Essential and non-essential element concentrations in human milk samples and the assessment of infants' exposure.

As the data concerning element concentrations in human milk (HM) samples and their intake by infants are lacking in Poland, the present study aimed to explore this issue. The material consisted of HM samples obtained from 30 exclusively breastfeeding mothers during 4-6 weeks postpartum. Additionally, to identify the factors that may potentially affect HM composition, information regarding maternal data (anthropometry, body composition, and diet) was also collected. Maternal diet was assessed with two methods-a food frequency questionnaire and 3-day dietary records. In total, 18 essential and non-essential elements were determined. For the elements analysis, we used inductively coupled plasma quadrupole mass spectrometry. Most of the elements (n = 11, 61%) were detected in all HM samples. In all HM samples tin concentration was higher (5.67 ± 2.39 µg/L) than the usual range reported by the World Health Organization (~ 1.0 µg/L). HM cadmium content was positively associated with maternal salty snacks intake (r = 0.502, p = 0.005), arsenic with whole-grain products intake (r = 0.37, p = 0.043), and mercury concentration with fruits and seeds/nuts consumption (r = 0.424, p = 0.042 and r = 0.378, p = 0.039, respectively). Higher HM lead concentration was predicted by maternal age (95% CI [0.94-0.97]), intake of fish (95% CI [1.01-1.03]), and vegetables (95% CI [1.02-1.06]). The highest infants' intake was observed for copper (35.24 ± 12.48) and the lowest for arsenic (0.076 ± 0.102). Infants' exposure to lead was associated with maternal frequency consumption of canned fish (p = 0.0045). There is a need to perform further research on this topic to maximize the benefits of breastfeeding by minimizing maternal and infant exposure to potentially toxic elements.

Liquid chromatographytandem mass spectrometry methods for determination of stanozolol and l6ß-hydroxy-stanozolol in animal urine.

Introduction: Because of the activities and effects they induce, hormones are prohibited for use for anabolic purposes in farm animals intended for slaughter, which is regulated in the European Union by relevant legal provisions. Therefore, there is an obligation to monitor residues of hormones in animals and food of animal origin to ensure consumer safety. A hormone banned but used formerly for fattening cattle, stanozolol, and its metabolite 16ß-OH-stanozolol are synthetic compounds that belong to a large group of steroid hormones. This study investigates residues of these compounds in animal urine. Material and Methods: From 2006-2022, 2,995 livestock urine samples were tested for stanozolol residues in Poland as part of the National Residue Monitoring Programme. A liquid chromatography-tandem mass spectrometry method to determine stanozolol and 16ß-OH-stanozolol in animal urine was developed and validated according to the required criteria. Urine sample analysis was based on enzymatic hydrolysis of hormones potentially present in it to the free form, extraction of them from the sample with a mixture of n-hexane and butyl alcohol, purification of an extract on an NH2 amine column and finally, instrumental detection. Results: The apparent recovery and precision parameters of the developed method were in line with the established criteria, while its decision limits CCα and detection capabilities CCß were lower than the recommended concentration for analytical purposes set at 2 µg L-1 (valid until December 15, 2022; currently set as 0.5 µg L-1). Conclusion: All examined samples were compliant with the evaluation criteria.

Survey of the enniatins and beauvericin in raw and UHT cow's milk in Poland.

Introduction: The enniatins A, A1, B and B1 (ENNs) and beauvericin (BEA) are structurally related compounds produced by Fusarium species. They occur as contaminants in cereals, such as wheat, barley and maize. They are called "emerging mycotoxins", because they have been reported in feed and food and their toxic effects are not fully known. Data on their levels in food (especially in milk) are limited. The study aimed to evaluate the occurrence of ENNs and BEA in milk. Material and Methods: A total of 103 bovine milk samples (76 of raw milk and 27 of UHT milk) were collected from different parts of Poland and analysed using liquid chromatography-tandem mass spectrometry. Results: Among the 76 raw milk samples, 31 (41%) and 15 (20%) samples were contaminated with ENN B and with BEA, respectively. No contamination with other enniatins was found. The highest concentration of BEA was found in raw milk and was 6.17 µg kg-1. Out of the 27 samples of UHT milk, 16 (59%) were contaminated with ENN B at concentrations ranging from 0.157 µg kg-1 to 0.587 µg kg-1 (limit of quantification (LOQ) 0.098 µg kg-1). Beauvericin was detected in 9 UHT milk samples (33%) at concentrations ranging from 0.101 µg kg-1 to 1.934 µg kg-1 (LOQ 0.095 µg kg-1). Conclusion: This study demonstrated constant but low milk contamination in Poland with ENN B and BEA. The analysis of milk samples revealed that the emerging mycotoxins ENN B and BEA were measured in trace amounts. It does not suggest any immediate risk to milk consumers; however, it is unknown whether long-term exposure to low levels of toxins may be harmful.

Inter-laboratory validation of liquid chromatography-tandem mass spectrometry multi-mycotoxin determination in animal feed - method transfer from the reference laboratory to regional laboratories.

Introduction: The results are presented of the inter-laboratory validation of a liquid chromatography-tandem mass spectrometry method for the determination of eight mycotoxins (aflatoxin B1, deoxynivalenol, fumonisin B1, fumonisin B2, ochratoxin A, toxin T-2, toxin HT-2 and zearalenone) in animal feeds. Material and Methods: This study was an essential part of the method's transfer from the National Reference Laboratory to six regional laboratories in Poland working in the official survey of mycotoxins in feed. The laboratories received a batch of standard solutions, blank samples and quality control materials on which to perform analysis with one procedure and different liquid chromatography-tandem mass spectrometry conditions. Results: The validation results show good precision (reproducibility coefficient of variation 3.7-20.5%) and accuracy of the method (recovery 89-120% and trueness 94-103%) and sufficient skills of the laboratory personnel. Conclusion: The study is an example of the successful transfer of the method among laboratories.

Residues of an anthelmintic veterinary drug (closantel) detected in red foxes (Vulpes vulpes) in Scotland.

The contamination of the environment by some veterinary medicines and their impact on wild animals is of increasing concern. However, there is a lack of information about their residues in wildlife. The sentinel animals most commonly used for monitoring the level of environmental contamination are birds of prey, and information on other carnivores and scavengers scarce. This study examined the livers from 118 foxes for residues of a range of 18 veterinary medicines (16 anthelmintic agents and 2 metabolites) used on farm livestock. The samples were collected from foxes, primarily in Scotland, shot during legal pest control activities conducted between 2014 and 2019. Closantel residues were detected in 18 samples, and the concentrations found ranged from 6.5 µgkg-1 to 1383 µgkg-1. No other compounds were found in significant quantities. The results show a surprising frequency and level of closantel contamination, raising concerns about both the route of contamination and the potential impacts on wild animals and the environment, such as the potential for significant wildlife contamination to contribute to the development of closantel-resistant parasites. The results also suggest that red fox (Vulpes vulpes) could be a useful sentinel species for detecting and monitoring some veterinary medicine residues in the environment.

Lead, cadmium, and other trace elements in the liver of golden eagles and white-tailed eagles: recent data from Poland and a systematic review of previous studies.

The golden eagle (Aquila chrysaetos) and the white-tailed eagle (Haliaeetus albicilla), being apex predators and facultative scavengers, can bioaccumulate different environmental contaminants, including toxic elements that may adversely affect their health. We analyzed the levels of cadmium (Cd), lead (Pb), and other metals and metalloids, including arsenic (As), barium (Ba), beryllium (Be), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), molybdenum (Mo), selenium (Se), thorium (Th), thallium (Tl), uranium (U), vanadium (V), and zinc (Zn) in liver samples taken from three golden eagles and 36 white-tailed eagles that were found dead across Poland to verify their exposure. We also used a systematic review to summarize the available literature data on Cd, Pb, and other studied elements in the liver of both eagle species. Analyses of trace elements in the liver samples of the Polish eagles revealed interspecific differences in Cd, Cu, and Mn and differences in Co, Mn, Tl, and Zn among study regions. All elements tested except Pb were below the suggested thresholds linked with adverse health effects in birds. The hepatic Pb found in almost half of all the tested individuals suggests environmental exposure to this toxic element. One of the tested white-tailed eagles had hepatic Pb above the threshold of sublethal poisoning. Although our results seem optimistic, as previous Polish studies showed a higher prevalence of birds with hepatic Pb exceeding the toxicity threshold, they indicate that exposure to this toxic metal could still pose an additional threat to the health of Polish eagles.

Does Deoxynivalenol Affect Amoxicillin and Doxycycline Absorption in the Gastrointestinal Tract? Ex Vivo Study on Swine Jejunum Mucosa Explants.

The presence of deoxynivalenol (DON) in feed may increase intestinal barrier permeability. Disturbance of the intestinal barrier integrity may affect the absorption of antibiotics used in animals. Since the bioavailability of orally administered antibiotics significantly affects their efficacy and safety, it was decided to evaluate how DON influences the absorption of the most commonly used antibiotics in pigs, i.e., amoxicillin (AMX) and doxycycline (DOX). The studies were conducted using jejunal explants from adult pigs. Explants were incubated in Ussing chambers, in which a buffer containing DON (30 µg/mL), AMX (50 µg/mL), DOX (30 µg/mL), a combination of AMX + DON, or a combination of DOX + DON was used. Changes in transepithelial electrical resistance (TEER), the flux of transcellular and intracellular transport markers, and the flux of antibiotics across explants were measured. DON increased the permeability of small intestine explants, expressed by a reduction in TEER and an intensification of transcellular marker transport. DON did not affect AMX transport, but it accelerated DOX transport by approximately five times. The results suggest that DON inhibits the efflux transport of DOX to the intestinal lumen, and thus significantly changes its absorption from the gastrointestinal tract.

An Interlaboratory Comparison Study of Regulated and Emerging Mycotoxins Using Liquid Chromatography Mass Spectrometry: Challenges and Future Directions of Routine Multi-Mycotoxin Analysis including Emerging Mycotoxins.

The present interlaboratory comparison study involved nine laboratories located throughout the world that tested for 24 regulated and non-regulated mycotoxins by applying their in-house LC-MS/MS multi-toxin method to 10 individual lots of 4 matrix commodities, including complex chicken and swine feed, soy and corn gluten. In total, more than 6000 data points were collected and analyzed statistically by calculating a consensus value in combination with a target standard deviation following a modified Horwitz equation. The performance of each participant was evaluated by a z-score assessment with a satisfying range of ±2, leading to an overall success rate of 70% for all tested compounds. Equal performance for both regulated and emerging mycotoxins indicates that participating routine laboratories have successfully expanded their analytical portfolio in view of potentially new regulations. In addition, the study design proved to be fit for the purpose of providing future certified reference materials, which surpass current analyte matrix combinations and exceed the typical scope of the regulatory framework.

Biomarkers of Deoxynivalenol, Citrinin, Ochratoxin A and Zearalenone in Pigs after Exposure to Naturally Contaminated Feed Close to Guidance Values.

This study applied multi-mycotoxin liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) methods to determine the biomarkers of exposure in urine and serum samples from a dose-response study with pigs. The 24 studied pigs were divided into three groups: a control and two experimental ones (with different levels of feed contamination). They were exposed to feed prepared from cereals contaminated with deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA) and citrinin (CIT) for 14 days. After that, both experimental groups received the same feed as the control group for the next 14 days to determine the kinetics of the disappearance of mycotoxin biomarkers. Urine samples were collected daily in the morning and blood samples-eight-times during the experiment. The study reported herein was the first prolonged exposure experiment for multiple mycotoxins like OTA and CIT in pigs. The urinary and serum levels of all biomarkers correlated well with the respective toxin intake; thereby demonstrating that they are suitable biomarkers of exposure in pigs. Urine is a good candidate to monitor DON, ZEN, OTA, CIT exposure while serum may be used to monitor DON, OTA and CIT. Additionally, OTA has even been quantified in both matrices in the experimental groups two weeks after changing the contaminated feed back to the control, this result differed from those produced by the other mycotoxins which were only quantified during the first two weeks. Therefore both matrices are suitable candidates to monitor prolonged OTA exposure in pigs.

LC-MS/MS Determination of 21 Non-Steroidal Anti-Inflammatory Drugs Residues in Animal Milk and Muscles.

The presented procedure combines experience from two LC-MS/MS methods previously developed by our team for NSAIDs determination in meat and milk. The novelty was a modification of sample preparation and combining LC-MS/MS method for milk and muscle. The clean-up procedure was investigated, leading to a change from SPE to dSPE with C18 bulk sorbent. Unlike most of the existing methods, chromatographic separation was achieved on a C8 chromatographic column. This method was developed and validated under European Commission Decision 2002/657/EC. Recovery for milk samples values between 86.3% to 108%, with the coefficient of variation, varied from 5.51% to 16.2%. The recovery for muscle was calculated to be between 85.0% and 109%, and the coefficient of variation was-4.73% to 16.6%. The validation results prove that the method is suitable for confirmatory purposes in milk and muscle. Of 452 samples tested in 2019 and 2020, two have been identified as non-compliant.

Mycotoxin Biomarkers in Pigs-Current State of Knowledge and Analytics.

Farm animals are frequently exposed to mycotoxins, which have many adverse effects on their health and become a significant food safety issue. Pigs are highly exposed and particularly susceptible to mycotoxins, which can cause many adverse effects. For the above reasons, an appropriate diagnostic tool is needed to monitor pig' exposure to mycotoxins. The most popular tool is feed analysis, which has some disadvantages, e.g., it does not include individual exposure. In recent years, the determination of biomarkers as a method to assess the exposure to mycotoxins by using concentrations of the parent compounds and/or metabolites in biological matrices is becoming more and more popular. This review provides a comprehensive overview of reported in vivo mycotoxin absorption, distribution, metabolism and excretion (ADME) and toxicokinetic studies on pigs. Biomarkers of exposure for aflatoxins, deoxynivalenol, ochratoxin A, fumonisins, T-2 toxin and zearalenone are described to select the most promising compound for analysis of porcine plasma, urine and faeces. Biomarkers occur in biological matrices at trace levels, so a very sensitive technique-tandem mass spectrometry-is commonly used for multiple biomarkers quantification. However, the sample preparation for multi-mycotoxin methods remains a challenge. Therefore, a summary of different biological samples preparation strategies is included in that paper.

Risk of residues of toltrazuril sulfone in eggs after oral administration - Could setting maximum residue limit be helpful?

A depletion study of toltrazuril and its metabolites was performed using 20 hens medicated via drinking water for two days in a dosage of 7 mg kg-1 per kg body weight. Afterward, eggs were collected for 42 days. Residues were analyzed in whole eggs and yolk and whites. Toltrazuril sulfone was found to be the most predominant in all matrices, the highest concentration was found in the yolk - 5567 µg kg-1, followed by whole eggs samples - 4767 µg kg-1 and egg whites - 532 µg kg-1. On last day toltrazuril sulfone were still detected - 22.5 µg kg-1. 70 days is required to concentration of toltrazuril sulfone reach zero. Administrating toltrazuril before the laying phase can pose a risk of residues of toltrazuril sulfone in eggs. Setting Maximum Residue Limit could reduce the risk of non-complaint samples and ensure the safety of consumers, but still requires 44 days of the withdrawal period.

Development of a Multimatrix UHPLC-MS/MS Method for the Determination of Paracetamol and Its Metabolites in Animal Tissues.

Paracetamol/acetaminophen (APAP) is one of the most popular pharmacologically active substances used as an analgesic and antipyretic agent. The metabolism of this drug occurs in the liver and leads to the formation of two main metabolites-glucuronic acid and sulfate derivate. Despite the wide use of paracetamol in veterinary medicine, a handful of analytical methods were published for the determination of paracetamol residues in animal tissues. In this paper, a multimatrix method has been developed for the determination of paracetamol and two metabolites-paracetamol sulfate (PS) and p-Acetamidophenyl ß-D-glucuronide (PG). A validation procedure was conducted to verify method reliability and fit purpose as a tool for analyzing acetaminophen and metabolites in muscle, liver, lung, and kidney samples from different species of animals. Established validation parameters were in agreement with acceptable criteria laid by the European legislation. The initial significant matrix effect was successfully reduced by implementing an internal standard-4-Acetamidophenyl ß-D-glucuronide-d3 (PG-d3, IS). The usefulness of the developed method was verified by analyzing samples from an experiment in which paracetamol was administrated to geese.

Development of a multi-mycotoxin LC-MS/MS method for the determination of biomarkers in pig urine.

An LC-MS/MS method has been developed for the sensitive and selective determination of 35 mycotoxins (biomarkers of exposure) in pig urine samples. Sample preparation includes creatinine adjustment (with the developed LC-UV method) with enzymatic hydrolysis of pig urine samples followed by liquid-liquid (LLE) extraction. The LLE protocol, as well as enzymatic hydrolysis for indirect mycotoxin glucuronides determination, was optimized in this study. Additionally, two other sample preparation protocols were compared with the developed LLE method: immunoaffinity columns and solid-phase extraction cartridges (Oasis HLB). The detection and quantification of the biomarkers were performed using triple quadrupole mass spectrometry.The method was validated with regard to the guidelines specified by the EMEA (European Medicines Agency). The extraction recoveries were higher than 60% for 77% of the analytes studied, with the intra- and inter-day relative standard deviation being lower than 20% for most of the compounds at four different concentration levels. The limits of quantification ranged from 0.1 ng/mL for zearalenone and sterigmatocystin to 8 ng/mL for nivalenol. To the best knowledge of the authors, the matrix effect was evaluated for the first time in this study for six different urine samples, and the coefficient of variation was found to be lower than 15% for most analytes studied. Finally, the developed method was applied to analyse 56 pig urine samples. Deoxynivalenol (1-20 ng/mL), zearalenone (0.1-1.5 ng/mL) and ochratoxin A (1.5-15 ng/mL) were the main analytes detected in these samples. Moreover, the co-occurrence of alternariol monomethyl ether and alternariol in pig urine is reported herein for the first time.

Simultaneous Determination of Deoxynivalenol, Its Modified Forms, Nivalenol and Fusarenone-X in Feedstuffs by the Liquid Hromatography-Tandem Mass Spectrometry Method.

A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3Ac-DON), 15-acetyldeoxynivalenol (15Ac-DON), DON-3-glucoside (DON-3Glc) nivalenol and fusarenone-X in feedstuffs. Different techniques of sample preparation were tested: solid-liquid-extraction, QuEChERS, solid phase extraction with OASIS HLB columns or immunoaffinity columns and a Mycosep 225 Trich column. None of the six immunoaffinity columns tested showed cross-reactivity to all of the mycotoxins. Surprisingly, the results show that if the immunoaffinity columns bound 3Ac-DON, then they did not bind 15Ac-DON. The most efficient sample preparation was achieved with a Mycosep 225 Trich column clean-up. The chromatography was optimised to obtain full separation of all analytes (including 3Ac-DON and 15Ac-DON isomeric form). The validation results show the relative standard deviations for repeatability and reproducibility varied from 4% to 24%. The apparent recovery ranged between 92% and 97%, and the limit of quantification described a 1.30 to 50 µg/kg range. The method trueness was satisfactory, as assessed by a proficiency test and analysis of reference material. A total of 99 feed samples were analysed by the developed method, revealing the presence of DON and DON-3Glc in 85% and 86% of examined animal feeds, respectively at concentrations between 1.70 and 1709 µg/kg. The ratios DON-3Glc to DON in the surveyed feedstuffs were from a low of 3% to high of 59%.

Could mycotoxigenic Fusarium sp. play a role in ulcerative dermal necrosis (UDN) of brown trout (Salmo trutta morpha trutta)?

Fusarium infections have been reported in aquatic animals, but are still poorly investigated in wild salmonids. The aim of the study was to determine the impact of the fungi and their toxins on the health status of brown trout (Salmo trutta morpha trutta) migrating from the Baltic Sea to the freshwater. Individuals from the wild brown trout population exhibiting ulcerative skin lesions were collected from the Slupia River in Poland and subjected to microbiological, histopathological, and hematological examinations, as well as toxicological analysis for a presence of mycotoxins. The results of microflora isolation from the brown trout skin samples revealed the presence of conditionally pathogenic bacteria and fungi classified by molecular techniques as Fusarium spp. Toxicological analysis allowed for detection of zearalenone (ZEN) in the liver, kidney, and gastrointestinal tract of the fish. In several cases, there was α-zearalenone (α-ZEL) identified at trace levels in the liver, as well as sterigmatocystin and enniatin B at low levels in the kidney and the liver. Histopathological examination revealed the presence of fungal hyphae disrupting the epidermis and penetrating into the necrotic dermis and hypodermis. The decreased values of the blood parameters, i.e., hemoglobin concentration (HGB), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and white blood cell count (WBC), were indicative of osmoregulation failure being a consequence of the skin damage. The results of the study provide new information regarding Fusarium sp. infection in brown trout and serve as the basis for further research on the potential impact of the fungi and their mycotoxins on the Baltic salmonid population, including their role in ulcerative dermal necrosis.

Dilute-and-Shoot HPLC-UV Method for Determination of Urinary Creatinine as a Normalization Tool in Mycotoxin Biomonitoring in Pigs.

A simple, rapid, and accurate HPLC-UV method was developed for the determination of creatinine in pig urine. Usually, it is determined in urine in biomonitoring of xenobiotics to correct for variations in dilutions of urine samples. The colorimetric method (based on Jaffe reaction), which was mainly used for this purpose in mycotoxin biomonitoring, is not a reliable approach for pig urine. Therefore, a novel and accurate HPLC method for creatinine determination was developed. The sample preparation was based on the dilute and shoot approach. An HPLC separation was performed with a porous graphitic carbon column with an aqueous mobile phase to achieve satisfactory retention time for creatinine. The method has been successfully validated, applied for the determination of creatinine in pig urine, and compared with other methods commonly used for that purpose-a colorimetric method based on Jaffe reaction and commercial ELISA test. The developed HPLC method shows the highest precision and accuracy for pig urine samples. Finally, the method was applied as a normalization tool in LC-MS/MS mycotoxin biomarkers analysis. The standardization to a constant creatinine level (0.5 mg/mL) enables similar matrix effects for eleven mycotoxin biomarkers for pig urine samples with different creatinine levels.

Effect of Pterostilbene, a Natural Analog of Resveratrol, on the Activity of some Antiepileptic Drugs in the Acute Seizure Tests in Mice.

Pterostilbene (PTE), a natural analog of resveratrol, is available both as a diet ingredient and a dietary supplement. The present study was undertaken to assess the effect of PTE on the activity of antiepileptic drugs in the acute seizure tests in mice, i.e., the intravenous pentetrazole (iv PTZ) seizure threshold, maximal electroshock (MES), and 6 Hz-induced psychomotor seizure tests. Our study revealed that PTE enhanced the anticonvulsant effect of clonazepam but did not change the activity of tiagabine in the iv PTZ test. In the MES test, PTE increased the effect of carbamazepine but did not affect the protective properties of topiramate, while in the 6-Hz test, we noted a significant enhancement of the activity of oxcarbazepine, but there were no changes in the activity of valproate. Interactions of PTE with carbamazepine and oxcarbazepine were pharmacokinetic, which was determined by the increase of concentration of these antiepileptic drugs both in the serum and brain. In contrast, interactions between PTE and clonazepam were pharmacodynamic since there were no changes in the concentration of clonazepam. Combined treatment with carbamazepine and PTE significantly attenuated muscular strength (estimated in the grip strength test) but did not change motor coordination (assessed in the chimney test) in mice. Other studied antiepileptic drugs and their combinations with PTE did not change these parameters. Further studies are required to evaluate the influence of PTE on the activity of anticonvulsant drugs to estimate the safety of using PTE by patients with epilepsy.

Multiple mycotoxins analysis in animal feed with LC-MS/MS: Comparison of extract dilution and immunoaffinity clean-up.

The aim of this study was a performance comparison of two clean-up procedures (dilutions versus immunoaffinity columns) in the simultaneous determination of eight mycotoxins (aflatoxin B1, deoxynivalenol, fumonisin B1 & B2, ochratoxin A, toxin T-2 & HT-2 and zearalenone) in the animal feed. After extraction the analytes were separated on a Kinetex Biphenyl column with a gradient elution using methanol/0.01 M ammonium acetate as a mobile phase and analyzed with the LC-MS/MS technique. Both of the procedures were validated by analysis of a series of spiked feed samples (n = 6) at three different concentration levels. Better signal to noise ratios were observed for immunoaffinity clean-up. The recoveries of analyses were in the range 88-110% for the dilution procedure and 78-120% for the immunoaffinity clean-up. The dilution procedure was more precise (coefficient of variation of the within-laboratory reproducibility for it was 7.8-22.4% in comparison to 12-35.5% for the immunoaffinity clean-up. The results show that both procedures fulfilled the requirements for mycotoxin analysis and can be used successfully in multi-analyte determination. Although the dilution procedure shows better precision and trueness, the immunoaffinity clean-up procedure can have advantages in more complex feed samples thanks to lower matrix effect and limits of detections.

Identification of metabolites of anticancer candidate salinomycin using liquid chromatography coupled with quadrupole time-of-flight and hybrid triple quadrupole linear ion trap mass spectrometry.

RATIONALE: Salinomycin is an ionophore antibiotic with potential anticancer activity. The history of its use in veterinary medicine shows large differences in species susceptibility to its toxicity. At the same time, the results of research to date suggest a correlation between the extent and pathways of ionophore biotransformation and its toxicity. The biotransformation pattern of salinomycin has not been studied so far. METHODS: Extracts from culture media of human hepatoma cells (HepG2) exposed to salinomycin were analysed with two mass spectrometry techniques. For the first one, micro-liquid chromatography coupled with a quadrupole time-of-flight (Q-TOF) mass spectrometer was used. In the second approach, high-performance liquid chromatography was coupled with a hybrid triple quadrupole linear ion trap. Both experiments were operated in positive electrospray ionization mode. To identify unknown salinomycin metabolites, information-dependent acquisition was applied. RESULTS: Metabolites identified with tandem mass spectrometry included hydroxylated, demethylated and hydroxylated-demethylated derivatives, in total 14 compounds. Using high resolution, only eight isomers of hydroxysalinomycin were detected. The efficiency of biotransformation was low, and so was the abundance of the signals; only for two metabolites did the signal exceed 1% of the salinomycin signal. The analysis of fragmentation patterns narrowed the structure combinations but the actual modification site could not be specified. CONCLUSIONS: Tandem mass spectrometry was more sensitive in the identification of salinomycin metabolites in comparison to the Q-TOF approach. Because of low efficiency of biotransformation of the applied model, the obtained fragmentation data are not sufficient to fully characterize the detected compounds. A study with more metabolically active primary hepatocytes is needed.