Methods in Molecular Biology (2020) 1980: 223–232
DOI 10.1007/7651_2017_109
© Springer Science+Business Media New York 2017
Published online: 21 November 2017
UPLC-ELSD Analysis of Algal Lipid Classes and
Derivatization of Bound and Free Fatty Acids and Sterols for
GC-MS Methods
Tuulikki Sepp € anen-Laakso, Heli Nygren, and Heiko Rischer
Abstract
Constituents of microalgae and sample preparation for UPLC-ELSD and GC-MS analyses are described.
Bound fatty acids from acylglycerols, alkylacylglycerols, galactosyldiacylglycerols, glycerophospholipids,
and sterol esters are derivatized by using transesterification with sodium methoxide to form fatty acid
methyl esters. Compounds containing free hydroxyl groups, either present originally or formed during
previous step, like free fatty acids, sterols, α-tocopherol, phytol, and nonesterified alkoxyglycerols, are
trimethylsilylated. The compounds in algal lipid extract are subsequently derivatized by these two steps.
Keywords Lipid classes, Evaporative light-scattering detection, Fatty acids, Gas chromatographymass spectrometry, Transesterification, Trimethylsilylation, Ultra performance liquid chromatography
1 Introduction
Both macro- and microalgae thrive in very diverse and extreme
environments and produce specific bioactive substances and complex lipids and fatty acids [1]. The considerable variation is being
utilized in selecting strains with adequate growth rate and accumulation of desired lipid quantities and compositions. Attention has
also been paid to the possibility to obtain high-added value chemicals for nutritional, cosmetic, and pharmaceutical uses [2]. The use
of versatile and novel extraction and analysis methods is a prerequisite for characterization and identification of the highly variable and
heterogeneous lipid constituents in algae.
Considerable differences in the lipid content (1–75%) and composition appear among various microalgae species and within the same
genus [2]. Besides synthesizing storage lipids, microalgae can be
induced to accumulate them under stress conditions, e.g., nitrogen
limitation. Screening of brackish and marine microalgae (19 species)
grown under different conditions revealed a significant dependency
between decrease in cell nitrogen and increase in lipids [3].
The biomass of the microalgae Chlorella vulgaris, for example, is
composed of lipids (18%, dry weight), protein (28%), ash (11%), and
223
DOI 10.1007/7651_2017_109
© Springer Science+Business Media New York 2017
Published online: 21 November 2017
UPLC-ELSD Analysis of Algal Lipid Classes and
Derivatization of Bound and Free Fatty Acids and Sterols for
GC-MS Methods
Tuulikki Sepp € anen-Laakso, Heli Nygren, and Heiko Rischer
Abstract
Constituents of microalgae and sample preparation for UPLC-ELSD and GC-MS analyses are described.
Bound fatty acids from acylglycerols, alkylacylglycerols, galactosyldiacylglycerols, glycerophospholipids,
and sterol esters are derivatized by using transesterification with sodium methoxide to form fatty acid
methyl esters. Compounds containing free hydroxyl groups, either present originally or formed during
previous step, like free fatty acids, sterols, α-tocopherol, phytol, and nonesterified alkoxyglycerols, are
trimethylsilylated. The compounds in algal lipid extract are subsequently derivatized by these two steps.
Keywords Lipid classes, Evaporative light-scattering detection, Fatty acids, Gas chromatographymass spectrometry, Transesterification, Trimethylsilylation, Ultra performance liquid chromatography
1 Introduction
Both macro- and microalgae thrive in very diverse and extreme
environments and produce specific bioactive substances and complex lipids and fatty acids [1]. The considerable variation is being
utilized in selecting strains with adequate growth rate and accumulation of desired lipid quantities and compositions. Attention has
also been paid to the possibility to obtain high-added value chemicals for nutritional, cosmetic, and pharmaceutical uses [2]. The use
of versatile and novel extraction and analysis methods is a prerequisite for characterization and identification of the highly variable and
heterogeneous lipid constituents in algae.
Considerable differences in the lipid content (1–75%) and composition appear among various microalgae species and within the same
genus [2]. Besides synthesizing storage lipids, microalgae can be
induced to accumulate them under stress conditions, e.g., nitrogen
limitation. Screening of brackish and marine microalgae (19 species)
grown under different conditions revealed a significant dependency
between decrease in cell nitrogen and increase in lipids [3].
The biomass of the microalgae Chlorella vulgaris, for example, is
composed of lipids (18%, dry weight), protein (28%), ash (11%), and
223