title: Development of a highly sensitive method for the quantification of estrone and estradiol in serum by liquid chromatography tandem mass spectrometry without derivatization.
abstract: Measurement of estrone (E1) and estradiol (E2) values <1 pg/mL (3.7 pmol/L) is necessary for postmenopausal, pediatric and male serum samples. Until now this was rarely reached and only through derivatization which can present problems for estradiol. A very sensitive LC-MS/MS method was developed avoiding derivatization, convenient for large-scale studies. The desired sensitivity and specificity were achieved using ESI negative mode, LLE and a 2D chromatography consisting of a trapping column and a second dimension reverse-phase C8 analytical column. A mixture of an aqueous solution of ammonium fluoride at 0.2mM and methanol was used on the analytical column to further increase the sensitivity. Serum LOQ was <0.5 pg/mL (1.9 pmol/L) for E2 and E1 and recoveries ranged from 95 to 105%. No carry-over was detectable. Inter assay CV's were 4.0% at 21 pg/mL (77 pmol/L) for E2, 7.6% at 25 pg/mL (93 pmol/L) for E1. Comparison with commercial direct estrogen assays (Roche Diagnostics E170 for E2, Bioline RIA for E1) exposed analytical unsuitability (due to a combined lack of sensitivity and specificity) for the assay of male, postmenopausal or pediatric samples.
authors.affiliation: Ghent University Hospital, Department of Hormonology, De Pintelaan 185, 9000 Gent, Belgium. tom.fiers@uzgent.be
authors.forename: Tom
authors.initials: T
authors.lastname: Fiers
authors.forename: Bruno
authors.initials: B
authors.lastname: Casetta
authors.forename: Brigitte
authors.initials: B
authors.lastname: Bernaert
authors.forename: Eric
authors.initials: E
authors.lastname: Vandersypt
authors.forename: Martine
authors.initials: M
authors.lastname: Debock
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Kaufman
mesh_terms: D064751:Ammonium Compounds; D002853:Chromatography, Liquid / Q000379:methods*; D004958:Estradiol / Q000097:blood*; D004970:Estrone / Q000097:blood*; D005459:Fluorides; D006801:Humans; D000432:Methanol; D000644:Quaternary Ammonium Compounds; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D064751:Ammonium Compounds; D000644:Quaternary Ammonium Compounds; D004970:Estrone; C024822:ammonium fluoride; D004958:Estradiol; D005459:Fluorides; D000432:Methanol