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.

The research article focuses on a method to separate and detect equine conjugated estrogen isomers, equilin sulfate and delta8,9-dehydroestrone sulfate, using a variation of liquid chromatography-mass spectrometry. This was achieved by using carbon-coated zirconia and porous graphitic carbon stationary phases.

Objective of the Research

• The main objective of this study was to develop a method to separate and detect the isomers, equilin sulfate and delta8,9-dehydroestrone sulfate, found in equine conjugated estrogens.
• These isomers are structurally identical with the exception of the position of a double bond in the steroid B-ring.

Methodology

• The researchers used a version of liquid chromatography-mass spectrometry (LC-MS) for their experiments. This technology uses high pressure liquid chromatography to separate mixtures which are then identified by mass spectrometry.
• The process involved using an acetonitrile-ammonium acetate buffer as the mobile phase with the compound being run on a C18 column.
• However, this initial technique did not result in the isolation of the two isomers. Consequently, they experimented with other compounds as alternative stationary phases, including alkyl-bonded silica, phenyl bonded silica, carbon-coated zirconia (Zr-CARB), and porous graphitic carbon (Hypercarb).

Findings

• No separations of the isomers were observed using C18 or alkyl-bonded silica based phases under a variety of mobile phase conditions.
• However, partial separations were achieved on phenyl bonded silica phases, with the separation resolution reaching 1.5 on a diphenyl phase.
• Ensuing use of two carbonaceous phases resulted in successful baseline separations. Resolution often surpassed three on graphitic carbon-coated zirconia (Zr-CARB), and reached as high as 19 on porous graphitic carbon (Hypercarb).
• Finally, the study presented an analysis of conjugated estrogens in a complex drug substance using the LC-MS on Hypercarb method, demonstrating a practical application of the technique developed.

Significance of the Research

• The success of this technique marks a substantial step forward in isolating and studying these isomers.
• The capability to separate these substances could allow for improved understanding of their individual biological effects, potential uses, and interactions within complex compounds.