Abstract
Serial coupled columns reversed-phase separations in high-performance liquid chromatography can be a useful tool for the analysis of complex real samples. The great difficulties found when analyzing complex carotenoid samples, due to the high natural variability of these compounds, as well as to the presence of carotenoid esters, are well documented. In the present contribution, the applicability of connecting two C30 columns to increase significantly the separation power, resolution and peak capacity for the analysis of carotenoids in a complex carotenoid sample, like sweet bell peppers, has been shown for the first time. By using LC coupled to PDA/APCI-MS detectors, 56 different carotenoids have been detected in red sweet bell peppers. By using two serial coupled C30 columns a peak capacity of 95.4 was obtained, compared with 73 achieved using a single column. Moreover, resolution greatly improved between different critical peaks when using two serial coupled C30 columns, compared with a single column. Interestingly, free carotenoids, mono-esters and diesters were quantitatively equally represented (around 33% for each different class) in red sweet bell pepper, showing, therefore, a value for the ratio of mono-esters/di-esters of around 1, which could be considered a parameter of typicality. Free β-carotene (12.6%), capsanthin-C14:0 (8.4%), and capsanthin-C12:0-C14:0 (8.9%) were the most abundant carotenoids in the three different classes of red sweet bell pepper. No carotenoid esters were detected in either yellow or green sweet bell peppers. The application of such methodology in the analysis of other complex carotenoid matrices could be a future objective of research.