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.

Some azo dyes, including Sudans I–IV and Para Red, are genotoxic and may be biotransformed to cancerogenic aromatic amines. They are banned as food and feed additives, but their presence has been detected in food. Aromatic amines are also considered potentially toxic. Online EC–MS is a promising tool to study the transformation mechanisms of xenobiotics such as azo dyes. The aim of the study was to investigate emulation of how azo dyes are enzymatically transformed to amines with EC–MS. The reduction reactions of five azo dyes (Sudans I–IV and Para Red) were conducted using a glassy carbon working electrode and 0.1% formic acid in acetonitrile. Reduction results were compared with the literature and in silico to select preliminary candidates for metabolites. The LC-MS/MS method was used to confirm results obtained by electrochemical reactor. A limited number of pre-selected compounds were confirmed as azo dyes metabolites – aniline for Sudan I, aniline and 4-aminoazobenzene for Sudan III, o-toluidine for Sudan IV, and 4-nitroaniline for Para Red. No metabolites were found for Sudan II. Electrochemistry–mass spectrometry was successfully applied to azo dyes. This approach may be used to mimic the metabolism of azo dyes, and therefore predict products of biotransformation.

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 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.

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: 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.

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: The objective of this study was to describe a pharmacokinetic–pharmacodynamic (PK/PD) approach for determination of a rational dosage of ampicillin (AMP) and depletion of the antibiotic residues in milk after intramammary administration to cows.Material and Methods: The cows came from different farms from the Lublin Province area. They (n = 9) received 5 g of the drug, containing 75 mg of AMP sodium in physiological solution, through a syringe tube by intramammary administration. Following single intramammary administration, the milk samples (5 mL) were collected after 2, 4, 6, 8, 10, 24, 36, 48, and 60 h. The liquid chromatography-mass spectrometry analysis was performed on the Agilent 1200 system connected to an AB Sciex API 4000™ mass spectrometer. The pharmacokinetic analysis of the concentrations of the antibiotic in milk was performed using software Phoenix® WinNonlin® 6.4. Calculations were made in non-compartmental (slopes, highest, amounts, and moments) and compartmental analysis.Results: The pharmacokinetic characteristics of AMP after intramammary administration indicate rapid elimination of the drug from milk. The mean residence time had a several-fold lower value than the designated elimination half-life and amounts to only 3.4 h. The concentration of the drug in the milk dropped relatively quickly and the process was very dynamic.Conclusion: The conducted research confirms the rationale of using the PK/PD model in order to verify the dosing regimen for other antibiotic groups and various indicators of the applied PK/PD model.

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.