Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and delta8,9-dehydroestrone sulfate, by liquid chromatography--electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases.
Equilin-3-sulfate and delta8,9-dehydroestrone-3-sulfate are two isomers found in equine conjugated estrogens that differ in structure only by the position of a double bond in the steroid B-ring. These geometric isomers were not resolved on a C18 column during the analysis of conjugated estrogen drug products by LC-MS using acetonitrile-ammonium acetate buffer as the mobile phase. While no separations of these two isomers were observed on C18 or other alkyl-bonded silica based phases using a variety of mobile phase conditions, partial separations were achieved on phenyl bonded silica phases with a resolution of 1.5 on a diphenyl phase, and baseline separations were readily achieved on two carbonaceous phases with resolutions routinely exceeding three on graphitic carbon-coated zirconia (Zr-CARB) and resolutions as high as 19 on porous graphitic carbon (Hypercarb). An examination of a selected few conjugated estrogens in the complex drug substance by LC-MS on Hypercarb is presented.
Reepmeyer
John C
JC
US Food and Drug Administration, Division of Pharmaceutical Analysis, St. Louis, MO 63101, USA. reepmeyerj@cder.fda.gov
Brower
James F
JF
Ye
Hongping
H
D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D004857:Equilin / Q000031:analogs & derivatives* / Q000302:isolation & purification; D004966:Estrogens, Conjugated (USP) / Q000302:isolation & purification*; D004970:Estrone / Q000031:analogs & derivatives* / Q000302:isolation & purification; D006108:Graphite; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D015040:Zirconium
C119735:8,9-dehydroestrone sulfate; D004966:Estrogens, Conjugated (USP); D004857:Equilin; D004970:Estrone; D006108:Graphite; D015040:Zirconium; C037875:equilin sulfate; C028541:zirconium oxide