Identification of carotenoid pigments and their fatty acid esters in an avian integument combining HPLC–DAD and LC–MS analyses

Yellow-orange-red ornaments present in the integuments (feathers, bare parts) of birds are often produced by carotenoid pigments and may serve to signal the quality of the bearer. Although carotenoid esterification in tissues is a common phenomenon, most of the work on avian carotenoids has been focused on the identification of free forms or have been done after sample saponification. Here we determined free and esterified carotenoid composition in a bird species with red ornaments: the red-legged partridge (Alectoris rufa). Carotenoids from leg integument were extracted and processed by TLC to separate three major carotenoid groups (free form, mono- and diesters with fatty acids), whereas saponified extracts gave only free forms of carotenoids. TLC fractions were then analyzed by HPLC–DAD with C18 phase column for a preliminary identification of carotenoid groups. The final characterization of free carotenoids and its esters with fatty acids was performed with direct extracts analyzed by LC–MS and LC–MS/MS with a C30 phase, always with a system coupled to DAD. The main carotenoid (λmax 478 nm and [M+H]+ at m/z 597.2) was identified as astaxanthin by comparison with standards. A second carotenoid (λmax between 440 and 480 nm and [M+H]+ at m/z 581.3) was not identified among any of the commercially available carotenoid standards, although it could correspond to pectenolone according to its fragmentation pattern. Both the unidentified carotenoid and astaxanthin formed monoesters with fatty acids, but only astaxanthin was in its diesterified form. Monoesters were mainly formed with palmitic, stearic, oleic and linoleic acids. Complementary analyses of fatty acid composition in partridge integument by GC–MS revealed high amounts of these and other fatty acids, such as myristic, arachidic and docosanoic acids. The combination of HPLC–DAD and LC–MS/MS spectra was especially useful to identify the carotenoids present in the esterified forms and the probable masses of the fatty acids included in them, respectively.