Development and validation of a method for determination of 17 endocrine disrupting chemicals in milk, water, blood serum and feed by UHPLC-MS/MS.

The concern for human exposure to bisphenol A (BPA) has led to the introduction of other bisphenols to be used as substitutes in industrial processes. These compounds show activity similar to BPA as endocrine disruptors and could be already widespread both in the environment and in food. To monitor their possible occurrence in the food chain, an analytical method based on affinity chromatography clean-up and UHPLC coupled to tandem mass spectrometry detection was developed and in-house validated according to European law, for simultaneous determination of 17 bisphenols in milk and blood serum from bovine and buffalo, in drinking water and in feed. The analytical performance parameters of the method for these matrices were determined. The results showed satisfactory precision in terms of relative standard deviation (3.3%-21.4%), overall good trueness as mean percentage recoveries (77.0%-119.4%), with the only exception of bisphenol PH and bisphenol S in milk and BPA diglycidyl ether in serum. The high specificity and sensitivity of the method allowed us to determine the analytes at very low concentrations, that is, 0.01-1.0 ng/mL in water, 0.1-2.0 ng/mL in milk, 0.01-1.0 ng/g in blood serum and 1.0-10.0 ng/g in feed.

Determination of 20 Endocrine-Disrupting Compounds in the Buffalo Milk Production Chain and Commercial Bovine Milk by UHPLC-MS/MS and HPLC-FLD.

Bisphenol A (BPA) and some of its analogues are well known as endocrine-disrupting chemicals (EDCs), i.e., compounds that can affect the physiological hormonal pathways in both animals and humans, causing adverse health effects. The intake of these substances through diet represents a public concern, compounded by the scarce data in the literature about contamination levels in food. In the framework of a research project, funded by the Italian Ministry of Health, we determined the contamination levels of BPA and 19 EDCs in the production chain of buffalo milk, analysing feed, drinking water, buffalo milk, and blood sera. Overall, 201 feed, 9 feed additive, 62 drinking water, 46 milk, and 190 blood serum samples were collected from 10 buffalo farms in the Campania region, Southern Italy, between 2019 and 2020, and analysed. Moreover, 15 retail bovine milk samples packaged both in Tetra Pak and in PET were analysed to further evaluate consumers' exposure to EDCs. The results of our work showed no contamination by EDCs in drinking water samples, whereas in 43% of all of the other samples from the farms at least one bisphenol was detected. The most abundant bisphenol detected was BPA (32% of the samples from the farms and 80% of the retail milk samples), thus proving that this compound is still widely used for plastic production.

A survey of Δ9-THC and relevant cannabinoids in products from the Italian market: A study by LC-MS/MS of food, beverages and feed.

In 2016, the European Commission recommended the Member States to monitor the content of Δ9-tetrahydrocannabinol and other cannabinoids in food and feed derived from hemp and in food of animal origin for possible transfer from feed. Thus, the Italian Ministry of Health implemented a monitoring plan. To this aim, nine cannabinoids in beverages and food for human consumption and in feed were determined. The method applied, based on rapid clean-up and LC-MS/MS determination, was previously developed and in-house validated, evaluating the analytical performance in the concentration ranges 2-50 µg/L for beverages, 0.020-0.500 mg/kg for food and 0.100-10.0 mg/kg for feed. Then, it was applied to determine the cannabinoids in 78 food, 16 beverage and 6 feed samples, collected from the Italian market since 2017. The results are herein reported, for evaluation of both product characteristics and compliance to national maximum limits. Some study cases are also described.

T-2 and HT-2 toxins in feed and food from Southern Italy, determined by LC-MS/MS after immunoaffinity clean-up.

A method to determine T-2 and HT-2 toxins, both in feed and food, is described. The method is based on sample clean-up by immunoaffinity columns, followed by triple quadrupole LC-MS/MS determination. It was in-house validated in compliance with the European Union legislation, in the concentration range 5.0-750 µg kg-1 and accredited according to the ISO/IEC 17025 standard, for the scope of official control of feed and food. The method was applied to monitor the contamination levels of T-2 and HT-2 toxins in 105 feed samples, both raw materials and compound feed and 17 cereals and derived products for human consumption, collected in Southern Italy between 2015 and 2019. T-2 toxin was quantified in 32 feed (30.5%) and in 2 food samples (11.8%). HT-2 toxin was quantified in 65 feed (61.9%) and in 3 food (17.6%) samples.

Bisphenol A, octylphenols and nonylphenols in fish muscle determined by LC/ESI-MS/MS after affinity chromatography clean up.

An analytical method is applied to simultaneously determine bisphenol A, 4-octylphenol, 4-tert-octylphenol, 4-nonylphenol and tert-nonylphenol in fish muscle. The method consisted of a cleanup on molecularly imprinted polymers cartridges (MIPs) followed by liquid chromatography coupled to a tandem mass spectrometer. The method is rapid, specific, reliable and very sensitive, allowing for quantification down to 0.5 ng/g for all studied compounds. After in-house validation, the method was employed to monitor the concentration levels of BPA and four target alkylphenols in 44 marine fish, collected from third countries in the frame of official control activities. The results showed that 27 samples (61.4%) were contaminated by at least one target compound. Bisphenol A was determined in 11 samples in the range 0.6-7.1 ng/g, 4-tert-octylphenol in 8 samples (0.5-1.6 ng/g) and tert-nonylphenol in 14 samples (0.8-8.9 ng/g). 4-Octylphenol and 4-nonylphenol were not detected at all.

A method to determine BPA, BPB, and BPF levels in fruit juices by liquid chromatography coupled to tandem mass spectrometry.

The monitoring of the contamination levels of bisphenol A (BPA) and its congeners bisphenol B (BPB) and bisphenol F (BPF) in foodstuffs is a necessary process for assessment of consumers' risk. After development and validation of a method using Strata® C18-E cartridge cleanup with detection by liquid chromatography coupled to tandem mass spectrometry, forty-six samples of fruit juices, sold on Italian markets, have been analysed to assess the concentration of BPA, BPB and BPF. BPB and BPF were not detected in any samples, BPA was found in 33 % of the samples. The observed levels ranged from 0.50 ng mL-1 to 2.85 ng mL-1. Potential Daily Intakes (PDI) of BPA for Italian populations were calculated by the budget method model. PDIs ranged from 0.012 to 0.285 µg kg-1 bw day-1. None of the calculated values exceeded the current temporary TDI of 4 µg kg-1 bw day-1.

BPA, BPB, BPF, BADGE and BFDGE in canned beers from the Italian market.

A survey of BPA, BPB, BPF, BADGE and BFDGE contamination in canned beers from the Italian market is reported. An analytical method for the determination of these five bisphenols down to 0.5 ng mL-1 using UPLC with fluorescence detection was developed and validated. A total of 40 canned beers were collected from the market in Southern Italy and analysed. The results showed that only 14 samples were contaminated at concentrations ranging from 0.5 to 2.5 ng mL-1 by at least BPA, BPF and BADGE. No contamination by BPB and BFDGE was detected. This survey suggests that canned beers from the Italian market should represent neither a relevant source of intake of bisphenols nor a risk for consumer's health.

A rapid method to determine nine natural cannabinoids in beverages and food derived from Cannabis sativa by liquid chromatography coupled to tandem mass spectrometry on a QTRAP 4000.

RATIONALE: Phytocannabinoids are natural compounds produced by Cannabis spp. Some of these compounds show psychotropic effects on humans and are therefore used as drugs of abuse. These compounds are present in food and beverages containing ingredients from hemp, and thus can reach consumers. The Italian Ministry of Health planned to evaluate the intake of cannabinoids from food containing hemp ingredients. Thus, we were asked to develop and validate a multi-residue test method to determine nine phytocannabinoids in beverages and food. METHODS: Nine natural phytocannabinoids, hereafter called cannabinoids, were cleaned up from food by solid-liquid extraction, while beverages were simply diluted prior to analysis. The cannabinoids were separated by reversed-phase high-performance liquid chromatography, and on-line determination was carried out by tandem mass spectrometry using a 4000 QTRAP mass spectrometer with a TurboIonSpray source, in multiple-reaction monitoring mode, using both positive and negative ionization. RESULTS: Each compound was determined down to 0.25 ng/mL in solvent. In-house validation was carried out; the mean recoveries ranged from 83.4% to 101.2% in food, and from 84.5% to 104.5% in beverages. The limits of quantification were 20 µg/kg for food and 2 µg/L for beverages. CONCLUSIONS: A reliable and rapid method for the identification and quantification of the psychotropic Δ9 -tetrahydrocannabinol, its non-psychoactive precursor Δ9 -tetrahydrocannabinolic acid A, and seven other cannabinoids was developed and validated, to monitor the content of these substances in food and beverages produced using hemp seeds, flour and oil as ingredients.

Determination of BPA, BPB, BPF, BADGE and BFDGE in canned energy drinks by molecularly imprinted polymer cleaning up and UPLC with fluorescence detection.

A new method for simultaneous determination of five bisphenols in canned energy drinks by UPLC with fluorescence detection, after clean up on molecularly imprinted polymers, is herein described. The method was validated at two concentration levels, calculating trueness, repeatability and within-laboratory reproducibility, specificity, linearity of detector response, the limits of quantifications and the limits of detection for each bisphenol. The method is specific, reliable and very sensitive, allowing for determination of bisphenol F diglycidyl ether (BFDGE), bisphenol A (BPA), bisphenol B (BPB), bisphenol F (BPF) and bisphenol A diglycidyl ether (BADGE) down to 0.50ng/mL; it was employed to determine contamination levels from these bisphenols in forty energy drinks of different brands, collected from the market in Naples. BPA was detected in 17 out of 40 samples (42.5%); in some energy drinks also BPF, BADGE and BFDGE were determined.

A novel method to determine valnemulin in feedingstuffs for several animal species by liquid chromatography-electrospray tandem mass spectrometry.

A new method specific for the determination and unambiguous confirmation of the antibiotic valnemulin in feed for all animal species is described. Simple clean-up based on solvent extraction and liquid partition is carried out prior to determination by liquid chromatography coupled to electrospray tandem mass spectrometry on a hybrid QTRAP 4000 system, in multiple reaction monitoring mode. Valnemulin is licensed in the European Union as a veterinary drug for use in feed for pigs and rabbits, but unavoidable carryover in feed for non-target species should be monitored. The method is rapid and reliable, and was validated in-house at 10, 100 and 750 ng g-1, evaluating the analytical performances according to Commission Regulation (EC) No. 882/2004. Good mean recoveries from 79.7% to 92.6%, and within-laboratory reproducibility RSDs, ranging from 6.2% to 12.1%, were measured; the limit of quantification at 10.0 ng g-1 also allows for sensitive evaluation of undesirable carryover in feed for non-target species. The results of monitoring 24 compound feeds for several animal species are also reported.

A developmental hepatotoxicity study of dietary bisphenol A in Sparus aurata juveniles.

Previous studies in rats have indicated that a diet enriched with Bisphenol A adversely effects metabolism and reproductive success. In rats exposed to BPA by maternal gavage, alteration in the developmental programming, higher obesity rates and reproductive anomalies were induced. Starting with this evidence, the aim of this study was to provide important insights on the effects induced by a BPA enriched diet, on the reproductive physiology and metabolism of juvenile fish, simulating the scenario occurring when wild fish fed on prey contaminated with environmental BPA. Seabream was chosen as model, as it is one of the primary commercial species valued by consumers and these results could provide important findings on adverse effects that could be passed on to humans by eating contaminated fish. A novel method for measuring BPA in the food and water by affinity chromatography was developed. Analysis of signals involved in reproduction uncovered altered levels of vtg and Zp, clearly indicating the estrogenic effect of BPA. Similarly, BPA up-regulated catd and era gene expression. A noteworthy outcome from this study was the full length cloning of two vtg encoding proteins, namely vtgA and vtgB, which are differently modulated by BPA. Cyp1a1 and EROD activity were significantly downregulated, confirming the ability of estrogenic compounds to inhibit the detoxification process. GST activity was unaffected by BPA contamination, while CAT activity was down regulated. These results collectively confirm the estrogenic effect of BPA and provide additional characterization of novel vtg genes in Sparus aurata.