7 Analytical Protocols in Carotenoid Analysis
169
7.2.5.2 Electrochemical Detection (ED)
ED is a detection technique suitable for those compounds with electroactivity and
carotenoids are electroactive and HPLC-ED systems are an alternative for their
analysis. The application of an electrical potential modifies the oxidation state of
the analytes, yielding hydrodynamic voltammograms, which are featured for each
compound. This technique is more sensitive than UV-visible detection but the number
of artifacts and interferences (background noise produced by solvent phase) considerably increases. Analytes are destroyed during the analysis, so that coupling with
a subsequent detection method is not allowed. Guaratini et al. (2009) revealed
the carotenoid profile in phytoplankton biomass (microalgae and cyanobacteria)
by HPLC-ED, and successfully compared the performance of this technique with
UV-visible detection.
7.2.6 Identification Methods
7.2.6.1 Mass Spectrometry (MS)
MS is an analytical technique where the analyst must develop the know-how in
different areas (hardware electronics, chemistry, experimental design, computing,
and big data management), and apply the best laboratory practices to fully take advantage of the potential capabilities of this tool. In the field of carotenoids analysis, MS is
mainly focused in the identification when spectrophotometry does not allow distinction between carotenoid structures with the same UV-visible spectra and similar chromatographic behavior, or when the acquisition of high-quality UV-visible spectrum
is not possible in low concentrated samples. Indeed, those carotenoid profiles where
unprecedent carotenoid structures or unknowns appear, and consequently the UVvisible spectrum does not provide enough information, MS is required for structural
elucidation. The reader is referred to excellent revisions dealing with the fundamentals of MS and the progress of the hardware and state-of -the-art set-ups currently
available, including those exclusively focused in carotenoids (Enzell and Wahlberg
1980; Britton et al. 1995; Dole 1997; de Rosso and Mercadante 2007; Rezanka et al.
2009; Rivera et al. 2011; van Breemen et al. 2012; Amorim-Carrilho et al. 2014),
while this section is dedicated to the identification strategy.
The first information contained in MS spectra acquired through soft-ionization
techniques (atmospheric pressure ionization) is the molecular weight of the protonated ion, denoted as [M+H]
+ , which allows to filter the elemental composition
among a huge amount of alternatives, the candidates, with the application of two
filtering rules: mass accuracy and isotopic pattern. Thus, when the mass accuracy
level is increased, the number of candidates is reduced, but still the list could be
reduced by the application of additional physicochemical information such as the
isotopic pattern. The experimental and theoretical isotopic patterns are compared,
and a correlation value is obtained for each candidate. Subsequently, the analyst must
169
7.2.5.2 Electrochemical Detection (ED)
ED is a detection technique suitable for those compounds with electroactivity and
carotenoids are electroactive and HPLC-ED systems are an alternative for their
analysis. The application of an electrical potential modifies the oxidation state of
the analytes, yielding hydrodynamic voltammograms, which are featured for each
compound. This technique is more sensitive than UV-visible detection but the number
of artifacts and interferences (background noise produced by solvent phase) considerably increases. Analytes are destroyed during the analysis, so that coupling with
a subsequent detection method is not allowed. Guaratini et al. (2009) revealed
the carotenoid profile in phytoplankton biomass (microalgae and cyanobacteria)
by HPLC-ED, and successfully compared the performance of this technique with
UV-visible detection.
7.2.6 Identification Methods
7.2.6.1 Mass Spectrometry (MS)
MS is an analytical technique where the analyst must develop the know-how in
different areas (hardware electronics, chemistry, experimental design, computing,
and big data management), and apply the best laboratory practices to fully take advantage of the potential capabilities of this tool. In the field of carotenoids analysis, MS is
mainly focused in the identification when spectrophotometry does not allow distinction between carotenoid structures with the same UV-visible spectra and similar chromatographic behavior, or when the acquisition of high-quality UV-visible spectrum
is not possible in low concentrated samples. Indeed, those carotenoid profiles where
unprecedent carotenoid structures or unknowns appear, and consequently the UVvisible spectrum does not provide enough information, MS is required for structural
elucidation. The reader is referred to excellent revisions dealing with the fundamentals of MS and the progress of the hardware and state-of -the-art set-ups currently
available, including those exclusively focused in carotenoids (Enzell and Wahlberg
1980; Britton et al. 1995; Dole 1997; de Rosso and Mercadante 2007; Rezanka et al.
2009; Rivera et al. 2011; van Breemen et al. 2012; Amorim-Carrilho et al. 2014),
while this section is dedicated to the identification strategy.
The first information contained in MS spectra acquired through soft-ionization
techniques (atmospheric pressure ionization) is the molecular weight of the protonated ion, denoted as [M+H]
+ , which allows to filter the elemental composition
among a huge amount of alternatives, the candidates, with the application of two
filtering rules: mass accuracy and isotopic pattern. Thus, when the mass accuracy
level is increased, the number of candidates is reduced, but still the list could be
reduced by the application of additional physicochemical information such as the
isotopic pattern. The experimental and theoretical isotopic patterns are compared,
and a correlation value is obtained for each candidate. Subsequently, the analyst must