Analysis of Testosterones Using a Core Enhanced Technology Accucore HPLC Column

October 2, 2011

The Application Notebook

The Application Notebook, The Application Notebook-10-02-2011, Volume 0, Issue 0

Thermo Fisher Scientific Application Note

This application note demonstrates the use of the Thermo Scientific Accucore RP-MS HPLC column for the fast analysis of three testosterones.


Accucore HPLC columns use Core Enhanced Technology to facilitate fast and high efficiency separations. The 2.6 µm diameter particles are not totally porous, but rather have a solid core and a porous outer layer. The optimized phase bonding creates a series of high coverage, robust phases. Accucore RP-MS uses an optimized alkyl chain length for more effective coverage of the silica surface. This coverage results in a significant reduction in secondary interactions and thus highly efficient peaks with very low tailing. The tightly controlled 2.6 µm diameter of Accucore particles provides much lower backpressures than typically seen with sub-2 µm materials.

11-ketotestosterone, 19-nortestosterone and epitestosterone are all related to testosterone. 11-ketotestosterone is an oxidized form of testosterone. 19-nortestosterone contains an additional methyl group in comparison to testosterone and epitestosterone and is an inactive epimer of testosterone. All three compounds are anabolic steroids, which promote protein synthesis within cells, particularly in muscles. 19-nortestosterone occurs naturally at low levels (0.4 ng/mL) in the body and is routinely tested for in athletes.

The separation of these three compounds and more significantly the speed of analysis are demonstrated in this application.

Experimental Conditions

Sample Preparation

Working standard contained 50 µg/mL of each testosterone in mobile phase

Thermo Scientific Column

Accucore RP-MS 2.6 µm, 100 × 2.1 mm (P/N 17626-102130) Measured pressure: 300 bar

Thermo Scientific Accela HPLC system

Column temp: 40 °C

Injection volume: 1 µL

Flow-rate: 0.6 mL/min

UV detection: 54 nm

Mobile Phase

60:40 (v/v) water/acetonitrile


Fisher Scientific HPLC grade water (P/N W/0106/17)

Fisher Scientific HPLC grade acetonitrile (P/N A/0626/17)

NSC Mass Spec Certified 2 mL clear vial with blue bonded PTFE silicone cap (P/N MSCERT4000-34W)


Figure 1: Chromatogram for 11-ketotestosterone (1), 19-nortestosterone (2) and epitestosterone (3) separated on an Accucore RP-MS 2.6 µm 100 × 2.1 mm column.

The analysis was carried out on an Accucore RP-MS 2.6 µm, 100 × 2.1 mm column. 11-ketotestosterone, 19-nortestosterone and epitestosterone eluted in under 2 minutes (Figure 1).

Replicate injections of the testosterone mix showed that Accucore RP-MS produced stable and reproducible results (Table 1).


Accucore RP-MS columns successfully separated 11-ketotestosterone, 19-nortestosterone and epitestosterone in less than 2 minutes, providing an excellent choice for the fast analysis of testosterones, allowing high sample throughput.

Table 1: Method precision (%RSD) for 11-ketotestosterone, 19-nortestosterone and epitestosterone (data calculated from six replicate injections).

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