64
enter vapor phase under conditions that will alter their molecular structure. Thus, this
method is sensitive to the polarity of the compound and requires derivatization steps
to improve volatility (Christie 1993). Fatty acids have been analyzed as fatty acid
methyl esters (FAMEs) using GC/GC-MS generated either by acid-catalyzed transesterification (using BF 3 /HCl/H 2 SO 4 ) or alkali-catalyzed transmethylation using 2 M
ethanolic/methanolic KOH of lipid extracts. Oxylipins and sterols have been mostly
analyzed by methylation with ethereal diazomethane and silylation with a mixture of
BSTFA (N,N-bistrimethylsilyl-trifluoroacetamide)/TMCS (trimethylchlorosilane)
(Bouarab et al. 2004; Choi et al. 2012; Kamenarska et al. 2004; Lion et al. 2006;
Wiesemeier et al. 2008). The major disadvantage of GC/GC-MS has been lower
sensitivity for less abundant species, and it yields information on the hydrolysis
products of lipids, not on the parent compounds, and thus, the identification of lipid
classes and the information of fatty acid main location cannot be retrieved.
NMR is a strong nondestructive and nonselective technique to identify a wide
variety of lipids without losing chemical information about the analyte environment
in biological systems (Gross and Han 2011). It provides unique information about
molecular structure and dynamics; however, its sensitivity and resolving power to
distinguish individual chemical species is limited and complicated due to considerable number of spin-coupled multiplets. NMR in seaweeds has been mainly
employed for structural characterization of purified bioactive lipid compounds and
novel oxylipins (Al-Fadhli et al. 2006; Choi et al. 2012; Kousaka et al. 2003; Todd
et al. 1993, 1994; Williams et al. 2007) instead of lipidomic studies. It is anticipated
that it may develop as a powerful lipidomic tool in the future through the synergistic
application of a solution-state and solid-state NMR approaches in seaweeds.
LC (or HPLC) is an analytical tool for separation of different subsets of lipid
molecules such as lipid classes, oxylipins, and sterols and is usually coupled with
evaporative light scattering detector (ELSD), UV, or MS, recently reviewed by Pati
et al. (2016). The lipid analysis is performed using normal phase (NP), reverse
phase (RP), or hydrophilic interactions (HILIC). In NPLC and HILIC, lipid molecules are distinguished by their hydrophilic properties and separate them according
to their polar head groups. NPLC and HILIC are suitable methods for the separation
of lipid classes and different groups of oxylipins (hydroxy, epoxy, oxo, and others).
RPLC distinguish lipid molecules by their hydrophobic properties and separates
them according to their length and unsaturation. RPLC coupled with MS is the most
widely used method for analysis of complex lipids (Al-Fadhli et al. 2006; El-Baroty
et al. 2011; Kendel et al. 2015; Kim et al. 2007; Williams et al. 2007) and oxylipins
(Barbosa et al. 2015; Bouarab et al. 2004; Choi et al. 2012; Collén et al. 2013;
Gaquerel et al. 2007; Küpper et al. 2009; Ritter et al. 2008, 2014,Weinberger et al.
2011) in various seaweeds including the species of Avrainvillea, Chondria,
Chondrus, Cymathere, Codium, Dilophys, Ectocarpus, Fucus, Gracilaria,
Laminaria, Padina, Plocamium, Sargassum, Solieria, Ulva, and others. Recently,
Jacquemoud and Pohnert (2015) developed a protocol for the comprehensive analysis of oxylipins (including extraction, purification, chromatographic, and mass
spectrometric procedures) for G. vermiculophylla that can be applicable to any
P. Kumari
enter vapor phase under conditions that will alter their molecular structure. Thus, this
method is sensitive to the polarity of the compound and requires derivatization steps
to improve volatility (Christie 1993). Fatty acids have been analyzed as fatty acid
methyl esters (FAMEs) using GC/GC-MS generated either by acid-catalyzed transesterification (using BF 3 /HCl/H 2 SO 4 ) or alkali-catalyzed transmethylation using 2 M
ethanolic/methanolic KOH of lipid extracts. Oxylipins and sterols have been mostly
analyzed by methylation with ethereal diazomethane and silylation with a mixture of
BSTFA (N,N-bistrimethylsilyl-trifluoroacetamide)/TMCS (trimethylchlorosilane)
(Bouarab et al. 2004; Choi et al. 2012; Kamenarska et al. 2004; Lion et al. 2006;
Wiesemeier et al. 2008). The major disadvantage of GC/GC-MS has been lower
sensitivity for less abundant species, and it yields information on the hydrolysis
products of lipids, not on the parent compounds, and thus, the identification of lipid
classes and the information of fatty acid main location cannot be retrieved.
NMR is a strong nondestructive and nonselective technique to identify a wide
variety of lipids without losing chemical information about the analyte environment
in biological systems (Gross and Han 2011). It provides unique information about
molecular structure and dynamics; however, its sensitivity and resolving power to
distinguish individual chemical species is limited and complicated due to considerable number of spin-coupled multiplets. NMR in seaweeds has been mainly
employed for structural characterization of purified bioactive lipid compounds and
novel oxylipins (Al-Fadhli et al. 2006; Choi et al. 2012; Kousaka et al. 2003; Todd
et al. 1993, 1994; Williams et al. 2007) instead of lipidomic studies. It is anticipated
that it may develop as a powerful lipidomic tool in the future through the synergistic
application of a solution-state and solid-state NMR approaches in seaweeds.
LC (or HPLC) is an analytical tool for separation of different subsets of lipid
molecules such as lipid classes, oxylipins, and sterols and is usually coupled with
evaporative light scattering detector (ELSD), UV, or MS, recently reviewed by Pati
et al. (2016). The lipid analysis is performed using normal phase (NP), reverse
phase (RP), or hydrophilic interactions (HILIC). In NPLC and HILIC, lipid molecules are distinguished by their hydrophilic properties and separate them according
to their polar head groups. NPLC and HILIC are suitable methods for the separation
of lipid classes and different groups of oxylipins (hydroxy, epoxy, oxo, and others).
RPLC distinguish lipid molecules by their hydrophobic properties and separates
them according to their length and unsaturation. RPLC coupled with MS is the most
widely used method for analysis of complex lipids (Al-Fadhli et al. 2006; El-Baroty
et al. 2011; Kendel et al. 2015; Kim et al. 2007; Williams et al. 2007) and oxylipins
(Barbosa et al. 2015; Bouarab et al. 2004; Choi et al. 2012; Collén et al. 2013;
Gaquerel et al. 2007; Küpper et al. 2009; Ritter et al. 2008, 2014,Weinberger et al.
2011) in various seaweeds including the species of Avrainvillea, Chondria,
Chondrus, Cymathere, Codium, Dilophys, Ectocarpus, Fucus, Gracilaria,
Laminaria, Padina, Plocamium, Sargassum, Solieria, Ulva, and others. Recently,
Jacquemoud and Pohnert (2015) developed a protocol for the comprehensive analysis of oxylipins (including extraction, purification, chromatographic, and mass
spectrometric procedures) for G. vermiculophylla that can be applicable to any
P. Kumari