Microwave-Assisted Saponification Conditions for MOH in Fats and Oils: An Update

What are mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) and why are they important to analyze?

Mineral oil hydrocarbons (MOH) are a diverse mixture of petroleum‑derived compounds, typically divided into two structural groups: mineral oil saturated hydrocarbons (MOSH)—linear, branched, or cyclic alkanes—and mineral oil aromatic hydrocarbons (MOAH), which contain one or more alkyl‑substituted aromatic rings.

From a toxicological standpoint, MOSH tend to accumulate in human tissues but have not been linked to clear harmful effects at current exposure levels. MOAH, however, present a greater concern. MOAH with three or more aromatic rings are regarded as potentially carcinogenic because of their structural resemblance to polycyclic aromatic hydrocarbons (PAHs). The health implications of MOAH with one or two aromatic rings, especially those with varying alkylation patterns, remain uncertain and require further study.

MOSH and MOAH can enter food through numerous pathways, and the European Food Safety Authority (EFSA) has highlighted potential concerns associated with this kind of contamination, especially for vulnerable groups like toddlers.¹

Are there new directives/regulations/legislation coming into effect in EU?

On 13 May 2026, the amendment to Regulation (EU) 2023/915 regarding maximum levels of MOAH in food received a favorable vote in the Standing Committee on Plants, Animals, Food and Feed (SCoPAFF), Section "Novel Foods and Toxicological Safety of the Food Chain." Contextually, Regulation (EC) No 333/2007 regarding the sampling method for MOH and the Commission Recommendation on the monitoring of MOH in food were approved. The legislation framework should be adopted from October 2026, with maximum levels for MOAH that will apply gradually from 2027, 2028, or 2030 (depending on the food category).²

What motivated you and your team to revisit and optimize the microwave-assisted saponification/extraction (MASE) conditions for MOSH and MOAH determination in fats and oils? What specific issues did you observe with the ISO 20122:2024 method that convinced you a revised analytical approach was necessary?

We initially decided to work on the saponification method for the extraction and enrichment of MOSH and MOAH because we participated in a series of interlaboratory tests in which discrepancies in the expected ratio of the internal standards, namely 1,3,5-tri-tert-butylbenzene (TBB) and 2-methylnaphthalene (2MN) (which is supposed to be 1.00), were observed whenever saponification was applied. This different behavior of internal standards added to the already high uncertainty associated with the MOAH determination, leading to variability across laboratories as high as 60%. Through careful standardization, the situation was improved; nevertheless, different and rather high acceptable values of the TBB/MN ratio were proposed (up to 1.25 in ISO 20122:2024).³ Additionally, the final protocols, as well as the Joint Research Centre (JRC) Guidance, were not crystal clear about which internal standard to use, leaving open the choice between TBB and MN (although they suggest using TBB). We considered this suggestion unacceptable to minimize uncertainty; therefore, we investigated the reason for the differing partition behavior of the two internal standards and arrived at an improved solvent composition that reduced the ratio to ~1.05 across different oils.⁴ The need to change the overall solvent composition of the official method was further corroborated by an independent study that reported results comparable to ours, suggesting solvent modifications very similar to the one we proposed.⁵

Nevertheless, as we began using this new protocol on a more routine basis across different matrices, oils, and fats, the PhD candidate, Aleksandra Gorska, observed that it sometimes failed to saponify the sample completely, prompting an additional investigation to understand why.⁶ It was thus decided to focus on the evaluation of the entire MOAH fraction, taking into account also the extraction efficiency of perylene, an internal standard used to mark the end of the elution of MOAH in liquid chromatography (LC) but difficult to study in real sample using monodimensional gas chromatography flame ionization detection (GC–FID) due to a combined effect: when intereferent compounds are present, either they coelute with perylene or, if epoxidation is applied to remove the interferences, perylene is also partially lost. For this reason, the entire study was performed using comprehensive two-dimensional gas chromatography with flame ionization detection (GC×GC–FID) to ensure chromatographic separation of perylene from interferences, thereby enabling reliable quantification and avoiding the need for epoxidation.

During your experiments, why did the original MASE conditions of 60 °C for 30 min fail to completely saponify certain samples, such as hydrogenated fats?

As mentioned previously, we have observed failures in efficient saponification, especially with more concrete oils, such as highly hydrogenated ones. This was mainly due to their high melting points, which required higher temperatures to reduce viscosity and facilitate contact between the saponifying solution and the entire sample. Investigating the saponification efficiency in more detail (using a simple gravimetric measurement of the residue), we found that the solvent proportion we proposed resulted in a lower saponification yield. Nevertheless, when concrete fats were treated, the ISO method also failed to saponify the matrices efficiently due to their markedly high melting points.

How did you determine that 120 °C for 20 min represented the optimal balance between complete saponification and reliable extraction performance?

We performed a design of experiments (DoE) accounting for the effects of time and temperature on the combined saponification yield and the internal standard ratio. A temperature of 120 °C for 20 min was selected as an extreme condition to ensure complete saponification across the broadest range of matrices.

Your study emphasizes internal standard ratios being as close as possible to 1.00. Could you explain why this value is analytically significant and how it reflects method reliability?

The selection of internal standards for the overall workflow was thoroughly conducted to serve as a watchdog over critical points throughout the procedure. The ratio was proposed as a guarantee that each critical point was under control and corresponded to the ratio of the nominal concentrations of the added internal standards.⁷ Therefore, a correct ratio is a fundamental control of the overall workflow. Accepting values up to 1.25 translates to an acceptable 25% variability, which adds to the uncertainty linked to normal manipulation repeatability and to the uncertainty in data integration and interpretation, which, in MOSH and MOAH analysis, has been estimated to account for up to 20%.⁸

You found that fatty acid composition influenced MOAH internal standard recoveries. What is your interpretation of the relationship between soap formation, matrix composition, and extraction efficiency in these systems?

This part of the study was really intriguing. Indeed, a trend was observed across the different edible oils and fats, showing a clear matrix effect. The only major difference across these samples was the fatty acid profile. It was hypothesized that the fatty acid composition could affect the physical properties of the soap formed during saponification, leading to a different partitioning of the internal standards.⁹

The samples were grouped into three main categories based on C18 fatty acid content: high, medium, and low. In the group with high C18 content (~90%), a clear improvement in the TBB/2MN ratio towards the expected 1.00 value was observed with increasing unsaturation. Per/2MN remained constant, meaning that its extraction proportionally improves following the behavior of 2MN, although remaining slightly discriminated (ratio = ~0.9). In the “medium C18 group,” a significant proportion of fatty acids is C16 (~40%). In this group, the TBB/2MN ratio is significantly less impacted. Finally, the group with the lowest abundance of C18 (~20%) but high abundance of short-chain fatty acids (C10-14 ~70%; C16 ~10%) showed the opposite behavior, with greater discrimination at higher degrees of unsaturation.

In conclusion, significant impacts of both chain length and degree of unsaturation were observed on soap properties and, consequently, on internal standard recoveries.

References

1. Schrenk, D.; Bignami, M.; Bodin, L.; et al. Update of the Risk Assessment of Mineral Oil Hydrocarbons in Food. EFSA J 2023, 21, e08215. DOI: 10.2903/j.efsa.2023.8215
2. European Commission—Catalogue of Measures Following Opinions on Mineral Oil Hydrocarbons (accessed 2026-06-08).
3. International Organization for Standardization (ISO). ISO 20122:2024. Vegetable Oils—Determination of Mineral Oil Saturated Hydrocarbons (MOSHs) and Mineral Oil Aromatic Hydrocarbons (MOAHs) with Online-Coupled High Performance Liquid Chromatography-Gas Chromatography-Flame Ionization Detection (HPLC-GC-FID) Analysis—Method for Low Limit of Quantification; ISO: Geneva, Switzerland, 2024.
4. Bauwens, G.; Purcaro, G. Improved Microwave-Assisted Saponification To Reduce the Variability of MOAH Determination in Edible Oils. Anal Chim Acta 2024, 1312, 342788. DOI: 10.1016/j.aca.2024.342788
5. Koch, M.; Hamscher, G.; Brühl, L. Towards a Better Understanding of Saponification for the Determination of Mineral Oil Hydrocarbons (MOH) in Edible Oils and Fats—Evaluation and Optimization of Current Protocols. Anal Chim Acta 2025, 1376, 344591. DOI: 10.1016/j.aca.2025.344591
6. Gorska, A.; Ferrara, D.; Albendea, P.; Cordero, C. E.; Purcaro, G. Update on the Microwave-Assisted Saponification Conditions for Mineral Oil Hydrocarbons Determination in Fats and Oils. Analyst 2025, 150, 5190–5200. DOI: 10.1039/D5AN00701A
7. Bratinova, S.; Hoekstra, E. Guidance on Sampling, Analysis and Data Reporting for the Monitoring of Mineral Oil Hydrocarbons in Food and Food Contact Materials; European Commission, Joint Research Centre: Luxembourg, 2023.
8. Bratinova, S.; Hoekstra, E.; Emons, H.; Hutzler, C.; Kappenstein, O.; Biedermann, M.; McCombie, G. The Reliability of MOSH/MOAH Data: A Comment on a Recently Published Article. J Verbrauch Lebensm 2020, 15, 285–287. DOI: 10.1007/s00003-020-01287-w
9. Hill, M.; Moaddel, T. Soap Structure and Phase Behavior. In Soap Manufacturing Technology, 2nd ed.; Spitz, L., Ed.; Elsevier: Amsterdam, The Netherlands, 2016; pp 35–54. DOI: 10.1016/B978-1-63067-065-8.50002-5