Analysis of carotenoids is very complex and demanding in terms of both separation and detection. In this article, an analytical strategy relying on high-performance liquid chromatography-photodiode array detection-tandem mass spectrometry (HPLC-PAD-MS/MS) is presented for the large-scale screening of these phytochemicals. Separation was realized by means of Non-Aqueous Reversed Phase (NARP) chromatography on a triacontyl stationary phase kept at subambient temperature, using a mobile phase compatible with atmospheric pressure chemical ionization (APCI). The standards of 14 analytes were used to optimize the method and to predict the chromatographic behaviour of untargeted carotenoids. MS and MS/MS data, obtained during Information Dependent Acquisition (IDA) experiments, were utilized to set up a sensitive HPLC- selected reaction monitoring (SRM) method. Relative abundance between SRM ion currents (ion ratio) allowed the MS distinction of structural isomers (for example, bicyclic, monocyclic and acyclic isomers), while the identification of geometrical isomers was based on Qratio and fine structure, as calculated from UV-vis spectra. The comparison of LC-PAD- SRM chromatograms, acquired after applying two different extraction procedures (matrix solid-phase dispersion and overnight cold saponification), allowed verifying that sweet peppers are a good source of xanthophylls, prevalently occurring as esterified forms. The overall strategy could identify more than 40 carotenoids in some sweet pepper varieties (Jolly, horn and sweet chili pepper) available on the Italian and European food market.
Keywords: Carotenoids; HPLC-PAD-MS, large-scale profiling; Non-aqueous reversed-phase chromatography; Nutraceuticals; Sweet pepper.
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