Analysing Testosterone in Human Serum by UHPLC Using High Efficiency Kinetex 1.7 µm C18 Core-Shell Columns

August 2, 2011

The Application Notebook

Volume 0, Issue 0

Phenomenex Application Note

Testosterone was extracted from human serum by strong anion exchange polymeric SPE and analysed using a Kinetex C18, 30 × 2.1 mm, 1.7 μm column and positive polarity ESI LC–MS–MS system. Kinetex sub-2 μm core-shell technology offers higher efficiencies than traditional sub-2 μm columns, producing greater chromatographic resolution, sensitivity and higher peak capacities.


Testosterone is an androgenic steroid responsible for the development of male reproductive organs, maintaining (or increasing) muscle mass and bone density. As anabolic steroids, testosterone has been used (or abused) to increase muscle mass and enhance the athletic performance. The concentration of testosterone is lower in female population than men and in general diminishes with advancing age. Monitoring body concentration of testosterone is an aid in diagnosing and treating disease state related to the hormonal imbalance.

Figure 1: The separation of 100 pg/mL standard of testosterone and epitestosterone extracted from human serum on Kinetex C18, 30 × 2.1 mm, 1.7 um using the LC gradient profile listed in Table 1. Testosterone retention time is 2.62 min, Epitestosterone 2.77 min and Int. Std is 2.61 min.

The analysis is based on a simple extraction method using strong anion exchange SPE (Strata-X-A) to produce a clean extract from human serum. Following the extraction, testosterone is derivatized to form an oxime which is then analysed in positive mode ESI LC–MS–MS under multiple-reactions-monitoring function (1). A short-length 30 mm, 1.7 um Kinetex C18 column efficiently separates testosterone from its isomeric form epitestosterone (Figure 1).

Experimental Conditions

The mobile phase consisted of 0.1% formic acid with 1 mM ammonium formate with no pH adjustment, in water (MP A) and acetonitrile (MP B). A typical LC gradient is used (Table 1) for the separation.

Table 1: LC gradient programme.

An AB Sciex API 5000 triple-quadrupole tandem mass spectrometer is used for analysis equipped with an ESI probe operating in positive polarity mode. Under an MRM mode, two channels were monitored for Testosterone and Testosterone-D3 (Table 2).

Table 2: MRM transitions used for data analysis.

Results and Discussions

As is demonstrated from the chromatogram, the Kinetex column provides a high degree of selectivity even in small dimensions to provide superior chromatographic separation. For further details or questions contact your Phenomenex sales representative.


1. M.M. Kushnir et al., Clinical Chemistry, 52(1), 120–128 (2006).

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