title: Sensitive routine liquid chromatography-tandem mass spectrometry method for serum estradiol and estrone without derivatization.
abstract: The need for a routinely applicable assay to measure low estradiol levels in adult men, postmenopausal women, and young adolescents was recently discussed in an Endocrine Society position statement. Our aim was to develop a sensitive liquid chromatography-tandem mass spectrometry method for estradiol and estrone in human serum without the need for derivatization or extended extraction protocols. After protein precipitation of serum with a mixture of methanol/acetonitrile (85/15) (v/v) containing isotopic internal standards (17β-estradiol-16,16,17-d 3 and estrone-2,3,4-(13)C), we quantified estradiol and estrone by two-dimensional liquid chromatography-tandem mass spectrometry with electrospray ionization in the negative mode monitoring 5 × 271.20→145.00 (17β-estradiol) and 269.20→145.00 (estrone). Sensitivity was increased by using fluoride and summation of 5 identical transitions for estradiol. Our method was analytically validated, compared against direct immunoassays using serum of 25 adult men, and clinically tested by measuring samples of 3 men at baseline and after chemical castration, 30 postmenopausal women and 15 patients receiving aromatase inhibitors. Total imprecision was below 20% for the low quality controls. Limit of quantification was 1.3 ng/L (4.8 pmol/L) for estradiol and 1.2 ng/L (4.4 pmol/L) for estrone. Estradiol in Certified Reference Material BCR-576 was within specified uncertainty limits. No significant ion suppression or interference was observed. Our method showed modest correlation with direct immunoassay for estradiol (r(2) = 0.64) but no correlation for estrone (r(2) = 0.12). Patient sample results were within expected ranges. In conclusion, we developed a routinely applicable liquid chromatography-tandem mass spectrometry method for estradiol and estrone measurement which is sensitive enough for use in men, postmenopausal women, and young adolescents.
authors.affiliation: Clinical Department of Laboratory Medicine, University Hospitals Leuven, Herestraat 49, 3000, Leuven, Belgium, steven.pauwels@uzleuven.be.
authors.forename: Steven
authors.initials: S
authors.lastname: Pauwels
authors.forename: Leen
authors.initials: L
authors.lastname: Antonio
authors.forename: Ivo
authors.initials: I
authors.lastname: Jans
authors.forename: Anneleen
authors.initials: A
authors.lastname: Lintermans
authors.forename: Patrick
authors.initials: P
authors.lastname: Neven
authors.forename: Frank
authors.initials: F
authors.lastname: Claessens
authors.forename: Brigitte
authors.initials: B
authors.lastname: Decallonne
authors.forename: Jaak
authors.initials: J
authors.lastname: Billen
authors.forename: Dirk
authors.initials: D
authors.lastname: Vanderschueren
authors.forename: Pieter
authors.initials: P
authors.lastname: Vermeersch
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002853:Chromatography, Liquid / Q000379:methods*; D004958:Estradiol / Q000097:blood*; D004970:Estrone / Q000097:blood*; D006801:Humans; D007118:Immunoassay; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004970:Estrone; D004958:Estradiol