Extraction of Testosterone and Other Steroid Hormones from Human Plasma Using ISOLUTE SLE+ 96-Well Plates - - Chromatography Online
Extraction of Testosterone and Other Steroid Hormones from Human Plasma Using ISOLUTE SLE+ 96-Well Plates

The Application Notebook

This application note describes an optimized and effective supported liquid extraction protocol for the sample preparation of a range of steroid hormones, including testosterone, from human plasma using ISOLUTE SLE+. Supported liquid extraction is an efficient and cost effective alternative to traditional liquid-liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation and significantly reduced sample preparation time. This simplified extraction method has significant analyte recoveries ranging from 90 to 107% with LOQs as low as 500 pg/mL.

Extraction Conditions

This application note outlines the procedure using the ISOLUTE SLE+ 200 Supported Liquid Extraction plate (part number 820-0200-P01) optimized for a 200 無 pretreated human plasma sample volume (other formats are available). Method parameters and dilution factors have been optimized to maximize recoveries and minimize ion suppression.

Sample pre-treatment: Dilute human plasma (100 無) 1:1 with HPLC-grade water (100 無).

Sample load: Load pre-treated sample (200 無) to plate followed by a pulse of vacuum to initiate flow and leave for five minutes.

Analyte elution: Elute with dichloromethane (1 mL) directly in to a deep well collection plate (1215203). Leave to flow under gravity for 5 min then apply short pulse of vacuum.

Post extraction: Evaporate to dryness at ambient temperature and reconstitute in 50% methanol (aq) (100 無). Vortex samples to ensure full reconstitution.

Additional information: Use of the reconstitution solvent combined with the vortex step addresses any issues that may arise from nonspecific binding of testosterone and other analytes. All samples were processed using Biotage VacMaster-96 sample processing manifold and evaporated to dryness on a SPE Dry 96 Dual.

Analytical Conditions

Instrument: Waters Acquity UPLC interfaced to a Quattro Premier XE triple quadrupole MS using electrospray ionization.

Column: Acquity BEH C18 100 2.1 mm 1.7 .

Mobile phase: A: 0.1% formic acid (aq)
                     B: 0.1% formic acid in methanol.
Time (min)    %B
Initial            50
0.8            50
4.2            81
4.3            100
5.3            100
5.8            50
6.8            50
Flow-rate: 0.40 mL/min.
Injection: 15 無 (partial loop, needle overfill).
Sample temp: 20 蚓.
Column temp: 40 蚓.
Source temp: 150 蚓.
Desolvation temp: 450 蚓.


Table 1: Steroid LOQs recorded using SLE+ extraction protocol.
Sample recoveries are >90% for all analytes with RSDs below 10%. Table 1 shows the range of LOQs recorded for each individual analyte.


This method demonstrates that an extensive suite of steroids can be extracted under the same conditions using a very simple supported liquid extraction protocol. Non-chlorinated elution solvents were also evaluated and although levels were not fully optimized it should be possible to substitute ethyl or butyl acetate for dichloromethane with only a slight reduction in performance.


1. A. Senior, L. Williams et al., Application Note AN 740 (available from http://www.biotage.com/applications/).

Biotage AB
Kungsgatan 76, SE-753, 18, Uppsala, Sweden
tel: +46 18 56 59 00 fax: +46 18 59 19 22
Website: http://www.biotage.com/


blog comments powered by Disqus
LCGC E-mail Newsletters
Global E-newsletters subscribe here:



Column Watch: Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments. LATEST: When Bad Things Happen to Good Food: Applications of HPLC to Detect Food Adulteration

Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: HPLC for Characterization and Quality Control of Therapeutic Monoclonal Antibodies

MS — The Practical Art: Kate Yu brings her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers. LATEST: Radical Mass Spectrometry as a New Frontier for Bioanalysis

LC Troubleshooting: LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan is able to highlight common problems and provide remedies for them. LATEST: How Much Can I Inject? Part I: Injecting in Mobile Phase

More LCGC Columnists>>

LCGC North America Editorial Advisory Board>>

LCGC Europe Editorial Advisory Board>>

LCGC Editorial Team Contacts>>

Source: The Application Notebook,
Click here