HPLC Separation of Anabolic Steroids on ZirChrom-PBD

HPLC Separation of Anabolic Steroids on ZirChrom-PBD

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The Application Notebook

The Application Notebook, The Application Notebook-09-01-2013, Volume 31, Issue 9

ZirChrom Separations, Inc.

This note shows the separation of three closely related anabolic steroids, boldenone, nandrolone, and testosterone, using a ZirChrom®-PBD column. A typical analysis of these compounds involves derivatization and subsequent quantitation by GC–FID or GC–MS, however these methods tend to be labor intensive, and analytically unreliable. Baseline resolution of all three compounds was obtained on ZirChrom®-PBD at slightly elevated temperature in under 10 min using isocratic conditions.


The rapid and accurate detection of anabolic steroids is crucial in today's sporting world. Historically the structural similarity of these compounds has made quantitative analysis by reversed-phase HPLC difficult at best. These steroids are very difficult to separate on silica ODS phases and provide nearly identical mass spectra. This method capitalizes on the unique temperature stability and surface chemistry of a zirconia-based stationary phase to achieve baseline resolution of these compounds in less than 10 min.


A mixture of anabolic steroids was separated at 60 °C using a ZirChrom®-PBD column (see Figure 1) using the following conditions.

Figure 1: Separation of anabolic steroids on ZirChrom®-PBD, 1-Boldenone, 2-Nandrolone, 3-Testosterone.

Column: 150 mm × 4.6 mm i.d. ZirChrom®-PBD

Mobile Phase: 15/85 ACN/Water

Flow Rate: 1.5 mL/min

Injection Vol.: 5 μL

Pressure Drop: 160 bar

Temperature: 60 °C with Metalox™ 200-C Column Heater

Detection: UV at 215 nm

This separation allows for clear identification and quantification of these compounds without the use of expensive MS detection. The separation also requires no complex buffers and uses a minimum of organic modifier.


(1) Walter Hyde, Iowa State University.

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