ИСТИНА

Postpartum endometritis and mastitis cause long-term infertility and development of inflammatory and degenerative processes in the reproductive organs and mammary glands of cows.This has a direct impact on the profitability of dairy cattle farming. To date a significant amount of high-efficient protocols for treatment of endometritis and mastitis have been developed. However, in their vast majority these protocols include various chemotherapeutic active ingredients, hormones et al. The last have high bioavailability, accumulate in the muscle tissue and internal organs of cows, excrete with milk and contribute in development of the multidrug-resistant micro-organisms [1]. Veterinary drug Argumistin® deprived of these disadvantages. The active component of Argumistin® is silver nanoparticles stabilized with biologically active quaternary ammonium compound – benzyldimethyl[3-(miristoilamino)propyl]ammonium chloride (miramistin) [2]. It is well known from long history of application in medicine that miramistin and colloidal silver possess a low bioavailability when applied topically. However, researches examining the residues of miramistin and silver in milk and blood plasma of cows remain very actual today, because the practice of widespread use of silver and miramistin (both together and separately) in the treatment of mastitis and endometritis of dairy cattle until now was not established well. Understanding the bioavailability of miramistin and silver after intramammary and intrauterine administration of Argumistin® (50 ppm of silver nanoparticles stabilized with 100 ppm of miramistin) is needed to control food safety of animal products.The purpose of research was to develop sensitive and reliable technique for quantitative determination of active components of Argumistin® (miramistin, silver) in cow blood plasma and milk, following by validation (evaluation of linearity, application range, limits of detection and quantitation (LOD and LOQ), precision and accuracy) and approbation for its subsequent application in different pharmacokinetic researches. For miramistin determination HPLC-MS/MS method was chosen. An Agilent 1290 system consisted of a vacuum degasser, ultra-high pressure binary pump, diode-array detector, thermo-controlled column compartment, auto sampler, and triple quadrupole mass-spectrometer (QqQ) Agilent 6460 with Agilent JetStream Electrospray Ionization Source (AJS ESI) was used for analysis. Mass Hunter software was used for data acquisition and processing. For silver determination ICP-AES method was chosen. Silver determination was conducted on ICP-AES spectrometer Agilent 5100. ICP-AM-6 (High Purity Standards 100 mg/L Ag) served as standard solution.The LOQ of miramistin was expressed as the minimum analyte amount that the instrumentation can detect with a signal-to-noise ratio (S/N) of 10. LOQ 1.5 µg L-1 was achieved (both for blood plasma and milk). The LOD of miramistin was expressed as the minimum analyte amount that the instrumentation can detect with a signal-to-noise ratio (S/N) of 3. LOD 0.5 µg L-1 was achieved (both for blood plasma and milk).The LOQ of silver was expressed as the minimum analyte amount that the instrumentation can detect with a signal-to-noise ratio (S/N) of 10. LOQ 2.4 µg/L was achieved (silver/milk). The LOD was expressed as the minimum analyte amount that the instrumentation can detect with a signal-to-noise ratio (S/N) of 3. LOD 0.7 µg/L was achieved (silver/milk). The LOQ was expressed as the minimum analyte amount that the instrumentation can detect with a signal-to-noise ratio (S/N) of 10. LOQ 1.6 µg/L was achieved (silver/plasma). The LOD was expressed as the minimum analyte amount that the instrumentation can detect with a signal-to-noise ratio (S/N) of 3. LOD 0.5 µg/L was achieved (silver/plasma). To proceed the development of HPLC-MS/MS and ICP-AES analytical techniques for determination of miramistin and silver two experimental groups of cows were formed, each consisting of 3 cows. In all experiments Argumistin® comprising of two active ingredients (100 ppm of miramistin and 50ppm of silver nanoparticles) was used. Animals of the first group received Argumistin® intramammary. Animals of the second group received Argumistin® intrauterine. Duration of the course of administration was determined from the results of clinical studies and instruction for use of the drug: intramammary administration for 3 days, one injection in one quarter of the udder after the morning milking once a day; duration of the intrauterine administration was 3 days, one injection once a day. When administered intramammary single dose was 10 ml. When administered intrauterine a single dose was 100 ml. Scheme of blood sampling from animals receiving Argumistin® intramammary and intrauterine: just before the first administration of the drug; 6 hours after the first drug injection; 12 hours after the first drug injection; 24 hours after the first drug injection; 6 hours after the second drug injection; 12 hours after the second drug injection; 24 hours after the second drug injection; 12 hours after the third drug injection; 48 hours after the third drug injection; 72 after the third drug injection. It was found that through the time of monitoring the concentration of miramistin and silver in all samples of blood plasma and milk of cows after intramammary and intrauterine administration of Argumistin® did not exceed LOQ (less than 1.5 ppb for miramistin and less than 1 and 5 ppb for silver in the milk and blood plasma respectively). Thus, in our case, colloidal silver and miramistin have demonstrated low bioavailability after intramammary and intrauterine administration of Argumistin®. Possible explanation of this fact lays in colloidal nature of Argumistin®. When administered intramammary and intrauterine miramistin being partially adsorbed on surface of silver nanoparticles via electrostatic interactions and coordination bonds does not absorbed into the systemic circulation and eliminated from the body of cows in milk and secretions. Opposite to common antibacterial drugs [3] leading to long-term milk and meat rejection (5-10 days) the active components of the Argumistin® have low bioavailability along with high therapeutic efficacy in the treatment of cows with mastitis and endometritis. Use of Argumistin® does not entail a withholding period, and after its intrauterine and intramammary administration meat and dairy products (from healthy quarters of the udder) can be used for food purposes without restrictions.