Denver, N., Khan, S., Stasinopoulos, I., Church, C., Homer, N. Z.M., Maclean, M. R. and Andrew, R. (2019) Derivatization enhances analysis of estrogens and their bioactive metabolites in human plasma by liquid chromatography tandem mass spectrometry. Analytica Chimica Acta, 1054, pp. 84-94. (doi: 10.1016/j.aca.2018.12.023) (PMID:30712596) (PMCID:PMC6363983)
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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 . 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. [Abstract copyright: Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.]
| Item Type: | Articles |
|---|---|
| Additional Information: | Supported by funding: BBSRC iCASE award (BB/N503691/1) for PhD funding and to the Graham Wilson Travel Scholarship for their award to fund commuting costs for two years of the project. We would also like to thank the Wellcome Trust (202794/Z/16/Z) and The BHF (PG/15/63/31659) for funding. |
| Keywords: | Derivatization, Estrogen, Estrogen metabolites, Liquid chromatography tandem mass spectrometry, Methylpiperazine. |
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Maclean, Miss Margaret and Denver, Nina |
| Authors: | Denver, N., Khan, S., Stasinopoulos, I., Church, C., Homer, N. Z.M., Maclean, M. R., and Andrew, R. |
| College/School: | College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health |
| Journal Name: | Analytica Chimica Acta |
| Publisher: | Elsevier |
| ISSN: | 0003-2670 |
| ISSN (Online): | 1873-4324 |
| Published Online: | 21 December 2018 |
| Copyright Holders: | Copyright © 2019 The Authors |
| First Published: | First published in Analytica Chimica Acta 1054:84-94 |
| Publisher Policy: | Reproduced under a Creative Commons License |
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