Application Note
Food and Beverage
Testing
Authors
Tatiana Cucu,
Christophe Devos, and
Frank David
RIC Technologies
Kortrijk, Belgium
Laurent Pascaud
Agilent Technologies, Inc.
Abstract
The presence of ethylene oxide (EtO) and 2-chloroethanol (2-CE) in sesame
seeds and other food commodities is an emerging food-safety concern. The first
notification about the presence of EtO in sesame seeds occurred in August 2020,
and since then over 800 new notifications have been published on the European
Rapid Alert Food and Feed Safety (RASFF) portal. To ensure that foods are safe
for consumption, food industry and enforcement agencies need reliable and
robust methods to detect EtO and 2-CE in foods at levels below the EU established
maximum residue limits (MRLs). The EU Reference Laboratories for Residues of
Pesticides have proposed a method for the determination of these contaminants
that uses Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) sample
preparation followed by gas chromatography/tandem mass spectrometry
(GC/MS/MS) analysis. However, laboratories encounter several challenges when
it comes to applying this method to the analysis of EtO and 2-CE in food products.
The high volatility of EtO requires the use of a dedicated column to ensure its
separation from potential acetaldehyde interferences. Second, QuEChERS extracts
often contain high amounts of nonvolatile material that can accumulate in the GC
inlet liner and column inlet, affecting the accuracy and robustness of the analysis.
Additionally, the analytical column can deteriorate due to the injection of samples
containing high-boiling-point compounds. This application note describes an
optimized and robust analytical method for the quantification of EtO and 2-CE in
sesame and curcuma that uses a dedicated setup based on an Agilent 8890 Gas
Chromatograph (GC) system, an Agilent 7010 Triple Quadrupole GC/MS (GC/TQ),
and a Gerstel MPS sampler with the Automated Liner Exchange option. This option
reduces downtime related to inlet maintenance, column exchange, and MS source
cleaning compared to the proposed EU method.
Simultaneous Analysis of Ethylene
Oxide and 2-Chloroethanol in Sesame
Seeds and Other Food Commodities:
Challenges and Solutions
2
Introduction
Ethylene oxide (EtO) is a disinfectant
that was banned in the EU as a pesticide
in 1991 due to its classification
as a category-1 carcinogen by the
International Agency Research of
Cancer (IARC).1 In September 2020,
Belgium added a notification on the
EU Rapid Alert System for Food and
Feed (RASFF) concerning EtO residues
in sesame seeds originating from
India. The levels detected substantially
exceeded the maximum residue level
(MRL) of 0.05 mg/kg for sesame seeds
set by Regulation (EU) 2015/868.2 The
notification resulted in increased testing
and controls, leading to withdrawals
and recalls of a significant number of
conventional and organic products in
many EU Member States. By February
of 2022, over 700 notifications related to
the occurrence of EtO, mostly in seeds
(particularly sesame seeds) and spices,
have been registered on the RASFF
portal. The presence of unauthorized
levels of EtO and its metabolite
2-chloroethanol (2-CE) is probably due
to its use for fumigation to control
insects and microorganisms (fungi and
bacteria) in dry food products such
as herbs, spices, nuts, and oily seeds.
After contact with food products, EtO
either interacts with the major matrix
constituents leading to the formation
of several metabolites, including 2-CE,
or dissipates through evaporation.
Because of the toxicity of EtO and 2-CE,
a joint residue definition for the two
components was introduced in 2008 in
Regulation (EC) No 149/2008.3 In 2015,
the EU-MRLs for spices were lowered
to 0.1 mg/kg. At the same time, the
MRLs for nuts, oil fruits, and oilseeds
were lowered to 0.05 mg/kg.2 To enforce
these regulations, accurate analysis
methods for these contaminants in food
are essential.
Various methods for the analysis of
EtO, or the sum of EtO and 2-CE, have
been published. Some of these methods
are based on the conversion of 2-CE
to EtO under alkaline conditions, an
approach that was later optimized
and used as an official standard in
Germany. Other methods are based
on the conversion of the EtO to 2-CE
under acidic conditions, followed by
extraction of 2-CE with ethyl acetate
and analysis by GC/MS. However, these
methods are time consuming, labor
intensive, and require the use of large
quantities of harmful solvents. Sample
preparation is essential in the analysis
of EtO and 2-CE. Therefore, in December
2020, EU Reference Laboratories for
Residues of Pesticides recommended a
single-residue method for the analysis
of EtO and 2-CE in sesame seeds that
uses QuEChERS extraction followed by
GC/MS/MS analysis.4 QuEChERS-based
GC methods generally involve
three steps:
1. Extraction using an organic solvent
and partitioning salts,
2. Sample cleanup with adsorbent
materials (dispersive sorbents), and
3. GC/MS/MS analysis.
However, food and feed matrices are
known to be extremely complex and
contain many interfering compounds
that can lead to ion suppression effects,
coelution, and severe contamination of
the analytical instrumentation from the
injector to the detector. In this application
note, several improvements to the
published EU Reference Laboratories
for Residues of Pesticides method are
proposed, including implementation of
an automated liner exchange option. The
option provides more accurate results
by allowing unattended exchange of
programmed-temperature-vaporization
(PTV) inlet liners during an extended
analytical sequence, which prevents
accumulation of the high amounts
of nonvolatile material present in
QuEChERS extracts. Also, the use of
precolumn backflushing protects the
analytical column, resulting in increased
robustness and higher productivity.
Materials and methods
Chemicals and reagents
HPLC-S gradient grade acetonitrile
was from Biosolve. Reverse-osmosis
water was prepared using a Millipore
water purification system. Samples
were generously provided by a
research partner or purchased in a
local supermarket. EtO in methanol
at 50 mg/mL was acquired from
Sigma-Aldrich. 2-CE was also purchased
from Sigma-Aldrich (part number 23000).
2-CE-D4 was purchased from TechLab
(part number 117067-62-6). EtO-D4
was synthesized in-house from 2-CE-D4
under alkaline conditions as described
in the Recommended Single Residue
Method.3 The purity and concentration
of the ETO-D4 was tested against an
EtO standard using GC-flame ionization
detection (FID).
Sample preparation overview
Food material: Samples
were homogenized using a
liquid-nitrogen-cooled grinder to avoid
loss of EtO and 2-CE.
Extraction: QuEChERS extraction kit, EN
15662 method (part number 5982-5650)
Dispersive SPE cleanup: QuEChERS
Dispersive Kit, Fruits and Vegetables with
Fats and Waxes, EN method (150 mg
PSA, 150 mg C18EC, 900 mg MgSO
4)
(part number 5982-5156)
3
QuEChERS extraction
QuEChERS extraction was performed
according to the EN 15662 procedure
as shown in Figure 1. Briefly, sample
(2 ±0.01 g) was weighed in 50 mL
centrifuge tubes and spiked as required.
Water (10 mL) was added to the
centrifuge tubes followed by capping and
vortexing for 1 minute or until the sample
was homogeneous. After the samples
were thoroughly wetted, acetonitrile
(10 mL) and 20 µL of internal standard
(EtO-D4 and 2-CE-D4 10 µg/mL) were
added to the centrifuge tubes along
with two ceramic homogenizers
(part number 5982-9313) to improve
the extraction efficiency of the target
compounds. The centrifuge tubes were
then capped and placed in a rotary
shaker for 15 minutes. The tubes were
then removed, the QuEChERS extraction
salts (4 g of MgSO
4,1 g of NaCl, 1 g of
NaCitrate, and 0.5 g of disodium citrate
sesquihydrate) were added, and the
tubes were shaken on the rotary shaker
for another 3 minutes. The samples
were then centrifuged for 5 minutes at
6,000 rpm, resulting in phase separation
between the aqueous and organic
solvents. Following centrifugation, the
upper acetonitrile layer (6 mL) was
transferred to QuEChERS Dispersive Kit
15 mL tubes (150 mg of PSA, 150 mg
of C18EC, and 900 mg of MgSO
4). The
tubes were vortexed for 30 seconds
followed by centrifugation at 5,000
rpm for 5 minutes. After centrifugation,
cleaned extracts were transferred to a
2 mL GC vial for analysis.
GC and GC/TQ instrument conditions
The GC and GC/TQ conditions and
method parameters are listed in Tables 1
and 2, respectively.
Figure 1. QuEChERS workflow for the extraction and cleanup of samples.
Weigh 2.00 ±0.01 g of sample into a 50 mL tube.
Add 10 mL of acetonitrile, IS, and extraction aids
and mix for 15 minutes.
Add extraction salts and mix for 3 minutes.
Centrifuge at 6,000 rpm for 5 minutes.
Add water and shake briefly to wet the sample.
Transfer 6 mL of supernatant to a 15 mL dSPE tube.
Agitate for 30 seconds.
Centrifuge at 5,000 rpm for 5 minutes.
Transfer the clear supernatant to a 2 mL vial for injection.
QuEChERS extraction step
Cleanup step
Table 1. GC method parameters.
Parameter
Value
Model
Agilent 8890 Gas Chromatograph
Injector
Gerstel CIS 4 with Automated Liner Exchange (ALEX) option
Injector Temperature
90 °C (0.8 min), 12 °C/s to 250 °C (14.3 min)
Injection Volume
2 µL; split 1:4
Liner Type
Glass wool (Gerstel p/n 010850-010-00)
Precolumn
5 m FS
Analytical Column
Agilent J&W HP-VOC GC, 30 m × 0.20 mm, 1.12 µm (p/n 19091R-303)
Carrier Gas
Helium
Analytical Column Flow
1 mL/min
Oven Gradient
45 °C (2 min), 50 °C/min to 220 °C (10 min)
Transfer Line Temperature
280 °C
Table 2. GC/TQ method parameters.
Parameter
Value
Model
Agilent 7010 triple quadrupole GC/MS
Source Temperature
230 °C
Quadrupole Temperature
150 °C
Collision Gas Flow
1.5 mL/min (N
2)
Quench Gas Flow
2.25 mL/min (He)
Time Events
0 min – detector ON
2.95 min – detector OFF
3.6 min – detector ON
MRM Transitions and
Retention Times
ETO-D4 (2.56 min): 48 & 16 (CE 40)
48 & 30 (CE 5)
ETO (2.57 min):
44 & 29 (CE 5)
44 & 28 (CE 5)
44 & 15 (CE 5)
2-CE-D4 (4.47 min): 86 & 33 (CE 5)
84 & 33 (CE 5)
2-CE (4.48 min):
80 & 44 (CE 0)
80 & 31 (CE 5)
80 & 43 (CE 0)
4
Calibration standard preparation
Stock standard solutions were prepared
by mixing the individual stock standard
solutions. Working standard solution
was created by making a dilution of
the stock solutions to 50 µg/mL EtO
and 84.2 µg/mL 2-CE in acetonitrile
that was stored at –18 °C until use.
For calibration curve development,
standards were mixed and prepared
fresh in acetonitrile in the range between
1 to 100 ng/mL and were used within
one day. Matrix-matched standards
were prepared at 10, 40, and 100 ng/mL
extract levels by spiking the QuEChERS
extracts of the blank sesame and
curcuma samples. Internal standards
were spiked into all samples (20 µL of a
10 µg/mL solution) and taken through
the entire QuEChERS workflow.
Results and discussion
Chromatography
Both EtO and 2-CE may occur in
food samples. The 2-CE metabolite
predominates, mainly because it is
less volatile than EtO. As a result,
simultaneous determination of
both analytes in a single method is
recommended. In addition, a dedicated
method for quantification of EtO
and 2-CE is proposed, rather than
integrating the analytes into an existing
multiresidue pesticides method. Specific
to the described method, the solvent
(acetonitrile) elutes between EtO and
2-CE, meaning that care should be taken
to protect the filament by programing
a "Detector OFF" time event in the MS
method between 2.95 and 3.6 minutes.
Also, optimal injection conditions are
needed to ensure a good peak shape
for the EtO. The cooled injection system
(CIS), a PTV-type inlet, was used to
perform cold split injection of samples.
Samples were introduced into the liner
without the intermediate evaporation
step that is typical of classical
split/splitless injectors. Sample transfer
from the liner into the analytical column
thus started the moment the injector was
heated. The well-controlled evaporation
process allowed for reproducible and
accurate injection, optimizing results for
the target analytes. Figure 2 shows the
chromatograms for EtO at the 5 ng/mL
level (equivalent to 25 µg/kg sample
level) and 2-CE at the 1 ng/mL level
(equivalent to 5 µg/kg sample level),
which are well below the EU-MRL levels
for spices and sesame seeds.
Figure 2. Chromatograms of EtO (MRM transition 44 & 29) at the
5 ng/mL level and 2-CE (MRM transition 80 & 44) at the 1 ng/mL level.
1.12
1.14
1.16
1.18
1.20
1.22
1.24
EtO
* 2.578
Acquisition time (min)
2.45
2.55
2.65
2.75
2.85
2.95
3.05
3.15
3.25
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2-CE
* 4.483
Acquisition time (min)
4.4
4.5
4.6
4.7
4.8
×105
×103
5
Acetaldehyde is often present in food
products, particularly in those that are
fatty. Because acetaldehyde and EtO
have very similar mass spectra and
retention indices, there are essentially
no MRM transitions available to select
for EtO if it coelutes with acetaldehyde.
Therefore, it is essential to prevent
their coelution during analysis. To
test the method for this concern,
acetaldehyde, EtO, and a mixture of
acetaldehyde and EtO were injected to
confirm chromatographic resolution on
the HP-VOC column used. As shown
in Figure 3, acetaldehyde eluted just
before EtO and baseline separation
was achieved.
Food matrices are extremely complex
and contain many interfering
compounds. Considering the
nonselective character of the QuEChERS
extraction method, the extracts obtained
are relatively dirty and could contain
trace amounts of water. The matrix
compounds can accumulate in the
injector, damage the analytical column,
and contaminate the detector. Therefore,
the method introduced two levels of
matrix protection with:
1. Automated Liner Exchange option to
allow unattended liner exchange, and
2. Integrated precolumn backflushing
to ensure that high-boiling-point
compounds do not reach the
analytical column, increasing column
lifetime and minimizing downtime
related to detector maintenance.
Method performance
EtO was quantified using calibration
curves ranging from 5 to 100 ng/mL,
with linear fit, no weighting, and without
including the origin. 2-CE was quantified
using calibration curves ranging from
0.84 to 84 ng/mL, with linear fit, 1/x
weighing, and without including the
origin. The R2 value was greater than
0.99 for both target compounds
(Figure 4) indicating excellent fit.
Figure 3. Chromatograms of the acetaldehyde (A), acetaldehyde and EtO mixture (B), and
EtO (C) showing baseline separation of the compounds.
1
2
3
2.40
1
2
3
2.57
2.40
1
2
3
2.57
Acquisition time (min)
2.3
2.5
2.7
2.9
3.1
3.3
3.5
3.7
3.9
4.1
4.3
4.5
×105
×105
×105
A
B
C
Relative concentration
0
0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8 5.2
Relative response
Relative response
0
1
2
3
4
5
6
7
R2 = 0.9985
R2 = 0.9981
2-CE
0
0.4
0.8
1.2
1.6
2.0
2.4
2.8
3.2
3.6
4.0
4.4
0
1
2
3
4
5
y = 1.174932x + 0.011209
EtO
y = 1.433383x – 0.121541
A
B
Figure 4. Calibration curves for EtO (A) and 2-CE (B).
6
To determine the chromatographic
stability of the method, extracts of
sesame seed and curcuma were
spiked with EtO and 2-CE to 10, 40, and
100 ng/mL, and injected repeatedly
from the same vial into the same liner.
Table 3 shows the absolute area and
recovery for EtO and 2-CE spiked into
the sample extracts at 40 ng/mL. A
small decrease in the absolute area of
EtO in sesame, and especially curcuma,
extract was observed. The decrease can
be explained by evaporation of EtO from
the vial, which was pierced several times
while stored in the sample tray at room
temperature. Despite this concern, the
repeatability of the recovery was 5 to 6%,
indicating that the deuterated internal
standard allowed correction for the loss.
Though less evaporation loss of 2-CE
was expected because it is less volatile
than EtO, due to liner activity there was
about a 40% reduction in the absolute
area between the first and fifth injection.
Some peak distortion was observed
after injection, particularly for extracts
containing interfering compounds
such as curcuma. This distortion
demonstrated that injection of relatively
dirty QuEChERS extracts affects
accurate analysis of 2-CE. Therefore,
use of the Automated Liner Exchange
(ALEX) option for the analysis of EtO
and especially 2-CE is recommended.
Using the ALEX option, the liners were
exchanged after 20 injections (as a
user-defined time event in the sequence),
minimizing accumulation of nonvolatile
material from the extracts in the liner
that could adversely affect method
accuracy. Also, the ALEX option allowed
unattended exchange of PTV inlet liners
during extended analytical sequences,
minimizing downtime related to manual
inlet maintenance while increasing
instrument robustness and productivity
for routine analyses.
To prevent high-boiling-point compounds
from reaching the analytical column
and contaminating the MS ion source,
the precolumn backflushing option
(using a purged Ultimate union) was
used. Shown in Figure 5, the full-scan
analysis of a curcuma extract performed
without backflushing demonstrates the
complexity of the injected food samples,
particularly for compounds eluting
after 5 minutes (which is after elution
of the target compounds). Without
precolumn backflushing, all the injected
matrix reaches the analytical column
Table 3. Absolute area and recovery of the EtO and 2-CE from sample extracts injected
repeatedly on the same liner.
EtO
2-CE
Sesame, 40 ng/mL Curcuma, 40 ng/mL Sesame, 40 ng/mL Curcuma, 40 ng/mL
Area
Recovery
(%)
Area
Recovery
(%)
Area
Recovery
(%)
Area
Recovery
(%)
Injection 1
230,904
87.2
258,262
92.9
56,959
100.65
66,763
97.81
Injection 2
240,094
98.7
227,977
84.7
51,690
103.81
51,575
96.67
Injection 3
241,489
101.5
185,710
78.7
43,990
103.92
44,970
98.12
Injection 4
221,448
95.8
221,869
85.5
37,175
101.54
38,618
98.70
Injection 5
207,740
94.9
178,201
81.7
34,618
104.89
40,934
95.73
Average
228,335
95.6
214,403
84.7
44,886
102.9
48,572
97.7
RSD
6.2
5.6
15.3
6.3
21.0
1.7
23.2
1.2
Figure 5. Chromatographic profile of the curcuma extract analyzed in scan mode without backflushing (A),
and with backflushing (B).
0
0.4
0.8
1.2
1.6
2.0
2.4
2.8
3.2
3.6
4.0
4.4
4.8
5.2
5.6
6.0
6.4
6.8
7.2
Acquisition time (min)
Acquisition time (min)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
DETECTOR OF
F
0
0.4
0.8
1.2
1.6
2.0
2.4
2.8
3.2
3.6
4.0
4.4
4.8
5.2
5.6
6.0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
DETECTOR OF
F
2-CE
×109
×105
A
B
7
and ultimately the MS ion source.
When backflushing begins immediately
after the transfer of the target analytes
to the analytical column, the sample
matrix has little to no influence on the
column, allowing for analysis of more
samples before maintenance is required
compared to a conventional setup.
The sample preparation method
described in this application note
was validated by performing recovery
experiments in triplicate at three
different concentrations: 0.05, 0.2, and
the 0.5 mg/kg sample-based level. The
recovery was between 84.5 to 100.6% for
EtO and 88.8 to 106.2% for 2-CE in the
sesame and curcuma samples (Table 4).
Representative food samples collected
in a local supermarket were analyzed
using the method (Table 5). None of the
commercial samples tested positive
for EtO, probably because the samples,
some of which had been thermally
treated, suffered losses during storage
or the thermal treatment. However, in
border-control programs for example,
under the correct sampling and storage
conditions, significant amounts of EtO
would be detected. All of the samples
tested positive for 2-CE, some at
levels above the MRL values for these
commodities (i.e. Sesame 1, Curcuma 1,
and plant material). Method accuracy
was tested using a sesame reference
material (kindly provided by a research
partner). The value obtained was
close to the average reported value of
4,660 µg/kg (27.3% CV).
Table 4. Recovery and relative standard deviation (RSD) of EtO and 2-CE in
sesame and curcuma samples spiked at different levels.
Matrix
Spike Level
(mg/kg)
Recovery EtO (%)
Recovery 2-CE (%)
Average
RSD % (n = 3)
Average
RSD % (n = 3)
Sesame
0.05
100.1
9.1
97.9
6.3
0.2
84.5
7.6
92.5
8.4
0.5
92.0
6.9
88.8
2.7
Curcuma
0.05
100.6
16.4
106.2
4.4
0.2
94.5
8.5
105.8
9.9
0.5
92.5
5.2
94.4
4.3
Table 5. Amounts of EtO and 2-CE detected in commercial samples.
Matrix
EtO
2-CE
Sum EtO and 2-CE*
µg/kg
µg/kg
µg/kg
Sesame, 1
n/d
224.7
122.9
Sesame, 2
n/d
75.7
41.4
Sesame, Reference Material
n/d
4,581**
2,505
Curcuma, 1
n/d
199.7
109.2
Curcuma, 2
n/d
12.7
6.9
Fresh Curcuma
n/d
2.3
1.3
Garlic Powder
n/d
9.2
5.0
Ginger Powder
n/d
27.7
15.2
Plant Material
n/d
9,653
5,280
Spices Mixture
n/d
31.2
17.1
Herbs Mixture
n/d
29.2
16.0
* Sum EtO and 2-CE = conc EtO + (concentration 2-CE in sample × 0.55)
(conversion factor based on conversion of molecular weights: 44/80 = 0.55)
** 4,660 µg/kg (27.3% CV)
DE46467947
This information is subject to change without notice.
© Agilent Technologies, Inc. 2022
Printed in the USA, June 15, 2022
5994-4942EN
Conclusion
The presented method meets the
requirements of the EU Reference
Laboratories for Residues of Pesticides
single-residue method that uses
QuEChERS extraction followed by
GC/MS/MS analysis for determination of
EtO and 2-CE sesame seeds. Recoveries
were within 85 to 106%, with good
repeatability for both EtO and 2-CE. LOQs
for sesame and curcuma (representative
of the spices food category), were lower
than the currently established MRLs.
Results were stable for both sesame
and curcuma samples, with RSDs for
triplicate extractions and analyses found
to be well below 20% for the recovery
experiments. The sample preparation
and optimized GC/MS/MS setup
based on the 8890 GC system, 7010B
GC/TQ, and Gerstel MPS sampler with
the Automated Liner Exchange option
provided reliable results and excellent
performance, demonstrating that the
method can be used to quantify EtO and
2-CE in sesame and spice samples at the
EU-regulated levels. The method has the
advantages of high sensitivity, selectivity,
accuracy, and sample throughput due to
reduced downtime for inlet maintenance,
analytical column exchange, and MS ion
source cleaning.
References
1. Tateo, F.; Bononi, M. Determination
of Ethylene Chlorhydrine as Marker
of Spices Fumigation With Ethylene
Oxide. Journal of Food Composition
and Analysis 2006, 19, 83–87
2. Regulation (EU) 2015/868 of 26 May
2015 amending Annexes II, III and V
to Regulation (EC) No 396/2005 of
the European Parliament and of the
Council as regards maximum residue
levels for 2,4,5-T, barban, binapacryl,
bromophos-ethyl, camphechlor
(toxaphene), chlorbufam,
chloroxuron, chlozolinate, DNOC,
di-allate, dinoseb, dinoterb,
dioxathion, ethylene oxide,
fentin acetate, fentin hydroxide,
flucycloxuron, flucythrinate,
formothion, mecarbam, methacrifos,
monolinuron, phenothrin, propham,
pyrazophos, quinalphos, resmethrin,
tecnazene and vinclozolin in or on
certain products. Off. J. Eur. Union L.
2015, 145, 1–71.
3. Regulation (EC) No 149/2008 of 29
January 2008 amending Regulation
(EC) No 396/2005 of the European
Parliament and of the Council by
establishing Annexes II, III and IV
setting maximum residue levels for
products covered by Annex I thereto.
Off. J. Eur. Union L. 2008, 58, 1–398.
4. EURL-SRM-Analytical Observation
Report: Analysis of Ethylene Oxide
and its metabolite 2-Chloroethanol
by the QuOil or the QuEChERS
method and GC-MS/MS. December
2020.
