quantify endogenous estrogens in human serum or plasma. Its application to a number of PAH cohorts from international collaborators generated evidence of elevated 16-hydroxylation in disease. The novel findings of elevated 16OHE2 in all female patient samples, of reduced concentrations following ERα antagonism in post-menopausal women, and of sex-dependent proliferative effects of this metabolite in PASMCs indicates its potential influence in the pathogenesis of PAH. They also provide evidence that this LC-MS/MS method may be utilised as a clinical tool to monitor estrogen concentrations in patients and for the analysis of estrogen-inhibition therapies in PAH