Application of comprehensive two-dimensional liquid chromatography to elucidate the native carotenoid composition in red orange essential oil

In the present work, the ability of a LC × LC-DAD/APCI-MS method developed at this laboratory to identify the native composition of carotenoid in an extremely complex matrix such as red orange essential oil was demonstrated. To carry out this task, two independent and orthogonal separation mechanisms were coupled through a 10-port switching valve that simultaneously collected the eluent from a microbore cyano column used as the first dimension in normal phase mode and injected it to a conventional reversed phase monolithic C18 column in the second dimension separation. By using this novel analytical technique together with the use of DAD and APCI-MS detectors it was possible to identify in the sample, without the need of any pretreatment, 40 different carotenoids. Among them, 16 carotenoid monoesters were identified, mainly -cryptoxanthin palmitate (C16:0), myristate (C14:0), and laureate (C12:0) as well as several lutein, violaxanthin, antheraxanthin, and luteoxanthin monoesters. Moreover, 21 carotenoid diesters composed by several antheraxanthin, luteoxanthin, violaxanthin, and auroxanthin diesters were found in the native carotenoid composition of the orange oil. The main carotenoid diesters were the laureate palmitate (C12:0, C16:0), myristate palmitate (C14:0, C16:0), and dipalmitate (C16:0, C16:0) diesters, although other diesters were also identified. Besides, two different free carotenes, -carotene and phytofluene, and a xanthophyll, lutein, were also determined. To the authors’ knowledge, this is the first time that carotenoid diesters are described and identified in orange essential oil. Likewise, it has been demonstrated that the LC × LC approach proposed in this study is capable of coping with the direct analysis and identification of a complex natural source of carotenoids such as the orange.