Application Notes
SFC Steroid Analysis by HPLC/ELSD
A mixture of four steroids, progesterone, testosterone, 17-hydroxyprogesterone and hydrocortisone was separated by reversed phase HPLC/ELSD using supercritical fluid chromatography.
Supercritical Fluid Chromatography Steroid Analysis Using an ELSD, Alltech Application Note 0047LE, July 7, 2000.
Modifiers and additives commonly used in supercritical fluid chromatography (SFC) interfere with FID or UV detection. An HPLC light scattering detector, the Alltech ELSD Mark III (Evaporative Light Scattering Detector) was modified to interface with an SFC system to evaluate performance of the ELSD in the reversed phase HPLC determination of steroids.
A mixture of four steroids, progesterone, testosterone, 17-hydroxyprogesterone and hydrocortisone was separated separated by gradient elution in around 10 minutes by reversed phase HPLC on a Deltabond CN, 5µm, 250 x 4.6mm column (Keystone Scientific, Inc) using methanol/carbon dioxide. The steroids were detected by evaporative light scattering using an Alltech ELSD Mark III (Evaporative Light Scattering Detector). Detection limits of 5-10ng were achieved.
In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
Key Analyte(s)
17-hydroxyprogesterone17-hydroxyprogesterone is a naturally produced steroid measured in blood serum to test for adrenal steroid deficiency. It is most often measured using HPLC.
hydrocortisone
progesterone
testosteroneTestosterone is an anabolic steroid produced by the body and manufactured synthetically. It is most often measured using GC/MS and HPLC.
Technique
HPLCHigh Performance Liquid Chromatography (HPLC)is a technique for separating components in a sample on the basis of interactions between the component, a liquid mobile phase and a solid stationary phase.
Source
Alltech Associates, Inc.Alltech Associates, Inc., one of the leading suppliers of HPLC columns, manufactures and distributes HPLC, GC and IC columns, accessories and consumables and maintains a large searchable database of application notes and chromatograms on its website.
