High-performance liquid chromatography profiling of the major carotenoids in Arabidopsis thaliana leaf tissue

Carotenoids are extremely sensitive to a variety of physico-chemical attacks which may have a profound effect on their characteristic properties, thereby influencing the accurate identification and quantification of individual compounds. In this light, a comprehensive summary of the pitfalls encountered and precautions to be administered during handling and storage of authentic standards and samples was found to be incomplete. Furthermore, acceptable baseline separation of trans-lutein from trans-zeaxanthin and between the cis- and trans-forms of neoxanthin and violaxanthin has not been satisfactorily demonstrated. Hence the most optimal sample preparation and analytical steps were determined and a sensitive and reproducible method for the quantitative HPLC profiling of the principal carotenoids found in plant leaf tissue was developed. A reverse-phase C30 column with a binary mobile solvent system was used for the baseline separation of eight of the major carotenoids and the two chlorophylls (a and b) within 18 min. These compounds were identified via the use of authentic standards, their spectral characteristics and HPLC-atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) confirmation. This method has been successfully applied for the quantification of plant pigments in Arabidopsis thaliana wild-type (WT) leaf tissue and in two A. thaliana non-photochemical mutants, namely npq1 and npq2. These mutants have previously been well-characterised and provided valuable reference data as well as acting as internal controls for the assessment of our new method.