title: Derivatization of estrogens enhances specificity and sensitivity of analysis of human plasma and serum by liquid chromatography tandem mass spectrometry.
abstract: 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.
authors.affiliation: Endocrinology, University/British Heart Foundation Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. Electronic address: A.M.M.Faqehi@sms.ed.ac.uk.
authors.forename: Abdullah M M
authors.initials: AMM
authors.lastname: Faqehi
authors.affiliation: Endocrinology, University/British Heart Foundation Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. Electronic address: D.F.Cobice@sms.ed.ac.uk.
authors.forename: Diego F
authors.initials: DF
authors.lastname: Cobice
authors.affiliation: Mass Spectrometry Core, Edinburgh Clinical Research Facility, Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. Electronic address: gnaredo@outlook.com.
authors.forename: Gregorio
authors.initials: G
authors.lastname: Naredo
authors.affiliation: Endocrinology, University/British Heart Foundation Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. Electronic address: C.S.mak@sms.ed.ac.uk.
authors.forename: Tracy C S
authors.initials: TCS
authors.lastname: Mak
authors.affiliation: Endocrinology, University/British Heart Foundation Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. Electronic address: ritaupreti@yahoo.com.
authors.forename: Rita
authors.initials: R
authors.lastname: Upreti
authors.affiliation: Endocrinology, University/British Heart Foundation Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. Electronic address: fraser.gibb@ed.ac.uk.
authors.forename: Fraser W
authors.initials: FW
authors.lastname: Gibb
authors.affiliation: Clinical Biochemistry, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh EH16 4SA, United Kingdom. Electronic address: g.j.beckett@ed.ac.uk.
authors.forename: Geoffrey J
authors.initials: GJ
authors.lastname: Beckett
authors.affiliation: Endocrinology, University/British Heart Foundation Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom; Mass Spectrometry Core, Edinburgh Clinical Research Facility, Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. Electronic address: b.walker@ed.ac.uk.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Walker
authors.affiliation: Mass Spectrometry Core, Edinburgh Clinical Research Facility, Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. Electronic address: n.z.m.homer@ed.ac.uk.
authors.forename: Natalie Z M
authors.initials: NZM
authors.lastname: Homer
authors.affiliation: Endocrinology, University/British Heart Foundation Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom; Mass Spectrometry Core, Edinburgh Clinical Research Facility, Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. Electronic address: ruth.andrew@ed.ac.uk.
authors.forename: Ruth
authors.initials: R
authors.lastname: Andrew
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001557:Benzenesulfonates / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D004958:Estradiol / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D004967:Estrogens / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D004970:Estrone / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D017698:Postmenopause; D017697:Premenopause / Q000097:blood; D011726:Pyridinium Compounds / Q000737:chemistry; D015203:Reproducibility of Results; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*; D055815:Young Adult
chemical_list: D001557:Benzenesulfonates; D004967:Estrogens; D011726:Pyridinium Compounds; D004970:Estrone; D004958:Estradiol; C066037:2-fluoro-1-methylpyridinium; C029501:4-toluenesulfonic acid
keywords: Derivatization; Estradiol; Estrone; Liquid chromatography; Mass spectrometry; Methylpyridinium ether