Determination of the Synthetic Antioxidant Ethoxyquin and Its Metabolites in Fish and Fishery Products Using Liquid Chromatography-Fluorescence Detection and Stable-Isotope Dilution Analysis-Liquid Chromatography-Tandem Mass Spectrometry.

The use of the synthetic antioxidant ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ) as a flame retardant in fish meal transported by sea is required by international authorities to prevent self-ignition. Because of extensive carry-over within the food chain, selective and sensitive analytical methods are required for investigations on human exposure and the safety of EQ and its metabolites. Therefore, a simple, fast, and rugged liquid-chromatography (LC) method was developed for the detection of EQ and its metabolites in fish and fishery products after liquid-liquid extraction using QuEChERS. For screening purposes, a fluorescence detector was used (LC-FLD) with the EQ-analogue methoxyquin serving as an internal standard. For stable-isotope dilution analysis by liquid chromatography-tandem mass spectrometry (SIDA-LC-MS/MS), deuterated analogues of EQ and its metabolites were synthesized for the first time and allowed for sensitive quantification and thus confirmation of screening results. Both methods were validated and successfully applied to commercially available fish samples.

Modelling of the feed-to-fillet transfer of ethoxyquin and one of its main metabolites, ethoxyquin dimer, to the fillet of farmed Atlantic salmon (Salmon salar L.).

Ethoxyquin (EQ) is an antioxidant supplemented to feed ingredients, mainly fish meal, which is currently under re-evaluation for use in the food production chain. EQ is partly metabolized into several metabolites of which the ethoxyquin dimer (EQDM) accumulates most in the farmed fish fillet. In this study, the feed-to-fillet transfer of dietary EQ and EQDM in Atlantic salmon fillet was investigated, and a physiologically based pharmacokinetic (PBPK-) two-compartmental model was developed, based on experimental determined EQ and EQDM uptake, metabolism, and elimination kinetics. The model was verified with an external data-set and used to simulate the long term (>1.5 years) EQ and EQDM feed-to fillet transfer in Atlantic salmon under realistic farming conditions such as the seasonal fluctuations in feed intake, growth, and fillet fat deposition. The model predictions showed that initial EQDM levels in juvenile fish are the driving factor in final levels found in food-producing animals, while for EQ the levels in feed, and seasonal variations were the driving factor for food EQ levels.

Subchronic dietary exposure to ethoxyquin dimer induces microvesicular steatosis in male BALB/c mice.

The use of the synthetic antioxidant ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline; EQ) in animal feed results in the presence of EQ residues and metabolites, including the EQ dimer (1,8'-bi(6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline); EQDM) in animal food products. To investigate the toxicity and dose-response of dietary exposure to EQDM, male BALB/c mice were exposed to one of six dietary doses of EQDM, ranging from 0.015 to 518 mg/kg body weight/day for 90 days. Doses above 10 mg/kg body weight/day affected whole body lipid metabolism resulting in increased liver weights and decreased adipose tissue mass. Metabolomic screening of livers revealed alterations indicating incomplete fatty acid ß-oxidation and hepatic oxidative stress. Histopathological evaluation and biochemical analyses of the liver confirmed the development of microvesicular steatosis and activation of the glutathione system. Hepatic protein profiling and pathway analyses suggested that EQDM-induced responses are mediated through activation of CAR/PXR nuclear receptors and induction of a NRF2-mediated oxidative stress response. Based on the development of microvesicular steatosis as the critical endpoint, a Reference Point for dietary EQDM exposure was established at 1.1 mg/kg body weight/day (BMDL10) from benchmark dose modelling. Applying an uncertainty factor of 200, an Acceptable Daily Intake of 0.006 mg EQDM/kg body weight was proposed.

Liquid chromatography-electrochemical detection for the determination of ethoxyquin and its dimer in pear skin and salmon samples.

Ethoxyquin (EQ) is widely used as a synthetic antioxidant in animal feed, an antiscalding agent in apples and pears and as a color preservative in some spices. Since the presence of EQ in food products could cause negative health effects it is necessary to develop reliable analytical methods to evaluate the risk of human exposure. In this work, a sensitive, selective and accurate method based on solid-liquid extraction followed by clean-up with solid sorbent and liquid chromatography-electrochemical detection analysis with boron doped diamond electrode (LC-EC) for the determination of ethoxyquin and its dimer (EQDM) in pear skin and salmon samples, was developed. The method was validated according to the European Commission guidelines. The main variables of extraction were accurately optimized. The amounts of solid sorbents for clean-up procedure were optimized by using experimental design. A Box-Behnken design to obtain the optimum conditions was applied. For validation, a matrix-matched calibration was established and a recovery assay with spiked samples was carried out. The limits of detection (LODs) found were 0.05 and 0.1mgkg-1 for EQ and its dimer, respectively. The precision (as relative standard deviation, RSD) was lower than 15% with recoveries of compounds close to 100% in spiked samples.

Comprehensive characterization of ethoxyquin transformation products in fish feed by traveling-wave ion mobility spectrometry coupled to quadrupole time-of-flight mass spectrometry.

Feed additives are typically used in intensive farming production over long periods, and hence, they can accumulate in farmed animal tissues. Concerns regarding the use of ethoxyquin as an antioxidant feed additive, have recently arisen due to its potential conversion into a series of transformation products (TPs). The aim of this work was to characterize the TPs of ethoxyquin in fish feed by a novel approach based on the use of traveling-wave ion mobility spectrometry (TWIMS) coupled to high-resolution quadrupole time-of-flight mass spectrometry (QTOFMS). First, ethoxyquin was oxidized under controlled conditions and the generated TPs were added to a comprehensive database. Atlantic salmon feeds were then screened for ethoxyquin TPs using both targeted and untargeted approaches. Twenty-seven TPs were tentatively identified during the oxidation experiments, fifteen of them also being present in the feed samples. In addition, ten other potential TPs were detected in fish feed following the untargeted approach. Thirty-one of these TPs have been reported for the first time in this work through the oxidation experiments and the feed samples. Therefore, this study provides valuable information on the oxidative fate of ethoxyquin in feed, which can be used for future evaluations of potential risk related to this additive.

Multiclass detection and quantitation of antibiotics and veterinary drugs in shrimps by fast liquid chromatography time-of-flight mass spectrometry.

A fast liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) method has been developed for simultaneous quantitative multiclass determination of residues of selected antibiotics and other veterinary drugs (benzalkonium chloride, ethoxyquin, leucomalachite green (LMG), malachite green (MG), mebendazole, sulfadiazine, sulfadimethoxine, sulfamethazine, sulfamethizole, sulfanilamide, sulfapyridine, sulfathiazole and trimethoprim) in shrimps. Different sample treatment methodologies were tested for the extraction of the targeted species based on either liquid partitioning with different solvents, solid-phase extraction or and matrix solid-phase dispersion. The final selected extraction method consisted of solid-liquid extraction protocol using acetonitrile as solvent followed by a clean-up step with primary secondary amine (QuEChERS). Recovery rates for the extraction of the selected multiclass chemicals were in the range 58-133%. Subsequent identification, confirmation and quantitation were carried out by LC-TOFMS analysis using a reverse-phase C(18) column with 1.8 µm particle size. The confirmation of the target species was based on accurate mass measurements of the protonated molecules ([M+H](+)) and their fragment ions, obtaining routine accuracy errors lower than 2 ppm in most cases. The optimized LC-TOFMS method displayed excellent sensitivity for the studied analytes, with limits of detection (LODs) in the range 0.06-7 µg kg(-1). Finally, the proposed method was successfully applied to the analysis of 12 shrimp samples collected from different supermarkets, showing the potential applicability of the method for ultratrace detection of these chemicals in such complex matrix.

Antioxidant profile of ethoxyquin and some of its S, Se, and Te analogues.

6-(Ethylthio)-, 6-(ethylseleno)-, and 6-(ethyltelluro)-2,2,4-trimethyl-1,2-dihydroquinoline-three heavier chalcogen analogues of ethoxyquin-were prepared by dilithiation of the corresponding 6-bromodihydroquinoline followed either by treatment with the corresponding diethyl dichalcogenide (sulfur derivative) or by insertion of selenium/tellurium into the carbon-lithium bond, oxidation to a diaryl dichalcogenide, borohydride reduction, and finally alkylation of the resulting areneselenolate/arenetellurolate. Ethoxyquin, its heavier chalcogen analogues, and the corresponding 6-PhS, 6-PhSe, and 6-PhTe derivatives were assayed for both their chain-breaking antioxidative capacity and their ability to catalyze reduction of hydrogen peroxide in the presence of a stoichiometric amount of a thiol reducing agent (thiol peroxidase activity). Ethoxyquin itself turned out to be the best inhibitor of azo-initiated peroxidation of linoleic acid in a water/chlorobenzene two-phase system. In the absence of N-acetylcysteine as a coantioxidant in the aqueous phase, it inhibited peroxidation as efficiently as alpha-tocopherol but with a more than 2-fold longer inhibition time. In the presence of 0.25 mM coantioxidant in the aqueous phase, the inhibition time was further increased by almost a factor of 2. This is probably due to thiol-mediated regeneration of the active antioxidant across the lipid-aqueous interphase. The ethyltelluro analogue 1d of ethoxyquin was a similarly efficient quencher of peroxyl radicals compared to the parent in the two-phase system, but less regenerable. Ethoxyquin was found to inhibit azo-initiated oxidation of styrene in the homogeneous phase (chlorobenzene) almost as efficiently (kinh = (2.0 +/- 0.2) x 106 M-1 s-1) as alpha-tocopherol with a stoichiometric factor n = 2.2 +/- 0.1. At the end of the inhibition period, autoxidation was additionally retarded, probably by ethoxyquin nitroxide formed during the course of peroxidation. The N-H bond dissociation enthalpy of ethoxyquin (81.3 +/- 0.3 kcal/mol) was determined by a radical equilibration method using 2,6-dimethoxyphenol and 2,6-di-tert-butyl-4-methylphenol as equilibration partners. Among the investigated compounds, only the tellurium analogues 1d and, less efficiently, 1g had a capacity to catalyze reduction of hydrogen peroxide in the presence of thiophenol. Therefore, analogue 1d is the only antioxidant which is multifunctional (chain-breaking and preventive) in character and which can act in a truly catalytic fashion to decompose both peroxyl radicals and organic hydroperoxides in the presence of suitable thiol reducing agents.

Comparative analysis of cytotoxic, genotoxic and antioxidant effects of 2,2,4,7-tetramethyl-1,2,3,4-tetrahydroquinoline and ethoxyquin on human lymphocytes.

2,2,4,7-Tetramethyl-1,2,3,4-tetrahydroquinoline (THQ) is a new synthetic compound with potential antioxidant activity. In this study, cytotoxic, genotoxic and antioxidant activities of THQ were studied on human lymphocytes with the use of the trypan blue exclusion assay, the TUNEL method, the comet assay and the micronucleus test. The activities of THQ were compared with those of a structurally similar compound-ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ), which is used in animal feeds as a preservative. Cytotoxic effects of THQ were observed after 1-h treatment at the concentration of 500 microM and after 24-h treatments at the concentrations of 250-500 microM. Although the micronucleus test did not reveal a genotoxic effect of THQ, in the comet assay the statistically significant increase in DNA damage was observed as compared with the control. On the other hand, the protection of human lymphocytes against DNA damage induced by hydrogen peroxide suggests an antioxidant activity of THQ. The comparative analysis of THQ and EQ activities performed in these studies revealed that THQ was less cytotoxic and less genotoxic than EQ. Slightly lower antioxidant activity of THQ was also shown in the comet assay when it was used at the lower studied doses (1-5 microM), but for the highest one (10 microM) its efficiency was similar to that of EQ. In the micronucleus assay THQ was more effective than EQ in protecting the cultured lymphocytes from clastogenicity of H2O2. We believe that THQ is worthy of further detailed studies on its antioxidant properties to confirm its usefulness as a preservative.

Fate and effect of the antioxidant ethoxyquin on a mixed methanogenic culture.

The potential inhibitory effect of ethoxyquin, an antioxidant commonly used as a preservative in the food processing industry (e.g., for stabilizing dissolved air flotation residuals), was evaluated at concentrations up to 300 mg/L using a mixed, mesophilic (35 degrees C) methanogenic culture and dextrin, peptone and methanol as the carbon source. A batch assay conducted with a range of ethoxyquin concentrations did not result in any inhibition up to an ethoxyquin concentration of 75 mg/L, but severe inhibition of methanogenesis was observed at concentrations higher than 150 mg/L. Ethoxyquin addition to a batch reactor with the same mixed, methanogenic culture, at ethoxyquin concentrations gradually increasing over 100 days, resulted in a transient and a complete inhibition of methanogenesis at ethoxyquin concentrations of 150 and 300 mg/L, respectively. Acidogens were not significantly impacted, whereas aceticlastic and methanol degrading methylotrophic methanogens were impacted the most. Acclimation of the methanogenic culture to ethoxyquin was not observed over an incubation period of more than 100 days. Long-term (>100 days) incubation at sub-inhibitory ethoxyquin concentrations did not result in ethoxyquin biotransformation. Similarly, ethoxyquin biotransformation was not evident over an 8-day aeration period in a laboratory-scale activated sludge reactor operated under fully aerobic conditions. Ethoxyquin phase distribution tests conducted with the mixed, methanogenic culture at 1.61 g/L volatile solids concentration and nominal ethoxyquin concentrations equal to or higher than 300 mg/L resulted in solid phase/liquid phase ethoxyquin ratios equal to or higher than 1.0. The combined effect of ethoxyquin recalcitrance under anaerobic conditions along with its phase distribution, which favors biosolids, will result in ethoxyquin accumulation in anaerobic treatment systems used by the food processing industry. Such accumulation may pose concerns relative to inhibitory effects in these treatment systems and the disposal of ethoxyquin-bearing biosolids.

Induction of chromosome aberrations in cultured human lymphocytes treated with ethoxyquin.

The chromosomal aberration test was employed to investigate the effect in vitro of a known antioxidant and food preservative, ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) on human chromosomes. The studies were undertaken because there are no published in vitro data on genotoxicity of EQ in mammalian cells and there are many reports pointing out that it may be harmful to animals and human beings. Lymphocytes obtained from three healthy donors were incubated with EQ (0.01-0.5mM) both with and without metabolic activation. Stability studies performed by HPLC analysis showed that EQ was stable under the conditions of the lymphocyte cultures. The results of the chromosome aberration assay showed that EQ induces chromosome aberrations: gaps and breaks as well as dicentrics and atypical translocation chromosomes.

High-performance liquid chromatography coupled with chemiluminescence nitrogen detection for the study of ethoxyquin antioxidant and related organic bases.

The Chemiluminescent Nitrogen Detector (CLND) for use with high-performance liquid chromatography (HPLC) allows for the low-level detection of nitrogen-containing compounds with simple quantitation. The nitrogen selective detector's equimolar response (i.e., equal response for nitrogen independent of its chemical environment) allows for any nitrogen-containing compound to be quantitated as long as the number of nitrogens are known. The HPLC-CLND provides a new detection method for analytes that are not available in large quantities or have unknown chemical or physical characteristics such as oxidation products, metabolites, or impurities. Ethoxyquin is a primary antioxidant that is used to preserve many food products and animal feeds. HPLC-CLND is used in the study of the oxidation products of ethoxyquin because limited quantities of these compounds are available and subsequent calibration curves are difficult to maintain. HPLC-CLND as a new method of detection has been evaluated for its equimolarity of response, linear range, limit of detection, and limit of quantitation.

Purification of ethoxyquin and its two oxidation products.

2,6-Dihydro-2,2,4-trimethyl-6-quinolone (QI) and 1,8'-bis(1, 2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) (DM) are two oxidation products of 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline (ethoxyquin, EQ). This paper describes several methods for the purification of technical grade EQ and for the production of pure QI and DM as standards with the purity required (>99%) for calibration of quantitative determination methods. EQ of high purity was obtained through vacuum distillation followed by a quick column chromatographic purification on silica gel. Preparative scale purity DM could be obtained through recrystallization from methanol, but QI could be purified only by a high-pressure liquid chromatographic method.

New quinolinic derivatives as centrally active antioxidants.

A series of new 1,2-dihydro and 1,2,3,4-tetrahydroquinolines, synthesized from the corresponding propargylaniline intermediates, have been developed as antioxidants for the potential treatment of pathologies implicating central oxidative stress.

Some oxidation products of ethoxyquin including those found in autoxidising systems.

2,4-Dimethyl-6-ethoxyquinoline (2), 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline nitroxide (3), 2,6-dihydro-2,2,4-trimethyl-6-quinone imine N-oxide (4), 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (5), 1.8' -di(1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) (6) and 1,2-dihydro-6-hydroxy-2,2,4-trimethylquinoline (7) have been prepared from 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline (1) (ethoxyquin) and their spectroscopic properties (UV, IR, mass and NMR) examined.