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

Estrogens circulate at concentrations less than 20pg/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...

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Bibliographic Details
Published in:	Talanta (Oxford) Vol. 151; pp. 148 - 156
Main Authors:	Faqehi, Abdullah M.M., Cobice, Diego F., Naredo, Gregorio, Mak, Tracy C.S., Upreti, Rita, Gibb, Fraser W., Beckett, Geoffrey J., Walker, Brian R., Homer, Natalie Z.M., Andrew, Ruth
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 01.05.2016 Elsevier
Subjects:	Adult Aged Aged, 80 and over Benzenesulfonates - chemistry blood serum Chromatography, Liquid - methods Derivatives Derivatization detection limit Estradiol Estradiol - blood Estradiol - chemistry Estradiol - isolation & purification Estrogens Estrogens - blood Estrogens - chemistry Estrogens - isolation & purification Estrone Estrone - blood Estrone - chemistry Estrone - isolation & purification Female Females Human Humans immunoassays Ionization ions Liquid chromatography Male males Mass spectrometry men Methylpyridinium ether Middle Aged moieties monitoring Postmenopause premenopause Premenopause - blood Pyridinium Compounds - chemistry quantitative analysis Reproducibility of Results Serums Solid Phase Extraction spectrometers Spectrometry, Mass, Electrospray Ionization tandem mass spectrometry Tandem Mass Spectrometry - methods women Young Adult 17αE2 D MRM SPE 3,4-[13C2E1 APCI E1 Derivatization E2 Estradiol LC-MS/MS LODs SD SNR MD ESI Methylpyridinium ether UHPLC HPLC CID RSD LOQs cps IS Liquid chromatography RME TEA d4E1 LC-MS d4E2 13C2E2 FMP-TS Estrone FA Mass spectrometry
ISSN:	0039-9140, 1873-3573, 1873-3573
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Summary:	Estrogens circulate at concentrations less than 20pg/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 0.2pg on-column and the method was linear from 1–400pg/sample. Measures of intra- and inter-assay variability, precision and accuracy were acceptable (<20%). The derivatives were stable over 24h at 10°C (7–9% degradation). Using this approach, E1 and E2, respectively were detected in human plasma and serum: pre-menopausal female serum (0.5mL) 135–473, 193–722pmol/L; male plasma (1mL) 25–111, 60–180pmol/L and post-menopausal female plasma (2mL), 22–78, 29–50pmol/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. [Display omitted] •Quantitative analysis of low amounts of estrone and estradiol in plasma and serum.•Quantitation across physiological range in men and pre- and post-menopausal women.•Methylpyridinium ether derivatives improve analytical specificity and sensitivity.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0039-9140 1873-3573 1873-3573
DOI:	10.1016/j.talanta.2015.12.062