Sensitising compounds caught by chemiluminescence chromatography

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  • Published: Jun 1, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Sensitising compounds caught by chemiluminescence chromatography

Improved analytical methods needed for linalyl acetate hydroperoxides

Linalyl acetate is an important fragrance chemical, found in a wide range of products, such as perfumes and soaps. It occurs in a number of essential oils, including lavender, bergamot and other citrus oils, while the synthetic compound is also an important item of commerce. Linalyl acetate has been associated with cases of allergic dermatitis, but studies have shown that these are actually caused by the hydroperoxides formed on oxidation. Oxidation of linalyl acetate can slowly occur on exposure to the air, giving rise to two main hydroperoxides: the 6-hydroperoxide and the 7-hydroperoxide. Each of these is a mixture of diastereomers. Traditionally, hydroperoxides have been measured in perfumery compounds using peroxide values (POVs) determined by iodometric titrations. Such methods will detect all the oxidising compounds present, whether or not they are sensitising agents. More accurate methods are necessary in order to give proper control of those compounds that may cause dermatitis. Chemiluminescence detection methods can be used to detect oxidising species by HPLC. Typical systems use luminol and a catalyst, such as the cytochrome c protein, to give a signal in the presence of hydroperoxides. The Firmenich workers have previously described using such a system with limonene and linalool hydroperoxides, and in the current paper use the same system for linalyl acetate hydroperoxides.

HPLC with chemiluminescence detection used for linalyl acetate hydroperoxides

A mixture of the 6 and 7-hydroperoxides was formed from linalyl acetate by catalytic oxidation with hydrogen peroxide. NMR showed a roughly 1:1 mixture of the two compounds was formed. HPLC was carried out using an Agilent 1100 system and a Phenomenex Luna C18(2) column. A three-solvent gradient system of methanol, acetonitrile and water was used for elution. A Hitachi Sensivate Elite post-column reactor was used for the reaction with luminol and cytochrome c, with a Jasco CL-2027 Plus chemiluminescence detector. A single peak was seen for the mixture of 6- and 7-hydroperoxides, with no separation of the positional isomers or the diastereomers. Samples for HPLC were prepared in a cold room in amber vials, and BHT (butylated hydroxytoluene) was added as an antioxidant. The sample headspace was flushed with nitrogen. These precautions were required to prevent hydrogen peroxide formation and other free radical reactions during sample preparation.

The response of the system was not quite linear, but it was not clear why. The limit of detection in the prepared sample solution was 0.00125 mmol/L, equivalent to 0.0625 mmol/L in the initial sample. It was found that the sensitivity tended to decrease on extended runs, but this could be overcome by cleaning the detector loop with dilute nitric acid in order to remove any precipitated material. A so-called ECHO injection (injection of the hydroperoxide standard with a time delay) could also be used to compensate for the downward trend in sensitivity. It was also found that different batches of cytochrome c gave a different response, necessitating recalibration.

The system was used to investigate the amount of linalyl acetate hydroperoxide in samples of bergamot oil and lime oil that had been shaken in air to induce oxidation. Clear separation of linalyl hydroperoxide from the hydroperoxides of limonene and linalool was achieved.

HPLC and chemiluminescence is a better hydroperoxide detection technique

The use of HPLC and chemiluminescence is a definite improvement over the old-fashioned iodometric techniques. The chemiluminescence apparatus allows for the specific detection of hydroperoxides, compounds that often give poor results with LC-MS. Further refinement of the conditions may lead to greater isomer separation. It is likely that the future will bring more regulations on hydroperoxide limits, so it is important that analytical techniques are adequate for the task.

Related Links

Flavour and Fragrance Journal, 2017, Early View paper. Calandra et al. An HPLC method for the detection of hydro peroxides derived from linalyl acetate in citrus oils, using post-column luminol-mediated chemiluminescence detection.

Flavour and Fragrance Journal, 2015, 30, 121-130. Calandra et al. An HPLC method for hydroperoxides derived from limonene and linalool in citrus oils, using post-column luminol-mediated chemiluminescence detection.

Biomedical Chromatography, 2003, 17, 96-105. Li et al. Chemiluminescence detection in HPLC and CE for pharmaceutical and biomedical analysis.

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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