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