Recent decades have witnessed the worrying phenomenon of a disproportionate increase in the use of performance-enhancing substances, in particular anabolic androgenic steroids (AAS), not only among high-level professional athletes, but also among all other athletes up to amateur ones [1
]. This is mainly related to the prompt availability of powerful AAS for the average user: in fact, due to the proliferation of online retail stores, these substances can be purchased on the web and shipped directly home through the postal service [2
]. The phenomenon has spread mostly in the framework of gym circuits, where athletes abuse AAS in order to increase muscle strength and size, and the lean-to-fat body mass ratio, thus amplifying the physical exercise outcomes [3
]. These results, however, also entail serious side effects: endocrine effects due to competition with cortisol, virilisation, acne, gynecomastia and testicular atrophy, increase of cardiovascular issues, liver complications [4
]. It has also been proven that individuals abusing AAS incur more easily in neuropsychiatric disorders such as anxiety, paranoia and depression [6
]. Although the possible role of AAS in causing either neurotoxicity or neuroprotection is still unclear, evidence of prevalent neurotoxic effects after administration of synthetic AAS is mounting [7
]. As a consequence, AAS use is strictly prohibited by the World Anti-Doping Agency (WADA) [10
], both in-competition and out-of-competition, and for this reason it is of the utmost importance to develop and update analytical tools to reliably detect their use and possible abuse. These tools could also be useful to study and clarify the activity of AAS administration on neuronal health and the effectiveness of prospective neuroprotective agents. In the recent past, gas chromatography (GC) coupled to mass spectrometry (MS) has been routinely applied to human urine for the screening and confirming analysis of AAS and metabolites [11
]. However, AAS analysis by GC-MS requires time consuming derivatisation steps and still suffers from sensitivity and selectivity issues for some steroids present at very low levels. Liquid chromatography (LC) coupled to tandem MS (MS/MS) is increasingly being used as an analytical tool alternative to GC-MS procedures. There are several reports regarding AAS analysis using liquid chromatography–tandem mass spectrometry (LC-MS/MS) in human samples [15
]. These methods are usually applied to urine samples for anti-doping analysis, since this matrix contains relatively high concentrations of drugs and/or their metabolites and its sampling is non-invasive. Nevertheless, classical urine sampling and handling requires storage and transportation at low and controlled temperatures, thus increasing the overall costs of analysis and the complexity of pre-analytical steps. Moreover, the possible occurrence of microorganisms in urine samples can alter AAS profiles, as it can trigger metabolism in stored samples [20
]. Sample stability over time is indeed a crucial aspect, especially in anti-doping activities when delayed sample re-analysis is required, like in the case of the so-called “B-sample”, an aliquot of the sample originally supplied by the athlete, kept by the laboratory in charge and analysed as a confirmation if a prohibited substance is found in the original sample [22
]. The purpose of this work is the development and comparison of two original urine microsampling and pretreatment techniques coupled to a fully validated LC-MS/MS method for the purpose of AAS analysis, with the aim of overcoming the current limitations arising from the use of large volumes of fluid samples and to develop new simplified procedures while maintaining high reliability and soundness of the resulting analytical data [23
]. Thirteen representative AAS have been considered, namely nandrolone, 1-androstenedione, dehydroepiandrosterone (DHEA), testosterone, epitestosterone, dihydrotestosterone (DHT), methandrostenolone, norethandrolone, mesterolone, clostebol, stanozolol, fluoxymesterone and danazol (see Table 1
and Table 2
This is the first study to describe a comparative DUS/VAMS methodology coupled to LC-MS/MS detection, as a feasible and reliable strategy to assess free AAS concentrations in dried urine microsamples. DUS and VAMS process parameters have been investigated in order to develop effective and reliable protocols. Moreover, urine microsamples were compared with conventional fluid urine analysis, in order to assess correspondence of the obtained quantitative results. Finally, stability studies on dried microsamples were carried out and the results compared to those of classic sample cryopreservation.
The comparative analytical approach for dried urine microsamples presented herein aims at providing data useful to implement simple and rapid analytical protocols, with potential immediate applicability in sport drug testing scenarios and in neurotoxicity or neuroprotection studies.