MSACL 2017 EU Abstract

Biological Variation of Testosterone, Dihydrotestosterone and their Ratio Determined in 30 Healthy Individuals with LC-MS/MS

Anna van der Veen (Presenter)
University Medical Center Groningen

Bio: My name is Anna van der Veen and I’m in the third year of my PhD. I’m working in the laboratory of professor Ido Kema (University Medical Center Groningen, The Netherlands, Department of Special Chemistry). This laboratory is specialized in the LC-MS/MS analysis of low molecular weight biomarkers, with special focus on neuroendocrine biomarkers, such as biogenic amines for routine patient care and research projects. My focus is on optimization and implementation of mass spectrometry assays for routine diagnostics and projects with special interest in androgens and the tryptophan pathway.

Authorship: A. van der Veen, H.J.R. van Faassen, W.H.A. de Jong, D.A.J. Dijck-Brouwer, I.P. Kema
Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, the Netherlands.

Short Abstract

For correct interpretation of laboratory data, information on biological variation is essential, including within-person variation (CVi), between-person variation (CVg), analytical variation (CVa) and the reference change value (RCV). Recently, in our laboratory the biological variation was determined for plasma testosterone (T), dihydrotestosterone (DHT) and the DHT/T-ratio using online-SPE coupled to LC-MS/MS in 30 healthy individuals over a period of 4 months with 4 weeks intervals. Comparable CVi’s and RCVs in men and women varied from 10-16% and 30-42%, respectively. The CVg was higher in women (35-45%) compared to men (19-31%). The data on biological variation shows a high degree of individual variation, illustrating its importance and careful interpretation of single measurements for T and DHT. The RCVs are valuable for the assessment of significant changes in patient follow-up.

Long Abstract


For correct interpretation of laboratory data, information on the biological variation is essential. This includes within-person biological variation (CVi), between-person biological variation (CVg) and analytical variation (CVa). When monitoring disease, serial measurements usually reflects clinical improvement or disease progression. Interpretation of data on changes over time in an individual patient can be complicated. Therefore the reference change value (RCV), the percentage change of a biomarker within a person that indicates a ‘true’ statistical change, can be determined.

With the introduction of LC-MS/MS, and the ability to measure testosterone (T) and dihydrotestosterone (DHT) in low concentrations, data on the CVa have changed. In order to accurately interpret measurements of T, DHT and their ratio, in both men and women, we assessed the biological variation of these androgens with a validated LC-MS/MS method.


Plasma total T and DHT were measured as part of the androgen profile in 30 apparently healthy subjects (15 males/15 females; aged between 21-63 years) with online-SPE coupled to LC-MS/MS. In short, 200µL plasma, 25µL internal standard solution (13C3-labeled T and DHT) and 25µL protein disrupting buffer were mixed together. After 30 minutes incubation, water was added to a total volume of 1mL. After centrifugation, 40µL was injected onto an automated solid-phase extraction system (Waters) with XBridge C8 OSM cartridges (Waters) in combination with a Kinetex C18 column (Phenomenex) and a Waters Xevo-TQS mass spectrometer. The blood samples were collected at a standardized time with four-week intervals from January to May. The CVa, CVi, CVg and RCV were calculated according to the method of Fraser and Harris (1) and Fokkema et al (2).


The CVi’s for T, DHT and the DHT/T-ratio were comparable in men and women. The CVi’s in men were 14% for T and DHT and 11% for the ratio. In women, the CVi’s were 16% for T, 11% for DHT and 10% for the ratio. The CVg’s were higher in women, T-35%, DHT-45% and the DHT/T-ratio 41%, compared to 20%, 31% and 19% in men respectively. The CVa’s in men were 2.0% for T, 2.7% for DHT and 1.7% for the DHT/T-ratio at mean concentrations of 15.6 nmol/L, 1.7 nmol/L and 0.11, respectively. In women, the CVa’s were 2.1% for T, 7.1% for DHT and 7.1% for the DHT/T-ratio at mean concentrations of 0.7 nmol/L, 0.3 nmol/L and 0.5, respectively. The RCVs were similar between men and women. In men, the RCVs were 41% for T, 40% for DHT, and 30% for the DHT/T-ratio. In women, the RCVs were 42% for T, 37% for DHT and 36% for the ratio.

Conclusions & Discussion

With an extensively validated LC-MS/MS method we established the biological variation of DHT, T and their ratio in a healthy population, including the RCVs. In general, the data on biological variation shows a high degree of inter-individual variation, illustrating its importance and careful interpretation of single measurements for T and DHT using population-based reference intervals. For the biological variation of the DHT/T ratio, as compared to the single T and DHT values, less variation was expected, as it reflects 5-alpha-reductase activity. However, this did not become apparent from our data. For clinical conditions associated with T and DHT, the calculated RCVs can be used for assessment of significant changes over time in the patient-status. The RCV data demonstrates that a change of approximately 40% represents a significant shift.

In conclusion, the biological variation determined with LC-MS/MS is valuable for the interpretation of T, DHT and DHT/T-ratio, and shows that these parameters can be useful in patient follow-up.

References & Acknowledgements:

(1): Fraser et al. Crit Rev Clin Lab Sci 1989, 27; 409-437

(2): Fokkema et al. Clin Chem 2006; 52: 1602-1603

Financial Disclosure

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