Determination of Multi-Class Antimicrobial Residues and Antimicrobial Resistance in Cow Milk and Feces Samples during Withdrawal Period.

The use of antimicrobials in livestock production and their effect on the development of antimicrobial resistance (AMR) is a global health problem for humans, animals and the environment. The aim of this study was to determine antimicrobial residue levels in milk and feces samples during the withdrawal period in dairy cattle administrated with a single dose of the drug, as well as to characterize the antimicrobial resistance patterns of Escherichia coli cultured from feces samples. In the study, dairy cows from three different farms in North Macedonia were included. Raw milk and feces samples were collected before drug administration (0 day) and on the 1st, 2nd, 3rd, 7th and 21st day after drug administration. The antimicrobial residues of oxytetracycline, enrofloxacin, amoxicillin, trimethoprim and procaine-benzylpenicillin were determined using a validated liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method involving stable isotopes. According to results obtained, the highest levels of analyzed antimicrobial residues were determined on the first day after drug administration, which then gradually decreased until their elimination (7th day). The highest AMR of E. coli (100%) was found in ß-lactam antimicrobials. Less exposure to broad-spectrum antimicrobials could be an important factor for reduction of AMR on dairy farms.

Multi-class/residue method for determination of veterinary drug residues, mycotoxins and pesticide in urine using LC-MS/MS technique.

BACKGROUND: Veterinary drugs are widely used in animals to prevent diseases and are a complex set of drugs with very different chemical properties. Multiclass and multi-residue methods for simultaneous detection of residues from veterinary drugs and contaminants in urine are very rare or non-existent. Therefore, the aim of this study was to develop and validate a sensitive and reliable quantitative LC-MS/MS method for simultaneous determination of a wide range of veterinary drug and pesticide residues and mycotoxins in bovine urine. This involved 42 veterinary drug residues (4 thyreostats, 6 anabolic hormones, 2 lactones, 10 beta agonists, 15 antibiotics, 5 sulphonamides), 28 pesticides and 2 mycotoxins. Stable isotopically labelled internal standards were used to facilitate effective quantification of the analytes. Analysis was performed in both positive and negative ionization modes with multiple reaction monitoring transitions over a period of 12 min. RESULTS: The parameters validated included linearity, limit of detection (LOD), limit of quantification (LOQ), detection capability (CCß), decision limit (CCα), stability, accuracy and precision. The process followed guidelines of the regulation 2021/808/EC. The calibration curves were linear with coefficient of correlation (R2) from 0.991 to 0.999. The LODs were from 0.01 to 2.71 µg/L, while the LOQs were from 0.05 to 7.52 µg/L. The CCα and CCß were in range 0.05-12.11 µg/L and 0.08-15.16 µg/L. In addition, the average recoveries of the spiked urine samples were from 71.0 to 117.0% and coefficient of variation (CV) < 21.38% (intraday and interday). CONCLUSION: A new isotopic LC-MS/MS method has been developed, validated and applied for identification and quantification of 72 residues of veterinary drugs and pesticides and other contaminants such as mycotoxins in bovine urine. The most appropriated sample preparation procedures involved sodium acetate buffer, enzymatic hydrolysis using ß-glucuronidase and cleanup solid phase extraction with OASIS SPE cartridges. The parameters were satisfactorily validated fulfilling requirements under Regulation 2021/808/EC. Consequently, the method could be used in routine analysis of bovine urine samples for simultaneous detection of veterinary drug and pesticide residues as well as contaminants such as mycotoxins.

Natural Products/Bioactive Compounds as a Source of Anticancer Drugs.

Cancer is one of the major deadly diseases globally. The alarming rise in the mortality rate due to this disease attracks attention towards discovering potent anticancer agents to overcome its mortality rate. The discovery of novel and effective anticancer agents from natural sources has been the main point of interest in pharmaceutical research because of attractive natural therapeutic agents with an immense chemical diversity in species of animals, plants, and microorganisms. More than 60% of contemporary anticancer drugs, in one form or another, have originated from natural sources. Plants and microbial species are chosen based on their composition, ecology, phytochemical, and ethnopharmacological properties. Plants and their derivatives have played a significant role in producing effective anticancer agents. Some plant derivatives include vincristine, vinblastine, irinotecan, topotecan, etoposide, podophyllotoxin, and paclitaxel. Based on their particular activity, a number of other plant-derived bioactive compounds are in the clinical development phase against cancer, such as gimatecan, elomotecan, etc. Additionally, the conjugation of natural compounds with anti-cancerous drugs, or some polymeric carriers particularly targeted to epitopes on the site of interest to tumors, can generate effective targeted treatment therapies. Cognizance from such pharmaceutical research studies would yield alternative drug development strategies through natural sources which could be economical, more reliable, and safe to use.

Determination of Veterinary Drug Residues, Mycotoxins, and Pesticide Residues in Bovine Milk by Liquid Chromatography Electrospray Ionisation -tandem Mass Spectrometry.

Introduction: Multi-class and multi-residue analyses are very complex procedures because of the physico-chemical properties of veterinary drug residues and other contaminants. The purpose of the study was to develop an analytical method for the sensitive determination of 69 analytes in bovine milk by liquid chromatography electrospray ionisation-tandem mass spectrometry. Material and Methods: Antimicrobial, anabolic hormone, lactone, ß-agonist, mycotoxin and pesticide residues were analysed in 120 raw milk samples from different dairy farms in North Macedonia. Stable isotopically labelled internal standards were used to facilitate effective quantification of the analytes. Results: The linear regression coefficients were higher than 0.99, the limits of detection ranged from 0.0036 to 47.94 µg/L, and the limits of quantification ranged from 0.053 to 59.43 µg/L. The decision limit values ranged from 0.062 to 211.32 µg/L and the detection capability from 0.080 to 233.71 µg/L. Average recoveries of the analytes spiked in raw milk were in the range of 70.83% to 109%, intra-day coefficient of variation (CV) values from 2.41% to 22.29%, and inter-day CV values from 3.48% to 23.91%. The method was successfully applied in the testing of bovine milk samples. In five samples residues were detected. They were sulfadimethoxine (in two samples), enrofloxacin, tetracycline and oxytetracycline and were at concentrations below the EU maximum residue limit. Conclusion: The method is useful for routine testing for this group of chemical hazards in bovine milk.

A new LC-MS/MS method for multiple residues/contaminants in bovine meat.

A multi-class and multi-residue/contaminant method for the determination of veterinary drug and pesticide residues and mycotoxins in bovine meat has been developed and validated. The veterinary drug residues/contaminants included antimicrobials, anabolic hormones, lactones, ß-agonists, mycotoxins, and pesticides. Isotopic labeled internal standards were included to compensate residual matrix effects. The calibrators used in the method demonstrated linearity with the R2 > 0.98. The decision limit (CCα) values were in the range from 0.067 to 2103.84 µg/kg, while the range for detection capability (CCß) was from 0.083 to 2482.13 µg/kg. The limit of detection (LOD) and limit of quantification (LOQ) were in the range from 0.059 to 291.36 µg/kg, and 0.081 to 328.13 µg/kg, respectively. The recovery of analytes ranged from 61.28% to 116.20%. The intra-day coefficient of variation (CV) was from 0.97 to 25.93% and the inter-day CV was 2.30-34.04%. The method has been used for the determination of 49 residues/contaminants in bovine meat. Application of the method in routine analysis in bovine samples, revealed in limited samples the presences of enrofloxacin, oxytetracycline and sulfadiazine at the concentration of 35.22 µg/kg, 27.35 µg/kg, and 36.20 µg/kg, respectively.

Fatty acid profile as a discriminatory tool for the origin of lamb muscle and adipose tissue from different pastoral grazing areas in North Macedonia - A short communication.

The fatty acid (FA) profiles of lamb muscle and adipose tissue originating from ten different grazing areas in North Macedonia and an assessment of whether the meat origin could be distinguished on the basis of FA profile are presented. Muscle and adipose tissues of three-month-old male lambs (crossbreds of autochthonous Ovcepolian and Württemberg) reared on spring pastures were studied. Statistically significant differences in the individual FA contents, FA groups and FA ratios were observed between lamb tissues from the different geographic regions. Canonical discriminant analysis showed there was a significant linear divergence between tissues from almost all examined regions. The greatest weight in the differentiation of the different areas showed fatty acid ratios, C18:3n3, C18:1n9c and C20:5n3 for the muscle tissue, as well as C18:1n9c, C18:2n6c, C16:1 and C17:1 for the adipose tissue. This showed that the FA composition of muscle and adipose tissue obtained from lambs reared on pasture could serve as a useful indicator to aid differentiation of its geographic origin but it should be confirmed through further replicated studies.

Surface water characterization of three rivers in the lead/zinc mining region of northeastern Macedonia.

Mine waste is recognized as being one of the most serious threats for freshwater ecosystems, and it still represents one of the greatest environmental concerns in Macedonia. The aim of our investigation was to obtain an in-depth understanding of mining influence on freshwater systems from water contamination to effects on aquatic organisms. In this study, we assessed the impact of active lead (Pb)/zinc (Zn) mines Zletovo and Toranica on the water quality of three rivers in northeastern Macedonia (Bregalnica, Zletovska, and Kriva rivers) based on data collected in spring and autumn of 2012. The Bregalnica River, near Shtip, was characterized mainly by weak contamination with arsenic, barium, iron, molybdenum, titanium, uranium, vanadium, nitrate, and phosphate, as well as critical faecal pollution, which alltogether could be connected to agricultural activities; however, an impact of the mines was not observed. Contrary, both the Zletovska and Kriva rivers showed a clear impact of Pb/Zn mines on water quality. In the Zletovska River, increased concentrations of cadmium (Cd), cobalt, cesium, copper, lithium, manganese (Mn), nickel, rubidium, tin, strontium, thallium, Zn, sulphates, and chlorides were found, especially in autumn (e.g., Cd 2.0 µg L(-1); Mn 2.5 mg L(-1); Zn 1.5 mg L(-1)). In the Kriva River, increased Cd (0.270 µg L(-1)) and Pb (1.85 µg L(-1)) concentrations were found only in spring, possibly due to sediment resuspension during greater water discharge. The selected sampling sites on the Bregalnica, Zletovska, and Kriva rivers were confirmed as being appropriate locations for further studies of mining waste's impact on freshwater ecosystems, the first one as a nonimpacted site and the other two as possible areas of increased exposure of aquatic organisms to metals.