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

Abstract

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, butylhydroxytolueneacetonitrilewater (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 µg/g, except for pig fat and cow's milk, and the RSD (n = 6) at 0.1 µg/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 µg/g, all less than the MRL (5 µg/g).

Ethoxyquin: An Antioxidant Used in Animal Feed

“…The levels of the parent compound (EQ) in meat of farmed animals are usually lower than MRL (Maximum Residue Level) [ 20 , 84 ], but EQ oxidation products are usually not controlled. It was shown that EQDM and other EQ residues can be present in different animal tissues [ 23 , 24 , 26 , 28 , 37 ].…”

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

“…Various procedures for the extraction of EQ and some of its oxidation products have been described in the literature, mostly comprising simple liquid extraction of homogenized samples using organic solvents , or the QuEChERS protocol. , Other methods make use of solid-phase extraction or include a further saponification step with sodium hydroxide for enhanced matrix disruption prior to liquid extraction. , However, because of the high fat content of certain fish species like salmon, the biggest challenge remains the efficient separation of the most lipophilic analyte, EQDM, from disturbing matrix components.…”

“…Notably, GC-FID and GC-MS used to be the methods of choice some decades ago, − , despite the drawback of the low temperature stability of EQ, which quickly degrades to DMEQ at the temperatures applied in the injector and the column oven during GC analysis . Today, LC-based methods can be considered state-of-the-art. ,,,, Moreover, the dihydroquinoline moiety of EQ and EQDM as well as the quinoline in DMEQ make these compounds excellent fluorophores, allowing for selective and sensitive detection using a fluorescence detector after LC separation (LC-FLD). The reported drawback that QI can hardly be detected by LC-FLD, as its quinone imine structure shows only weak fluorescence, can easily be overcome by postcolumn reduction of QI to DEQ (Figure ).…”

“…Consequently, appropriate analytical methods are required in order to investigate levels of EQ and its metabolites in fish and fishery products for human consumption. Several methods for this purpose have been described in the literature, including gas chromatography with flame-ionization detection (GC-FID) and mostly liquid chromatography with fluorescence detection (LC-FLD) or more recently electrochemical (LC-EC) detection. ,,− Also coupling of the respective chromatographic systems with mass spectrometry (GC-MS and LC-MS) is common. ,, Quantification of analytes in these methods is either performed by external calibration or with the aid of internal standards like quinoline or methoxyquin (1,2-dihydro-6-methoxy-2,2,4-trimethylquinoline, MQ), the latter allowing for correction of analyte extraction losses during sample preparation. , However, until now, no method has been reported applying stable-isotope dilution analysis (SIDA) using stably labeled isotopomers of the respective analytes as internal standards, although SIDA can be considered state-of-the-art in modern residue analysis. , A reason for this might be the lack of commercially available stably labeled isotopomers, which in SIDA allow the compensation of extraction losses or matrix effects during mass spectrometry because of their almost identical physicochemical properties.…”

“…4,7,13−18 Also coupling of the respective chromatographic systems with mass spectrometry (GC-MS and LC-MS) is common. 13,16,19 Quantification of analytes in these methods is either performed by external calibration or with the aid of internal standards like quinoline or methoxyquin (1,2-dihydro-6-methoxy-2,2,4-trimethylquinoline, MQ), the latter allowing for correction of analyte extraction losses during sample preparation. 15,17 However, until now, no method has been reported applying stable-isotope dilution analysis (SIDA) using stably labeled isotopomers of the respective analytes as internal standards, although SIDA can be considered state-of-the-art in modern residue analysis.…”