The subject of this study is the validation of a high-performance liquid chromatography method for the analysis of amino acids in feed. The contents of amino acids were determined in maize, soybean, soybean meal, as well as in their mixtures enriched with different amounts of methionine, threonine and lysine. The method involves the acid hydrolysis of the sample (6 h at 150ºC), automated derivatisation of amino acids with the aid of o-phthaldialdehyde and 9-fluorenylmethyl chloroformate reagents, separation on the ZORBAX Eclipse-AAA column, and detection using a diode-array detector. The method is characterized by high specificity (the difference between the retention times of the feed samples and standard mixtures are below 1.7 %), wide linear range (from 10 to 1000 nmol cm-3, r2 = 0.9999), high accuracy (recovery 93.3-109.4 %), and the precision of the results (RSD below 4.14 % in case of repeatability and below 4.57 % in the case of intermediate precision). The limit of detection and the limit of quantification are in the range 0.004-1.258 μg cm-3 and 0.011-5.272 μg cm-3, respectively. The results demonstrate that the procedure can be used as a method for the determination of the composition of primary amino acids of feed proteins. [Projekat Ministarstva nauke Republike Srbije, br. 31081

Emerging mycotoxins such as moniliformin (MON), enniatins (ENs), beauvericin (BEA), and fusaproliferin (FUS) may contaminate maize and negatively influence the yield and quality of grain. The aim of this study was to determine the content of emerging Fusarium mycotoxins in Serbian maize from the 2016, 2017, and 2018 harvests. A total of 190 samples from commercial maize production operations in Serbia were analyzed for the presence of MON, ENs, BEA, and FUS using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The obtained results were interpreted together with weather data from each year. MON, BEA, and FUS were major contaminants, while other emerging mycotoxins were not detected or were found in fewer samples (<20%). Overall contamination was highest in 2016 when MON and BEA were found in 50–80% of samples. In 2017 and 2018, high levels of MON, FUS, and BEA were detected in regions with high precipitation and warm weather during the silking phase of maize (July and the beginning of August), when the plants are most susceptible to Fusarium infections. Since environmental conditions in Serbia are favorable for the occurrence of mycotoxigenic fungi, monitoring Fusarium toxins is essential for the production of safe food and feed.

Aspergillus flavus is the main producer of aflatoxin B1, one of the most toxic contaminants of food and feed. With global warming, climate conditions have become favourable for aflatoxin contamination of agricultural products in several European countries, including Serbia. The infection of maize with A. flavus, and aflatoxin synthesis can be controlled and reduced by application of a biocontrol product based on non-toxigenic strains of A. flavus. Biological control relies on competition between atoxigenic and toxigenic strains. This is the most commonly used biological control mechanism of aflatoxin contamination in maize in countries where aflatoxins pose a significant threat. Mytoolbox Af01, a native atoxigenic A. flavus strain, was obtained from maize grown in Serbia and used to produce a biocontrol product that was applied in irrigated and non-irrigated Serbian fields during 2016 and 2017. The application of this biocontrol product reduced aflatoxin levels in maize kernels (51–83%). The biocontrol treatment had a highly significant effect of reducing total aflatoxin contamination by 73%. This study showed that aflatoxin contamination control in Serbian maize can be achieved through biological control methods using atoxigenic A. flavus strains.

In recent years climate changes recorded in temperate regions of Europe have led to aflatoxin (AF) contamination of maize. Thus, the aim of this study was to investigate the influence of weather conditions on levels of aflatoxin B (AFB1), aflatoxin B (AFB2), aflatoxin G (AFG1) and aflatoxin G (AFG2) in 180 maize samples collected from the main maize-growing regions (Western Bačka, North Banat, South Banat and Central Serbia) in Serbia after harvest in 2015. The concentrations of AFs were determined by a validated HPLC method with post-column derivatisation and fluorescence detection (HPLC-FLD). The presence of AFB1, AFB2, AFG1 and AFG2 was detected in 57.2%, 13.9%, 5.6% and 2.8% of maize samples in the concentration ranges of 1.3-88.8 µg kg, 0.60-2.8 µg kg, 1.8-28.5 µg kg and 2.1-7.5 µg kg respectively. The recorded smaller amount of precipitation and especially higher air temperatures during the summer of 2015 were favourable for AF production, which resulted in 32.2% and 21.1% of samples being unsuitable for human consumption, since AFB1 and the sum of AFs concentrations were above 5.0 and 10.0 µg kg respectively. Furthermore, the findings in this study indicate that the microclimate conditions in the investigated regions had a great influence on the contamination frequency of maize with AFs. The highest percentage of samples unsuitable for human consumption, considering both AFB1 and total AFs content were 72.5% and 51.5% respectively from Central Serbia, whilst the lowest percentages of 15.6% and 6.2% respectively were found in Western Bačka. These findings confirmed that maize should be continuously monitored in order to protect human and animal health from the harmful effects caused by AFs contamination.

Presence of aflatoxin M1 (AFM1) in milk should be continuously controlled in order to protect the population from risks associated with its proven toxicity and carcinogenicity. During recent years, there has been an increase in demand for development of sensitive, accurate, simple and fast method which is reliable for detection of AFM1 at low concentrations found in milk samples. For that purpose, enzyme linked immunosorbent asssay (ELISA), high performance liquid chromatography with fluorescence detector (HPLC-FLD) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) were optimized and validated in order to apply them for AFM1 analysis in naturally contaminated milk samples, and to assess the closeness of agreement between results of three different methods. The obtained validation parameters indicate that all three methods are suitable for determination of AFM1 in milk samples. The statistical analysis of variance between the methods and the obtained correlation coefficients indicate that there is a strong correlation between methods. All three methods are satisfactory in meeting the requirements for official control purposes. To the best of author's knowledge, this study represents the first report of an investigation and comparison of ELISA, HPLC-FLD and HPLC-MS/MS methods for determination of AFM1 in naturally contaminated milk samples.