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Derivatization of estrogens enhances specificity and sensitivity of analysis of human plasma and serum by liquid chromatography tandem mass spectrometry

Lookup NU author(s): Professor Brian WalkerORCiD


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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.

Publication metadata

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


DOI: 10.1016/j.talanta.2015.12.062

PubMed id: 26946022


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