with carotenoids and research has been primed towards enhancing
the abundance of carotenoids and specific isoforms in agriculture
and protected cropping [12, 13], aquaculture industries [14], as
well as in skin medicines and micronutrient supplements
[15]. Since enzymatic and nonenzymatic oxidation as well as cis/
trans isomerization (triggered by light and temperature) affect
cleavage and carotenoid configuration, their stability, bioavailability, and biological functions can be difficult to elucidate
[16, 17]. Therefore, the identification, characterization, and absolute quantification of different carotenoid isomers can be crucial to
resolve complex metabolic phenomena in plants and animals.
In plants, carotenoid biosynthesis and storage can vary in different organs [18, 19] during development (young vs. old leaves
and stage of fruit ripening) [20, 21], and in response to changes in
temperature [22], light intensity and/or spectra [23], oxidative
stress [24], drought [25, 26], as well as the presence of biotic
factors like pathogens [27] or mutualistic organism like mycorrhizal fungi [28]. Depending on the plant tissue and plastid sink,
carotenoids may not be present in their free forms and some
additional processes can be required to enable quantification. For
instance, carotenoids present in callus and seed tissues are highly
esterified, which hinders their extraction and identification using
chromatographic methods like high-performance liquid chromatography (HPLC). A process known as saponification is necessary
to remove fatty acids bound to free carotenoids. Additional details
about this process can be found elsewhere [29].
Carotenoid extraction can be performed using organic solvents
like hexane, acetone, tetrahydrofuran (THF), or mixed solvents like
acetone–ethyl acetate (3:2, v/v) [30], hexane–isopropanol (1:1,
v/v) [31], THF–methanol (1:1, v/v) [32], and ethanol–hexane
(4:3, v/v) [33]. Here we refer the reader to a detailed report
describing frequently used carotenoid extraction methods
[34]. Individual carotenoids have different chemical properties in
terms of their molecular weights, structure, ionic charge, and
hydrophobicity. When carotenoid extracts are passed through a
reverse-phase column using an HPLC solvent gradient, these
chemical properties enable carotenoid separation by retention
time. The efficiency of HPLC separation methods depend on the
chemistry of both stationary and mobile phases which are comprised of a column and organic solvents, respectively. Due to the
similarity of some geometric and positional carotenoid isomers,
chromatographic separation can be a demanding task. There is a
range of carotenoid extraction methods, columns, and reverse
phase solvent gradients reported [35]. Therefore, selecting the
correct combination of organic solvents for extraction, reversephase column chemistry, and the solvent gradient improve the
chromatographic technique leading to a higher resolving power in
carotenoid binding and separation. This can improve detection of
146
Yagiz Alagoz et al.
the abundance of carotenoids and specific isoforms in agriculture
and protected cropping [12, 13], aquaculture industries [14], as
well as in skin medicines and micronutrient supplements
[15]. Since enzymatic and nonenzymatic oxidation as well as cis/
trans isomerization (triggered by light and temperature) affect
cleavage and carotenoid configuration, their stability, bioavailability, and biological functions can be difficult to elucidate
[16, 17]. Therefore, the identification, characterization, and absolute quantification of different carotenoid isomers can be crucial to
resolve complex metabolic phenomena in plants and animals.
In plants, carotenoid biosynthesis and storage can vary in different organs [18, 19] during development (young vs. old leaves
and stage of fruit ripening) [20, 21], and in response to changes in
temperature [22], light intensity and/or spectra [23], oxidative
stress [24], drought [25, 26], as well as the presence of biotic
factors like pathogens [27] or mutualistic organism like mycorrhizal fungi [28]. Depending on the plant tissue and plastid sink,
carotenoids may not be present in their free forms and some
additional processes can be required to enable quantification. For
instance, carotenoids present in callus and seed tissues are highly
esterified, which hinders their extraction and identification using
chromatographic methods like high-performance liquid chromatography (HPLC). A process known as saponification is necessary
to remove fatty acids bound to free carotenoids. Additional details
about this process can be found elsewhere [29].
Carotenoid extraction can be performed using organic solvents
like hexane, acetone, tetrahydrofuran (THF), or mixed solvents like
acetone–ethyl acetate (3:2, v/v) [30], hexane–isopropanol (1:1,
v/v) [31], THF–methanol (1:1, v/v) [32], and ethanol–hexane
(4:3, v/v) [33]. Here we refer the reader to a detailed report
describing frequently used carotenoid extraction methods
[34]. Individual carotenoids have different chemical properties in
terms of their molecular weights, structure, ionic charge, and
hydrophobicity. When carotenoid extracts are passed through a
reverse-phase column using an HPLC solvent gradient, these
chemical properties enable carotenoid separation by retention
time. The efficiency of HPLC separation methods depend on the
chemistry of both stationary and mobile phases which are comprised of a column and organic solvents, respectively. Due to the
similarity of some geometric and positional carotenoid isomers,
chromatographic separation can be a demanding task. There is a
range of carotenoid extraction methods, columns, and reverse
phase solvent gradients reported [35]. Therefore, selecting the
correct combination of organic solvents for extraction, reversephase column chemistry, and the solvent gradient improve the
chromatographic technique leading to a higher resolving power in
carotenoid binding and separation. This can improve detection of
146
Yagiz Alagoz et al.