• Application Note

Development of a Clinical Research Method for the Measurement of Testosterone and Dihydrotestosterone

Development of a Clinical Research Method for the Measurement of Testosterone and Dihydrotestosterone

  • Martijn van Faassen
  • Ido Kema
  • Department of Laboratory Medicine, University Medical Center, Groningen

For research use only. Not for use in diagnostic procedures.

This is an Application Brief and does not contain a detailed Experimental section.


This application brief demonstrates to develop a method to measure Testosterone and Dihydrotestosterone (DHT) from plasma samples.


Direct measurement of androgens is made possible without derivatization using LC-MS with online SPE.


Testosterone is a steroid hormone from the androgen group and  is the principal male sex hormone and an anabolic steroid (Figure 1).  

In men, approximately 5% of testosterone undergoes 5α-reduction to form the more potent androgen, dihydrotestosterone (DHT) (Figure 1).  

Figure 1: Structure of Androgen Hormones. Dihydrotestosterone (DHT) is the product of the reduction of the double bond in the a ring of testosterone by the enzyme 5-alpha reductase.

Measuring the male steroid hormones and related metabolites, particularly at the low levels found in pediatrics and females, has proven to be a major challenge. Immunoassays for steroids often suffer from poor sensitivity and specificity. While traditional LC-MS assays require derivatization procedures and/or long and complicated sample preparation protocols.

In the current study, a research method for the measurement of testosterone and DHT in plasma was developed. This method allows for the direct measurement of these two androgens at physiological levels in male and female samples by LC-MS without the need for derivitization. Moreover the method makes use of automated sample prep, minimizing sample pretreatment.


Method details:

LC-MS System Configuration:


Mass Spectrometer: Xevo TQ-S


ACQUITY UPLC BEH C18, 1.7 μm, 2.1 x 50 mm

Sample Preparation:



MassTrak C8 OSM Cartridge

Sample Pre-Treatment:

200 μL Plasma

20 μL Internal Standard (d3 Testosterone; 13C3 DHT) 300 μL Zinc Sulfate:MeOH Solution; Centrifuge to pellet protein; remove supernatant

After addition of zinc sulfate:MeOH we filled up to 1.0mL prior to centrifugation. 25 uL was then injected (no transfer of supernatant → needle adjusted so stays above pellet).

Dilute to 1 ml with Water

Inject 25 μL of final sample

Chromatographic conditions

Solvent A:

10% MeOH+0.05% Formic Acid

Solvent B:

MeOH+0.05% Formic Acid

Flow rate:

0.4 ml/min

















Online SPE conditions


Step 1: 0.5 ml MeOH @ 4 ml/min

Step 2: 2.0 ml Magic Mix* @ 3 ml/min


1.0 ml Water @ 4 ml/min

Sample loading:

1.0 ml Water @ 2 ml/min

Cartridge wash:

Step 1: 0.5 ml 80% 0.2% NH4OH/20% MeOH @ 2 ml/min

Step 2: 0.5 ml 70% 0.2% Formic Acid/30% MeOH @ 2 ml/min


Elution Time: 0.8 minutes

Run Time: 0.8 minutes

Clamp flush:

Step 1: 2.0 ml Magic Mix* @ 3 ml/min

Step 2: 1.0 ml Water @ 3 ml/min

* Magic Mix= MeOH/ACN/IPA/Water+0.2% Formic Acid

Results and Discussion

Figure 2: SPE Method Development. SPE sample preparation was facilitated by using an online SPE system and Design of Experiments (DoE) principles to manipulate multiple SPE variables simultaneously. This approach allowed for identification and optimization of significant sample preparation method development variables very quickly.
Figure 3: SPE Method Development Results. Use of the Advanced Method Development (AMD) operation mode of the online SPE system to optimize sample preparation. AMD allowed a number of SPE conditions to be tested rapidly to identify the optimal SPE conditions for these two analytes. Also, the SPE method is also helping with separation of testosterone and DHT. Testosterone is breaking through, while DHT is retained under the conditions examined.
Figure 4: Separation and measurement of testosterone and DHT in male plasma samples (Sample from Male between 20 and 65 years of age). Testosterone and DHT are readily separated and quantitated.
Figure 5: Separation and measurement of testosterone and DHT from Female plasma sample from pre-menopausal females. Testosterone can be measured at pM range in these samples, but DHT is below the LOQ in female samples.


The wide variation in testosterone and DHT concentrations as well as the inherent difficulties in measuring these molecules, has made it difficult to develop test methods for them. 

To measure these molecules successfully by LC-MS requires an effective sample preparation method. Using an online SPE system greatly accelerated the development of an LC-MS method by quickly identifying and utilizing optimized SPE conditions for isolating these androgens from plasma samples.

The focus of the clinical research method described herein was on quickly and efficiently developing an integrated SPE sample preparation method with high sensitivity mass spectrometry to simultaneously measure testosterone and DHT. The method developed uses online SPE to provide a high quality sample that is relatively free from sample matrix effects. By using LC-MS augmented with online SPE, the direct measurement of androgens, even at low levels, is possible.

The clinical research method developed here provides: 

  • Simultaneous analysis of testosterone and DHT in a single assay
  • Simple sample preparation and androgen detection without derivatization
  • Fast analysis time of < 5 min/sample
  • Measurement of testosterone and DHT in male and female samples; where applicable
  • Highly efficient SPE sample preparation integrated with LC-MS

720004829, January 2014

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