Stojanovska dimzoska, Biljana

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Stojanovska dimzoska, Biljana

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COMPARISON OF EFFICIENCY OF TWO TYPES SPE COLUMNS FOR DETECTION OF BETA AGONISTS IN LIVER WITH LC-MS/MS METHOD
(Faculty of Veterinary Medicine - Skopje, 2024-09-22)
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Jovanov, Stefan
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Angeleska, Aleksandra
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Beta-agonists are a large group of chemical compounds that can be used as drugs to treat bronchial asthma and other pulmonary diseases, due to their spasmolytic properties. Moreover, they are use as tocolytics and heart tonics. Beta-agonists can be illegally used in meat producing animals to accelerate growth and increase muscle mass, thereby increasing total red meat yield. Consumption of meat from treated animals incur a risk to human health, because the residues from beta agonists accumulate and persist in animal tissues. Due to adverse side effects in human, the Commission of European Communities has banned the use of these compounds in slaughter animals. Тhere are various extraction methods for the analysis of beta agonists. The aim of this study was to compare the efficiency of elution procedure of ten beta agonists from liver with two types of solid-phase extraction (SPE) columns. The sample preparation procedure includes homogenization of liver samples, double extraction with phosphate buffer, enzymatic hydrolysis with β-glucoronidasa/aril sulfatase from Helix pomatia and solid phase extraction with two types of SPE columns: Discovery® DSC-MCAX (300 mg, 6 ml) and Strata Screen-C (500 mg, 6 ml). The analyses were performed with liquid chromatography tandem mass spectrometry (LC-MS/MS). The validation of the method was done according to the criteria established in Commission Decision 2002/657. Accuracy (recovery) and precision of the method were used for comparison of the efficiency of the proposed SPE columns. The recovery of the method using DSC MCAX columns ranges from 75.20 % to 109.4 %. A higher recovery can be observed with Strata Screen-C for some β-agonists. For clenbuterol at spiking levels of 0.25 and 0.75 ng/g, the recoveries were 129.0 % and 130.61 %, respectively, while for terbutaline at the spiking levels of 0.25 and 0.5 ng/g, the recoveries were 244.80 % and 275.76 %, respectively. Zilpaterol was not detected using Strata Screen-C columns. The coefficient of variation ranges from 3.43 % to 15.73 % (DSC-MCAX columns) and from 4.07% to 33.57 % (Strata Screen-C). The difference in the efficiency of the two types of columns is a result of the phases they contain. DSC-MCAX columns contain C8 (octyl), while Strata Screen-C columns contain C18 (octadecyl) phase. In the elution of samples, the C8 phase does not retain all analytes of interest. According to the obtained results DSC-MCAX columns fulfil the criteria, while Strata Screen-C columns don’t meet the criteria established in the 2002/657/EC.
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DEOXYNIVALONOL IN PIG FEED: AN OVERVIEW ON OCCURENCE IN THE PERIOD OF FIVE YEARS (2019-2024)
(Faculty of Veterinary Medicine - Skopje, 2024-09-22)
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Dushica, Koceva
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Gordana, Ilievska
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Angeleska, Aleksandra
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Deoxynivalenol (DON) belongs to the large group of mycotoxins called trichothecenes, occurring predominantly in cereal grains such as wheat, barley, oats, rye and maize. DON is primarily produced by the pathogenic Fusarium fungi which grow on the cereals in the field, preferably at temperate climates as they are commonly found in Europe. Cereal grains intended for food and feed may also become contaminated during storage. Deoxynivalenol can make animals refuse feed even at low doses, and at larger concentrations, it can cause vomiting, leading to growth depression, increase an animal's susceptibility to infections, cause diarrhea, and hemorrhage. DON also has been connected with the human disease ATA (alimentary toxic aleukia). DON is chemically stable and extent resistant to thermal processing. Because it is difficult to neutralize or remove DON from feed, it is important to detect contamination by these trichothecene mycotoxin. At least 40 countries have set maximum levels (MRLs) or guidelines for DON in animal feed on a global scale, ranging from 0.9 to 12 mg/kg. Total of 25 complementary feedingstuffs for fattening pigs, 28 feed samples for sows, 19 samples for piglets, 10 corn and 10 barley samples were analyzed in our laboratory for presence of DON. ELISA screening method was used for determination, which is validated (in compliance with 2002/657/EC) and accredited (ISO 17025). The results were evaluated according to Macedonian legislation (Official Gazette 53/2013) which are in accordance with European regulations (2006/576/EU). All cereal samples (corn and barley) were with DON concentration below LOD (45 µg/kg). Ten (10) out of 28 samples for sows (35.7%) were in concentration range of 65 – 898.74 µg/kg. DON was found in 6 out of 25 samples for fattening pigs (24%) in concentration range of 40.63 – 800 µg/kg. Both of these categories of pig feed fulfill the criteria set in the regulations (5 mg/kg for complementary and complete feedingstuffs). Concerning the feed for piglets, 6 samples out of 19 (31.5%) shown presence of DON in the concentration range of 77.3 – 143 µg/kg. MRL for pigs is set on 0.9 mg/kg. Although all feedingstuffs for pigs do not surpassed MRLs values proposed within the regulation, it is important to acknowledge the existence of DON, and mycotoxins in general, in feed samples. The strategies to be undertaken in the future should be aimed at prevention, elimination and detoxification, as three basic approaches in the fight against mycotoxins and their entry into food chain.
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A STUDY ON THE PRESENCE OF SOME HEAVY METALS AS FEED ADDITIVES IN ANIMAL NUTRITION IN THE REPUBLIC OF NORTH MACEDONIA
(Faculty of Veterinary Medicine - Skopje, 2024-09-22)
Vangelica, Enimiteva
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Dushica, Koceva
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Aleksandra, Angeleska
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Essential heavy metals Mn, Co, Cu, and Zn are important in livestock production in trace amounts due to their physiological functions such as hormone synthesis, oxygen and electron transportation, fertility, antioxidant defense, and immunity. However, their presence beyond a certain limit in the biological system potentiates toxicity and provokes adverse effects in animals. These metals are administered by feed, where they are added in the form of chelates or organic compounds. In the European Union, Mn, Co, Cu, and Zn utilization in animal nutrition is ruled by Regulation (EC) No. 1831/2003. So far, there is no data on the presence of metals as feed additives in the nutrition of livestock in North Macedonia. As a result, this study aimed to determine the levels of Mn, Co, Cu, and Zn in complete feeds and evaluate their amounts relative to the maximum permitted levels under the legislation mentioned above. Between May 2023 and April 2024, we collected 109 complete feed samples, including 37 for cattle, 33 for pork, and 39 for poultry, from farms and feed distributors in North Macedonia. Samples were analyzed according to EN 15763:2009 with inductively coupled plasma-mass spectrometry (ICP-MS), after microwave-assisted mineralization. We performed the method validation following the ICH Q2 Guideline. The average determined levels of Mn, Co, Cu, and Zn are 0.440, 0.367, 1.648, and 0.524 mg/kg, respectively. The maximum determined amounts are 0.969 mg/kg for Mn, 0.698 mg/kg for Co, 7.160 mg/kg for Cu, and 3.562 mg/kg for Zn. We observe no violation of the maximum permitted levels; however, we must consider some precautions regarding the Co content, as the average concentration hovers around 37%, and the maximum 70% of the permitted level sat at 1 mg/kg. Regarding Mn, Cu, and Zn, the determined average and maximal concentrations are far below the maximum permitted levels. In conclusion, the determined levels of Mn, Co, Cu, and Zn within this study pose no risk to the health of the food-producing animals. Moreover, the presence of the aforementioned metals in animal nutrition is not of concern for the environment, provided that their maximum permitted levels are respected.
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Multi-class analysis of antimicrobial substances in poultry feed at cross-contamination levels by UHPLC-MS/MS – Method establishment, validation, and application
(Society of Chemists and Technologists of Macedonia, 2025-12-24)
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Koceva, Dushica
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Angjeleska, Aleksandra
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Stojković, Goran
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The Commission Delegated Regulation (EU) 2024/1229 establishes maximum limits (MLs) for the cross-contamination of antimicrobial substances in feed intended for food-producing animals. This study presents the development and in-house validation of two multi-class analytical methods for detecting antimicrobial substances in poultry feed at cross-contamination levels, using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Methods with and without solid-phase extraction (SPE) purification were developed for 26 antimicrobial substances across eight classes, covering a concentration range of 5–300 μg kg–1. The methods were validated for parameters in-cluding selectivity, limit of quantification, within- and between-day precision, accuracy, decision limit, matrix effect, and short-term analyte stability in the corresponding extracts. Both methods met the required performance criteria for detecting antimicrobial substances at cross-contamination levels, except for reduced sensitivity to lincomycin in the SPE method. Between-day precision was below 18 % and 19 %, with recoveries ranging from 95 to 103 %, for the method without SPE, and from 88 % to 101 % for the method with SPE, respectively. The analytes remained stable after two days of storage in the dark un-der two temperature conditions. These methods were applied to 39 poultry feed samples, revealing that approximately 41 % contained one or more antimicrobial substances, some of which exceeded the cross-contamination MLs. This study underscores the importance of simple, rapid, sensitive, and reliable analytical methods for controlling the presence of antimicrobials at cross-contamination levels. Such methods enable stringent control to prevent uncontrolled antimicrobial use in poultry production systems, thereby mitigating the development of antimicrobial resistance.
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Monitoring Heavy Metals, Mycotoxins, Coccidiostats and Microbial Contamination in Animal Feed: A North Macedonia Study (2018-2022)
(Walter de Gruyter GmbH, 2025-06-24)
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Enimiteva, Vangelica
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Angelovski, Ljupco
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Feed contamination can occur at every stage of the feed chain, including manufacturing, storage, transport, and utilization. Ensuring feed safety is fundamental for livestock health, animal performance, and food safety throughout the feed-to-food chain. The study aimed to make qualitative and quantitative assessment of microbiological and chemical hazards in animal feed in North Macedonia from 2018 to 2022. A total of 1,629 feed samples were analysed for heavy metals, mycotoxins, and coccidiostats, whereas 598 samples were examined for microbial contamination. The results indicate a significant prevalence of heavy metals, with lead (Pb) detected in 100% of the samples, although mostly within regulatory limits. Mycotoxins, notably ochratoxin A (OTA) and aflatoxin B1 (AFB1), were also identified, with non-compliance in 10.88% of cattle feed for AFB1. Coccidiostat residues exceeded the maximum levels in 4.6% of the samples. Microbiological analysis revealed that 1.34% of feed samples were contaminated with Salmonella spp., and 3.8% tested positive for sulphitereducing clostridia. The findings on both microbiological and chemical hazards indicate their potential to threaten the feed safety chain. Accordingly, this study emphasizes the need for continuous comprehensive feed safety monitoring and the enforcement of stringent safety regulations to safeguard animal and public health in North Macedonia.

Stojanovska dimzoska, Biljana

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