age-related macular degeneration (AMD) and cataracts), enhancement of the immune response, and cell protection against free
radicals [3, 4].
Thus, the qualitative and quantitative analysis of carotenoids is
of paramount importance for the investigation of all these associated aspects. Chromatography, and especially HPLC, has always
been the choice methodology for attaining this task. In fact, the
invention and development of the initial chromatography technique by Mikhail Tswett in the early 1900s was closely linked to
the study of chloroplast pigments [5, 6]. The first HPLC application for the separation of carotenoids was reported by Stewart and
Wheaton in 1971 for the investigation of the carotenoid composition of Citrus extracts [7]. Since then, important advances and
innovations have been made in the HPLC technique, including
the development of new stationary phases well as sensible, selective
and robust detectors (mainly DAD and MS detectors), which have
been applied to the carotenoid research field [8]. Consequently, a
vast diversity of HPLC methods can be found in the carotenoid
literature. The protocol here presented has been used routinely in
the author’s lab during the last 25 years for analysing carotenoids in
great variety of plant and food matrices [9]. This method was firstly
developed for the separation and quantification of the complex
carotenoid profile in red pepper fruits (Capsicum annuum) and
derived products such as paprika powder and oleoresin [9]. The
HPLC method uses a C18 reverse-phase and a binary gradient
composed by acetone and deionized water, providing a rapid
(β-carotene elutes at 21 min) and very reproducible separation.
The optimised conditions have given excellent results when used
for the major carotenoid analysis in a wide variety of samples such as
green vegetables, apple fruits, potato tubers, cereal grains, dates,
strawberry fruits, rose hips, sarsaparilla berries, fungi extracts, and
orange juice [9–20]. Additionally, the present HPLC conditions
have been applied to the analysis of carotenoids in fungi and bacteria [21, 22], and in animal samples such as plasma, egg-yolk, and
skin [23–25].
2 Materials
Reagents should be at least of analytical grade, unless stated
otherwise.
2.1 Sample
Pretreatment
Plant material and food-related samples can be analyzed either in
fresh (solid or liquid) or dehydrated state (see Note 1). Solid and
semisolid materials, such as fruits and vegetables, should be
chopped and mixed with a household blender right before extraction. In the case of harder material (i.e., cereals and other seeds) a
lab mill can be used (see Note 2).
118
Da ´ maso Hornero-Me ´ ndez
radicals [3, 4].
Thus, the qualitative and quantitative analysis of carotenoids is
of paramount importance for the investigation of all these associated aspects. Chromatography, and especially HPLC, has always
been the choice methodology for attaining this task. In fact, the
invention and development of the initial chromatography technique by Mikhail Tswett in the early 1900s was closely linked to
the study of chloroplast pigments [5, 6]. The first HPLC application for the separation of carotenoids was reported by Stewart and
Wheaton in 1971 for the investigation of the carotenoid composition of Citrus extracts [7]. Since then, important advances and
innovations have been made in the HPLC technique, including
the development of new stationary phases well as sensible, selective
and robust detectors (mainly DAD and MS detectors), which have
been applied to the carotenoid research field [8]. Consequently, a
vast diversity of HPLC methods can be found in the carotenoid
literature. The protocol here presented has been used routinely in
the author’s lab during the last 25 years for analysing carotenoids in
great variety of plant and food matrices [9]. This method was firstly
developed for the separation and quantification of the complex
carotenoid profile in red pepper fruits (Capsicum annuum) and
derived products such as paprika powder and oleoresin [9]. The
HPLC method uses a C18 reverse-phase and a binary gradient
composed by acetone and deionized water, providing a rapid
(β-carotene elutes at 21 min) and very reproducible separation.
The optimised conditions have given excellent results when used
for the major carotenoid analysis in a wide variety of samples such as
green vegetables, apple fruits, potato tubers, cereal grains, dates,
strawberry fruits, rose hips, sarsaparilla berries, fungi extracts, and
orange juice [9–20]. Additionally, the present HPLC conditions
have been applied to the analysis of carotenoids in fungi and bacteria [21, 22], and in animal samples such as plasma, egg-yolk, and
skin [23–25].
2 Materials
Reagents should be at least of analytical grade, unless stated
otherwise.
2.1 Sample
Pretreatment
Plant material and food-related samples can be analyzed either in
fresh (solid or liquid) or dehydrated state (see Note 1). Solid and
semisolid materials, such as fruits and vegetables, should be
chopped and mixed with a household blender right before extraction. In the case of harder material (i.e., cereals and other seeds) a
lab mill can be used (see Note 2).
118
Da ´ maso Hornero-Me ´ ndez