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®

• Pavel V. Postnikov
• Andrey V. Polosin
• Elena S. Mochalova
• Zurab G. Ordzhonikidze
• Dmitriy B. Nikityuk
• Viktor A. Tutelyan

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 15^th (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 above-mentioned 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.

Keywords: Emidonol®; meldonium; doping control; milk consumption; high-performance liquid chromatography-tandem mass spectrometry

Emidonol (3-(2,2,2-trimethylhydrazinium) propionate-2-ethyl-6-methyl-3-hydroxypyridine disuccinate) is a Russian veterinary drug with antioxidant, membrane-protective and antihypoxic effects [1]. It is a complex chemical structure consisting of methyl-ethyl-hydroxypyridine disuccinate and meldonium (Fig. 1). Its use in small and large cattle, domestic animals and poultry is permitted by Russian Federal Service for Veterinary and Phytosanitary Surveillance both for prophylactic purposes and for hypoxia treatment in the form of 5, 10 and 20% solutions [2]. The drug is available for purchase without a prescription in veterinary pharmacies and pet stores.

For use in cattle, the recommended concentration of the drug is 10%. When entering the animal's body, Emidonol^® undergoes biotransformation with the formation of meldonium and mexidol (emoxypine) molecules. According to a number of authors [3], the effect of mexidol can also lead to increased productivity and endurance in athletes and is comparable to the effect of doping drugs. Thus, dairy and meat products become contaminated with a prohibited substance included in the WADA Prohibited List in accordance with Article S4, paragraph 4 “Metabolic modulators” [4].

To date, several articles have been published on the topic of determining this prohibited substance in foods for doping control purposes, but the studied samples were either meat and milk [5] or urine of volunteers after consuming pasteurized milk with the added meldonium [6] using ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry (UHPLC-HRMS) [5] or high-resolution tandem mass spectrometry (HPLC-MS/HRMS) [6, 7]. In our study, volunteers once have consumed samples (900 ml) 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. Then, qualitative determination of meldonium in collected during 48 hours urine samples was carried out using HPLC-MS/MS.

According to information from the Russian Anti-Doping Agency RUSADA, from 2003 to 2021, up to 30.3% of the total number of positive samples contained meldonium and it was the most widely used doping substance [8, 9]. Therefore, the problem of food contamination with meldonium and its detection is extremely important both for Russian athletes and for the anti-doping community as a whole.

The aim of the research was to study the possibility of qualitative determination of meldonium in urine samples after a single consumption of large amount of milk from cows that had undergone a 15-day course of the veterinary drug Emidonol^® 10% using the HPLC-MS/MS method, and to assess the duration of excretion of the metabolic modulator prohibited in sports, taking into account the WADA identification criteria.

Material and methods

Samples for analysis and reagents

To conduct the preventive injection course for cows, the veterinary drug Emidonol^® 10% (“NVC Agrovetzashita” LLC, Russian Federation) was used, purchased in a pharmacy chain. The drug is sold through pharmacy chains and is approved for the use as an over-the-counter drug. Dosages were administered according to the instructions for the drug depending on the weight of the animals for 15 days every morning. Milk samples were collected on the 15^th last day of the course in sterile plastic bottles with a volume of 1 liter.

The research involved urine samples from volunteers, collected over 48 hours, before and after a single intake of 900 ml of fresh cow's milk in the morning. Urine samples from several volunteers (n=4) who had not previously taken meldonium, any dietary supplements, as well as had not consumed milk and dairy products, meat products within 4–5 days before submitting a blank sample and during the studies. Their ages were 35–52 years, body weight 65–93 kg, gender was not taken into account. Urine samples were collected in sterile medical containers with a capacity of 100 ml, labeled with the date and time of collection, stored at a temperature of +4 ºС or frozen at -20 ºС until sample preparation.

The work does not contradict the Declaration of Helsinki¹, and written consent was obtained from volunteers for the use of their biological material for conducting research.

¹ The World Medical Association, Declaration of Helsinki [Internet source] Available at: https://www.wma.net/wp-content/uploads/2018/07/DoH-Oct2008.pdf (Accessed date 30th of August 2024)

Deuterated meldonium (meldonium-d3, certified standard) (TLC PharmaChem Inc., Canada) was used as an internal standard, from which a solution with a concentration of 1 mg/ml was prepared. To prepare positive control urine samples and solutions with a meldonium concentration of 10, 100, 1000 ng/ml, a reference standard of meldonium dihydrate (European Pharmacopoeia, Meldonium Dihydrate CRS batch 1) (manufactured by EDQM, France) was used; a stock solution with the concentration of 1 mg/ml was also prepared from it, which was added to the negative control urine sample.

The following was used for the experiments: methanol, acetonitrile, acetic acid – all HPLC grade manufactured by JT Baker™ (Netherlands); water HPLC grade (Thermo Scientific Chemical, USA); ammonium acetate (purity of at least 99.9%) was obtained from Sigma-Aldrich (USA). Compressed argon 5.0 with a purity of at least 99.999%. Deionized water with a specific resistance of 18.2 MOhm×cm (Millipore, USA) was used to prepare buffer solutions.

Auxiliary equipment and materials

Crimper, decapper, 1.5 ml glass vials (Macherey-Nagel GmbH & Co, Germany), automatic variable volume pipettes 0.5–10, 20–200, 100–1000, 500–5000 µl (Eppendorf, Germany) and tips for them, benchtop centrifuge with a bucket rotor for 1.5–2 ml test tubes Centrifuge 5430 (Eppendorf, Germany), 1.5 ml polypropylene test tubes (Eppendorf, Germany), 15 ml and 50 ml falcon test tubes (Greiner Bio-One, Austria), OHaus Discovery DV215CD analytical balance (accuracy 5 digits), Vortex liquid shaking apparatus.

Sample preparation

For the experiments, 100 μl of blank urine and urine samples after milk intake, as well as 100 μl of negative and positive control urine samples were added in 1.5 ml Eppendorf tubes. Then 900 μl of diluent were added (to prepare the diluent, 22 μl of an internal standard solution with a concentration of 0.1 mg/ml (d3-meldonium) were added to a 100 ml volumetric flask and brought to the mark with methanol). Then samples were shaken and centrifuged for 10 minutes at 14 000 g. 900 μl of the supernatant were collected in glass vials, capped and analysed. Sample preparation of cow's milk samples for assessing meldonium concentrations was carried out in the same way.

To prepare mobile phase A, 15 ml were removed from a 2.5 L bottle of water and 2.5 ml of concentrated acetic acid and 12.5 ml of a 2 M ammonium acetate solution were added. Mobile phase B was acetonitrile.

Parameters of instrumental analysis by high-performance liquid chromatography/tandem mass spectrometry

HPLC-MS/MS analysis was performed using an Ultimate 3000 liquid chromatograph coupled to a TSQ Vantage triple quadrupole mass spectrometer (ThermoFisher Scientific, USA) with electrospray ionization source with heated atomizing gas flow in positive ion registration mode (ESI+). For the analysis, a Cortecs UPLC HILIC 2.1×100 column with a particle size of 1.6 μm and a Cortecs UPLC HILIC 2.1×5 precolumn with a particle size of 1.7 μm (Waters, USA) were used. Flow rate 0.3 ml/min. Injected sample volume 10 μl. Column thermostatting at 40 ºС. Gradient elution program:

Results and discussion

Before studying the excretion of meldonium with urine following the consumption of a single dose of cow's milk collected after a course of 10% emidonol injection, the concentrations of the prohibited metabolic modulator in milk samples were assessed. At the same time, the content of emidonol biotransformation products in milk can be affected by many factors that were not taken into account in this study, such as the metabolic characteristics of each individual, diet, volume of liquid consumed, etc. It is important to note that the milk used for the experiment was not pasteurized or exposed to high temperatures. The following assessed concentrations of meldonium in milk samples were obtained: sample 1 – 3700 ng/ml, sample 2 – 3200 ng/ml, sample 3 – 1400 ng/ml, sample 4 – 1350 ng/ml. The first two milk samples were from morning milking, samples 3 and 4 were from evening milking.

Fig. 2 shows the graphs of meldonium excretion in the urine collected over 48 hours after a single morning intake of 900 ml of milk by the volunteers, who did not consume other dairy products or meat. The abscissa axis shows the excretion time, the ordinate axis shows the assessed meldonium concentration expressed in ng/ml. The meldonium concentration was estimated by the two longest-lived and most stable SRM transitions (147.1>59.1, 147.1>58.1), the detection limit was about 2.5 ng/ml. As in the case of milk samples, the concentration of meldonium in urine samples may vary depending on the characteristics of a person’s metabolism, excretory capacity of the kidneys, the amount of fluid consumed, etc. The presented data shows that the maximum concentrations of meldonium in the body are reached 5-10 hours after drinking milk. Thus, in volunteer 1 it was about 400 ng/ml, volunteer 2 – 310 ng/ml, volunteer 3 – 160 ng/ml, volunteer 4 – 260 ng/ml. Then a sharp drop in concentration was observed and over the next 24 hours it decreased from about 20 ng/ml (12–13 hours after intake) to 2.5–5 ng/ml and below (after 36–40 hours). In volunteer 2, the assessed concentrations of meldonium remained at a level of ~5 ng/ml for just over 42 hours.

Meldonium, being a metabolic agent and antihypoxant, affects the body's recovery processes, activating glycolysis, which occurs without additional oxygen consumption, stabilizes the mitochondrial potential. It is noted that when taking such a single dose of milk, volunteers did not feel any effect, increased performance or vigor, which is not surprising, since therapeutic doses of meldonium are about 500-1000 mg per day, while its amount obtained from consuming even a large volume of contaminated milk is significantly (orders of magnitude) lower.

According to WADA Technical Document TD2023IDCR², certain chromatographic and mass spectrometric criteria must be met to confirm the identity of the analyte in the sample. Thus, the chromatographic retention time of meldonium, in this case 5.00 min, in the sample must not differ by more than 1% or ±0.1 min from that in the reference sample. For MS/MS determination, at least two diagnostic ions must be obtained (i.e. two SRM transitions – precursor ion and product ion). The signal-to-noise ratio (S/N) for all diagnostic peaks must be greater than 3:1. The relative intensity of the peaks of each diagnostic ion must not differ from those in the reference sample by more than the value (expressed in % or in absolute values) specified in TD2023IDCR².

² WADA Technical document – TD2023IDCR [Internet source] Available at: https://www.wada-ama.org/sites/default/files/2023-02/td2023idcrv1.1_eng_final.pdf (Accessed date 30th of August 2024)

Under the influence of collision energy, the meldonium precursor ion with m/z 147.1 (C6H15N2O2+) dissociates to form four product ions with m/z 42.1 (C2H4N+), m/z 58.1 (C3H8N+), m/z 59.1 (C3H9N+) as a result of the loss of the trimethylamine residue and m/z 132.1 (C5H12N2O2+) (Fig. 3).

C. Görgens et al. [7] found that fragmentation due to the loss of one methyl group also resulted in the formation of a fragment with m/z 132.1. Indeed, when analyzing the pure medicinal product or urine samples from volunteers after a single injection of meldonium (2–5 ml of the drug intramuscularly at a concentration of 100 mg/ml), a peak is recorded for the SRM transition 147.1>132.1, the intensity of which allows it to be used for identification. However, when analyzing urine samples after drinking milk containing Emidonol^® biotransformation products, the intensity of the peak of this transition is 2–3 orders lower than the peaks of other transitions; the peak is recorded only in urine samples with maximum concentrations (from 100–150 ng/ml and above), collected 4–12 hours after drinking large amounts of milk when used samples obtained from morning milking (and collected 6–9 hours after drinking samples from evening milking). It is possible that when consuming small amounts of milk, the peak of the SRM transition 147.1>132.1 will be difficult to register or will be absent altogether [10]. Registration of certain peaks depends on the stage of meldonium elimination.

If we evaluate the obtained results according to the WADA criteria, then meldonium, which is a product of emidonol biotransformation, is reliably determined in urine samples of volunteers 4-12 hours after consuming a single dose of 900 ml of milk using followed 5 SRM transitions 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) (Fig. 4).

After more than 12 h, the peak of the SRM transition 147.1>132.1 (17) is no longer detected. It should be noted that the peaks of two other SRM transitions 147.1>147.1 (15) and 147.1>42.1 (60) were recorded up to 14-15 h (data not shown), but then their use for identification becomes problematic due to high interference and the presence of extraneous peaks. As mentioned earlier, the most stable and “long-lived” transitions are 147.1>59.1 (17) and 147.1>58.1 (17). Their peaks are reliably identified in urine samples 36-40 h after ingestion of large doses of milk (Fig. 5), and in one of the volunteers over 42 h. For the determination of meldonium in trace amounts, it is preferable to use the HPLC-MS/HRMS method.

S. Cristoni et al. noted [10] that according to the European Union Directive 2002/657/EC³ for substances belonging to Group A (anabolic and prohibited substances), such as meldonium, the substance must be identified using at least four points (SRM transitions), as reported by Görgens et al. [7], which is contrary to the methodology used in anti-doping analysis, where 3 points are used (ions with m/z 58.0654, m/z 59.0732 and m/z 147.1133). In our research, five SRM transitions were used to detect meldonium, although the use of even three ones is not contrary to TD2023IDCR³.

³ Commission Decision 2002/657/EC implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results [Internet source] Available at: https://fishquality.ru/assets/files/Documents%20on%20activities/Legislation/EU/Решение%202002-657.pdf (Accessed date 30^th of August 2024)

According to the data presented in the article [5] by A. Temerdashev et al., even heat treatment of milk does not guarantee complete elimination of meldonium. Based on the results of the study, the authors note that both fresh and pasteurized milk contain meldonium, and its content in the same milk sample before and after pasteurization may differ by approximately 2 times. It is also noted that the prohibited metabolic modulator can be determined in samples of cows' milk approximately 7 days after the end of the Emidonol^® course.

S. Guddat et al. [6] added meldonium to 100 ml of pasteurized milk (50 μg absolute) and studied the elimination profile after consumption by volunteers for one and five consecutive days. The authors found that the accumulation effects of low-dose meldonium over five consecutive days were insignificant (approximately 2.5 times compared to a single dose) and urine samples were negative (provided “blank” urine specimens) after 24 h. However, in our case, repeated consumption of large amounts of meldonium-contaminated milk may have a significant effect.

The results of our studies show that when large amounts of milk are consumed, the assessed concentrations of meldonium in urine exceed the minimum required performance level (MRPL) of 100 ng/ml⁴. In general, analyzing the data presented above, it can be said with confidence, that consumption of large amounts of fresh unpasteurized cow's milk collected after a 15-day course of treatment with Emidonol^® 10% veterinary drug is guaranteed to result in an adverse analytical finding result according to WADA criteria within 12 hours [when scanning in SRM mode using 5 transitions – 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)], and within about 36–40 hours [when scanning in SRM mode using 2 transitions – 147.1>59.1 (17), 147.1>58.1 (17)].

⁴ WADA Technical document – TD2022MRPL [Internet source] Available at: https://www.wada-ama.org/sites/default/files/2022-01/td2022mrpl_v1.1_eng_0.pdf (Accessed date 5th of August 2024)

Conclusion

As a result of the first studies on urine samples of volunteers after a single consumption of a large amount of milk from cows treated with the veterinary drug Emidonol 10%, it was found that maximum concentrations of meldonium in urine can reach 160–400 ng/ml 5–10 hours after a single intake of 900 ml, which significantly exceeds the MRPL level of 100 ng/ml. It was shown that an unfavorable result of analysis in accordance with the WADA criteria for determining meldonium by HPLC-MS/MS can be recorded, depending on the scanning conditions used in the SRM mode, even 40–42 hours after milk consumption.

The data obtained indicate that, apparently, a certain waiting period should be established for productive farm animals during the period of therapeutic or prophylactic treatment with the above-mentioned veterinary drug, and professional athletes should refrain from consuming large amounts of fresh cow's milk in order to avoid a positive doping test for meldonium.

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