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The fragrant terpenes limonene and linalool can form skin sensitizing hydroperoxides upon prolonged exposure to air. Recently, high frequencies of positive patch tests to oxidized linalool and limonene were reported from multiple dermatological centres. However, there is a lack of data indicating potential sources of consumer exposure to sensitizing doses of terpene hydroperoxides which explains this frequent contact allergy. Within the IDEA project (International Dialogue for the Evaluation of Allergens; http://ideaproject.info/), a taskforce was formed to drive analytical method development and evaluation. In an inter-laboratory study in five laboratories, a method based on hydroperoxide reduction combined with GC–MS was tested for reproducibility. Blinded samples of commercial fine fragrances were spiked with four different hydroperoxides. In samples spiked with 100–200 μg/ml, an average recovery of 86–105% with a relative standard deviation between laboratories of 7.4–22% was found. In samples spiked with 20–50 μg/ml, the recovery was 85–91%. The reduction approach offers a transferable and reproducible method to indirectly detect low levels of hydroperoxides, at least in fine fragrances. Ideally, one would prefer to directly detect the parent hydroperoxide. Therefore the same samples were further tested with three LC-based methods directly detecting the parent hydroperoxide. LC coupled to chemiluminescence, LC-Q-TOF-MS or LC-orbitrap-MS were used. Results indicate that with specific gradients a separation of the four analytes and quantification in the fragrance matrix can be achieved. Results of this method evaluation study present a toolbox of methods to detect terpene hydroperoxides to further investigate consumer exposure.

Upon prolonged exposure to air, limonene is prone to oxidation. Hydroperoxides formed upon oxidation are strong skin sensitizers in animal tests and give positive patch tests in human dermatitis patients. However, there is no analytical evidence indicating potential sources from which the public can be exposed to sufficient quantities of limonene hydroperoxide for induction of skin sensitization to occur. It has been proposed that fragranced products might lead to such exposure. Here, we developed analytical methods using mass spectrometry (MS) in combination with gas chromatography (GC)-MS and liquid chromatography (LC)-MS to detect limonene-2-OOH. GC-MS has a low sensitivity, whereas LC-MS was found to be a sensitive method to detect this hydroperoxide. However, selectivity of LC-MS is low in complex fine fragrances owing to limited separation and unspecific ions. We developed an additional method based on selective reduction of limonene-2-OOH to carveol by triphenylphosphine (PPh3). Quantitative carveol formation from the hydroperoxide in the presence of PPh3 could be verified in hydroperoxide-spiked fragrances. These methods were then applied to stability studies. We found a high stability of limonene in eaux de toilette and the PPh3-reduction assay gave no indication of hydroperoxide formation in repeatedly opened, half-empty bottles stored for 9 months. Aged fine fragrances retrieved from consumers contained an average of 1990 µg/g limonene, but only traces of carveol were formed in a minority of the samples in the presence of PPh3. Control samples were spiked with trans-limonene-2-OOH. Quantitative trans-carveol formation in the presence of PPh3 indicated that the method would detect the hydroperoxide in these consumer products if it was present at higher concentrations. In conclusion, limonene-2-OOH may be detected in complex fragrance samples by selective reduction followed by GC-MS. However, we could not find evidence for significant limonene-2-OOH formation in aged fine fragrance samples. Copyright © 2014 John Wiley & Sons, Ltd.

The chemical structure of human body odorants and the enzymatic pathways for their release from amino acid conjugates by skin bacteria have been elucidated in recent years. These findings open up theoretical avenues for an added layer of malodour prevention by blocking the cleavage of malodour precursors. Previously, we had validated in vitro an alternative substrate that releases a fragrance molecule instead of a malodour acid upon the action of a corynebacterial Nα-acyl-glutamine-aminoacylase. In order to assess whether this concept delivers a perceivable reduction of malodour in vivo, a number of studies were performed: (1) a controlled clinical study with expert assessment, (2) a blinded study with consumer self-assessment of a placebo versus an active formulation in left–right comparison, and (3) a longitudinal consumer study comparing two doses of the active versus a placebo formulation. A statistically significant benefit for malodour reduction was observed in all three studies, this benefit consistently seen on panellists with a higher self- or expert-perceived malodour, whereas there was no difference in panellists with a lower level of malodour formation. In addition, malodour formation was higher in panellists with noticeable cleavage of the alternative substrate, indicating a correlation between enzyme activity and malodour levels. These results indicate that the targeted enzyme is a relevant source of malodour on panellists suffering from strong body odour, and that malodour reduction on these panellists is possible by specifically targeting this enzyme. Whereas the absolute reduction of malodour with the tested substrates was relatively small, the studies presented here clearly validate Nα-acyl-glutamine-aminoacylase as a target for deodorant actives, and they demonstrate that a significant benefit can be achieved especially in individuals developing stronger axillary odour. Copyright © 2013 John Wiley & Sons, Ltd.