Application Notes
Steroid Conjugates by HPLC/ELSD
Three steroid conjugates, estrone 3-sulphate, b-estradiol 17-(b-D glucaronide) and 5b-pregnane-3a, 20a diol glucaronide were separated by reversed phase HPLC/ELSD.
Steroid Conjugates, Alltech Application Note 0042E, July 7, 2000.
A mixure of three steroid conjugates, estrone 3-sulphate, b-estradiol 17-(b-D glucaronide) and 5b-pregnane-3a, 20a diol glucaronide (PdG) were separated by gradient elution in around 20 minutes by reversed phase HPLC on an Alltima C18, 5µm, 150 x 2.1mm column (Part No. 88370) using water/methanol. The steroids were detected by evaporative light scattering using an Alltech ELSD Mark III (Evaporative Light Scattering Detector) and by UV at 210 nm. The ELSD baseline was more stable than that from the UV determination. The response factors for each were nearly the same except for PdG which was barely detected at all by UV due to its lack of sufficient chromophores for absorption.
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)
5b-pregnane-3a, 20a -diol -glucuronide5b-pregnane-3a, 20a -diolglucuronide is a steroid and a progesterone metabolite most frequently measured in urine using HPLC techniques.
b-estradiol 17-(b-D glucaronide)
estrone 3-sulphate
Equipment
Alltech Alltima Reversed-Phase ColumnsAlltech Alltima Reversed Phase columns are a line of HPLC columns in the Alltech HP family. The Alltech Alltima family are high quality general purpose columns. They are made with spherical silica, polymerically bonded and double endcapped for long life
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.
