Use of dried blood spots in doping control analysis of anabolic steroid esters
Graphical abstract
Introduction
Dried blood spots (DBS), a sampling technique for whole blood specimens dried on a piece of filter paper, has a more than 50-year long tradition. The first application of this microvolume sampling method using blood from a heel prick was reported in 1963 by Robert Guthrie [1] introducing an assay for the diagnostic investigation of phenylketonuria in newborns. Subsequently, further applications were suggested especially for neonatal screening of metabolic disorders [2], [3], [4]. In the last decades DBS analysis by liquid chromatography coupled to mass spectrometry was firmly established and the number of procedures enormously increased. Various applications are described for disease surveillance (e.g. cancer, diabetes), preclinical drug development, toxicological and pharmacokinetic studies, therapeutic drug monitoring (TDM), and metabolomic profiling [4], [5], [6], [7], [8], [9], [10], [11]. At this point, however, the DBS sampling technique has never been implemented in routine doping control analysis, although there are a couple of promising application examples [12], [13], [14], [15], [16]. Compared to conventional venous blood sampling, the collection of a volume of typically 20 μl of capillary blood from a heel, ear or finger prick is minimally invasive [17], [18]. Further benefits involve the fastness including the possibility for automation of the sample processing [10], [19], robustness against manipulation [5], [9], [14] and enhanced stability of the cellulose-fixed analytes at room temperature. Stability improvement may be attributed to the elimination of humidity via dehydration and the inactivation of enzymatic degradation [4], [5] as opposed to conventional sample matrices (urine, plasma, serum). This, as well as the small sample volume, results in simplified storage and shipment conditions. However, a few challenges have to be overcome, namely the comparably time consuming sample preparation and the extraction of the analytes from the cellulose-based matrix. The small sample volume of typically 20 μl of blood requires high sensitivity of the mass spectrometer and the applied analytical method [9].
The anabolic steroid testosterone is prohibited since 1976 by the International Olympic Committee (IOC) [20] but is still one of the most frequently detected doping agents [21]. Testosterone intake induces an increase of lean body mass and strength as well as a shortened recovery time [22]. Because testosterone is metabolically instable after oral application, prodrugs in form of the steroid esters are administered orally for doping purposes. Additionally sustained release is obtained via esterification of testosterone and its analogues when administered intramuscularly. In the systemic circulation the esters are hydrolyzed by blood esterases and become fully effective. Due to the ester cleavage in blood, concentrations of the intact esters are rather low. Pharmacy offers multiple formulations of the anabolic steroid esters, some of which are approved drugs used in the therapy of hypogonadism [22].
Traditionally the abuse of anabolic agents derived from the endogenous steroid hormone testosterone is screened for by means of the steroid profile (including the ratio of testosterone to epitestosterone (T/E ratio > 4)) in urine. This screening assay poses great challenges for doping control laboratories. The differentiation between the exogenously administered testosterone and testosterone occurring physiologically represents a major difficulty. Therefore suspicious results are verified or falsified by the use of carbon isotope ratio mass spectrometry (IRMS) [23], [24], [25].
An enormous benefit is achieved by the application of other sampling matrices such as blood or hair since a direct and unequivocal proof of the intact anabolic steroid esters is possible. A screening assay for testosterone esters in human plasma was presented by Forsdahl et al. [26], others reported the detection of the target analytes in equine serum [27] and human hair [28]. Conventional venous cannula sampling is an invasive method with the need for physicians or phlebotomists for sample collection. To cope with these challenges the present approach highlights the application of the DBS sampling technique in the analysis of anabolic steroid esters in doping control analysis for the first time. A screening method was developed for nandrolone and eight anabolic steroid esters deriving from testosterone, nandrolone and trenbolone using high performance liquid chromatography (HPLC) coupled to high resolution tandem mass spectrometry (HR–MS/MS). The identification and determination of the target compounds was conducted in targeted MS/MS mode.
Section snippets
Chemicals and reagents
Testosterone acetate, testosterone cypionate, testosterone isocaproate, testosterone phenylpropionate and nandrolone were obtained from Sigma (Schnelldorf, Germany). Nandrolone phenylpropionate was purchased from the National Measurement Institute (Sydney, Australia) and trenbolone enanthate from Steraloids (New Port, USA). Andriol® Testocaps 40 mg were obtained from Essex Pharma GmbH (Munich, Germany). Deuterium labeled nandrolone [29], d3-nandrolone caproate and d3-nandrolone undecanoate were
Results and discussion
To our best knowledge this paper outlines for the first time a screening method to detect intact anabolic steroid esters from DBS. The typically short half-life of the esters in the systemic circulation and the consequently low concentration levels at sampling time, as well as the small sample volume obtained in DBS, require a highly sensitive method in order to substantiate the analytes for a reasonable period of time. To permit the simplified shipment of DBS samples (e.g. room temperature,
Conclusion
DBS sampling has been found to be a promising technique in sports drug testing. Hitherto, only a few application examples have been suggested demonstrating the capability for the analysis of small molecules with diverse chemical properties and moreover for peptides with relevance for doping controls [12], [13], [14], [15], [16], [45]. However, in sports drug testing the DBS sampling technique has not been established so far.
In the present approach a screening method for the detection of
Acknowledgements
The study was carried out with support of the Manfred Donike Institute for Doping Analysis (Cologne, Germany), Antidoping Switzerland (Bern, Switzerland), and the Federal Ministry of the Interior of the Federal Republic of Germany (Berlin, Germany).
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