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Geometrical isomers of carotenoids behave differently in aspects like stability towards oxidants, bioavailability, vitamin A activity and specificity for enzymes. The availability of HPLC methods for their detailed profiling is therefore advisable to expand our knowledge on their metabolism and biological role. In this paper the development of a methodology to determine the highest number of geometrical isomers of major carotenoids in humans (phytoene, phytofluene, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene) is described. To assess its usefulness with biological samples both postprandial human plasma and lung samples from ferrets were analysed. Up to 48 isomers of the main human carotenoids were separated in 62min. This is to the best of our knowledge the report of the highest number of carotenoid geometrical isomers separated with a HPLC method. Twenty-six different …