Industrial processes are no less than a huge enigma, encompassing plethora of natural as well as synthetic routes that involve use of countless chemicals – the ones that are important, the ones that are useful yet harmful, the ones whose greener alternative will take time in execution. One such chemical that finds use in wide array of applications is ethylene oxide, a chemical that has garnered interest from different sectors of the industry. Let us try to draw a 360-degree view on how ethylene oxide analysis has become one of most talked topics in the analytical world.

About Ethylene Oxide

Ethylene oxide (EO), also recognized with IUPAC nomenclatures like 1,2-epoxyethane, di-methylene oxide, or oxirane is a colorless, odorless and flammable gas from the class of organic compounds called epoxides. EO and its derivatives act as building blocks in various chemical manufacturing industries and find use from the areas of upholstery, household/industrial cleaning agents and cosmetics, to adhesives and anti-freezing sectors. However, it’s major application is in the food industry and related sectors as:
• preservative (in dry herbs, spices, cereals, seeds, etc.) due to its anti-bacterial and anti-fungal properties, it helps in maintaining a good shelf life without interfering with nutritional and organoleptic profiles. Disinfection by EO is preferred when the items are susceptible to high temperature processes.
• fumigant pesticide to limit the growth of pests and insects in crops. It is also a registered anti-microbial pesticide under EPA.
• sterilizing agent (e.g., medical devices and equipment, masks, etc.) for the products that may suffer deterioration by strong chemicals, heat and/or moisture.

The problem

Despite such wide usage across various segments, there is an alarming concern due to the toxic effects of ethylene oxide on humans. Persistent and chronic exposure of EO causes nausea, vision irritation, depression in CNS and has the capabilities to damage DNA leading to mutagenic effects. International Agency for Research on Cancer (IARC) has categorized EO as human carcinogen. Further to this, ethylene oxide under aerated conditions gets quickly converted to 2-chloroethanol (2-CE), another toxic compound that negatively affects cardiac, liver and kidney functions. Hence, quality control check-points upon its use and quantitation has become critical to curb/prevent the health hazard it is capable of causing.

Surge in demand for EO analysis, why?

Scientific data and evidence validating health hazards on the exposure of EO has led to strict regulations and check on its usage across the globe. The noteworthy fact is that EO is banned in EU since 1991. However, its use continues in other parts of the world, including US, India, and Asia-American subcontinents. Presently, EU has set maximum residue limit (MRL) of 0.02 to 0.1 mg/kg for various commodities.

Tracing back the timeline of product recalls, it was in August 2020 that Rapid Alert System for Food and Feed (RASFF) highlighted EO content crossing the stated limits in the sesame seed and related products from India. This marked the start of alerts and by July 2022, there have been close to 120 notifications by RASFF regarding the same, extending the product scanner beyond seeds to various raw materials, spices, even finished products like ice-creams, cheese, spreads and supplements. In the most recent amendment of the EU Regulation No 231/2012, effective from 1st Sep 2022 the MRL of EO is at 0.1 mg/kg in additives.

All the food regulatory authorities as well as the food manufacturing companies are now mandated to critically monitor EO levels for unhindered exports/imports and prevent further recalls. The commodities currently being analyzed for EO content are cereals, pulses, nuts, seeds, herbs, spices, bakery-based products, malt-based products, coffee and cocoa related products, edible oil and oil seeds, fruits, and vegetables. As per regulations, total EO is reported as the sum of the two components EO and 2-CE expressed as EO.

Analytical challenges in quantitation of EO

Such dynamic changes in lower limits with diverse range of commodities and products necessitates a sensitive, robust, repeatable yet future proof method of analysis for EO. Gas chromatography with mass spectrometer detector (GC-MS/MS) is the most widely used analytical technique used for determination and quantitation of EO in various commodities. In the coming section, discussed are the challenges associated with the analysis of EO and how Thermo Scientific™ TRACE™ 1600 series GC along with Thermo Scientific™ TSQ™ 9610 MS/MS provides a comprehensive, advanced, and future ready workflow to address these challenges.

Plug and play modules for IC Injector & detector modules

Ethylene oxide inherently has difficult chemical and physical properties, for instance its high volatility (boiling point 10.7 deg C) and small size, make the sample preparation critical to prevent its loss due to fast evaporation. Any loss during this process could lead to mis-estimation. High volatility also poses difficulty in separation and retention in routine GC column chemistry. Usually in polar columns, it would elute just near to void time, too fast to be properly detected. In other mid-polar to non-polar columns, the separation doesn’t even take place or is too weak. In addition to this, there is high matrix interference which further can lead to wrong values in results.

Low molecular weight (44 m/z) and further lower SRM transition (44 -> 29 and 44->14) of EO makes it more prone to interferences of common organic small molecules in the sample matrix. One such most found interference is from acetaldehyde (CH3CHO) that shares the same precursor ion 44 m/z and same daughter ion transitions. Hence, for an accurate and precise estimation of EO, the method should ensure prominent separation and no coelution between these analytes. Another difficulty arises when the matrices being tested upon comprise of pigments, diverse phytochemicals, and complex compounds. Such commodities need extraction process that ensures high repeatability and recovery.

Analytical laboratory establishing EO analysis facility face high sample load due to increased demand. Such critical processes, high ROI expectation from labs and faster analysis time, create a complex cross play of layered difficulties, adding on to existing ones. The instruments under use need to be productive with least downtime, indicating a higher maintenance cost and efforts. In summary, to combat multimodal challenges, GC-MS/MS system along with the sample preparation process need to be sensitive, selective, robust, and reproducible offering maximum uptime with integration of innovative technologies. The system should be user friendly in operation and maintenance, ensuring affordable cost of analysis and time.

Optimized end-to-end comprehensive and future ready GC-MS/MS EO analysis

Recently developed and validated EO analysis method on TSQ 9160 GC-MS/MS (Image 1) not only successfully overcomes the above challenges but also makes sure to deliver high sensitivity with lower injection volumes. Triple quadrupole mass spectrometer utilizes Advanced electron ionization (AEI) source with dual filament that offers focused ion beam, more transmission of ions, in turn high ionization efficiency. It is a tool free wireless source that makes it smooth and easy for the users to operate. The repeatability and precision of injections is maintained using Thermo Scientific™ TriPlus™ RSH autosampler with advanced automation technology. This smart autosampler is well equipped with cooling racks to keep the samples at lower temperatures to avoid any losses of EO due to evaporation or high volatility, especially during unattended analysis. This method abides by the SANTE/11312/2021 and meets all the QC and analytical parameters guidelines.

Thermo Scientific TSQ 9610-GC-MS/MS
Image 2: Thermo Scientific TSQ 9610-GC-MS/MS

Another important expectation of the testing laboratories from the method is that it should be applicable to wide range of complex matrices. In such situations, to increase sensitivity, the common practice in analytical laboratories is to increase the injection volume. This may lead to huge background noise and interferences impeding the quality of results. With the innovative ion optics of TSQ 9610 GCMSMS in combination with AEI source and higher dynamic range XLXR detector, this method shows exceptional results in varied matrices with lower injection volumes (1µl), encompassing wide linearity of concentration range from 0.002 mg/L to 5mg/L in samples (Image 2a).

Image 2a & 2b

Due to incorporation broader range in linearity, high concentration samples do not require dilution or re-injection. In turn, labs increase sample throughput and save time. The column TG-624Sil MS with higher thickness of stationary phase helps in increasing the specificity and retention of highly volatile EO from interferences like AA (Image 2b).

Image 3 & Image 4

To test the robustness and applicability of the method in complex samples, different set of commodities (e.g., xanthan, sesame seed, safflower, Vanilla bean, cinnamon, citrus herb, etc.) were considered (Image 3), spiked with EO and 2-CE and were tested. Demonstration of robustness at high sample throughput is one of the most important requirements in the food testing laboratories. Around 230 injections in consecutive 3 days were set in a sequence containing ten sample extracts. (Image 4) The results showed no deviation in the peak shape, %RSD was around 8.8 with 0.01 min negligible deviation in RTs which is well under SANTE guideline of 0.1 min. Hence, this method not only meets the ultra-sensitivity and quantitation mandate, but also ensures the best quality data in terms of linearity, dynamic range, and repeatability, along with high productivity. To further strengthen the solution base, the method was used to test ready-to-use spice samples and the results were coherent in terms of results quality (Image 5).

Image 4 & Image 5

In addition to food commodities, EO analysis also need to be performed in medical equipment, high volume routine accessories like masks, etc. Hence, another method was developed for a fast, cost-effective HS- analysis of ethylene oxide and 2-chloroethanol in surgical-style face masks using GC-HS-FID technique (Image 6). Usually post the manufacturing process and before it comes to end consumer, these masks are sterilized using EO. Although EO’s use is banned in US for such commodities, it is advisable to get the products tested. GBT 16886.7-20156, ISO 10993-77 and GB 19083-20108 have set a specific limit of 10 μg/g in face masks. The results show excellent chromatographic resolution of the analytes, gaussian peak shapes and well-defined separations between EO and 2-CE, with R2 values >0.998 and AvCF %RSD <4, indicating accurate and precise quantification. Better sensitivity than regulatory requirement (as per GB 19083-2010) was obtained with minimum detection limit of 0.03 μg/g (EO) and 0.10 μg/g (2-CE).

Image 6

In summary, with the continuous change in regulatory requirements and expansion of commodities that need to be analyzed for EO quantitation, analytical laboratories need a complete setup from sample preparation till data analysis. The workflow should not only provide selectivity and sensitivity but should also be future proof to meet upcoming challenges. Laboratories look for time saving and energy saving sustainable solutions that help them achieve high productivity, fast turnaround time and get quick ROI. This article highlighted a comprehensive landscape on the current situation of ethylene oxide analysis, its challenges and how Thermo Fisher Scientific can help the users meet the regulatory requirements with end-to-end GC-MS/MS solution.


• SANTE/11312/2021
• Application note : Document Connect (
• Application note: A fast, cost-effective HS-GC method for the analysis of ethylene oxide in surgical-style face masks (
• 9610 Technical Note: Document Connect (
• Increased Laboratory Efficiency with the TSQ 9610 GC-MS/MS in a Food and Environmental Analytical Laboratory Document Connect (
• EPA website: Ethylene Oxide (EtO) | US EPA
• Ethylene Oxide (EtO) Explained | US EPA
• Document Display | NEPIS | US EPA

About the Authors:
1. Parul Thakur
Product Marketing Manager, India, GC/GCMS
Thermo Fisher Scientific India
2. Sunil Kumar T.
Marketing Manager, India – Mass Spectrometry & Food Safety
Thermo Fisher Scientific India
3. Dr. Dasharath Oulkar
Manager, India Customer Application Center, Food & Beverage
Thermo Fisher Scientific India


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An editor by day & dreamer at night; passionately involved with both print and digital media; Pet lover; Solo traveller.

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