Purification and use of carotenoid standards to quantify cis-trans geometrical carotenoid isomers in plant tissues

Reverse-phase high-performance liquid chromatography (HPLC) is a preferred method used to identify and quantify carotenoids. Here, we describe a straightforward, reliable, and cost-effective protocol to purify and develop individual carotenoid standards for absolute quantification of carotenoids, including selected cis-trans (geometric) isomers. Analytical techniques to extract, purify and collect individual carotenoids using an HPLC system equipped with a Diode Array Detector (DAD) and fraction collector are described. Carotenoids were separated and identified by their characteristic ultraviolet-visible (UV-Vis) absorption spectra and individually isolated based on their retention times using a C30 column. This chapter outlines how to prepare standard calibration curves using known quantities of purified and/or commercially available carotenoids. A series of molar extinction and slope coefficients for phytoene, phytofluene,.-carotene, neurosporene, pro-lycopene, all trans-lycopene, lutein, ss-carotene, zeaxanthin, antheraxanthin, violaxanthin, neoxanthin, capsanthin, capsorubin and ss-cryptoxanthin are defined to enable absolute quantification of their abundance in plant, animal, and bacterial tissues. Different approaches for reporting carotenoid abundance by absolute concentration, relative composition, and/or using ratios of different pigments are provided as a convenient resource for carotenoid researchers.

Automated summary

This article describes a method using reverse-phase high-performance liquid chromatography (HPLC) to purify and develop individual carotenoid standards for absolute quantification. The article provides detailed analytical techniques for extraction, purification, separation, and identification of individual carotenoids. It also outlines the preparation of standard calibration curves and different approaches for reporting carotenoid abundance.