Meldonium residues in milk: A possible scenario for inadvertent doping in sports?

Lately, the veterinary drug Emidonol® has been discussed as a possible scenario for inadvertent doping in sports. Emidonol® is approved for use in livestock breeding, exhibiting antihypoxic and weak sedative effects. The veterinary drug rapidly dissociates into meldonium, a substance prohibited in sports, and is excreted largely in its unchanged form into urine. To investigate if residues of meldonium in edible produce may result in adverse analytical findings in sports drug testing, a pilot study was conducted with three volunteers consuming a single dose of 100 ml meldonium-spiked milk at a concentration of 500 ng/ml (Study 1), and multiple doses of 100 ml of meldonium-spiked milk (500 ng/ml) on five consecutive days (Study 2). In the single dose study, urinary meldonium concentrations peaked between 2 and 6 h post-administration with maximum values of 7.5 ng/ml, whereas maximum meldonium concentrations of 18.6 ng/ml were determined after multiple doses 4 h post-administration. All samples were analyzed using an established and validated protocol based on HILIC-HRMS/MS.

Fluorescence chemosensing of meldonium using a cross-reactive sensor array.

In this paper, we report a fluorescent sensor array approach for the urinary detection of a prohibited substance in sports, meldonium. Four chemosensors with ethidium bromide scaffolds were employed in this method. The interaction between meldonium and chemosensors was investigated by different techniques, such as ultraviolet-visible absorption and fluorescence spectroscopy, nuclear magnetic resonance, and mass spectrometry. Molecular dynamics simulation was also used to elucidate and support the interaction mechanisms between meldonium and the chemosensors. Differential responses obtained from the sensor array enabled the qualitative and quantitative analyses of meldonium with low error values. This method was able to detect and quantify meldonium at the nM level, fulfilling the requirements of minimum performance defined by the World Anti-Doping Agency.

Application of hydrophilic interaction chromatography for simultaneous separation of six impurities of mildronate substance.

The possibility of separating the impurities of mildronate, an antiischemic drug, by hydrophilic interaction chromatography (HILIC) was investigated on different polar stationary phases (silica, amino, cyano and zwitterionic sulfobetaine). The investigations have shown that HILIC is a useful alternative to reversed phase and ion-pair chromatography. The impact of HILIC separation conditions (acetonitrile content, buffer pH in mobile phase) on retention and selectivity has been systematically studied. Importance of these factors was found to be dependent on the structural properties of solutes. A HILIC method using a zwitterionic sulfobetaine stationary phase was developed and validated to determine six impurities in the drug substance. The method was validated in terms of specificity, limit of quantitation, limit of detection, linearity, accuracy and precision.

Analysis of free malondialdehyde in photoirradiated corn oil and beef fat via a pyrazole derivative.

Malondialdehyde (MA) formed in linolenic acid, linoleic acid, corn oil and beef fat upon photoirradiation was determined by gas chromatography (GC). The MA produced was reacted with methylhydrazine to give 1-methylpyrazole and was subsequently analyzed on a GC equipped with a nitrogen-phosphorus specific detector and a fused silica capillary column. MA values determined by this method correspond to free or unbound MA levels. Linolenic and linoleic acids produced 867 micrograms MA/g and 106 micrograms MA/g, respectively. Oleic and stearic acids did not produce detectable levels of MA upon photoirradiation. Amounts of MA produced after eight hour irradiations of corn oil and beef fat were 56.24 micrograms/g and 25.01 micrograms/g, respectively. Some photoreaction products in irradiated corn oil also were identified as methylhydrazine derivatives.

New method for the determination of 1,1-dimethylhydrazine residues in apples and peaches.

A new method for determining 1,1-dimethylhydrazine (UDMH) in peaches and apples is presented. The method consists of extraction with L-ascorbic acid, derivatization with 2-nitrobenzaldehyde to the corresponding hydrazone, and cleanup on an alumina column. The hydrazone derivative is determined by gas chromatography using an electron-capture detector. Recoveries were determined from 10 to 100 ppb. Stability of the UDMH residues on frozen peaches was investigated, and results indicate that the residues degrade even while frozen.

Tumorigenic effect of 4-methylphenylhydrazine hydrochloride in Swiss mice.

4-Methylphenylhydrazine hydrochloride was administered as 10 weekly subcutaneous injections of 140 microgram/g body weight and as 7 weekly intragastric instillations of 250 microgram/g body weight in physiological saline to randomly bred Swiss mice. Treatments given subcutaneously resulted in induction of lung tumors in incidences of 36% in females and 44% in males, while intragastric treatment caused a 40% incidence in females. In addition, it gave rise to blood vessel tumors by intragastric route in incidences of 32% in females and 18% in males. In the two physiological saline-treated control groups, the lung tumor incidence (combined) was 20% in females and 21% in males, while the blood vessel tumor incidence (combined) was 7% in females and 6% in males. Histopathologically, the lesions were classified as adenomas and adenocarcinomas of the lungs, and angiomas and angiosarcomas of blood vessels. 4-Methylphenylhydrazine was postulated to be a metabolite of 4-hydroxymethylphenylhydrazine, an ingredient of the commonly eaten mushroom Agaricus bisporus. The implications are discussed with respect to the tumorigenesis data.