title: Development and validation of a high-sensitivity liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with chemical derivatization for the determination of ethinyl estradiol in human plasma.
abstract: An ultra-sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the analysis of oral contraceptive ethinyl estradiol (EE) was developed and validated over the curve range of 2.5-500 pg/mL using 1 mL of human plasma sample. Ethinyl estradiol and the internal standard, ethinyl estradiol tetra-deuterated (EE-d4), were extracted from the plasma matrix with methyl t-butyl ether, derivatized with dansyl chloride and then back-extracted into hexane. The hexane phase was evaporated to dryness, reconstituted and injected onto the LC/MS/MS system. The chromatographic separation was achieved on a Luna C(18) column (50 x 2 mm, 5 micro m) with an isocratic mobile phase of 20:80 (v/v) water:acetonitrile with 1% formic acid. The offline derivatization procedure introduced the easily ionizable tertiary amine function group to EE. This greatly improved analyte sensitivity in electrospray ionization and enabled us to achieve the desired lower limit of quantitation at 2.5 pg/mL. This high sensitivity method can be used for therapeutic drug monitoring or supporting bio-equivalence and drug-drug interaction studies in human subjects.
authors.affiliation: Covance Laboratories Inc., 3301 Kinsman Boulevard, Madison, WI 53704, USA. wilson.shou@covance.com
authors.forename: Wilson Z
authors.initials: WZ
authors.lastname: Shou
authors.forename: Xiangyu
authors.initials: X
authors.lastname: Jiang
authors.forename: Weng
authors.initials: W
authors.lastname: Naidong
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D003619:Dansyl Compounds / Q000737:chemistry*; D004997:Ethinyl Estradiol / Q000097:blood*; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D003619:Dansyl Compounds; D007202:Indicators and Reagents; D004997:Ethinyl Estradiol; C029038:dansyl chloride