temperature and should have an UV/Vis spectrophotometric
detector (if possible a diode array detector (DAD)).
6. Similar reversed-phase C18 columns from other suppliers can
be used, however selectivity may change in some cases and
elution order can be affected. For example, in our column
zeaxanthin elutes earlier than lutein, but in most C18 columns
zeaxanthin elutes right after lutein. Therefore, elution order
should be checked during method implementation by using
either carotenoid standards or natural extracts with well-known
carotenoid profile.
7. Traditionally, few carotenoids have been commercially available
in pure form. Consequently, authentic reference carotenoid
compounds have been isolated from natural sources with
well-known carotenoid profile and where the aimed compound, if possible, is a major one. A combination of solvent
partition and chromatographic techniques (TLC, open column
chromatography, analytical and semipreparative HPLC, etc.)
are used for this purpose. Guidance for the selection of appropriate natural sources, and recommendations for analytical
procedures for isolation, purification and identification are
found in the literature [8, 9, 27–31]. In the last decade, reliable
commercial suppliers have become available (e.g., CaroteNature GmbH, Extrasynthese SAS, and Sigma-Aldrich). Highly
experienced carotenoid labs still prefer to isolate their own
carotenoid standards since in most cases the purity is higher
than the commercial ones.
8. Follow adequate sampling procedures for guaranty representativeness as well as to minimize errors during quantification. For
carotenoids this has been recently reviewed by Rodrı ´guezAmaya [30].
9. In order to improve the extraction of carotenoids, dehydrated
and lyophilized samples should be reconstituted with water up
to the level present in the raw material. As a general rule five
parts of water are added for one part of dry sample.
10. Select a homogenization speed so that the formation of foam is
minimized and consequently reduce the degradation pigments
by decreasing contact with air.
11. β-Apo-8
0 -carotenal is the most common carotenoid used as
IS. The carotenoid used as IS needs to conform to some
important requirements, the first one being its absence in the
sample to be studied. The full of requirements for carotenoid
IS, as well as guidance in their use can be found in Khachik [8].
12. The dispersing tool used for homogenization is cleaned during
every successive extraction.
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Da ´ maso Hornero-Me ´ ndez
detector (if possible a diode array detector (DAD)).
6. Similar reversed-phase C18 columns from other suppliers can
be used, however selectivity may change in some cases and
elution order can be affected. For example, in our column
zeaxanthin elutes earlier than lutein, but in most C18 columns
zeaxanthin elutes right after lutein. Therefore, elution order
should be checked during method implementation by using
either carotenoid standards or natural extracts with well-known
carotenoid profile.
7. Traditionally, few carotenoids have been commercially available
in pure form. Consequently, authentic reference carotenoid
compounds have been isolated from natural sources with
well-known carotenoid profile and where the aimed compound, if possible, is a major one. A combination of solvent
partition and chromatographic techniques (TLC, open column
chromatography, analytical and semipreparative HPLC, etc.)
are used for this purpose. Guidance for the selection of appropriate natural sources, and recommendations for analytical
procedures for isolation, purification and identification are
found in the literature [8, 9, 27–31]. In the last decade, reliable
commercial suppliers have become available (e.g., CaroteNature GmbH, Extrasynthese SAS, and Sigma-Aldrich). Highly
experienced carotenoid labs still prefer to isolate their own
carotenoid standards since in most cases the purity is higher
than the commercial ones.
8. Follow adequate sampling procedures for guaranty representativeness as well as to minimize errors during quantification. For
carotenoids this has been recently reviewed by Rodrı ´guezAmaya [30].
9. In order to improve the extraction of carotenoids, dehydrated
and lyophilized samples should be reconstituted with water up
to the level present in the raw material. As a general rule five
parts of water are added for one part of dry sample.
10. Select a homogenization speed so that the formation of foam is
minimized and consequently reduce the degradation pigments
by decreasing contact with air.
11. β-Apo-8
0 -carotenal is the most common carotenoid used as
IS. The carotenoid used as IS needs to conform to some
important requirements, the first one being its absence in the
sample to be studied. The full of requirements for carotenoid
IS, as well as guidance in their use can be found in Khachik [8].
12. The dispersing tool used for homogenization is cleaned during
every successive extraction.
130
Da ´ maso Hornero-Me ´ ndez