Interlaboratory evaluation of methods to quantify skin sensitizing hydroperoxides potentially formed from linalool and limonene in perfumes

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 mu g/ml, an average recovery of 86-105% with a relative standard deviation between laboratories of 7.4-22% was found. In samples spiked with 2050 mu g/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.