Identification of meldonium in the urine of volunteers using high-performance liquid chromatography-tandem mass spectrometry after consumption of milk of cows treated with a preventive course of the veterinary drug Emidonol®

Abstract

Emidonol is a Russian antioxidant drug, widely used in veterinary medicine both for prophylactic purposes and under pathological conditions associated with oxygen deficiency. The product of its biotransformation in animals is meldonium, which is a metabolic modulator and has been included on the Prohibited List by the World Anti-Doping Agency (WADA) since 2016. In the presented research, volunteers once consumed samples of milk from cows that had undergone a 15-day course of the veterinary drug Emidonol® 10%, obtained from one of the farms in the Moscow region. The purpose of our research was to study the possibility of qualitative determination of meldonium in urine samples after drinking a large amount of milk using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and to assess duration of urinary excretion of the prohibited in sport metabolism modulator in accordance with WADA identification criteria. Material and methods. Milk samples were collected from the cows on the 15th (last) day of the injection course of the veterinary drug Emidonol® 10%. Urine samples from volunteers, collected before and within 48 hours after a single intake of 900 ml of fresh cow's milk have been examined. The volunteers (n=4, aged 35-52 years, body weight 65-93 kg, gender was not taken into account) had not previously taken meldonium, any dietary supplements, as well as milk, dairy and meat products within 4-5 days before submitting a blank sample and during the study. Sample preparation of urine specimens was carried out using the "dilute and shoot" method. The HPLC-MS/MS was used for analysis. Meldonium identification was carried out in selective reaction monitoring (SRM) mode using the following transitions and collision energies: 147.1>147.1 (15), 147.1>132.1 (17), 147.1>59.1 (17), 147.1>58.1 (17), 147.1>42.1 (60). Results. It was found that meldonium is reliably determined in urine samples of volunteers after 12 hours when identified by HPLC-MS/MS using the 5 abovementioned SRM transitions, and for 36-40 hours using the transitions 147.1>59.1 (17), 147.1>58.1 (17) after a single consumption of 900 ml of milk. The peak concentrations occur at 5-10 hours after administration (estimated concentration in urine from 160 to 400 ng/ml) with a subsequent decrease to 2.5-5 ng/ml and below after 36-40 hours. The excretion profiles of the prohibited modulator in urine are presented. Conclusion. The principal possibility of qualitative determination of meldonium in urine samples of volunteers over 36-40 hours with a single consumption of large quantities of milk from cows that have undergone a course of treatment with the veterinary drug Emidonol® 10% was demonstrated.