are based on pioneer studies on mass spectrometry of carotenoids
[3–8], which have been recently reviewed [9]. Hence, fast atom
bombardment (FAB) ionization has been replaced by atmospheric
pressure ionization (API) methods for the ionization of most natural products, and such improvement reached the carotenoids.
Although electrospray ionization (ESI) and atmospheric pressure
chemical ionization (APCI) show both advantages and drawbacks
for ionization of carotenoids, the use of APCI source has probably
become the standard choice. Similarly, ion trap (IT) and time of
flight (TOF) are the conventional hardware designs for mass analysis at the ionization downstream, IT for structural elucidation
through ion accumulation of selected protonated molecular ions
which are subjected to MS
n analysis (tandem-in-time), and TOF for
acquisition of accurate mass and isotopic pattern values by filtering
protonated molecular ions (tandem-in-space). Anyhow, with the
modern soft ionization techniques, tandem mass spectrometry has
emerged as an essential strategy, as in addition to increase the
accuracy of mass measurements, it allows to figure out certain
reaction patterns during the fragmentation process of the protonated molecular ions. Accordingly, the final aim is to allocate qualifier ions that simplify the identification process. Furthermore, it is
possible to reach high-resolution mass spectrometry (hr-MS) with
the modern equipment, so that the possibility of inaccurate identifications is considerably reduced [10]. These analytical advancements empower the performance of metabolomics analysis, and
two approaches can be pursued: targeted or untargeted. The former applies when the study is focused in known carotenoids, while
the latter is performed if the analysis comprised the study of all
carotenoids present, either known or unraveled structures. In any
case, the complexity of metabolomics studies demands the existence of specific workflows to consistently analyze carotenoids from
different sources. The present protocol describes a specific workflow for mass spectrometry analysis of carotenoids, with special
emphasis in the assistance of post-processing software. Although
the protocol has been focused on licensed software, free applications with comparable capabilities can be downloaded on the
internet.
2 Materials
2.1 Equipment
1. Homogenizer, filter paper, separating funnel.
2. Rotary evaporator.
3. LC-MS system and software for instrument control.
4. Analytical column.
5. Computer and software for MS data analysis.
136
Antonio Pe ´ rez-Ga ´ lvez et al.
[3–8], which have been recently reviewed [9]. Hence, fast atom
bombardment (FAB) ionization has been replaced by atmospheric
pressure ionization (API) methods for the ionization of most natural products, and such improvement reached the carotenoids.
Although electrospray ionization (ESI) and atmospheric pressure
chemical ionization (APCI) show both advantages and drawbacks
for ionization of carotenoids, the use of APCI source has probably
become the standard choice. Similarly, ion trap (IT) and time of
flight (TOF) are the conventional hardware designs for mass analysis at the ionization downstream, IT for structural elucidation
through ion accumulation of selected protonated molecular ions
which are subjected to MS
n analysis (tandem-in-time), and TOF for
acquisition of accurate mass and isotopic pattern values by filtering
protonated molecular ions (tandem-in-space). Anyhow, with the
modern soft ionization techniques, tandem mass spectrometry has
emerged as an essential strategy, as in addition to increase the
accuracy of mass measurements, it allows to figure out certain
reaction patterns during the fragmentation process of the protonated molecular ions. Accordingly, the final aim is to allocate qualifier ions that simplify the identification process. Furthermore, it is
possible to reach high-resolution mass spectrometry (hr-MS) with
the modern equipment, so that the possibility of inaccurate identifications is considerably reduced [10]. These analytical advancements empower the performance of metabolomics analysis, and
two approaches can be pursued: targeted or untargeted. The former applies when the study is focused in known carotenoids, while
the latter is performed if the analysis comprised the study of all
carotenoids present, either known or unraveled structures. In any
case, the complexity of metabolomics studies demands the existence of specific workflows to consistently analyze carotenoids from
different sources. The present protocol describes a specific workflow for mass spectrometry analysis of carotenoids, with special
emphasis in the assistance of post-processing software. Although
the protocol has been focused on licensed software, free applications with comparable capabilities can be downloaded on the
internet.
2 Materials
2.1 Equipment
1. Homogenizer, filter paper, separating funnel.
2. Rotary evaporator.
3. LC-MS system and software for instrument control.
4. Analytical column.
5. Computer and software for MS data analysis.
136
Antonio Pe ´ rez-Ga ´ lvez et al.