Monitoring of Endogenous Steroids in Female Dried Blood Spot Samples (Continuation of 17D18MS)

Code: 21T04MS 

Blood steroid profiling using ultra-high performance liquid chromatography (UHPLC)-MS/MS has been proposed as a potential additional evidence to support the scenario of an endogenous prohibited substance administration. Moreover, the blood matrix is more informative for the correlation between hormone concentration and the physiological responses. Serum steroid profiling has been proven particularly useful for T detection in female subjects in whom menstrual fluctuations may lead to a great source of variation for urinary biomarkers disrupting the sensitivity of their longitudinal monitoring. In the clinical context, profiling of a panel of steroids represent also a great asset for the characterization of possible alterations of steroid metabolism in endocrine syndromes.

A primary drawback of this approach based on blood matrix, principally in the anti-doping context, is that it requires invasive venous blood sampling. Specialized health-care personnel usually perform this procedure and samples have to be transported under specific conditions in a short time window involving important logistic costs. To reduce the large expenses associated with the collection and shipment of this matrix, dried blood spots (DBS) with the transfer of a limited volume of capillary blood onto a filter paper or similar matrix offer a convenient and more affordable alternative. The applicability of DBS in the anti-doping context has been investigated in various studies for either direct detection of prohibited substances or indirection detection through potential biomarkers . In particular, a method using microsampling and GC-MS/MS was recently developed for the quantification of T and eight synthetic anabolic androgenic steroid (AAS). However, this method was limited to the quantification of only one endogenous AAS (EAAS) and could therefore be hardly applied in the clinical context for the monitoring of steroidogenesis disorders.

Main Findings

Additional biomarkers and alternative matrices need to be investigated to improve the detection capability for doping practices with testosterone (T), especially in female athletes. The blood steroid profile consisting of the longitudinal monitoring of serum T and DHT concentrations was proposed as a complementary approach, demonstrating a high sensitivity for the detection of T gel administration in women. A primary drawback of the application of serum steroid profiling is that it requires invasive venous blood sampling and sample collection by a trained phlebotomist. Moreover, these biological specimens have to be transported under cooled temperature conditions within a short timeframe, which all increase the total costs of sample collection. Dried blood spots (DBS), which are based on the transfer of a limited volume of capillary blood onto a filter paper or similar matrix, could tackle these obstacles offering a convenient and more affordable alternative. This process benefits from minimal invasiveness, simplicity of sample collection, facilitated transport and storage conditions, and reduced costs that could allow for more frequent sampling for anti-doping programs. In this study, we developed and validated a UHPLC-MS/MS method for the simultaneous determination of eleven free and eight conjugated steroids in DBS matrix. This method was then used for the analysis of DBS samples collected in 14 healthy women during a normal menstrual cycle (control phase) followed by a 28days testosterone gel treatment (treatment phase). Results were compared with those obtained from serum matrix. The analysis of women samples demonstrated a high correlation between DBS and serum concentrations for most compounds in control phase. In treatment phase, higher testosterone concentrations were observed in capillary than in venous DBS, suggesting a possible interference resulting from testosterone contamination on finger(s) used for gel application. Steroid profiling in capillary DBS represents a simple and efficient strategy for monitoring endogenous steroid concentrations and their fluctuation in clinical context of steroid-related disorders, or for the detection of testosterone abuse in anti-doping. Ultimately, DBS may be used as screening method allowing for more frequent sampling and for targeting blood (serum) and urine sample collection that would be used for a full steroid profile and for GC/C/IRMS analysis. The increased sampling would provide a better estimation of natural baseline variability of a given athlete and would provide a better resolution of a doping picture.

Salamin O, Nicoli R, Xu C, Boccard J, Rudaz S, Pitteloud N, Saugy M, Kuuranne T. Steroid profiling by UHPLC-MS/MS in dried blood spots collected from healthy women with and without testosterone gel administration. J Pharm Biomed Anal 204, 2021, 114280, https://doi.org/10.1016/j.jpba.2021.114280