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Sarela77
Team TFS
Team TFS

090121 Image 1 Blog.jpg

 

Introduction

 

Ethylene Oxide (EtO) has been banned within the EU since 1991. Countries like Germany had already banned it in 1981. But it is still used in countries like India, the U.S. and Canada to kill bacteria and fungus on food such as sesame seeds, oil seeds in general, nuts and guar gum. Since July 2021, all products in the EU containing EtO above the LOQ of 0.1 mg/kg had to be withdrawn from the market. This decision was made by the EU committee.

 

This applies not only to raw materials, but also to finished food products. Guar gum (E410) can be found in ice cream, jams, meat and baking products. In France, already hundreds of ice creams have had to be withdrawn from the market.

 

The BfR (Federal Bureau of risk management) stated that EtO can be linked to cancer and shows a mutagenic effect. In regard to sesame, 34 contaminated seeds can contain enough EtO to be a serious health risk.

 

The EU stated a LOD of 0.05 mg/kg, which contains the sum of EtO and its metabolite 2-Chlorethanol, expressed as EtO.

 

Now why is this suddenly so important? There were several statements/publications online in 2020 claiming that EtO can be used to sterilize COVID-19. This may be right for medical devices, however, food is a different story.

 

Suddenly, additional food groups are exposed to EtO and — even in finished products — EtO was measured way above the LOQ in lots of finished products. Complete food lots had to be removed from the market.

 

How can you measure EtO?

 

There are two methods available today.

 

The first method is based on a GC-MS method from November 1999. [1]

This German Governmental method describes a procedure that determines Ethylene Oxide as the sum of Ethylene Oxide and 2-Chloroethanol. It is based on the transition of 2-Chloroethanol to Ethylene Oxide followed by the derivatization with iodide and the detection of the formed Iodidiethanol with GC. The method is very time-consuming; only six samples per day can be prepared by an experienced person. However, it is a well-proven and precise method.

 

The second method is based on a GC-MS/MS method developed in December 2020.

The detection limit of EtO that needs to be accomplished is 0.05 mg/kg. To reach this requires a special sample preparation and a GC-MS/MS method in SRM (Single-Reaction-Mode). The CVUA in Stuttgart (Germany) published an EURL-SRM in December 2020, utilizing a Thermo Scientific™TRACE™1310 gas chromatograph and a Thermo Scientific™ TSQ™ 9000 GC triple quadrupole.

 

Let’s start with the sample preparation

 

As mentioned in the CVUA’s single residue method for the analysis of Ethylene Oxide and its metabolite 2-Chloroethanol the QuEChERS EN 15662 method and GC-MS/MS were used. Therefore 2 g of sample, into which standards and Acetonitrile are added, and the sample is vortexed for five minutes. The EN 15662 extraction kit contains 4 g of anhydrous MgSO4, 1 g of NaCl, 1 g of Trisodium Citrate and 500 mg of Disodium Citrate. After shaking for one minute the sample is centrifuged at 5000 rpm for five minutes. 1 ml supernatant is transferred into a microcentrifuge tube containing 150 mg MgSO4, 25 mg PSA and 25 mg C18. It is vortexed for 30 seconds and afterwards centrifuged at 5000 rpm. The supernatant is transferred into a GC vial.

 

GC-MS analysis

 

Those parameters were developed for a Thermo Scientific™ ISQ™ 7000 GC single quadrupole system in a German food control lab.

 

Analytical parameters

 

Injector

PTV (Programmable Temperature Vaporizing )

Injector Mode

Splitless

Injection Volume

1µl

Flow Control Mode

Constant Flow

Column Flow

1.00 ml/min

Carrier Gas

Helium 6.0

Total Run Time

30 min

GC Program

50 °C, 1 min (hold) with 25 °C/min, to 200 °C, 23 min (hold)

Column

Carbowax, 30 m, 0.25 mm I.D., 0.25 µm film

 

GC-MS/MS parameters

 

Method Type

Selected-Ion-Monitoring (SIM)

MS transfer line temperature

250 °C

Ion source temperature

250 °C

Ionization

EI

m/z Iodidiethanol

RT 6.44 min

127, 128, 141, 142, 172                                

 

Results

 

The following figures show the results on a sesame seed sample, generated by following the German § 35 LMBG method.

 

090121 Image 2 Fig 1A.png

 

Fig.1: Real sesame seed sample containing 0.406 mg/kg Ethylene Oxide.Fig.1: Real sesame seed sample containing 0.406 mg/kg Ethylene Oxide.

 

Fig.2: Calibration curve for Iodidiethanol. The method LOQ was determined with 0.02 mg/kg.Fig.2: Calibration curve for Iodidiethanol. The method LOQ was determined with 0.02 mg/kg.

 

GC-MS/MS analysis

 

The CVUA in Stuttgart details a Thermo Scientific™TRACE™1310 gas chromatograph, a Thermo Scientific™ TSQ™ 9000 GC triple quadrupole and a TriPlus RSH autosampler.

Analysis can be performed on a TG-624 MS 30 m, 0.32 mm I.D X 0.25 mm film.

The following method and data were developed in a Thermo Fisher Scientific lab, based on the method by the CVUA Stuttgart with spiked sesame seed samples.

 

Analytical parameters

 

Injector

SSL (Split/Splitless)

Injector mode

Split

Injection volume

1µl

Injector Temperature

280 °C

Flow Control Mode

Constant Flow

Column Flow

2.00 ml/min

Carrier Gas

Helium 6.0

Split Ratio

5:1

Purge Flow

5 ml/min

Split Flow

10 ml/min

Total Run Time

25 min

GC Program

40 °C, 2 min (hold) with 5 °C/min, 40-90 °C, 50 °C/min 90-225 °C, 10.3 min (hold)

 

GC-MS/MS parameters

 

Method Type

t-SRM mode

MS transfer line temperature

230 °C

Ion source temperature

250 °C

Ionization

EI

SRM transitions

Ethylene Oxide     Q1 m/z   Q3 m/z   CE (V)

                                 44             14              20

                                 44             28                5

                                 44             29                5

 

2-Chloroethanol   Q1 m/z   Q3 m/z   CE (V)

                                 80             31               5

                                 80             43               5

                                 82             31               5

 

Results

Fig. 3: Total Ion Chromatogram (TIC) at 0.025 mg/kg EtO and 2-CE.Fig. 3: Total Ion Chromatogram (TIC) at 0.025 mg/kg EtO and 2-CE.

 

Fig. 4: Matrix-matched calibration curve for EtO and 2-CE in the range of 0.005-0.2 mg/kg.Fig. 4: Matrix-matched calibration curve for EtO and 2-CE in the range of 0.005-0.2 mg/kg.

 Fig. 5: Sensitivity observed for EtO and 2-CE in the matrix matched standard at 0.025 mg/kg.Fig. 5: Sensitivity observed for EtO and 2-CE in the matrix matched standard at 0.025 mg/kg.

 

Conclusion

 

  • EtO becomes more and more of a topic in the EU. Since lots of products need to be withdrawn from the market on a daily base, the importance to control for EtO is clear.
  • The risk assessment from the BfR shows why it is necessary to protect consumers from dangerous levels of EtO.
  • As this blog post shows, there are proven methods available that detect EtO to the required levels in all kind of food matrices.
  • A disadvantage of the § 64 LFGB method is the time required for the sample preparation. Only six samples can be processed per day, however it is a very precise method.
  • The advantage of the GC-MS/MS method is the speed due to the QuEChERS EN 15662 method. It provides enough specificity and sensitivity for the detection of EtO and its metabolite 2-Chloroethanol.
  • This is a plug and play method that is already established in the GC and food market.

 

[1] Amtliche Sammlung von Untersuchungsverfahren nach §64 LFGB; Gaschomatographische Bestimmung von Ethylenoxid und 2-Chloroethanol in Gewürzen