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© 2016, Elsevier B.V. All rights reserved. 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 (<20%). The derivatives were stable over 24 h at 10 °C (7-9% degradation). Using this approach, E1 and E2, respectively were detected in human plasma and serum: pre-menopausal female serum (0.5 mL) 135-473, 193-722 pmol/L; male plasma (1 mL) 25-111, 60-180 pmol/L and post-menopausal female plasma (2 mL), 22-78, 29-50 pmol/L. Thus FMP derivatization, in conjunction with LC-MS/MS, is suitable for quantitative analysis of estrogens in low abundance in plasma and serum, offering advantages in specificity over immunoassay and existing MS techniques.
Author(s): Faqehi AMM, Cobice DF, Naredo G, Mak TCS, Upreti R, Gibb FW, Beckett GJ, Walker BR, Homer NZM, Andrew R
Publication type: Article
Publication status: Published
Journal: Talanta
Year: 2016
Volume: 151
Pages: 148-156
Print publication date: 01/05/2016
Online publication date: 29/12/2015
Acceptance date: 23/12/2015
ISSN (print): 0039-9140
ISSN (electronic): 1873-3573
Publisher: Elsevier
URL: https://doi.org/10.1016/j.talanta.2015.12.062
DOI: 10.1016/j.talanta.2015.12.062
PubMed id: 26946022
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