Pyrrolizidine alkaloids (PAs) are probably the most widespread toxins of natural origin. More than 6,000 plant species produce these toxic compounds. Bees can forage on flowers of plants producing PAs, which leads to contamination of honey with the toxic compounds. To determine the contamination of honey with PAs, a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed. PAs were extracted with 0.05 M sulphuric acid and purified with MCX cartridges. A solvent mixture consisting of ethyl acetate, methanol, acetonitrile, ammonia, and triethylamine (8:1:1:0.1:0.1, v/v) was used to wash alkaloids from the cartridges. After evaporation the residues were reconstituted in water and methanol mixture and subjected to LC–MS analysis. The developed method was validated according to SANTE/11945/2015 requirements. The recovery was from 80.6% to 114.5%. The repeatability ranged from 2.3% to 14.6%, and the reproducibility was from 4.9% to 17.7%. A new method for the determination of PAs in honey has been developed and validated. All evaluated parameters were in accordance with the SANTE/11945/2015 guidance document. Out of 50 analysed honey samples, 16 (32%) were positive for the content of at least one PA.

Pyrrolizidine alkaloids (PAs) are probably the most widespread toxins of natural origin. More than 6,000 plant species produce these toxic compounds. Bees can forage on flowers of plants producing PAs, which leads to contamination of honey with the toxic compounds. To determine the contamination of honey with PAs, a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed.

Introduction: The main problem in determination of chloramphenicol in food of animal origin is a large number of matrices. The main target of this study was to create a method for determination and confirmation of chloramphenicol in products and food of animal origin. Material and Methods: Each 5 g matrix sample was mixed with 5 mL of water and 10 mL of acetonitrile/ethyl acetate, homogenised, and centrifuged. The organic layer was evaporated and redissolved in 6 mL of 4% NaCl. The extract was cleaned up by SPE technique. Chloramphenicol was analysed by LC-MS/MS in electrospray mode. Results: The procedure was validated according to the Commission Decision No. 2002/657/EC. The apparent recoveries were in the range of 92.1% to 107.1% with a repeatability less than 11.0% (4.4%-11.0%) and within-laboratory reproducibility below 13.6% (4.7%-13.6%). Conclusion: The method was successfully validated and proved to be efficient, precise, and useful for quantification of chloramphenicol in more than 20 different matrices.

Introduction: The main problem in determination of chloramphenicol in food of animal origin is a large number of matrices. The main target of this study was to create a method for determination and confirmation of chloramphenicol in products and food of animal origin. Material and Methods: Each 5 g matrix sample was mixed with 5 mL of water and 10 mL of acetonitrile/ethyl acetate, homogenised, and centrifuged. The organic layer was evaporated and redissolved in 6 mL of 4% NaCl. The extract was cleaned up by SPE technique. Chloramphenicol was analysed by LC-MS/MS in electrospray mode. Results: The procedure was validated according to the Commission Decision No. 2002/657/EC. The apparent recoveries were in the range of 92.1% to 107.1% with a repeatability less than 11.0% (4.4%-11.0%) and within-laboratory reproducibility below 13.6% (4.7%-13.6%). Conclusion: The method was successfully validated and proved to be efficient, precise, and useful for quantification of chloramphenicol in more than 20 different matrices.

Introduction: In the European Union the use of steroid growth promoters is prohibited under Council Directive 96/22/EC. For effective control of illegal use of natural steroids, highly sensitive analytical methods are required, because sex hormones can be present in very low concentrations in biological samples. The aim of the study was to develop a confirmatory method for the detection of testosterone in bovine serum at ppt level. Material and Methods: 17β-testosterone and internal standards of 17β-testosterone-d2 were extracted from serum samples with a mixture of tert-butyl methyl ether/petroleum ether and were directly analysed by an LC/MS/MS on QTRAP 5500 instrument with a TurboIon-Spray source operating in a positive ionisation mode. Chromatographic separation was achieved on the analytical column Inertsil® ODS-3 with an isocratic elution using mobile phase consisting of acetonitrile, methanol, and water. Method validation has been carried out in accordance with the Commission Decision 2002/657/EC. Results: The method was characterised by good recovery (82%) and precision (R.S.D 17 %). Decision limit (CCα) and detection capability (CCβ) was 0.05 μg L⁻¹ and 0.09 μg L⁻¹ respectively. The method met the criteria set out in Commission Decision 2002/657/EC for the purpose of confirmation in terms of retention time and ion ratio in the whole range of its application. Conclusions: The developed method is specific and sensitive, suitable for measuring the natural level of testosterone in blood of cattle and for use in routine control programme for the detection of this hormone in bovine serum.

Introduction: In the European Union the use of steroid growth promoters is prohibited under Council Directive 96/22/EC. For effective control of illegal use of natural steroids, highly sensitive analytical methods are required, because sex hormones can be present in very low concentrations in biological samples. The aim of the study was to develop a confirmatory method for the detection of testosterone in bovine serum at ppt level.

Pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs) are natural contaminants of honey and respectively hepatoxic and neurotoxic compounds. Because honey is a popular constituent of the human diet, it is relevant to warrant the safety of the product. For that reason, a method for simultaneous determination of PAs and TAs in honey based on liquid chromatography– mass spectrometry was developed. The analytical protocol used sulphuric acid extraction and solid-phase extraction purification. The developed procedure was subjected to validation in terms of linearity, selectivity, repeatability, reproducibility, limits of quantification and determination, matrix effect and uncertainty. A total of 29 honey samples were analysed for the determination of PAs and TAs. All the evaluated validation parameters fulfilled the requirements of European Commission Decision 2002/657/EC. At least one of the monitored alkaloids was determined in 52% of the samples. Among the most abundant alkaloids were echimidine, intermedine and lycopsamine. The total PA concentrations ranged from 2.2 to 147.0 μg kg⁻¹. Contrastingly, none of the monitored TAs was detected in the analysed samples. An assessment of the dietary exposure to PAs from the consumption of the contaminated honeys showed that three of them would pose a risk to consumers, especially if they were children. A sensitive method suitable for simultaneous determination of PAs and TAs in honey was developed and validated. The analysis of 29 honey samples for PAs and TAs revealed that honey destined for retail could pose a risk to consumers.

Pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs) are natural contaminants of honey and respectively hepatoxic and neurotoxic compounds. Because honey is a popular constituent of the human diet, it is relevant to warrant the safety of the product. For that reason, a method for simultaneous determination of PAs and TAs in honey based on liquid chromatography– mass spectrometry was developed.

The transfer of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs) from a contaminated environment into the food chain is a serious consumer safety problem. As part of the Polish National Surveillance Program of PCDD/Fs and PCBs in food of animal origin, a concentration of PCDD/Fs of 4.61 ± 0.75 pg WHO-TEQ/g fat was determined in a sample of free-range eggs, which exceeded the permitted limit of 2.5 pg WHO-TEQ/g. The aim of the study was to investigate the source of the egg contamination and the risk for the eggs’ consumers. Eggs, muscles, feed and soil from the place where backyard waste burning had been carried out in the past and ash from a household stove tipped onto the paddock were analysed using the isotope dilution technique with high-resolution gas chromatography coupled with high-resolution mass spectrometry. The concentration in ash was low at 0.20 pg WHO-TEQ/g and the congener profile did not indicate the source of contamination. The dioxin content in soil from the backyard waste-burning site was 2.53 pg WHO-TEQ/g dry matter (d.m.) and the soil’s profile of PCDD/F congeners matched the profile of the contaminated eggs. By reason of the congener profile similarity, the investigation concluded, that the cause of the contamination was the backyard waste-burning site soil which the animals had access to. Frequent consumption of contaminated eggs from the analysed farm could pose a health risk due to chronic exposure, especially for vulnerable consumers.

The transfer of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs) from a contaminated environment into the food chain is a serious consumer safety problem. As part of the Polish National Surveillance Program of PCDD/Fs and PCBs in food of animal origin, a concentration of PCDD/Fs of 4.61 ± 0.75 pg WHO-TEQ/g fat was determined in a sample of free-range eggs, which exceeded the permitted limit of 2.5 pg WHO-TEQ/g. The aim of the study was to investigate the source of the egg contamination and the risk for the eggs’ consumers.

Antimicrobial resistance is a global health threat. It has been studied in humans and domestic animals, but there is a lack of data on wild animals. The objective of this study is the elucidation of its patterns in Staphylococcus spp. isolated from wild mammals of the Autonomous Community of Aragón (Spain). A total of 103 mammals (Artiodactyla, Carnivora, Chiroptera, Erinaceomorpha, and Lagomorpha) were studied. A recovery centre provided 32 and hunting 71. Nasal and faecal samples yielded 111 staphylococci, which were identified by matrix-assisted laser desorption/ionization–time of flight mass spectrometry. A susceptibility test to 11 antibiotics was carried out, and statistical analysis was performed. Some differences were detected in bacterial prevalence depending on how the mammal fed. Artiodactyla, mainly hunted, were predisposed to carry coagulase-positive staphylococci. The staphylococci species recovered were resistant to at least two classes of antibiotics, and were disseminated in all of the geographical areas studied. Resistant staphylococci are widely distributed in the wild mammals in the areas of the study, but the resistance quantified in them is lower than that to be expected if the use of antibiotics in farms had a direct influence on the wildlife and its environment. On the other hand, resistance to antibiotics restricted to human use was widely disseminated in various wild animal species.

Antimicrobial resistance is a global health threat. It has been studied in humans and domestic animals, but there is a lack of data on wild animals. The objective of this study is the elucidation of its patterns in Staphylococcus spp. isolated from wild mammals of the Autonomous Community of Aragón (Spain).

Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by many plant species. Due to their toxicity PAs can pose a risk to human and animal health. To detect the toxic compounds in feed materials a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed. PAs were extracted with sulphuric acid and purified with cation exchange cartridges. A newly developed solvent mixture consisting of ethyl acetate, methanol, acetonitrile, ammonia, and triethylamine was used to wash alkaloids from the cartridges. After evaporation the residues were reconstituted in water and methanol mixture and subjected to LC-MS analysis. The developed method was validated according to SANTE/11945/2015 guidelines. The recovery was from 84.1% to 112.9%, the repeatability ranged from 3.0% to 13.6%, and the reproducibility was from 4.8% to 18.9%. A sensitive and selective method for determination of PAs in feed materials has been developed and validated. All evaluated validation parameters were in accordance with EU Reference Laboratories document no. SANTE/11945/2015. Almost 41% of the analysed feed samples were positive for the presence of at least one PA.

Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by many plant species. Due to their toxicity PAs can pose a risk to human and animal health. To detect the toxic compounds in feed materials a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed.

Introduction: Studies of anabolic hormone residues in the tissues of slaughter animals have been carried out in Poland for more than 25 years. During the period of 2011 to 2015, a total of 35 387 samples from different animal species were tested in the National Residue Control Programme for the presence of residues of compounds that cause hormonal effects, as listed in Annex 1 of Directive 96/23/EC. Material and Methods: The research was conducted in the National Reference Laboratory and eight regional laboratories in departments of veterinary hygiene located throughout the country. Urine, muscle tissue, serum, kidney fat, and drinking water were the targeted matrices. Test methods based on instrumental techniques such as gas and liquid chromatography coupled with mass spectrometry were applied, as well as enzyme-linked immunosorbent assays (ELISA). Results: The concentration of detected hormones exceeded the decision limits in 30 samples, the consequence of which was 41 non-compliances with current applicable criteria. The hormones found present pseudo-endogenous (nortestosterone and boldenone) only, while synthetic hormones were not identified. Conclusion: The non-compliant findings constitute a small percentage (0.085%) of the five-year analysis compilation. On this basis the related food produced in Poland can be accepted as safe for human consumption with regard to the hormone residues tested.

Introduction: Studies of anabolic hormone residues in the tissues of slaughter animals have been carried out in Poland for more than 25 years. During the period of 2011 to 2015, a total of 35 387 samples from different animal species were tested in the National Residue Control Programme for the presence of residues of compounds that cause hormonal effects, as listed in Annex 1 of Directive 96/23/EC.

Introduction: To identify novel pathways involved in the pathogenesis of ketosis, an isobaric tag for relative and absolute quantitation/mass spectrometry was used to define differences in protein expression profiles between healthy dairy cows and those with clinical or subclinical ketosis.Material and Methods: To define the novel pathways of ketosis in cattle, the differences in protein expression were analysed by bioinformatics. Go Ontology and Pathway analysis were carried out for enrich the role and pathway of the different expression proteins between healthy dairy cows and those with clinical or subclinical ketosis.Results: Differences were identified in 19 proteins, 16 of which were relatively up-regulated while the remaining 3 were relatively down-regulated. Sorbitol dehydrogenase (SORD) and glyceraldehyde-3-phosphate dehydrogenase (G3PD) were up-regulated in cattle with ketosis. SORD and G3PD promoted glycolysis. These mechanisms lead to pyruvic acid production increase and ketone body accumulation.Conclusion: The novel pathways of glycolysis provided new evidence for the research of ketosis.

Introduction: To identify novel pathways involved in the pathogenesis of ketosis, an isobaric tag for relative and absolute quantitation/mass spectrometry was used to define differences in protein expression profiles between healthy dairy cows and those with clinical or subclinical ketosis.

Introduction: The present study is a comprehensive overview of the natural occurrence of 17β-oestradiol and testosterone in serum of cattle in Poland. Material and Methods: The serum samples (n = 826) were collected from cattle within five years. The samples were examined for the presence of oestradiol and testosterone using ELISA or gas chromatography with mass spectrometry. Results: In 98 samples (24%) 17β-oestradiol was detected above decision limits of applied methods, including five samples over the recommended concentration of 0.1 μg L⁻¹. Of the serum samples taken from cows (≤18 months of age), 95 and 99 percentiles of the animals had 17β-oestradiol concentration below 0.027 and 0.086 μg L⁻¹ and of samples from cows over 18 months of age - below 0.059 and 0.125 μg L⁻¹ respectively. Calculated values for bulls (≤18 months of age) were 0.025 and 0.034 μg L⁻¹ and for the animals older than 18 months of age - 0.035 and 0.041 μg L⁻¹. The natural presence of testosterone was detected in 201 serum samples (48.7%). According to the obtained data, 95% and 99% of cows (≤18 months of age) serum samples had testosterone concentration below 0.05 and 0.23 μg L⁻¹ and the animals over 18 months of age - 0.30 and 0.49 μg L⁻¹, respectively. For bulls these values did not depend on the age of the animals and were in the ranges of 5 - 6.3 μg L⁻¹ (95%) and 11.4 - 12.1 μg L⁻¹ (99%). Conclusion: Our study showed that the threshold values for these hormones in plasma of cattle designated years ago are correct, but they need to be supplemented for animals older than 18 months.

Introduction: The present study is a comprehensive overview of the natural occurrence of 17β-oestradiol and testosterone in serum of cattle in Poland. Material and Methods: The serum samples (n = 826) were collected from cattle within five years. The samples were examined for the presence of oestradiol and testosterone using ELISA or gas chromatography with mass spectrometry. Results: In 98 samples (24%) 17β-oestradiol was detected above decision limits of applied methods, including five samples over the recommended concentration of 0.1 μg L-1. Of the serum samples taken from cows (≤18 months of age), 95 and 99 percentiles of the animals had 17β-oestradiol concentration below 0.027 and 0.086 μg L-1 and of samples from cows over 18 months of age - below 0.059 and 0.125 μg L-1 respectively. Calculated values for bulls (≤18 months of age) were 0.025 and 0.034 μg L-1 and for the animals older than 18 months of age - 0.035 and 0.041 μg L-1. The natural presence of testosterone was detected in 201 serum samples (48.7%). According to the obtained data, 95% and 99% of cows (≤18 months of age) serum samples had testosterone concentration below 0.05 and 0.23 μg L-1 and the animals over 18 months of age - 0.30 and 0.49 μg L-1, respectively. For bulls these values did not depend on the age of the animals and were in the ranges of 5 - 6.3 μg L-1 (95%) and 11.4 - 12.1 μg L-1 (99%). Conclusion: Our study showed that the threshold values for these hormones in plasma of cattle designated years ago are correct, but they need to be supplemented for animals older than 18 months.