Marigold flowers (Tagetes patula and Calendula officinalis) were chosen for analysis because they are the most often used source of lutein and its isomer zeaxanthin for the production of food supplements on the Czech market. Direct extraction and extraction with alkaline hydrolysis were compared to detect free or bound carotenoids. For carotenoid separation, C18 and C30 columns were used. A new method for determination of carotenoid content in food supplements in form of capsules has been developed and validated. All matrices were analysed by high-performance liquid chromatography with diode array detection (HPLC-DAD). It has been found that alkaline hydrolysis is required for both Marigold flowers and food supplements to release lutein from ester bonds to fatty acids. In Calendula officinalis lutein in the concentration of 807—1472 mg·kg−1 of dry matter was detected. Tagetes patula has been identified as a better lutein source with the content of 5906—8677 mg·kg−1 of dry matter. It has been found that the content of lutein and zeaxanthin in commercial food supplements (Lutein Complex Premium and Occutein Brillant) is consistent with the declared quantity. Linearity of the HPLC-DAD method ranged from 0.1—20 μg·mL−1 with the limit of quantification (LOQ) of 1.7 mg·kg−1 for lutein in Marigold flowers and 200 mg·kg−1 in food supplements. Repeatability was 2.3 % for lutein in all tested matrices.
