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

Article abstract of DOI:10.1021/acs.jafc.9b01508

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.

Full text of DOI:10.1021/acs.jafc.9b01508

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New Analytical Methods
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
Andreas Kranawetvogl, and Paul Wilhelm Elsinghorst
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01508 • Publication Date (Web): 16 May 2019
Downloaded from http://pubs.acs.org on May 16, 2019
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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
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Andreas Kranawetvogl,† Paul W. Elsinghorst*,†,‡
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† Central Institute of the Bundeswehr Medical Service Munich, Ingolstädter Landstrasse 102,
85748 Garching, Germany
‡ Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn,
An der Immenburg 4, 53121 Bonn, Germany
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* Corresponding author:
Priv.-Doz. Dr. Paul W. Elsinghorst, Central Institute of the Bundeswehr Medical Service
Munich, Ingolstädter Landstrasse 102, 85748 Garching, Germany; Phone: +49-89-3755849-
5310, Fax: +49-89-3755849-5209, e-mail: paulelsinghorst@bundeswehr.org
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Abstract
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. Due to extensive carry-over within the food
chain, selective and sensitive analytical methods are required for investigations on human
exposure and safety of EQ and its metabolites. Therefore, a simple, fast and rugged liquid
chromatography method was developed for 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
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.
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Keywords
ethoxyquin, Skraup-Doebner-von Miller quinoline synthesis, fishery products, metabolite,
fluorescence detection, stable isotope dilution analysis
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Introduction
The antioxidant ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ) was first
described by the German chemist Emil Knoevenagel in 1921, when working on the
condensation of aromatic amines with aliphatic ketones.1 Although initially used as a rubber
additive to impede cracking from isoprene oxidation, EQ has also a long history of application
as an antioxidant in feed, especially for poultry and fish.2 Fish meal, because of its high content
in polyunsaturated fatty acids, is prone to oxidation and requires stabilization to preserve its
high nutritional value. Moreover, international regulations by the International Maritime
Organization require addition of EQ to fish meal and fish scrap when transported by sea to
prevent spontaneous combustion.3 Currently, minimum levels of EQ at the time of consignment
are set at 100 mg/kg (100 ppm).
Previous studies revealed that EQ and metabolites thereof are detectable in various
tissues of fish fed with EQ-containing feed, indicating that human exposure may occur as a
result of carry-over.4–7 Notably, the EQ dimer (6,6’-diethoxy-2,2,2’,2’,4,4’-hexamethyl-1’,2’-
dihydro-2H-1,8’-biquinoline, EQDM) has been reported as the main metabolite showing
considerably higher concentrations than EQ itself.6 Other metabolites formed by
demethylation/rearomatization (2,4-dimethyl-6-ethoxyquinoline, DMEQ) or the de-ethylation
product hydroxyquin (1,2-dihydro-6-hydroxy-2,2,4-trimethylquinoline, DEQ) and its
corresponding quinone imine (2,6-dihydro-2,2,4-trimethyl-6-quinolone, QI) have also been
detected, but often at negligible intensity. Recently, even more oxidation products were
reported to be present in salmon feed, of which several were shown to carry-over into fish
including EQDM as the most abundant but also QI.8,9
Safety of EQ was assessed by the European Food Safety Authority (EFSA), which found
EQ to be neither genotoxic nor carcinogenic and not to cause developmental toxicity in
offspring.10 However, despite its high antioxidant capacity EQ may also act as a prooxidant
facilitating formation of reactive oxygen species that are known for their adverse health effects,
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in particular at higher concentrations.10,11 Typical symptoms observed in different animals after
oral administration of EQ were liver and kidney damages as well as changes in the
gastrointestinal tract. With respect to the toxicity of EQ metabolites data are incomplete except
for EQDM, whose toxicological profile is considered to reflect that of EQ. Toxicity of QI has
not been investigated yet, however, it is assumed to be capable of forming covalent DNA
adducts due to its quinone imine structure and might thus be mutagenic and carcinogenic.10
Due to this considerable lack of data the European Commission decided to suspend the
authorization of EQ as a feed additive in June 2017, pointing out that the suspension should be
reviewed upon submission and evaluation of additional data with focus on consumer
exposure.12 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 using
fluorescence (LC-FLD) or more recently electrochemical (LC-EC) detection.4,7,13–18 Also
coupling of the respective chromatographic systems with mass spectrometry (GC-MS, LC-MS)
are 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 stable-labeled isotopomers of the
respective analytes as internal standards, although SIDA can be considered state of the art in
modern residue analysis.20,21 A reason for this might be the lack of commercially available
stable-labeled isotopomers, which in SIDA will allow to compensate extraction losses or matrix
effects during mass spectrometry because of their almost identical physicochemical properties.
Therefore, deuterium-labeled isotopomers of EQ and its metabolites were synthesized
and a stable isotope dilution analysis liquid chromatography-tandem mass spectrometry (SIDA-
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LC-MS/MS) method was developed for highly selective and sensitive determination of the
analytes in fish and fishery products. Additionally, a simple, fast and rugged LC-FLD method
was established for screening of samples prior to confirmation of residue contents using SIDA-
LC-MS/MS. Both methods were validated and successfully applied to a set of commercially
available salmon samples.
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Materials and Methods
Chemicals and Reagents. 4-Ethoxyaniline (≥ 98%, for synthesis) was purchased from Sigma-
Aldrich (Taufkirchen, Germany). 4-Methoxyaniline (≥ 98%, for synthesis), ethyl acetate
(Emsure®, p.a.), ethanol (absolute, p.a.), ammonium acetate (Emsure®, ACS reagent),
hydrochloric acid (Titripur®, 2 M), solid sodium hydroxide (≥ 97%), sodium hydroxide
(Titrisol®, 1 M) and anhydrous sodium sulfate (Emsure®, p.a.) were purchased from Merck
(Darmstadt, Germany). Petroleum ether (boiling range 40-60 °C, Chemsolute®, p.a.) was
obtained from Th. Geyer (Renningen, Germany). Crystalline iodine (≥ 99.5%) and tert-butyl
hydroperoxide (70% aqueous solution) were from Thermo Fisher (Karlsruhe, Germany).
Toluene (Chromasolv®, p.a.) was purchased from Honeywell Riedel-de-Haën (Seelze,
Germany) and d6-acetone from Deutero (Kastellaun, Germany). Acetonitrile (MeCN, LC
grade) was purchased from VWR (Darmstadt, Germany) and ascorbic acid (ACS reagent, p.a.)
and ammonium ferrous sulfate hexahydrate (≥ 99%, ACS reagent) were from Fluka (Buchs,
Switzerland). Water (LC grade) was prepared using a Milli-Q® Gradient water purification
system from Merck. ALUGRAM® Xtra SIL G/UV254 (silica gel 60, layer thickness 0.2 mm)
plates for thin-layer chromatography, silica gel 60 (0.063–0.2 mm) for column
chromatography, the Chromabond® QuEChERS citrate extraction mix (Mix I) as well as the
Chromabond® QuEChERS clean-up mix (Mix VI) were obtained from Macherey-Nagel
(Düren, Germany).
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Instrumentation. A syringe pump KDS 100 Legacy from KD Scientific Inc. (Holliston, MA,
USA) was used for continuous addition of reagents for synthesis of d10-EQ and MQ as described
below. Melting points were determined using a capillary tube apparatus B-540 from Büchi
(Essen, Germany). GC-MS analysis for compound characterization was performed on an 7890B
GC system (injector: splitless, 250 °C; carrier gas: He; column: HP-5 MS,
30 m × 0.25 mm × 0.25 μm; temperature gradient, min/°C: 0/60, 2/60, 21/250, 22.5/280,
35/280) coupled to a 5977A mass spectrometer (EI, scan range m/z 50-500) from Agilent
Technologies (Waldbronn, Germany). NMR spectra were recorded on an Avance III (1H NMR:
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600 MHz, C NMR: 150 MHz) and DRX 500 (1H NMR: 500 MHz, C NMR: 125 MHz)
spectrometer from Bruker (Rheinstetten, Germany) and chemical shifts δ are given in parts per
million referring to the signal center using the respective solvent peaks for reference (CDCl3,
7.26/77.0 ppm; d6-DMSO, 2.50/39.5 ppm). To characterize the spin multiplicity, the following
abbreviations are used: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; q, quartet; m,
multiplet.
d10-Ethoxyquin (d10-EQ). In a round-bottom flask 4-ethoxyaniline (2 g, 14.6 mmol) and iodine
(50 mg, 0.20 mmol) were dissolved in toluene (6.5 mL) under vigorous stirring. After purging
with nitrogen, d6-acetone (4.68 g, 73 mmol) was added to the refluxing reaction mixture using
a syringe pump (0.26 g/h, 18 h). Subsequently the reaction mixture was filtered through silica
using petroleum ether/ethyl acetate 80:20 v/v (500 mL) and the filtrate was evaporated in vacuo.
Purification of the residue was achieved by column chromatography using petroleum
ether/ethyl acetate 90:10 v/v (detection: i) blue fluorescence upon excitation at λ = 365 nm, ii)
fast degradation to a brownish product under the influence of daylight) to provide d10-EQ
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(1.69 g, 51%) as a yellowish oil. GC-MS: purity > 94%. H NMR (d6-DMSO) δ: 1.13 (m,
2JH,D = 9.5 Hz, 2-CH3, 24% H), 1.26 (t, 3H, 3JH,H = 6.9 Hz, CH2CH3), 1.84 (m, 4-CH3, 11% H),
3.88 (q, 2H, 3JH,H = 6.9 Hz, CH2CH3), 5.29 (m, 3-H, 13% H), 5.31 (s, 1H, NH), 6.39 (m, 8-H,
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11% H), 6.53 (m, 2H, 5/7-H); C NMR (d6-DMSO) δ: 14.90 (CH2CH3), 17.56 (m, 4-CH3),
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29.52 (m, 2-CH3), 50.58 (m, C-2), 63.49 (CH2CH3), 110.14 (C-5), 112.65 (m, C-8), 114.52 (C-
7), 121.37 (C-4a), 127.32 (C-4), 129.11 (m, C-3), 138.38 (C-8a), 149.61 (C-6).
d7-Dimethylethoxyquinoline (d7-DMEQ). d10-EQ (0.5 g, 2.20 mmol) was heated to 180 °C
for 30 min under vigorous stirring. The crude reaction product was purified by column
chromatography using petroleum ether/ethyl acetate 50:50 v/v (detection: blue fluorescence
upon excitation at λ = 254 nm) and recrystallization from petroleum ether to obtain d7-DMEQ
(10 mg, 2%) as light brown crystals, m.p. 83-84 °C, lit.22 86-87 °C (non-deuterated). GC-MS:
purity > 95%. 1H NMR (CDCl3) δ: 1.49 (t, 3H, 3JH,H = 7.0 Hz, CH2CH3), 2.58 (m, 2-CH3, 14%
H), 2.65 (m, 4-CH3, 26% H), 4.15 (q, 2H, 3JH,H = 7.0 Hz, CH2CH3), 7.10 (s, 3-H, 11% H), 7.15
(d, 1H, 4JH,H = 2.7 Hz, 5-H), 7.33 (d, 1H, 4JH,H = 2.7 Hz, 7-H), 7.96 (app. d, 3JH,H = 9.2 Hz, 8-
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H, 13% H); C NMR (CDCl3) δ: 14.80 (CH2CH3), 18.17 (4-CH3), 24.16 (2-CH3), 63.76
(CH2CH3), 102.97 (C-5), 121.30 (C-7), 122.65 (C-3), 127.39 (C-4a), 129.90 (C-8), 142.91 (C-
4), 143.12 (C-8a), 155.62 (C-6), 156.50 (C-2).
d18-Ethoxyquin dimer (d18-EQDM). A solution of d10-EQ (0.51 g, 2.25 mmol) in ethanol
(50 mL) was cooled to 0 °C and an equimolar amount of tert-butyl hydroperoxide (70%,
0.31 mL) and aqueous ammonium ferrous sulfate hexahydrate (0.88 g in 20 mL) were added
under vigorous stirring. The mixture was allowed to react at 0 °C for 18 h before it was acidified
(1 M HCl, 50 mL) and extracted with petroleum ether (3 × 50 mL). The combined extracts were
washed (1°M HCl, 2 × 25 mL), dried using Na2SO4 and evaporated in vacuo. The remaining
residue was purified by column chromatography using petroleum ether/ethyl acetate 90:10 v/v
(detection: i) blue fluorescence upon excitation at λ = 365 nm, ii) fast degradation to a reddish
product under the influence of daylight) to obtain d18-EQDM (0.35 g, 69%) as a yellowish solid,
m.p. 109-110 °C, lit.22 107-109 °C (non-deuterated). GC-MS: purity 100%. 1H NMR (CDCl3)
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δ: 0.93 (m, JH,D = 11.7 Hz, 2’-H, 27% H), 1.14 (m, JH,D = 11.2 Hz, 2’-H, 24% H), 1.18 (m,
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2JH,D = 11.2 Hz, 2-H, 31% H), 1.27 (m, JH,D = 11.7 Hz, 2-H, 33% H), 1.37 (t, 3H,
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3JH,H = 7.0 Hz, 8-OCH2CH3), 1.39 (t, 3H, JH,H = 7.0 Hz, 8’-OCH2CH3), 2.01 (m,
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2JH,D = 9.7 Hz, 4/4’-H, 18% H), 3.95 (q, 2H, JH,H = 7.0 Hz, 8-OCH2CH3), 3.96 (q, 2H,
3JH,H = 7.0 Hz, 8’-OCH2CH3), 5.37 (s, 3’-H, 29% H), 5.44 (s, 3-H, 54% H), 6.06 (d,
3JH,H = 8.8 Hz, 8-H, 13% H), 6.49 (d, 1H, 4JH,H = 2.9 Hz, 7-H), 6.68 (d, 1H, 4JH,H = 2.5 Hz, 7’-
H), 6.72 (d, 1H, 4JH,H = 2.5 Hz, 5’-H), 6.76 (d, 1H, 4JH,H = 2.9 Hz, 5-H); 13C NMR (CDCl3) δ:
15.03 (CH2CH3), 15.07 (CH2CH3), 56.56 (C-2), 63.95 (CH2CH3), 64.27 (CH2CH3), 109.99 (C-
7), 110.76 (C-5’), 113.71 (C-5), 114.70 (m, C-8), 117.40 (C-7’), 124.32 (C-4a), 124.62 (C-8a’),
127.83 (C-4a’), 127.95 (C-4/4’), 128.48 (C-4/4’), 130.61 (C-3/3’), 130.98 (C-3/3’), 137.89 (C-
8a), 150.35 (C-6’), 150.86 (C-6).
d10-Quinone imine (d10-QI). The combined acidic layers obtained following the procedure for
the synthesis of d18-EQDM were basified (4 M NaOH, 50 mL) and extracted with petroleum
ether (5 × 50 mL). After washing (1 M NaOH, 2 × 25 mL) and drying with Na2SO4, the solvent
was evaporated in vacuo and the crude residue purified by column chromatography using
petroleum ether/ethyl acetate 50:50 v/v (detection: i) fluorescence extinction upon excitation at
λ = 254 nm, ii) yellow spots) to obtain d10-QI as an auburn solid (73 mg, 17%), m.p. 47-49 °C,
lit.22 52-53 °C (non-deuterated). GC-MS: purity > 90%. H NMR (CDCl3) δ: 1.38 (m,
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2JH,D = 9.7 Hz, 2-CH3, 29% H), 1.95 (m, 4-CH3, 14% H), 6.33 (s, 1H, 5-H), 6.44 (s, 3-H, 10%
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H), 6.65 (m, 1H, 7-H), 7.19 (d, JH,H = 9.9 Hz, 8-H, 12% H); C NMR (CDCl3) δ: 16.97 (4-
CH3), 28.01 (2-CH3), 61.60 (C-2), 120.64 (C-5), 126.64 (C-8a), 131.51 (C-4), 133.25 (C-7),
140.34 (C-3), 145.44 (C-8), 154.76 (C-4a), 188.46 (C-6).
Methoxyquin (MQ). In a round-bottom flask 4-methoxyaniline (1.8 g, 14.6 mmol) and iodine
(50 mg, 0.20 mmol) were dissolved in toluene (6.5 mL) under vigorous stirring. After purging
with nitrogen acetone (4.24 g, 73 mmol) was added to the refluxing reaction mixture using a
syringe pump (0.24 g/h, 18 h). Subsequently the reaction mixture was filtered through silica
using petroleum ether/ethyl acetate 80:20 v/v (500 mL) and the filtrate was evaporated in vacuo.
Purification of the residue was achieved by column chromatography using petroleum
ether/ethyl acetate 90:10 v/v (detection: i) blue fluorescence upon excitation at λ = 365 nm, ii)
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fast degradation to a brownish product under the influence of daylight) to provide MQ (1.65 g,
55%) as a yellowish oil. GC-MS: purity > 96%. 1H NMR (d6-DMSO) δ: 1.16 (s, 6H, 2-CH3),
1.88 (d, 3H, 4JH,H = 1.4 Hz, 4-CH3), 3.64 (s, 3H, OCH3), 5.30 (m, 1H, 3-H), 5.33 (s, 1H, NH),
6.41 (m, 1H, 3JH,H = 8.8 Hz, 8-H), 6.53 (dd, 1H, 3JH,H = 8.7 Hz, 4JH,H = 2.8 Hz, 7-H), 6.55 (app.
s, 1H, 5-H); 13C NMR (d6-DMSO) δ: 18.19 (4-CH3), 30.19 (2-CH3), 50.96 (C-2), 55.43 (OCH3),
109.27 (C-5), 113.00 (C-8), 113.87 (C-7), 121.36 (C-4a), 127.52 (C-4), 129.56 (C-3), 138.42
(C-8a), 150.49 (C-6).
Sample preparation. Samples for LC-FLD and SIDA-LC-MS/MS analysis were obtained
from local retailers and originated either from Norwegian aquaculture or from the North-East
Pacific. Fillets were cut into pieces and homogenized using a knife mill (Grindomix GM 200;
Retsch, Haan, Germany). Samples (5 g) were exactly weighed into amber glass centrifuge tubes
(50 mL) and spiked either with MQ (500 µL, 1 µg/mL, MeCN) for LC-FLD analysis or a
mixture of d10-EQ, d7-DMEQ, d18-EQDM and d10-QI (500 µL, 1 µg/mL for d10-EQ, d7-DMEQ
and d10-QI, 100 ng/mL for d18-EQDM, MeCN) in the case of SIDA-LC-MS/MS. Subsequently,
water was added (6,5 mL) and samples were shaken vigorously by hand (1 min) prior to
addition of MeCN (9,5 mL) and further mixing (15 min, 200 rpm) using a laboratory shaker
(KS 501 digital; IKA® Labortechnik, Staufen, Germany). Subsequently, QuEChERS citrate
extraction mix (Mix I) was added and the centrifuge tubes were shaken vigorously by hand
(1 min) before samples were centrifuged (3345 × g, room temperature (rt), 5 min) using a
laboratory centrifuge (Megafuge 1.0R; Heraeus, Hanau, Germany). Finally, an aliquot of the
supernatant (6 mL) was transferred into centrifuge tubes (15 mL) containing QuEChERS clean-
up mix (Mix VI) followed once more by vigorous shaking (1 min) by hand and another
centrifugation step (3345 × g, rt, 5 min). An aliquot (1 mL) of the supernatant was transferred
into amber glass vials and subjected directly to LC-FLD or SIDA-LC-MS/MS analysis.
LC-FLD analysis. Sample analysis was performed using an Infinity 1200 series LC-FLD
system (Agilent Technologies) consisting of an autosampler, a binary pump, and a column
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thermostat coupled to a fluorescence detector. Post-column derivatization was achieved using
an Infinity 1260 isocratic pump (Agilent Technologies) and an LC-MS effluent optimizer
(LEO) module (Gerstel, Mülheim, Germany). Samples (10 µL) were separated at 40 °C on a
Kinetex® EVO C18 column (100 mm × 4.6 mm, 2.6 µm; Phenomenex, Aschaffenburg,
Germany) protected by a precolumn (SecurityGuard Ultra cartridge for C18 ultra-high
performance LC; Phenomenex) using gradient elution with solvent A (10 mM ammonium
acetate) and solvent B (MeCN) at a flow of 500 μL/min. The following gradient was applied,
min/% B: 0/40, 10/80, 10.2/100, 14.8/100, 15/40, 20/40. Within each run the six-way valve
installed in the column oven was switched from configuration 1 to configuration 2 after 9 min
and backwards after 20 min (see Supporting Information, Figure S1) allowing for regular
backflushing and thus cleaning of the precolumn. Aqueous ascorbic acid (0.1% w/v) was
continuously added post-column at a flow of 50 µL/min using the LEO module to increase
detection sensitivity by reduction of QI to DEQ. The following parameters were used for
detection: excitation wavelength 235 nm; emission wavelength, min/nm: 0/440, 6/365, 8/440;
photomultiplier gain 16; time constant 4 s. Data acquisition, processing, and analysis were
performed using ChemStation (Rev. B.04.03, Agilent Technologies).
SIDA-LC-MS/MS. An Infinity 1260 series LC-MS/MS-system (Agilent Technologies) was
used consisting of an autosampler, a binary pump, and a column thermostat coupled to a 6460
triple quadrupole mass spectrometer equipped with an Agilent Jet Stream ion source.
Chromatographic parameters including sample volume, column type, column temperature,
solvent composition, flow, gradient and time-dependent valve switching were identical to those
described above for LC-FLD. The following parameters were used for detection by mass
spectrometry: gas temperature 300 °C, gas flow 9 L/min, nebulizer 15 psi (1.03 × 105 Pa),
sheath gas temperature 350 °C, sheath gas flow 12 L/min, nozzle voltage +500 V, capillary
voltage +3500 V. MS/MS transitions were recorded in dynamic multiple reaction monitoring
mode with the following parameters including expected retention time (tR) ± corresponding
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time window, fragmentor voltage (FV), collision energy (CE), and cell acceleration voltage
(CAV): QI m/z 188.1 → 145.0 (tR 4.3 ± 2 min, FV 100 V, CE 24 V, CAV 4 V), d10-QI
m/z 198.2 → 152.1 (tR 4.2 ± 2 min, FV 85 V, CE 32 V, CAV 4 V), DMEQ m/z 202.1 → 174.0
(tR 6.6 ± 2 min, FV 110 V, CE 24 V, CAV 4 V), d7-DMEQ m/z 209.2 → 181.1 (tR 6.5 ± 2 min,
FV 125 V, CE 24 V, CAV 4 V), EQ m/z 218.2 → 203.0 (tR 10.2 ± 2 min, FV 80 V, CE 8 V,
CAV 4 V), d10-EQ m/z 228.2 → 210.1 (tR 10.0 ± 2 min, FV 80 V, CE 8 V, CAV 4 V), EQDM
m/z 433.3 → 216.1 (tR 15.3 ± 2 min, FV 135 V, CE 24 V, CAV 4 V), d18-EQDM
m/z 451.4 → 225.0 (tR 15.2 ± 2 min, FV 130 V, CE 28 V, CAV 4 V). Data acquisition,
processing, and analysis were performed with MassHunter Workstation (version B.08.00,
Agilent Technologies).
Method validation. Linearity was assessed by spiking blank samples (wild salmon) with EQ,
DMEQ, EQDM and QI at six different levels resembling the natural occurrence in samples
from aquaculture: 0, 40, 80, 120, 160, and 200 µg/kg. For LC-FLD analysis the internal
standard MQ was added to each sample at a concentration of 100 µg/kg, as it was the case with
the deuterated isotopomers d10-EQ, d7-DMEQ and d10-QI for analysis by SIDA-LC-MS/MS.
However, due to extensive ion suppression at higher concentrations EQDM was added at 1/10
of the aforementioned levels (0, 4, 8, 12, 16, and 20 µg/kg), including the internal standard d18-
EQDM, which was spiked at a concentration of 10 µg/kg. All spiked samples were
subsequently extracted as described above and analyzed by LC-FLD or SIDA-LC-MS/MS.
Linear calibration curves were obtained by plotting the peak area ratios (analyte/internal
standard) versus the concentration ratios (analyte/internal standard). Limits of detection (LOD)
and quantification (LOQ) were derived from the obtained calibration lines by calculation
according to ISO 11843-2.23
Matrix effects on ionization were evaluated in a separate experiment by dividing the
slope of a corresponding calibration curve obtained from spiked blind matrix by the slope from
solvent-based calibration, where a ratio < 1 indicates ion suppression and a ratio > 1 ion
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enhancement.24 Recovery rates were subsequently derived for each linearity level from either
solvent-based (LC-FLD, MeCN) or matrix-matched calibration (SIDA-LC-MS/MS) data. For
estimation of intraday precision, six replicates were prepared by spiking blank samples to the
respective LOQ (Table 1) and analyzed within one day using MQ (LC-FLD) or the respective
deuterated isotopomers (SIDA-LC-MS/MS) as internal standards for recovery correction. The
calculated coefficients of variation served as a measure for precision.
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Results and Discussion
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Synthesis
Although the product of a rather simple condensation reaction, EQs exact chemical structure
has puzzled researchers for many years. Initially referred to as “aceton-p-phenetidil”,1 i.e., the
condensation product of p-phenetidine and acetone obtained in the presence of iodine, it was
not until 1961 that Elliott & Yates were finally able to unambiguously identify its structure as
1,2-dihydro-2,2,4-trimethylquinoline, thus refuting the idea of an “acetone anil” formation.25
Despite this problem significant effort has been put into optimizing synthetic strategies for EQ
production to meet industrial demand. Today, the underlying reaction mechanism is known as
the Skraup-Doebner-von Miller quinoline synthesis, named after three chemists that had
already worked on this topic in the early 1880s.26,27 It has been applied not only for the efficient
synthesis of EQ and closely related analogues like MQ, but also for the corresponding
metabolites recently discovered in fish and fishery products.22,28
Notably, the yield of EQ may be increased by slow and continuous addition of acetone
into the flask by means of a syringe pump, which is most presumably due to the reduced
evaporation of unreacted acetone despite the required high temperature of 110 °C (refluxing
toluene).29 Accordingly, application of d6-acetone provided deuterium-labeled EQ and
subsequently deuterium-labeled metabolites (Figure 1). Comparison of the NMR spectra of
unlabeled EQ and the product obtained from the reaction with d6-acetone clearly demonstrated
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the success of isotopic labeling. However, in contrast to initial expectations deuteration did not
only occur at carbon 3, 11, 12, and 13 but also at C-8, which was also confirmed by the NMR
spectra of deuterated DMEQ, EQDM and QI (Figure 2a). Residual proton NMR signals at
these deuteration sites allowed for estimation of deuterium incorporation ranging from 46% at
certain positions of deuterated EQDM to 89% at certain sites of deuterated EQ and DMEQ.
Additionally, GC-MS of the deuterated products revealed a quite broad isotopic distribution
(Figure 2b), which is in accordance with the aforementioned varying deuteration efficiency
and may be explained by H/D exchange processes during synthesis.
Indeed, using 4-ethoxyaniline and d6-acetone as starting materials the initial step is most
likely formation of the corresponding Schiff base (see Supporting Information, Figure S2).30
Water released by this condensation reaction may then participate in H/D exchange reactions
either with unreacted d6-acetone or any of the deuterated intermediates. Moreover, the resulting
Schiff base is considered to subsequently react with its enamine in a kind of a self-condensation
followed by cyclization and elimination of deuterated 4-ethoxyaniline.30,31 The latter most
likely serves as a source of deuterium leading to the observed labeling of deuterated EQ at
position C-8. In fact, EQ was shown to exist partly as a free radical produced by
dehydrogenation of the quinoline nitrogen through free radicals or simply UV light.32 Because
of its aromatic ring system, EQ is capable of stabilizing this radical through delocalization with
one of the corresponding resonance structures bearing the radical at position C-8 (see
Supporting Information, Figure S3). Quenching of this radical by deuterium donors like
deuterated 4-ethoxyaniline, d6-acetone or D2O and rearomatization by imine-enamine
tautomerism may produce EQ deuterated at C-8 as observed by NMR spectroscopy. The well-
established dimerization of EQ through a covalent bond between C-8 and the quinoline nitrogen
(EQDM) serves not only as an explanation for its antioxidant properties but also corroborates
this hypothesis.22,32,33
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Sample preparation
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 solvents13,34 or the QuEChERS protocol.35,36 Other methods make use of solid
phase extraction17 or include a further saponification step with sodium hydroxide for enhanced
matrix disruption prior to liquid extraction.4,14 However, due to 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.
In our hands, neither liquid extraction in combination with a freeze-out step nor solid-
phase extraction did sufficiently remove co-extracted lipids. Alternative saponification
followed by extraction provided sufficiently lipid-free extracts, but chromatography was
impaired by co-eluting matrix components in case of EQ and EQDM. We also considered to
apply gel permeation chromatography, a common sample preparation technique for lipid
removal, but did not succeed in sufficient separation of EQDM. Finally we opted for the
QuEChERS method obtaining high recovery rates for all analytes, albeit being aware that the
extracts still contained considerable amounts of lipophilic matrix components. Therefore, in
order to reduce the matrix load and thus extend the lifetime of the analytical column a
precolumn was used serving as a kind of “matrix trap”. Continuous cleaning of this precolumn
was achieved by operating the chromatographic system in a backflush mode (for details see
Materials and Methods as well as Supporting Information, Figure S1). In this way, lipophilic
matrix components are prevented from reaching the analytical column as they are first trapped
on the precolumn (configuration 1) and then flushed away by the inverted eluent flow
containing more than 76% MeCN (configuration 2). Altogether, the combination of quickly
preparing samples using the QuEChERS protocol and continuously preventing the protective
precolumn from matrix overcharging allowed for high-throughput sample analysis without
affecting the lifetime of the analytical column.
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Sample analysis using LC-FLD
Several chromatographic methods have been reported in the literature for the analysis of EQ
and some of its metabolites. Notably, GC-FID or GC-MS used to be the methods of choice
some decades ago,15–17,19 despite the drawback of a low temperature stability of EQ, quickly
degrading to DMEQ at temperatures applied in the injector or the column oven during GC
analysis.17 Today, LC-based methods can be considered state of the art.4,7,13,14,18 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,4 however,
can easily be overcome by post-column reduction of QI to DEQ (Figure 1). We succeeded in
enhancing method sensitivity for QI detection by infusing 0.1% (w/v) aqueous ascorbic acid at
1/10 of the total flow prior to fluorescence detection.
The resulting LOD and LOQ of QI (detected as DEQ) were therefore of the same order
of magnitude as for the lead compound EQ (Table 1). Moreover, comparison of the obtained
LOD and LOQ values for EQ and EQDM demonstrated the suitability of the developed method
for the analysis of commercial fish samples. Lundebye et al. reported on levels of EQ in fillets
of different fish species ranging from 9.5 to 55 µg/kg and of EQDM either in the range
> 700 µg/kg or not detectable.7 The latter values of EQDM are in line with the findings of He
& Ackman, who found EQDM levels in fish meals and feeds at least four times higher than in
fillets.34 In contrast, no values have been reported in the literature for QI, mainly because levels
found were always lower than the respective LOQs.4
Analysis of a small set of fish fillets including wild salmon caught in the North-East
Pacific and salmon obtained from Norwegian aquaculture allowed distinction between these
two types of commercial fish using the developed LC-FLD method. Whereas wild salmon did
not show any additional peaks (Figure 3a), there were distinct peaks in salmon from
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aquaculture indicating the presence of EQ, EQDM and a further compound of unknown identity
(Figure 3b). Notably, a highly abundant but also unknown compound termed UMEQ
(unknown metabolite of EQ) has already been described in previous studies by Bohne et al.4–6
but it remains unclear, whether this compound is identical to the one observed in the present
study. Analysis of wild salmon spiked with EQ, MQ, EQDM, QI and DMEQ demonstrated the
suitability of the developed gradient for sufficient separation of all analytes and the internal
standard MQ (Figure 3c).
Unfortunately, recovery rates of QI remained low despite correction using the internal
standard MQ. Significant differences in polarity between the more lipophilic MQ, EQ, DMEQ
or EQDM and the much more polar QI containing a basic nitrogen in its quinone imine moiety
may explain this behavior. Actually, this difference in polarity is the key feature allowing for
efficient separation from EQDM by liquid-liquid extraction during synthesis (see above).
Application of stable-labeled isotopomers of EQ and its metabolites, especially QI, in course
of SIDA-LC-MS/MS was expected to solve this problem.
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Sample analysis using SIDA-LC-MS/MS
Validation data obtained by SIDA-LC-MS/MS of EQ and its metabolites clearly showed the
advantages of this methodology compared to LC-FLD (Table 1). Both, LODs and LOQs of QI,
DMEQ and EQDM were significantly lower, especially for EQDM, where LOD and LOQ
accounted for only 1/10 of the levels obtained for LC-FLD. Results for EQ were slightly higher,
but still in comparable range and sufficiently low for analysis of commercial fish samples. Most
probably this observation results from EQ being thermally unstable leading to in-source
degradation to DMEQ during electrospray ionization.17 Evaluation of matrix effects on
ionization revealed a significant matrix influence, especially for the more lipophilic analytes
(QI: -9.9%, DMEQ: 6.1%, EQ: -13.9%, EQDM: -67.8%), which was successfully compensated
by combining a matrix-matched calibration with SIDA. Even more important, SIDA
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significantly improved the recovery rate of QI, as the almost identical polar properties of QI
and its internal standard d10-QI provided an efficient correction for additional extraction losses
resulting in recoveries of > 78% compared to < 35% obtained by LC-FLD (Table 1). Moreover,
recovery rates reached almost 100% as the corresponding isotopomers of EQ, EQDM and
DMEQ were applied for SIDA (Table 1).
Concluding, the developed SIDA-LC-MS/MS method is simple, fast, rugged and highly
sensitive and exceeds existing methods for the quantification of EQ and its metabolites in fish
and fishery products. First-time synthesis of the deuterated isotopomers of EQ and its
metabolites did not only allow for SIDA but provided additional insight into the complex
mechanism of the Skraup-Doebner-von Miller quinoline synthesis. The accompanying LC-
FLD procedure proofed sufficiently selective and sensitive to serve as a reliable screening
method followed by SIDA-LC-MS/MS for confirmation if required. Analysis of an extensive
set of commercial samples will allow for monitoring current levels of EQ and its metabolites in
fish and fishery products. These data will support assessment of human exposure due to carry-
over and facilitate surveillance of the current ban of EQ use as an antioxidant in fish meal and
feed.
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Supporting Information
LC valve wiring diagram for backflush operation; proposed mechanisms for the formation of
deuterated EQ (PDF)
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Acknowledgements
We gratefully thank J. Lang (Central Institute of the Bundeswehr Medical Service Munich) for
his excellent technical support.
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(36) EU Reference Laboratory for Single Residue Methods (EURL-SRM), CVUA Stuttgart.
Analysis of ethoxyquin and its metabolites in fish using the QuEChERS method.
October 5, 2016.
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Figures
H
8
5
NH2
N
N
8a
4a
7
6
2
a
b
3
RO
RO
EtO
4
R = Et
EQ R = Et
DMEQ
R = Me
MQ R = Me
c
EtO
H
N
N
d
N
H
N
HO
O
EtO
DEQ
QI
EQDM
519
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Figure 1
Synthesis of (deuterated) ethoxyquin and its (deuterated) metabolites. Reagents and conditions:
a) iodine, (d6-)acetone, toluene, 110 °C, 18 h; b) 180 °C, 1.5 h; c) (NH4)2Fe(SO4)2 (aq.), tert-
butyl hydroperoxide (70%, aq.), EtOH, 0 °C, 18 h; d) ascorbic acid (0.1%, aq.), rt. Carbons
marked with an ● are potential deuteration sites.
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530
531
532
533
534
535
Figure 2
Characterization of the reaction product obtained from the synthesis of deuterated EQDM (d-
EQDM). (a) Comparison of 1H NMR spectra of EQDM and d-EQDM reveals that deuteration
also occurs at position C-8. Apart from reduced intensity of the corresponding 8-H signal at δ
6.06 ppm the multiplicity of the signal at δ 6.49 ppm belonging to the adjacent 7-H is reduced
from a doublet of doublets to a simple doublet. (b) GC-MS analysis of both compounds
demonstrates that the reaction product obtained from the synthesis of d-EQDM does not only
consist of one isotopomer but represents a complex mixture of deuterated analogues including
d16- to even d20-EQDM. However, d18-EQDM is the main component as the corresponding
signal at m/z 450.5 in the mass spectrum shows the highest intensity.
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537
538
539
540
541
Figure 3
Analysis of commercial fish fillets using LC-FLD. Wild salmon (a) is clearly distinguishable
from breeding salmon (b) due to the absence of peaks at tR 10.7 min (EQ), tR 15.4 min (EQDM)
and a further peak at tR 14.8 min (unknown compound). The chromatogram in (c) was obtained
from wild salmon spiked with 80 µg/kg QI, DMEQ, EQ, EQDM and 100 µg/kg MQ.
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Tables
543
544
Table 1
Validation parameters obtained for both LC-FLD and SIDA-LC-MS/MS of EQ and its metabolites in wild salmon samples.
LC-FLDa
LOQ
SIDA-LC-MS/MSa
LOD
(µg/kg)
RR
(%)
P
(%)
LOD
(µg/kg)
LOQ
(µg/kg)
RR
(%)
P
(%)
R2
R2
(µg/kg)
QI
17.2
2.7
61.0
26.5 - 34.8
91.3 - 92.9
100.0 - 103.5
82.2 - 94.5
13.7
9.7
12.7
2.2
8.6
1.1
45.9
8.4
78.3 - 85.3
98.5 - 101.0
91.9 - 100.0
95.6 - 103.1
11.5
1.1
8.1
4.6
DMEQ
EQ
10.2
25.6
44.1
≥ 0.99
≥ 0.99
6.8
5.3
31.9
4.0
EQDM
12.2
14.4
545
a R2: coefficient of determination, LOD: limit of detection, LOQ: limit of quantification, RR: recovery range, P: intraday precision.
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Table of Contents Graphics
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