Estrogens are fractionated into estradiol and estrone by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The LC-MS/MS method employs an organic extraction to remove water-soluble conjugates and to allow for concentration of the specimen. The method is free from interference and represents a reference methodology.
17 Beta-estradiol and estrone are extracted from 0.5 mL of serum with the organic solvent methylene chloride. Deuterated 17 beta-estradiol-d5 and estrone-d4 are added to each specimen before the liquid extraction and serve as internal standards. After derivatization with dansyl chloride, HPLC is used prior to introduction of the derivatized sample extract into the MS/MS.(Bidlingmaier F, Wagner-Barnack M, Butenandt O, Knorr D: Plasma estrogens in childhood and puberty under physiologic and pathologic conditions. Pediatr Res 1973;7(11):901-907) The calibration utilizes an 8-point standard curve over a concentration range of 0 to 600 pg/mL.(Anari MR, Bakhtiar R, Zhu B, et al: Derivatization of ethynylestradiol with dansyl chloride to enhance electrospray ionization: application in trace analysis of ethynylestradiol in Rhesus monkey plasma. Anal Chem 2002;74,4136-4144)