Determination of ethoxyquin by ultra-high performance liquid chromatography with tandem mass spectrometry and a Singapore survey of ethoxyquin residues in eggs, egg products and poultry.

In this study, an advanced ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was developed for quantifying ethoxyquin (EQ). The approach employed a distinctive antioxidant added extraction step designed to prevent ethoxyquin decomposition and maintain analytical precision. This method effectively determines residue levels of EQ in eggs, processed egg products, poultry muscle, salmon, and liquid milk. The method was shown to have a limit of quantitation (LOQ) for eggs, milk, salmon, and chicken muscle of 1.5 µg/kg, 1.9 µg/kg, 2.1 µg/kg, and 1.2 µg/kg, respectively. The recoveries of EQ ranged from 79.2% to 107.6%, with a relative standard deviation (RSD) below 8.4%. A surveillance study for the presence of EQ in different types of eggs and poultry muscle available in Singapore was conducted and a total of 140 samples were tested. EQ residues in all samples were found to be below the U.S. Food and Drug Administration (FDA) MRLs of 500 µg/kg. Some samples of salted and preserved eggs from China were detected with higher concentration of EQ.

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.

Identification of ethoxyquin and its transformation products in salmon after controlled dietary exposure via fish feed.

Ethoxyquin (EQ) is an additive present in fish feed and its fate in fish should be carefully characterized due to food safety concerns regarding this compound. Therefore, the objective of this work was to identify the transformation products (TPs) of EQ in Atlantic salmon. Salmon in independent tanks were given feed containing ethoxyquin concentrations of 0.5 mg/kg, 119 mg/kg or 1173 mg/kg for 90 days. After the feeding trial, salmon fillets were extracted in acetonitrile and analyzed by liquid chromatography with traveling-wave ion mobility spectrometry coupled to high resolution mass spectrometry (UHPLC-TWIMS-QTOFMS). EQ was transferred from the feed to salmon fillets and 23 TPs were characterized, resulting from dimerization, oxygenation, cleavage, cleavage combined with oxygenation, cleavage combined with conjugation, and other uncategorized alterations. Moreover, EQ and some TPs were also detected in commercial salmon randomly sampled from different Norwegian fish farms. This study confirmed that the dimer 1,8'-EQDM was the main TP of EQ and, together with previous research, brought the overall number of characterized TPs to a total of 47.

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.

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.

Levels of synthetic antioxidants (ethoxyquin, butylated hydroxytoluene and butylated hydroxyanisole) in fish feed and commercially farmed fish.

Several synthetic antioxidants are authorized for use as feed additives in the European Union. Ethoxyquin (EQ) and butylated hydroxytoluene (BHT) are generally added to fish meal and fish oil, respectively, to limit lipid oxidation. The study was conducted to examine the concentrations of EQ, BHT and butylated hydroxyanisole (BHA) in several commercially important species of farmed fish, namely Atlantic salmon, halibut and cod and rainbow trout, as well as concentrations in fish feed. The highest levels of BHT, EQ and BHA were found in farmed Atlantic salmon fillets, and were 7.60, 0.17 and 0.07 mg kg(-1), respectively. The lowest concentrations of the synthetic antioxidants found were in cod. The concentration of the oxidation product ethoxyquin dimer (EQDM) was more than ten-fold higher than the concentration of parent EQ in Atlantic salmon halibut and rainbow trout, whereas this dimer was not detected in cod fillets. The theoretical consumer exposure to the synthetic antioxidants EQ, BHA and BHT from the consumption of farmed fish was calculated. The contribution of EQ from a single portion (300 g) of skinned fillets of the different species of farmed fish would contribute at most 15% of the acceptable daily intake (ADI) for a 60 kg adult. The consumption of farmed fish would not contribute measurably to the intake of BHA; however, a 300 g portion of farmed Atlantic salmon would contribute up to 75% of the ADI for BHT.

Determination of ethoxyquin by high-performance liquid chromatography with fluorescence detection and its application to the survey of residues in food products of animal origin.

An analytical method using HPLC with fluorescence detection (HPLC-FL) has been developed and applied for the survey of residue levels of ethoxyquin in a variety of food products of animal origin. HPLC was performed using a silica octadecylsilane column, butylhydroxytoluene-acetonitrile-water (0.05 + 800 + 200, v/v/v) mobile phase, and detection at excitation and emission wavelengths of 370 and 415 nm, respectively. HPLC/MS was used to confirm whether a chromatographic peak was ethoxyquin. The LOQ of the foods was 0.01 microg/g, except for pig fat and cow's milk, and the RSD (n=6) at 0.1 microg/mL of the standard solution was 1.12%. The accuracy of the calculated data of the standard solution was within the range of 94.0 to 101.2%. Recoveries of ethoxyquin from the food products of cattle, pigs, chickens, and salmon were more than 71.0% with an RSD of < 9.3%, except for chicken liver at different concentration levels, including the lower LOQ, the maximum residue limit (MRL), and in some tissues, twice the MRL. Residue levels of ethoxyquin in 33 commercially available food products of animal origin that were purchased on the west side of the Tokyo metropolitan area were surveyed. Contents of ethoxyquin residues in three chicken fat samples by the HPLC-FL method were 0.08, 0.03, and 0.04 microg/g, all less than the MRL (5 microg/g).

Simultaneous quantitative determination of the synthetic antioxidant ethoxyquin and its major metabolite in Atlantic salmon (Salmo salar, L), ethoxyquin dimer, by reversed-phase high-performance liquid chromatography with fluorescence detection.

A method for simultaneous quantitative determination of ethoxyquin (EQ) and its major metabolite in Atlantic salmon tissues, ethoxyquin dimer (EQ dimer), has been developed. The separation was achieved on tandem coupled phenyl-hexyl and C18 columns by 2-phase gradient elution with acetonitrile-ascorbic acid-acetic acid-diethyl amine organized in a 23.5 min sequence. Compounds were extracted with hexane from samples saponified in ethanol-NaOH and protected from air- and light-mediated oxidation by addition of saturated ethylenediaminetetraacetic acid, ascorbic acid, and pyrogallol. The identity of peaks was confirmed by spiking samples with standards verified by proton nuclear magnetic resonance spectrometry, mass spectrometry, and high-performance liquid chromatography. The detection limit (at 358/433 nm) of matrix-spiked EQ was 0.02 and 0.06 microg/L for EQ dimer, with 0.5 g sample weighed and resuspension in 0.5 mL hexane. Linearity was in the range of 0.2-175 microg/L for EQ and 0.3-5100 microg/L for EQ dimer. Two more ubiquitous compounds were identified as de-ethylated EQ and quinone imine. Totally, 14 peaks sharing spectral properties of EQ were separated in a single run, including a major peak present in all muscle samples, termed unknown metabolite of EQ (UMEQ). The concentrations of EQ, EQ dimer, and de-ethylated EQ, as well as concentrations of UMEQ (in arbitrary units), in the muscle were correlated to the amount of EQ fed to the salmon, thus indicating their possible metabolic origin. The pattern of 14 peaks in the muscle showed high specificity and could be used to discriminate between wild salmon and salmon fed EQ-supplemented feed. This method will be a useful tool for studying EQ metabolism and kinetics, and for the routine surveillance of residual levels of dietary EQ in farmed Atlantic salmon.

[Performance study of analytical method for ethoxyquin in fruit].

A performance study of an analytical method of ethoxyquin (ET) in apples and pears was conducted. In the proposed method, the sample was homogenized with thiourea and ET was extracted with acetone instead of hexane used in the official analytical method for ET, in order to improve the extraction efficiency. Furthermore, dibutyl hydroxytoluene was added in the test solution to prevent the decomposition of ET. For evaluation of the method. ET spiked into apples and pears at the level of 0.2 microgram/g was determined in replicate in six laboratories. Mean recoveries from apple, pear and Japanese pear were 85.3, 83.0 and 83.9%, respectively. Repeatability relative standard deviation values were 3.8-4.7% and reproducibility relative standard deviation values were 7.8-11.3%. The detection limits were 0.001-0.025 microgram/g in the six laboratories; this value may reflect instrument performance. ET spiked into apples and pears at the level of 0.2 microgram/g was detected by both LC/MS and GC/MS.

Residue levels of ethoxyquin, imazalil, and iprodione in pears under cold-storage conditions.

A gas chromatographic method is presented for the simultaneous determination of the antiscald ethoxyquin and the fungicides imazalil and iprodione in peel and pulp of Blanquilla pears. Fruits were cold-stored in commercial chambers in normal atmosphere and in controlled atmosphere with low oxygen content (oxygen and carbon dioxide were held at 2.5% and 1.5%, respectively). The method uses gas-liquid chromatography (GLC) with an alkaline flame ionization detector (detector of N-P, NPD) and allows the detection of the mentioned compounds to minimum levels of 0.08-0.12 mg/kg in fresh fruit. With this system the evolution of residues in fruit was monitored throughout the period of cold storage. In the surveys carried out the residue levels of these compounds were found to be below the limits allowed by the legislation of European Union. For the three studied products residues in pulp are lower and disappear more quickly than in peel.

Determination of ethoxyquin in feeds by liquid chromatography: collaborative study.

Ethoxyquin is a chemical antioxidant used in feeds, ingredients, fats, and oils. A liquid chromatographic (LC) method for determination of ethoxyquin was developed. The method involves acetonitrile extraction of the sample and isocratic C18 reversed-phase chromatography with ammonium acetate buffer-acetonitrile as mobile phase and fluorescence detection. A collaborative study of the determination of ethoxyquin in various meals and extruded pet foods was conducted by The Iams Company Research Laboratory. Eleven laboratories analyzed 16 samples (including 2 blind duplicates) consisting of 7 meat meals and 9 extruded pet foods. Sample means ranged from 0.25 to 289 ppm. Repeatability standard deviations ranged from 0.08 to 3.2 ppm, and repeatability relative standard deviations ranged from 4.5 to 32%. Reproducibility standard deviations ranged from 0.12 to 13 ppm, and reproducibility relative standard deviations ranged from 4.5 to 55%. The LC method for determination of ethoxyquin in feeds has been adopted first action by AOAC INTERNATIONAL.

A reverse-phase high-performance liquid chromatographic determination of nicarbazin residues in eggs.

A reverse-phase high-performance liquid chromatographic (HPLC) method was employed for analysis of nicarbazin [1:1 mixture of 4,4'-dinitrocarbanilide (DNC) and 2-hydroxy-4, 6-dimethyl-pyrimidine] in chicken eggs. Nicarbazin residues were analysed by determining the DNC of nicarbazin. HPLC of the DNC portion of nicarbazin was performed with a reverse-phase mu-Bondapak C18 column, using a mobile phase of acetonitrile-water (7:3, v/v). A variable-wavelength detector set at 340 nm, 0.02 AUFS, and a recorder set at 4 mm/min were used for the detection. The standard curve for nicarbazin was linear within the range 0.05-2.0 micrograms/ml. The recovery of nicarbazin added to eggs was 90.2%. The detection limit of nicarbazin in this analytical method was 0.005 micrograms/ml. Nicarbazin was detected in 10% of eggs obtained by feeding chickens with a diet contaminated with nicarbazin within the range 0.07 to 1.39 micrograms/g, but it was not detected in eggs obtained commercially.

Comparison of HPTLC and HPLC procedures for the determination of certain xenobiotic residues in apples and pears.

HPTLC was used to check for residues of benomyl, carbendazim, ethoxyquin and thiabendazole in apples and pears. The method used showed good precision with a percentage coefficient of variation of less than 5%; recoveries were always greater than 90%. The limits of determination using HPTLC were always at least four times lower than Italian statutory limits. Selectivity with respect to other matrix components was excellent for all fruit varieties tested. Comparison with HPLC confirmed the validity of the results.

Exposure to agricultural treatment residues: some simple statistical considerations based on monitoring data for ethoxyquin on apples.

The data examined here concern ethoxyquin levels, mainly measured in apple samples from the retail market. In this study about three hundred measurements, carried out on apples in Northern-Central Italy and recently published, are examined, with the purpose of defining the main characteristics of value statistical distribution and variability and to optimize sampling criteria. The analysis did not indicate the presence of significantly high levels or consequently significantly high risks; nevertheless, it pointed out some important aspects in data collection and evaluation. First, it appeared that the statistical distribution of data is typically nonsymmetrical, non-Gaussian, and characterized by a tail extending toward the high values. The logarithmic transformation of data appeared useful in order to obtain a Gaussian distribution. As a rule, the data variability appeared to be high. The frequency and distribution of values above the analytical threshold resulted in significant change, both in time and in space. The percentage of positive values (above the analytical threshold) appeared to vary up to a factor of 2-3, as analogously did their geometric mean.(ABSTRACT TRUNCATED AT 250 WORDS)

Reverse phase high pressure liquid chromatography and fluorescence detection of ethoxyquin in milk.

A high pressure liquid chromatographic (HPLC) method has been developed for the determination of ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethyl-quinoline) in milk. Milk solids are precipitated by adding acetonitrile, and the water-acetonitrile supernate is washed with hexane to remove fat. Addition of sodium chloride causes the water-acetonitrile solution to separate into an aqueous phase and an acetonitrile phase, thus separating ethoxyquin from most water-soluble impurities. A large volume of water is then added to the acetonitrile layer and ethoxyquin is partitioned into hexane, which is removed at reduced pressure. The residue is dissolved in the mobile phase and analyzed on a 4.6 mm id X 250 mm Ultrasphere ODS column using fluorescence detection (excitation 230 nm; 418 nm cutoff filter). Water-acetonitrile with a diethylamine-acetic acid buffer is the mobile phase. Recoveries from samples fortified at 1, 5, and 10 ppb averaged 78% with a coefficient of variation of 5.0%. Low levels (less than 1 ppb) of apparent ethoxyquin were found in commercial milk samples that were analyzed by using the method.

High pressure liquid chromatographic determination of ethoxyquin in paprika and chili powder.

A method is described for the determination of ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) in paprika and chili powder. Ethoxyquin is extracted from the spice with hexane and partitioned into 0.3N HCl. After adjusting the solution to pH 13-14, ethoxyquin is extracted into hexane, and the hexane layer is evaporated to dryness. An acetonitrile solution of the residue is then analyzed by reverse phase high pressure liquid chromatography with detection at 254 nm. The mobile phase is water-acetonitrile with ammonium acetate buffer. Recoveries from samples fortified at 50, 100, and 200 ppm averaged 92% with a coefficient of variation of 2.3%. The method was applied to a number of commercial samples of paprika and chili powder. Ethoxyquin was found in paprika samples at levels up to 63 ppm and in chili powder samples at levels up to 20 ppm.