Laura Tretzel

Use of dried blood spots in doping control analysis of anabolic steroid esters.

Dried blood spot (DBS) sampling, a technique for whole blood sampling on a piece of filter paper, has more than 50-years tradition, particularly in the diagnostic analysis of metabolic disorders in neonatal screening. Due to the minimal invasiveness, straightforwardness, robustness against manipulation and fastness DBS sampling recommends itself as an advantageous technique in doping control analysis. The present approach highlights the development of a screening assay for the analysis of eight anabolic steroid esters (nandrolone phenylpropionate, trenbolone enanthate, testosterone acetate, testosterone cypionate, testosterone isocaproate, testosterone phenylpropionate, testosterone decanoate and testosterone undecanoate) and nandrolone in DBS. The detection of the intact esters allows an unequivocal proof of the administration of conjugates of exogenous testosterone and its derivatives. Precise, specific and linear conditions were obtained by means of liquid chromatography high resolution/high accuracy mass spectrometry. Sensitivity in the low ppb range was accomplished by the preparation of the methyloxime derivatives of the target compounds. Labeled internal standards (d3-nandrolone, d3-nandrolone caproate and d3-nandrolone undecanoate) were applied to compensate for the broad range in chain length of the esters. The assay presented here outlines the application of DBS for the analysis of anabolic steroid esters in doping controls for the first time providing great potential to simplify the proof of exogenous administration of testosterone.

Sports drug testing using complementary matrices: Advantages and limitations.

Today, routine doping controls largely rely on testing whole blood, serum, and urine samples. These matrices allow comprehensively covering inorganic as well as low and high molecular mass organic analytes relevant to doping controls and are collecting and transferring from sampling sites to accredited anti-doping laboratories under standardized conditions. Various aspects including time and cost-effectiveness as well as intrusiveness and invasiveness of the sampling procedure but also analyte stability and breadth of the contained information have been motivation to consider and assess values potentially provided and added to modern sports drug testing programs by alternative matrices. Such alternatives could be dried blood spots (DBS), dried plasma spots (DPS), oral fluid (OF), exhaled breath (EB), and hair. In this review, recent developments and test methods concerning these alternative matrices and expected or proven contributions as well as limitations of these specimens in the context of the international anti-doping fight are presented and discussed, guided by current regulations for prohibited substances and methods of doping as established by the World Anti-Doping Agency (WADA). Focusing on literature published between 2011 and 2015, examples for doping control analytical assays concerning non-approved substances, anabolic agents, peptide hormones/growth factors/related substances and mimetics, β2-agonists, hormone and metabolic modulators, diuretics and masking agents, stimulants, narcotics, cannabinoids, glucocorticoids, and beta-blockers were selected to outline the advantages and limitations of the aforementioned alternative matrices as compared to conventional doping control samples (i.e. urine and blood/serum).

Analyses of Meldonium (Mildronate) from Blood, Dried Blood Spots (DBS), and Urine Suggest Drug Incorporation into Erythrocytes

Initially developed in the late 1970s for veterinary applications due to proposed growth-promoting effects in animals [5], meldonium has become an approved drug in selected Eastern European countries and is the subject of ongoing clinical trials focusing the compound’s anti-ischemic and cardioprotective properties [2, 3, 12, 15] as well as potential applications regarding diabetes, neurodegenerative disorders, and bronchopulmonary diseases. In the context of athletic performance, beneficial effects on the individuals’ physical working capacity, increased endurance performance, and accelerated recovery after physical activity were discussed [4, 10, 11], mentioning oral doses of meldonium of up to 2.0 g per day over 2–3 weeks in the course of pre-competition preparation phases [4]. In 2015, the World Anti-Doping Agency (WADA) initiated a one-year monitoring program [22] regarding the prevalence of meldonium (mildronate) in doping controls. Obtained data demonstrated a considerable extent of meldonium use by athletes [8, 16], which was further corroborated by a significant number of declarations of use and analytical findings at the Baku 2015 European Games [18]. Subsequently, the WADA Prohibited List that became effective in January 2016 [24] classified meldonium as banned under S4 (Hormone and Metabolic Modulators). Pharmacokinetic properties of meldonium were reported for singleand multiple-dose administration studies with healthy volunteers [25], where the drug’s elimination was monitored in plasma over 24 h post-administration and characterized by nonlinear pharmacokinetics. To date, doping controls are based on urine and blood as test matrices, and a variety of alternative matrices including amongst others dried blood spots (DBS) and dried plasma spots (DPS) have been considered lately [20]. Consequently, the knowledge about factors that potentially influence the elimination of target analytes is of particular importance to sports drug testing and, to the best of our knowledge, the role of erythrocytes and their ability to affect detection windows of meldonium in doping controls (e. g., by incorporation) has not been investigated. Therefore, in the context of a pilot study, DBS, whole blood (Na2EDTA), and urine samples were collected from 2 healthy male volunteers who orally administered meldonium either as single dose (500 mg) or as multi-dose (3 × 500 mg/day over a period of 6 consecutive days). The study protocol was approved by the local ethics committee of the National Institute for Sports Research (Bucharest, Romania, approval number #162/2016), written consent was obtained from the study participants, and the study was conducted in accordance with ethical standards in sports medicine and exercise science [9]. DBS samples were collected prior to and post-administration up to 16 days using standard DBS collection cards (Whatman DMPK-C, GE Heathcare Europe, Freiburg, Germany), dried at room temperature, and stored at + 4 °C in a plastic bag with desiccant until analysis. Na2-EDTA-stabilized whole blood specimens (3.5 mL) were sampled within the multi-dose study on day 4 and day 28 post-administration, and aliquots (4 × 20 μL) were immediately spotted on DBS cards. Further, following centrifugation of the blood samples at 1 000 × g for 15 min at 10 °C, the plasma was separated from the red blood cell (RBC) fraction, and 200 μL of the RBCs (retained for deposit onto DBS cards) was subsequently washed twice with 600 μL of phosphate-buffered saline (pH 7.4). The obtained plasma and washed erythrocytes were spotted onto DBS cards (four 20 μL aliquots each) and were also stored at + 4 °C in a plastic bag with desiccant until analysis. Online sample preparation of DBS was performed using a DBS card autosampler (DBSA) directly coupled to an automated solid-phase extraction (SPE) cartridge exchange module (SPExos) (Gerstel GmbH, Mulheim a.d.R., Germany). The sample preparation protocol was adapted from a previous application and was optimized to meet the current requirements [21]. In brief, the spots were extracted by means of flow-through desorption technology using 1 200 μL of acetonitrile/water (70:30, v/v), which included the online-addition of stable isotope-labeled meldonium (triply deuterated, TRC Toronto, Canada) as internal standard. Sample purification was performed by means of online-SPE using hydrophilic interaction liquid chromatography (HILIC) SPE cartridges. The target compounds were eluted onto the analytical column (Hypersil Gold C8, 2.1 mm × 30 mm, 1.9 μm particle size) via the LC mobile phase applying a gradient program with A: 5 mM ammonium acetate buffer (pH 3.5) and B: acetonitrile. LC-HR-MS/MS analysis was performed with a Thermo Dionex Ultimate 3000 liquid chromatograph interfaced to a Q Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany). Data were acquired in full scan mode with concomitant targeted higher energy collisional dissociation (HCD) experiments (precursor ion: m/z 147.1126, normalized collision energy: 40). The total sample-to-sample cycle time was 13 min. In addition to blood sampling, post-administration urine specimens were collected over a period of up to 49 days. These samples were subjected to analysis using a hydrophilic interaction liquid chromatography-high resolution high accuracy mass spectrometry approach (HILIC-HR-MS) published previously [8]. The analytical method for DBS measurements was validated for qualitative result evaluation purposes according to current guidelines of the International Standard for Laboratories (ISL) of the World Anti-Doping Code (WADC) [23]. Investigated parameters included specificity, carry-over, LOD (20 ng/mL), robustness, matrix interferences, and linearity (0–2 000 ng/mL), which allowed for estimating meldonium concentration levels in DBS by means of calibration curves prepared and analyzed with each batch of cards. Based on the method validation results ( ●▶ Table 1), the fitness-for-purpose of the assay was demonstrated. A total of 8 DBS samples collected prior to and up to 16 days post-administration of a single-dose (500 mg) of meldonium were analyzed using the automated isotope-dilution mass spectrometric approach. Maximum concentration levels were

Detection of testosterone esters in blood

Injections of synthetic esters of testosterone are among the most common forms of testosterone application. In doping control, the detection of an intact ester of testosterone in blood gives unequivocal proof of the administration of exogenous testosterone. The aim of the current project was to investigate the detection window for injected testosterone esters as a mixed substance preparation and as a single substance preparation in serum and plasma. Furthermore, the suitability of different types of blood collection devices was evaluated. Collection tubes with stabilizing additives, as well as non-stabilized serum separation tubes, were tested. A clinical study with six participants was carried out, comprising a single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido(®)) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanon(®)). Blood was collected throughout a testing period of 60 days. The applied analytical method for blood analysis included liquid-liquid extraction and preparation of oxime derivatives, prior to TLX-sample clean-up and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. All investigated testosterone esters could be detected in post-administration blood samples. The detection time depended on the type of ester administered. Furthermore, results from the study show that measured blood concentrations of especially short-chained testosterone esters are influenced by the type of blood collection device applied. The testosterone ester detection window, however, was comparable.

Dried blood spots (DBS) in doping controls: a complementary matrix for improved in- and out-of-competition sports drug testing strategies

A drop of whole blood dried on filter paper (Dried Blood Spots, DBS) represents an aspiring technique for minimally invasive sample collection in a multitude of analytical disciplines, e.g., therapeutic drug monitoring, preclinical drug development and diagnostic analysis of metabolic disorders in newborns. DBS sampling is characterized by cost-effectiveness, straightforwardness, robustness and facilitated storage and shipment conditions. The present investigation was conducted to highlight the opportunities arising from the implementation of DBS as a complementary matrix in doping control programs. Being frequently abused, three model compounds were chosen to represent the classes of anabolic agents (stanozolol and dehydrochloromethyltestosterone) and stimulants (pseudoephedrine). A quantitative method was developed and validated for the detection of the target analytes from DBS using liquid chromatography coupled to high resolution/high accuracy tandem mass spectrometry. The imprecision of the assay amounted to <8% for intraday and <18% for day-to-day measurements. Highly purified DBS sample extracts exhibited no ion suppression effects due to interfering matrix components and provided limits of detection of 20 pg mL−1 for stanozolol and 0.8 ng mL−1 for DHCMT and pseudoephedrine, respectively, notwithstanding an overall recovery of 26%. Deuterium-labeled internal standards were used to yield reliable quantitative results (accuracy 84–125%). The stability of the analytes was shown for at least 28 days at room temperature. The proof-of-principle for the method presented was substantiated by means of the analysis of authentic specimens obtained from administration studies with stanozolol, DHCMT and pseudoephedrine. The results provided, to the best of our knowledge, unprecedented detection windows for the tested anabolic agents accomplished by DBS sampling to support out-of-competition control efforts for the tested anabolic agents. Furthermore, the unambiguous proof of pharmacologically relevant blood concentrations at given urinary analyte levels is noteworthy for the improvement of in-competition controls, e.g., with regard to stimulant analysis.

Monitoring 2‐phenylethanamine and 2‐(3‐hydroxyphenyl)acetamide sulfate in doping controls

2-Phenylethanamine (phenethylamine, PEA) represents the core structure of numerous drugs with stimulant-like properties and is explicitly featured as so-called specified substance on the World Anti-Doping Agency (WADA) Prohibited List. Due to its natural occurrence in humans as well as its presence in dietary products, studies concerning the ability of test methods to differentiate between an illicit intake and the renal elimination of endogenously produced PEA were indicated. Following the addition of PEA to the Prohibited List in January 2015, retrospective evaluation of routine doping control data of 10 190 urine samples generated by combined gas chromatography-mass spectrometry and nitrogen phosphorus-specific detection (GC-MS/NPD) was performed. Signals for PEA at approximate concentrations > 500 ng/mL were observed in 31 cases (0.3%), which were subjected to a validated isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) test method for accurate quantification of the target analyte. Further, using elimination study urine samples collected after a single oral administration of 250 mg of PEA hydrochloride to two healthy male volunteers, two tentatively identified metabolites of PEA were observed and evaluated concerning their utility as discriminative markers for PEA intake. The ID-LC-MS/MS approach was extended to allow for the simultaneous detection of PEA and 2-(3-hydroxyphenyl)acetamide sulfate (M1), and concentration ratios of M1 and PEA were calculated for elimination study urine samples and a total of 205 doping control urine samples that returned findings for PEA at estimated concentrations of 50-2500 ng/mL. Urine samples of the elimination study with PEA yielded concentration ratios of M1/PEA up to values of 9.4. Notably, the urinary concentration of PEA did increase with the intake of PEA only to a modest extent, suggesting a comprehensive metabolism of the orally administered substance. Conversely, doping control urine samples with elevated (>50 ng/mL) amounts of PEA returned quantifiable concentrations of M1 only in 3 cases, which yielded maximum ratios of M1/PEA of 0.9, indicating an origin of PEA other than an orally ingested drug formulation. Consequently, the consideration of analyte abundance ratios (e.g. M1/PEA) is suggested as a means to identify the use of PEA by athletes, but further studies to support potential decisive criteria are warranted.

Formation of the diuretic chlorazanil from the antimalarial drug proguanil - implications for sports drug testing.

Chlorazanil (Ordipan, N-(4-chlorophenyl)-1,3,5-triazine-2,4-diamine) is a diuretic agent and as such prohibited in sport according to the regulations of the World Anti-Doping Agency (WADA). Despite its introduction into clinical practice in the late 1950s, the worldwide very first two adverse analytical findings were registered only in 2014, being motive for an in-depth investigation of these cases. Both individuals denied the intake of the drug; however, the athletes did declare the use of the antimalarial prophylactic agent proguanil due to temporary residences in African countries. A structural similarity between chlorazanil and proguanil is given but no direct metabolic relation has been reported in the scientific literature. Moreover, chlorazanil has not been confirmed as a drug impurity of proguanil. Proguanil however is metabolized in humans to N-(4-chlorophenyl)-biguanide, which represents a chemical precursor in the synthesis of chlorazanil. In the presence of formic acid, formaldehyde, or formic acid esters, N-(4-chlorophenyl)-biguanide converts to chlorazanil. In order to probe for potential sources of the chlorazanil detected in the doping control samples, drug formulations containing proguanil and urine samples of individuals using proguanil as antimalarial drug were subjected to liquid chromatography-high resolution/high accuracy mass spectrometry. In addition, in vitro simulations with 4-chlorophenyl-biguanide and respective reactants were conducted in urine and resulting specimens analyzed for the presence of chlorazanil. While no chlorazanil was found in drug formulations, the urine samples of 2 out of 4 proguanil users returned findings for chlorazanil at low ng/mL levels, similar to the adverse analytical findings in the doping control samples. Further, in the presence of formaldehyde, formic acid and related esters, 4-chlorophenyl-biguanide was found to produce chlorazanil in human urine, suggesting that the detection of the obsolete diuretic agent was indeed the result of artefact formation and not of the illicit use of a prohibited substance.