Detection of anabolic androgenic steroids in serum and dried blood spots

Background

Anabolic androgenic steroids (AAS) is a collective term used for substances that include synthetic substances that exert an anabolic and androgenic effect in various degrees including testosterone (T). Anabolic androgenic steroids are prohibited in sport and in some countries even illegal. In society, AAS is widely used by sport enthusiasts. Urine is the standard matrix of choice when analysing AAS. Due to the wide detection window in urine, misuse of AAS can be detected several months after a single use. To detect AAS, long-term metabolites can be analysed whereas the biomarker T/epitestosterone (T/E) ratio is used to detect T doping. However, there are limitations using urine samples. Testosterone esters are a form of AAS that are used therapeutically but can be abused due to its potent anabolic and androgenic effect. There are no methods in healthcare today to detect T esters, except for the T/E ratio in urine.

Aims

The overall aim of study I and II was to develop and validate a method on liquid chromatography tandem mass spectrometry (LC-MS/MS), to detect AAS including T esters in serum and dried blood spot (DBS) samples. As a proof-of- concept, the method was used to test the applicability of AAS detection in clinical samples.

Results and Conclusion

The results of both studies show that serum and DBS are suitable matrices for analysis of AAS and can be an appropriate complement to urinary testing. The methods have high sensitivity with a lower limit of quantification (LLOQ) at 0.05- 0.5 ng/ml in study I and a LLOQ at 0.4 ng/ml for the majority of AAS in study Il. In serum, AAS was found in 80% of the samples where positive urine findings had previously been confirmed. When comparing T concentrations measured with LC- MS/MS and immunochemistry assay, there was a notable difference in the measured concentration in samples positive for T esters. This indicates a cross- reaction in T ester positive samples when T is analysed with an immunochemistry assay. In the T enanthate study we could see a clear correlation between positive findings and a T/E ratio >10 except in one individual who was homozygous for UGT2B17 deletion. In study II, the DBS method quantified 72% of the samples above LLOQ even after six months of storage at 20 ℃. Dried blood spots proved to be a matrix with high stability that can improve sample storage in terms of logistics and offers low invasiveness regarding sample taking.

List of scientific papers

I. Bahare Makvandi, Anton Pohanka, Mats Bergström, Annica Börjesson, Mikael Lehtihet, Lena Ekström, Yufang Zheng. Detection of anabolic androgenic steroids in serum samples. Drug testing and analysis, vol 15(6), (2023): 678-688. https://doi.org/10.1002/dta.3476

II. Yufang Zheng, Bahare Makvandi, Anton Pohanka, Mikael Lehtihet, Lena Ekström. Detection of Anabolic Androgenic Steroids, including steroid- esters, in dried blood spots. [Manuscript]