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Analysis of Ethoxyquin and its Metabolites

in Salmon Using QuEChERS


Anne Benkenstein, Simone Adam, Sabine Mgle, Ellen Scherbaum and
Michelangelo Anastassiades
E-Mail: Anne.Benkenstein@cvuas.bwl.de
EURL-SRM
Introduction Extraction method at a glance:
Cryogenic milling with dry ice, add AA 1 g/100 g Add AA 1 g/100 g sample
Ethoxyquin (EQ) is a quinoline-based antioxidant, or

but it is still listed as a fungicide with pre- and


Weigh Add 1 mL AA-Mix Solution
5 g sample homogenate in 50 mL centrifuge tube (0.15 mg AA and 0.15 mg Sodium Ascorbate per mL Water)
if not added during milling
post-harvest applications, mainly on pears, to
(5.05 g in case AA was added during milling)

reduce scald. It is also used in paprika powder Add 100 L internal standard

and curcuma to reduce color loss due to oxidation Adjust water content of sample to 10 mL
salmon: add 6.5 g of water (if AA was added during milling)

of carotenoids. or 5.5 g of water (if AA-Mix solution was added)

There are also numerous feed-related applications Add 10 mL ACN

with indirect food relevance such as the use in fish Shake thoroughly for 15 min.

feed to prevent lipid-autoxidation. Add QuEChERS salts and shake for 1min.

Legal aspects
Centrifuge at 4000 rpm for 5 min.

dSPE-cleanup (25 mg/mL C18, 25 mg/mL PSA, 0.15 g/mL MgSO4)

EQ is an approved feed additive in this field and shake for 1 min. and centrifuge at 4000 rpm for 5 min.
QuEChERs salts:
4 g MgSO4,
is listed in the EU register on feed additives (Reg. Transfer 1 mL extract to vials
1 g NaCl,
1 g Na3-Citrate dihydrate,

1831/2003/EC) as E324. It has a permissible level


0.5 g Na2-hydrogen citrate
sesquihydrate
LC-MS/MS analysis

of 150 mg/kg in fish feed (Reg. 2316/1998/EC).


Fish is not yet regulated by Reg. 396/2005/EC, so
Results
no MRLs exist at this time. The recoveries for EQ and its metabolites
depending on different variations of AA addition to
EQ and Metabolites wild salmon blank are shown in the figure below.
EQ degrades to a multitude of metabolites but AA prior to cryo-milling 102
0.1 ppm
EQI

AA prior to ectraction byQuEChERS 96


only a few of them are commercially available as AA to the final extract 11
EQ metabolites

standards: Ethoxyquin-dimer (EQDM), 2,2,4- AA prior to cryo-milling 99


0.1 ppm
DHEQ

AA prior to ectraction byQuEChERS 103


Trimethyl-quinolinon (EQI) and Dihydroethoxyquin AA to the final extract 76
(DHEQ). Some of these metabolites also exhibit AA prior to cryo-milling 86
0.1 ppm
EQDM

AA prior to ectraction byQuEChERS


antioxidant properties themselves. EQDM is the AA to the final extract
79
87
most prominent metabolite in salmon and as it AA prior to cryo-milling 103
0.025 ppm 0.1 ppm

is more stable than EQ it may serve as a good AA prior to ectraction byQuEChERS 103
Ethoxyquin

AA to the final extract 71


indicator of salmon fed with EQ-containing feed. AA prior to cryo-milling 100
AA prior to ectraction byQuEChERS 108
Ethoxyquin Ethoxyquin dimer AA to the final extract 85
(EQ) (EQDM) 0 20 40 60 80 100 120 140
Recovery (%), n=5

In recovery experiments on wild Atlantic salmon


AA showed a strong protective effect on EQI
Dihydroethoxyquin (losses w/o AA ca 90%). Protection of EQ and
(DHEQ)
DHEQ was weaker (losses w/o AA ca 25%).
Ethoxyquin quinone imine EQDM was more stable and not notably affected
(EQI)
by the addition of AA.
In contrast, the impact of AA on EQ extraction
yields from farmed salmon was minimal. We
assume that this is due to the high levels of other
Analytical method antioxidants added to fish feed and accumulating
Our studies mainly focused on salmon, and in farmed fish. To be on the safe side, addition of
extractions were done by the QuEChERS method AA prior to QuEChERS extraction or even during
using 5 g homogenate to reduce the influence of cryogenic milling is still recommended.
lipids on the recoveries. As in a previous study on Using AA in the extraction step, validation of EQ,
pears (see analytical observation on Ethoxyquin, EQDM, DHEQ and QI in wild salmon showed Salmon 10
Salmon 09
0.039
0.008
0.26
0.29
EQ

EURL-SRM website) the impact of ascorbic acid satisfactory recoveries and RSDs. Partitioning of Salmon 08 0.005 0.38
EQDM

0.002
EQDM (LogKow 6.2) into the lipid phase during
Salmon 07
(AA) was checked by adding it:
0.27
Salmon 06 0.010 0.59

a) prior to cryogenic milling, QuEChERS resulted in lower recoveries. Salmon 05


Salmon 04
0.067
0.041
0.42
0.96
b) prior to extraction by QuEChERS,
Analysis of Samples from the Market
Salmon 03 0.014 0.69
0.010
c) to the final extract (no protection during
Salmon 02
0.11
Salmon 01 0.080
Several salmon samples and shrimp samples from
0.58
milling and extraction) 0.00 0.20 0.40 0.60 0.80 1.00 1.20

the market were analyzed. All farmed salmon Residues (mg/kg)

The extracts were measured via LC-MS/MS (ESI- samples contained both EQ and EQDM, but not
positive mode). EQI and DHEQ. The concentrations of EQDM
were significantly higher than those of EQ.
Method details: www.eurl-pesticides.eu
None of the wild salmon and shrimps samples
contained EQ or its metabolites. EPRW 2016
Summary
On wild salmon, addition of AA showed a strong protective effect on EQI and a weaker effect on EQ
and DHEQ. EQDM seemed unaffected.
In farmed salmon the impact of AA on EQ extraction yields was minimal, but AA addition prior or
during QuEChERS extraction is still recommended to be on the safe side.
All tested samples of farmed salmon showed EQ and EQDM residues. EQDM levels were higher.