Semiquantitative determination of meldonium and emoxypine in human urine by HPLC–MS/MS after receiving a single therapeutic dose of Brainmax® and milk from cows receiving a preventive course of Emidonol®

Objectives . Emidonol® is a veterinary drug used to treat pathological conditions associated with hypoxia in cattle. In addition tomeldonium, which is included in the Prohibited List of the World Anti-Doping Agency, the biotransformation product of Emidonol® in animals is the antioxidant and antihypoxant emoxypine, which can act as a marker of contamination of food products with the above-mentioned widely known metabolic modulator. The study set out to semiquantitatively determine emoxypine and meldonium levels, as well as to compare the excretion profiles of these substances by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) in urine samples of volunteers after receiving a single oral administration of a therapeutic dose of Brainmax® and after consuming a large amount of milk from cows that had received a prophylactic course of Emidonol®. Methods . Sample preparation of urine samples for the determination of meldonium was carried out using the “dilute and shoot” approach. Enzymatic hydrolysis with β-glucuronidase followed by purification by solid-phase extraction was used to determine emoxypine. Identification of meldonium and emoxypine was carried out by HPLC–MS/MS under conditions of electrospray ionization with registration of positively charged ions in the selective reaction monitoring (SRM) mode for the following transitions and collision energies: for meldonium, 147.1 > 147.1 (15), 147.1 > 132.1 (17), 147.1 > 58.1 (17), 147.1 > 59.1 (17), 147.1 > 42.1 (60); for emoxypine, 138.1 > 138.1 (7), 138.1 > 123.1 (15), 138.1 > 110.1 (20), 138.1 > 95.1 (20). Results. The possibility of simultaneous identifying meldonium and emoxypine obtained after enzymatic hydrolysis with β-glucuronidase in urine samples of volunteers after oral intake of single dose of Brainmax® and consuming a large amount of Emidonol®-contaminated milk using the HPLC–MS/MS method with different numbers and variants of SRM transitions was demonstrated. Differences in the excretion profiles of these substances were found after ingestion of large amounts of contaminated milk and a single oral dose of Brainmax® 15–18 h later and further. After taking contaminated milk 12 h or more later, emoxypine is detected in concentrations 5 or more times higher than meldonium concentrations and is excreted for a longer period of time. Conversely, after taking a single dose of Brainmax®, which contains both substances, the content of meldonium in urine samples of volunteers 15–18 h after taking it is several times higher in relation to emoxypine. The constant ratio of estimated concentrations of meldonium and emoxypine in Emidonol® was found to be approximately 1 : 2. Conclusions . Identification of meldonium in the presence of emoxypine in urine under certain conditions can be used to distinguish contamination of food products with a prohibited metabolic modulator from intentional ingestion of real doping.