Ethylene oxide (EO) has a broad array of applications across many industries. One of the most important is as a fumigant pesticide for the preservation of dry food products, such as seeds, milled cereals, spices, and herbs. However, upon consumption, ethylene oxide can have significant impacts on human health, adversely affecting the nervous system and mucous membranes, and exhibiting mutagenic and carcinogenic potential. Moreover, in food, EO readily degrades into 2-chloroethanol (2CE), which is itself considered toxic.

Such health concerns have driven a spate of strict regulations on EO’s usage in food production across the globe. Most notably, EO is now banned for use in food in many countries, including all of those in the European Union (EU), where it has been banned since 1991. Currently, the EU has set maximum residue levels (MRLs) for EO at 0.02 to 0.1 mg/kg, depending on the commodity, where EO is defined as the sum of both EO and 2CE (Reg. (EU) 2015/868). Despite the ban, there have been a number of recent reports of the presence of EO residue in food products in the EU, primarily owing to inconsistent regulations globally. Between January 1 and July 31 in 2022 alone, there were 119 EO contamination alerts published in the Rapid Alert System for Food and Feed (RASFF).

This volume of alerts demonstrates the critical importance of more accurate and frequent monitoring of food for contamination with EO and its degradation products. Using current analytical methods, however, EO analysis is incredibly challenging. In this article, we will provide an overview of the current difficulties of EO determination and explore how these can be effectively overcome using an optimized gas chromatography with tandem mass spectrometry (GS-MS/MS) workflow.

Grappling with Ethylene Oxide Determination

Because of the significant health risks posed by EO residues and owing to the low permissible MRLs set by the EU, methods for the analysis of food products for EO residues must be sensitive, precise, and accurate. At the same time, food testing laboratories must have the capability to meet the continuous and growing testing demand. In practice, this means that food laboratories must deliver an increase in sample throughput and shorter analysis turnaround times.

However, meeting these requirements using current methods—typically triple quadrupole gas chromatography mass spectrometry (GC-MS)—is challenging on several fronts due to the lower MRLs stipulated and the inherent hurdles of EO analysis.

Many of the challenges in EO analysis stem from the inherent physical and chemical properties of EO itself. For example, EO is a highly volatile compound, with a boiling point of just 10.7°C. If careful precautions are not taken during sample preparation, analysts risk EO evaporation, which could lead to an underestimation of the extent of EO contamination. This means potentially unsafe food samples could make it to consumers. EO’s volatility also means that it cannot be retained at all on some generic chromatographic columns and is only weakly retained by others. EO, therefore, elutes shortly after the void time, bringing a risk of interferences from other poorly retained compound that are present, which means that EO cannot be separated from the matrix. As a result, there is a significant risk of either missing EO residues or inaccurately determining the level to be safe in a given sample, when in fact it is not.

The low molecular weight of EO and its fragmentation products presents another analytical hurdle—increased susceptibility to interferences. One notable compound that commonly interferes with EO is acetaldehyde (AA), which has the same ion transitions as EO. Because of these non-selective ion transitions, insufficient chromatographic separation of the analytes (i.e., co-elution) can lead to overestimation of EO contamination in a sample. In the worst-case scenario, analysts can get a false negative result from the interference between EO and AA.

Then there are the general challenges associated with the analysis of dried food samples. Food samples are complex matrices, meaning extracts are typically rich in a plethora of co-extracted compounds. This presents two critical challenges for the analytical laboratory looking to achieve sensitive and selective EO analysis: First, continually running “dirty” samples through a GC-MS system can cause contamination of all parts of the chromatographic system, triggering the need for increased maintenance, greater down-time, and more costly operations. Second, running complex samples through instruments can directly impact the quality of the results, leading to poor chromatographic performance, retention time shifts, variable peak areas, and degraded peak shapes.

In the case of EO analysis using common methods, the impact of complex matrices is amplified by the fact that, to compensate for poor sensitivity and selectivity, larger sample volume injections of approximately 2 µL are required.

As explained, current EO analysis is exceptionally difficult. The array of hurdles involved means analytical labs can typically only run 15 to 20 samples before maintenance or system cleaning is required. The increased downtime means samples are sitting around longer, exposing them to greater EO evaporation risk. When throughput is stifled in this way, operational- and lab cost-efficiency also suffers greatly, driving up prices that could also result in lower rates of testing.

An Optimized GC-MS/MS Workflow For Ethylene Oxide Analysis

To overcome these common challenges in EO determination, an advanced GC-MS/MS method has recently been developed. Unlike traditional approaches, this method uses triple quadrupole mass spectrometry with an expanded linear dynamic range, as well as an advanced electron ionization (AEI) source that delivers a focused ion beam with enhanced transmission. Combined, these deliver increased sensitivity, selectivity, linearity, and robustness relative to current GC-MS approaches. Critically, the increase in sensitivity means that sample injection volumes can be reduced.

More specifically, the method uses a Thermo Scientific TRACE 1610 GC system coupled to a Thermo Scientific TSQ 9610 triple quadrupole GC-MS/MS system, which is equipped with a Thermo Scientific Advanced Electron Ionization (AEI) source. Samples are injected using a Thermo Scientific TriPlus RSH autosampler. EO evaporation from samples during unattended analysis is eliminated owing to a specially designed cooled rack, which keeps samples at 7°C. Samples were prepared following the QuOil-Method (CEN/TS 17062:2019 modified) with one amendment: 3g of the samples were used rather than 2g. The method was validated according to “analytical quality control and method validation procedures for pesticide residues analysis in food and feed” (Document Nº SANTE/11312/2021).

Advanced GC-MS/MS: Sensitive, Selective, and Linear EO Determination

When evaluated using standard solutions, the optimized method exhibited exceptional performance across several domains.

Greater sensitivity for smaller injection volumes. As noted above, the lack of sensitivity in current methods has meant that most la

Figure 1. Ion transitions for EO (left) and 2CE (right). Standard solutions of 0.002 mg/L concentration were used. Expected ion ratios for EO and 2 CE are 7% and 100%, respectively. When looking at the SANTE criteria, which allow for 30% variability, the ratios for EO must fall within 4.9%-9.1% and for 2 CE 70%-130%. Courtesy of Thermo Fisher Scientific.

boratories need to use larger 2 µL injection volumes, which exacerbates the problems associated with multiple injections of complex sample matrices. Thanks to the enhanced sensitivity and careful method optimization, however, the method being investigated can deliver exceptional sensitivity, even at 1 µL injection volumes.

Figure 1 shows that all ion transitions were characterized by high signal-to-noise ratios, and ion ratios were highly stable, meeting the variability criteria of DG SANTE guidelines.   

The ability to drastically reduce complex sample injection volume significantly reduces the burden on instruments, meaning equipment will need to be vented and cleaned less frequently, and throughput can be boosted as a result.

Superior selectivity to handle AA interference. As well as showing high sensitivity, this optimized method delivered exceptional selectivity. This is primarily due to the specific chemistry of the stationary phase and increased stationary phase thickness of the column, which means it can better retain highly volatile analytes such as EO and its residues. Co-elution of analytes with similar ion transitions can thus be mitigated, leading to better selectivity and more accurate results.

Figure 2 shows that the column enabled a good separation of EO and AA, with a retention time difference of more than 0.1 minute. As a result, despite the non-selective ion transitions, the risk of AA interfering with EO results was eliminated.

Figure 2. Superior chromatographic separation mitigates the impact of non-selective ion transitions, demonstrated here with the clear separation of AA and EO. Courtesy of Thermo Fisher Scientific.

Broad linearity reduces the need for dilutions. The method showed excellent linearity across both EO and 2CE, made possible by the extended linear dynamic range of the mass spectrometer’s detector. In the investigated concentration range of 0.002 mg/L to 5 mg/L (corresponding to 0.007 mg/kg to 16.5 mg/kg in the sample), all back-calculated concentrations were within ± 20% of the true concentrations, meeting the criterion of the DG SANTE guidelines (see Figure 3).

A method that can deliver such a broad linear range means that samples with a high concentration of analyte do not need to be diluted and reinjected. For the lab, this translates to reduced analysis time, which opens the possibility of greater throughput.

Real-World Robustness

While the method excelled in evaluations of sensitivity, selectivity, and linearity using standard solutions, challenging the method using complex food samples, at high throughput, is required to fully demonstrate its applicability to the food testing laboratory.

With that in mind, the method was used to evaluate 10 different food samples encompassing a range of foods. Here, external calibration curves were applied for both EO and 2CE, and samples were spiked with deuterated 2CE to provide an additional internal standard suitable for 2CE. Reference concentrations were also obtained in a laboratory accredited under ISO/IEC 17025:2005, using the same optimized GC-MS/MS method.

Figure 3. EO (A) and 2CE (B) calibration curves, both in the concentration range of 0.002 mg/L to 5mg/L (corresponding to 0.007 mg/kg to 16.5 mg/kg in the sample). Courtesy of Thermo Fisher Scientific.

While EO was not detected in any sample (which is common given its instability), 2CE was detected in all samples. Excellent results agreement was seen between our laboratory and the external laboratory (see Figure 4). Additionally, results from the internal standard also matched the external calibration curve as such, demonstrating the suitability of the quantitation method for real food analysis.

To meet the high-throughput needs of food testing laboratories, analytical methods must provide long-term, maintenance-free operation, which is no easy feat given the repeated injections of complex samples required. To evaluate whether this new method could deliver on this critical need, a sequence containing the 10 samples was injected continuously over three days. This amounted to a total of 230 injections. During this period, there was no instrument maintenance, tuning, or other interruption of the system.

Figure 4. Quantitation of 2CE using real food samples. Courtesy of Thermo Fisher Scientific.

The method exhibited remarkable robustness across the injections. Evaluation of the peak characteristics of isotopically labelled 2CE showed the system’s function was highly stable, with no degradation of chromatographic separation. Excellent con­­sistency of peak areas was also observed (relative standard deviation ± 8.8%), and retention times deviated by less than 0.01 minute, an order of magnitude lower than the limit set by SG SANTE guidelines (see Figure 5). This was all achieved despite there being visible sample matrix residue in the liner at the end of the experiment.

As demonstrated, the reduced sample volume enabled by the sensitivity of triple quadrupole mass spectrometry and an enhanced AEI source ensures a highly robust method.

Figure 5. Response of isotopically labeled 2CE standard in every 10th injection of the sequence, spanning 230 injections in total. Courtesy of Thermo Fisher Scientific.

Advanced GC-MS/MS: Better Analysis, Better-Protected Health

The risk posed by EO and its residues in food is significant but the inherent properties of EO, the performance of current analytical methods in the context of low MRLs, and the ever-increasing demands of the food testing sector are making EO determination unnecessarily slow and costly.

However, an optimized GC-MS/MS method that uses chromatography columns that can separate volatile analytes, an enhanced AEI source, and the sensitivity of triple quadrupole mass spectrometry can alleviate these challenges. The method described here does just this, delivering exceptional sensitivity, selectivity, linearity, and robustness to meet the needs of food testing labs.

In adopting such methods, laboratories will be able to detect EO and its residues more accurately, reduce method duration for increased throughput, and significantly increase the cost effectiveness of their operations. Ultimately, this means better protection of human health, and safer food products for consumers.

Dr. Rajski is product specialist for gas chromatography mass spectrometry for Thermo Fisher Scientific. Reach him at [email protected].

Ethylene Oxide Analysis for Food Safety
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