Chapter 11
cis/trans Carotenoid Extraction, Purification, Detection,
Quantification, and Profiling in Plant Tissues
Yagiz Alagoz, Namraj Dhami, Chris Mitchell, and Christopher I. Cazzonelli
Abstract
Reverse phase high-performance liquid chromatography (HPLC) is the method of choice used in
biological, health, and food research to identify, quantify, and profile carotenoid species. The identification
and quantification of cis- and/or trans-carotene and xanthophyll isomers in plant tissues can be affected by
the method of sample preparation and extraction, as well as the HPLC column chemistry and the solvent
gradient. There is a high degree of heterogeneity in existing methods in terms of their ease, efficiency, and
accuracy. We describe a simple carotenoid extraction method and two different optimised HPLC methods
utilizing C18 or C30 reverse-phase columns. We outline applications, advantages, and disadvantages for
using these reverse phase columns to detect xanthophylls and cis-carotenes in wild-type photosynthetic
leaves and mutant dark-grown etiolated seedlings, respectively. Resources are provided to profile individual
species based upon their spectral properties and retention time, as well as quantify carotenoids by their
composition and absolute levels in different plant tissues.
Key words Carotenoid, cis-carotene, Molar coefficient, Composition, Plants, HPLC, C30, C18
1 Introduction
Carotenoids are secondary metabolites comprised of 40 carbon
atoms synthesized from isoprene subunits connected head-to-tail.
They are hydrophobic compounds, stored within the lipophilic
environment of organelle membranes from plastid organelles. Carotenoids are mainly biosynthesized in plants, algae as well as some
photosynthetic bacteria and fungi. In plants, carotenoids are
required for photosynthesis and photoprotection [1]. They can be
cleaved into smaller apocarotenoids, that function as phytohormones and/or signaling metabolites to mediate developmental
processes (e.g., root and shoot branching), plant-microorganism
associations, and intracellular as well as organelle communications
[2–7]. In humans, carotenoids serve as antioxidants to protect
against some cancer types [8], cardiovascular, neurodegenerative,
and eye diseases [9–11]. There is a high nutritional value associated
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_11,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
145
cis/trans Carotenoid Extraction, Purification, Detection,
Quantification, and Profiling in Plant Tissues
Yagiz Alagoz, Namraj Dhami, Chris Mitchell, and Christopher I. Cazzonelli
Abstract
Reverse phase high-performance liquid chromatography (HPLC) is the method of choice used in
biological, health, and food research to identify, quantify, and profile carotenoid species. The identification
and quantification of cis- and/or trans-carotene and xanthophyll isomers in plant tissues can be affected by
the method of sample preparation and extraction, as well as the HPLC column chemistry and the solvent
gradient. There is a high degree of heterogeneity in existing methods in terms of their ease, efficiency, and
accuracy. We describe a simple carotenoid extraction method and two different optimised HPLC methods
utilizing C18 or C30 reverse-phase columns. We outline applications, advantages, and disadvantages for
using these reverse phase columns to detect xanthophylls and cis-carotenes in wild-type photosynthetic
leaves and mutant dark-grown etiolated seedlings, respectively. Resources are provided to profile individual
species based upon their spectral properties and retention time, as well as quantify carotenoids by their
composition and absolute levels in different plant tissues.
Key words Carotenoid, cis-carotene, Molar coefficient, Composition, Plants, HPLC, C30, C18
1 Introduction
Carotenoids are secondary metabolites comprised of 40 carbon
atoms synthesized from isoprene subunits connected head-to-tail.
They are hydrophobic compounds, stored within the lipophilic
environment of organelle membranes from plastid organelles. Carotenoids are mainly biosynthesized in plants, algae as well as some
photosynthetic bacteria and fungi. In plants, carotenoids are
required for photosynthesis and photoprotection [1]. They can be
cleaved into smaller apocarotenoids, that function as phytohormones and/or signaling metabolites to mediate developmental
processes (e.g., root and shoot branching), plant-microorganism
associations, and intracellular as well as organelle communications
[2–7]. In humans, carotenoids serve as antioxidants to protect
against some cancer types [8], cardiovascular, neurodegenerative,
and eye diseases [9–11]. There is a high nutritional value associated
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_11,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
145