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

Nina Denver, Shazia Khan, Ioannis Stasinopoulos, Colin Church, Natalie ZM. Homer, Margaret R. MacLean, Ruth Andrew

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

LanguageEnglish
Pages84-94
Number of pages11
JournalAnalytica Chimica Acta
Volume1054
Early online date21 Dec 2018
DOIs
Publication statusPublished - 25 Apr 2019

Fingerprint

Plasma (human)
Liquid chromatography
Metabolites
Tandem Mass Spectrometry
Liquid Chromatography
Mass spectrometry
liquid chromatography
metabolite
Estrogens
mass spectrometry
plasma
health and disease
hypertension
Plasmas
Pulmonary Hypertension
ACE 2
Health
Derivatives
Biological Phenomena
Electrospray ionization

Keywords

  • derivatization
  • estrogen
  • estrogen metabolites
  • liquid chromatography tandem mass spectrometry
  • methylpiperazine

Cite this

Denver, Nina ; Khan, Shazia ; Stasinopoulos, Ioannis ; Church, Colin ; Homer, Natalie ZM. ; MacLean, Margaret R. ; Andrew, Ruth. / Derivatization enhances analysis of estrogens and their bioactive metabolites in human plasma by liquid chromatography tandem mass spectrometry. In: Analytica Chimica Acta. 2019 ; Vol. 1054. pp. 84-94.
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Derivatization enhances analysis of estrogens and their bioactive metabolites in human plasma by liquid chromatography tandem mass spectrometry. / Denver, Nina; Khan, Shazia; Stasinopoulos, Ioannis; Church, Colin; Homer, Natalie ZM.; MacLean, Margaret R.; Andrew, Ruth.

In: Analytica Chimica Acta, Vol. 1054, 25.04.2019, p. 84-94.

Research output: Contribution to journalArticle

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T1 - Derivatization enhances analysis of estrogens and their bioactive metabolites in human plasma by liquid chromatography tandem mass spectrometry

AU - Denver, Nina

AU - Khan, Shazia

AU - Stasinopoulos, Ioannis

AU - Church, Colin

AU - Homer, Natalie ZM.

AU - MacLean, Margaret R.

AU - Andrew, Ruth

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