Abstract
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.
Highlights
Estradiol and estrone circulate at low pico/femtomolar levels, ranging between 0.1 and 530 pg mLÀ1 (0.36e1945 pmol LÀ1) depending on age, sex and menstrual status [1,2]
The lack of a suitable analytical method hampers the investigation of several estrogen sensitive diseases such as breast cancer, uterine cancers and various cardiovascular diseases [4,5] where differential estrogen metabolism is thought to contribute [6,7] to mechanisms underpinning poor patient prognosis and outcomes [2]
A number of studies have demonstrated that imbalances in estrogen metabolism results in either proliferative or anti-proliferative effects in the vasculature (Fig. 1B) [8,9]
Summary
Estradiol and estrone circulate at low pico/femtomolar levels, ranging between 0.1 and 530 pg mLÀ1 (0.36e1945 pmol LÀ1) depending on age, sex and menstrual status [1,2]. They are metabolized by CYP450 enzymes, generating bioactive hydroxy and methoxy metabolites (Fig. 1A). Derivatization improves sensitivity by addition of a permanently or readilycharged group, allowing higher ionization efficiency and increased sensitivity by MS. In turn this permits shorter extraction protocols and smaller sample volumes [14].
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