7 Analytical Protocols in Carotenoid Analysis
163
the method to reach high analytical standards. Indeed, several good laboratory practices are extremely recommended in the application of quality-assurance procedures,
because this practice reduces the number of factors introducing uncertainty in the
analytical data. Hence, the use of integrated extinction coefficients, application of
batch quantification limit, statistical procedures applied for estimation of linearity of
the calibration curves, and purity of the standards significantly increase the quality
of the method and deserve attention of the analyst as well (Claustre et al. 2004).
The recent evolution of the column packings from spherical particles with diameter of 3–5 μm to sub-2 μm particles has produced an extraordinary increment in
the efficiency of the liquid chromatography regarding time and cost of the analysis,
and greater separation power and performance of the results. The design of such
column products has been tied with the manufacture of instruments able to deliver
pressures higher than the conventional 400 bar (Mazzeo et al. 2005). Some of the
methods detailed in Table 7.1 operate under UHPLC conditions, while those annotated with HPLC settings are susceptible to be transferred to the UHPLC procedure
with the use of convenient tools (most of them are available in the web sites of
column dealers). The reader is also referred to specific publications regarding the
theory and equations applied in the transfer of methods from HPLC to UHPLC
conditions (Guillarme et al. 2007, 2008). As it was mentioned above, these developments are convenient in the hyphenation of liquid chromatography with mass
analyzers to take full advantage of the resolving power and high sensitivity that
features mass spectrometry. Some methods that were not specifically developed for
carotenoid analysis in phytoplankton biomass should be considered by the reader,
such as Guzman et al. (2012) aimed for determination of the photosynthetic pigments
in Brassica oleracea vegetables, the chromatographic separation established by Li
et al. (2012) that provides a complete scenario of different carotenoid isomers in
tomatoes, and the method published by Chauveau-Duriot et al. (2010) that achieves
the simultaneous determination of carotenoids and vitamins A and E in different
food sources. Although the advances in UHPLC have attractive characteristics for
the high-throughput screening of carotenoids, still the high selectivity of C 30 HPLC
columns deserves attention for thorough analysis of undetermined carotenoid profile
(Bijttebier et al. 2014), which may occur in unraveled phytoplankton species. Nevertheless, column packings with C 30 material developed for UHPLC conditions are
also available (Zoccali et al. 2017). The reader will find valuable information in the
work published by Turcsi et al. (2016) where a database containing the separation
characteristics of carotenoids in C 18 and C 30 stationary phases is provided. Hence, the
retention and elution order of ca. 90 carotenoids is detailed considering their structural features for hydrocarbons and polar carotenoids, optical isomers, geometrical
isomers, and epoxy-carotenoids.
Application of multidimensional LC (LC × LC) has been proposed as an excellent alternative to analyze the carotenoid profile in complex extracts, particularly
in the study of carotenoid esters. In this set-up, the extract is analyzed in the 2-D
independently, by using a switching valve as a transfer system between the columns
(Dugo et al. 2006, 2008a). Moreover, the number of analytical conditions increases
as the set-up may include normal-phased columns coupled to reverse-phased ones,
163
the method to reach high analytical standards. Indeed, several good laboratory practices are extremely recommended in the application of quality-assurance procedures,
because this practice reduces the number of factors introducing uncertainty in the
analytical data. Hence, the use of integrated extinction coefficients, application of
batch quantification limit, statistical procedures applied for estimation of linearity of
the calibration curves, and purity of the standards significantly increase the quality
of the method and deserve attention of the analyst as well (Claustre et al. 2004).
The recent evolution of the column packings from spherical particles with diameter of 3–5 μm to sub-2 μm particles has produced an extraordinary increment in
the efficiency of the liquid chromatography regarding time and cost of the analysis,
and greater separation power and performance of the results. The design of such
column products has been tied with the manufacture of instruments able to deliver
pressures higher than the conventional 400 bar (Mazzeo et al. 2005). Some of the
methods detailed in Table 7.1 operate under UHPLC conditions, while those annotated with HPLC settings are susceptible to be transferred to the UHPLC procedure
with the use of convenient tools (most of them are available in the web sites of
column dealers). The reader is also referred to specific publications regarding the
theory and equations applied in the transfer of methods from HPLC to UHPLC
conditions (Guillarme et al. 2007, 2008). As it was mentioned above, these developments are convenient in the hyphenation of liquid chromatography with mass
analyzers to take full advantage of the resolving power and high sensitivity that
features mass spectrometry. Some methods that were not specifically developed for
carotenoid analysis in phytoplankton biomass should be considered by the reader,
such as Guzman et al. (2012) aimed for determination of the photosynthetic pigments
in Brassica oleracea vegetables, the chromatographic separation established by Li
et al. (2012) that provides a complete scenario of different carotenoid isomers in
tomatoes, and the method published by Chauveau-Duriot et al. (2010) that achieves
the simultaneous determination of carotenoids and vitamins A and E in different
food sources. Although the advances in UHPLC have attractive characteristics for
the high-throughput screening of carotenoids, still the high selectivity of C 30 HPLC
columns deserves attention for thorough analysis of undetermined carotenoid profile
(Bijttebier et al. 2014), which may occur in unraveled phytoplankton species. Nevertheless, column packings with C 30 material developed for UHPLC conditions are
also available (Zoccali et al. 2017). The reader will find valuable information in the
work published by Turcsi et al. (2016) where a database containing the separation
characteristics of carotenoids in C 18 and C 30 stationary phases is provided. Hence, the
retention and elution order of ca. 90 carotenoids is detailed considering their structural features for hydrocarbons and polar carotenoids, optical isomers, geometrical
isomers, and epoxy-carotenoids.
Application of multidimensional LC (LC × LC) has been proposed as an excellent alternative to analyze the carotenoid profile in complex extracts, particularly
in the study of carotenoid esters. In this set-up, the extract is analyzed in the 2-D
independently, by using a switching valve as a transfer system between the columns
(Dugo et al. 2006, 2008a). Moreover, the number of analytical conditions increases
as the set-up may include normal-phased columns coupled to reverse-phased ones,