gas chromatography (GC) or GC-MS [18, 19]. After transesterification with sodium methoxide, the algal lipid extracts contain variable
amounts of hydroxyl group-containing compounds like FFAs, nonesterified alkoxyglycerols, sterols, α-tocopherol, and phytol.
Subsequent transesterification and trimethylsilylation enable the
GC analysis of these compounds. The mixture contains then
FAMEs, trimethylsilyl (TMS) derivatives, and squalene and other
hydrocarbons.
2 Materials
2.1 Samples,
Standards, and
Chemicals
1. 10 mg freeze-dried Euglena gracilis (single-cell eukaryote
algae) or Chlorella pyrenoidosa (freshwater green algae) biomass
from bioreactors.
2. External standards including squalene (SQ), wax ester (WE),
sterol ester (SE), triacylglycerol (TAG), free sterol (ST), diacyland monoacylglycerol (DAG, MAG), free fatty acid (FFA),
alkyldiacylglycerol ether (DAGE), mono- and digalactosyldiacylglycerol (MGDG, DGDG), ceramide (Cer), cerebroside,
cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine
(PC), sphingomyelin (SPH), lysoPE, and lysoPC (see Note 1).
3. The internal standard mixture for GC-MS analyses of fatty
acids and sterols consists of heptadecanoic acid (C17:0) in the
form of free fatty acid (FFA) and triacylglycerol (TAG, triheptadecanoate). The samples are spiked with the standards before
extraction at concentration levels of 10–30 μg FFA and
50–70 μg TAG. For identification, commercial fatty acid
methyl ester (FAME) and FFA mixtures can be used.
4. Extraction solvents: chloroform/methanol (2:1, v/v), dichloromethane (DCM), petroleum ether bp. 40–60
C, hexane, and
15 mM saline solution (NaCl).
5. Derivatization reagents: sodium methoxide in methanol
(NaOMe, 0.5 M), sodium hydrogen sulfate (NaHSO 4 , 15%), Nmethyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and trimethylchlorosilane (TMCS).
2.2 UPLC-ELSD
System
1. Acquity
® H-class Ultra Performance LC (UPLC) with a quaternary solvent manager (Waters Corporation, Wexford,
Ireland).
2. Evaporative
light-scattering
detector
(ELSD)
(Waters
Corporation).
3. LC column: Spherisorb Silica column (2.1 Â 100 mm, 3 μm,
narrow bore) (Waters Corporation).
4. Solvent A: iso-octane (2,2,4-trimethylpentane) and tetrahydrofuran (99:1, v/v) (see Note 2).
UPLC-ELSD Analysis of Algal Lipid Classes and Derivatization of Bound and. . .
225
amounts of hydroxyl group-containing compounds like FFAs, nonesterified alkoxyglycerols, sterols, α-tocopherol, and phytol.
Subsequent transesterification and trimethylsilylation enable the
GC analysis of these compounds. The mixture contains then
FAMEs, trimethylsilyl (TMS) derivatives, and squalene and other
hydrocarbons.
2 Materials
2.1 Samples,
Standards, and
Chemicals
1. 10 mg freeze-dried Euglena gracilis (single-cell eukaryote
algae) or Chlorella pyrenoidosa (freshwater green algae) biomass
from bioreactors.
2. External standards including squalene (SQ), wax ester (WE),
sterol ester (SE), triacylglycerol (TAG), free sterol (ST), diacyland monoacylglycerol (DAG, MAG), free fatty acid (FFA),
alkyldiacylglycerol ether (DAGE), mono- and digalactosyldiacylglycerol (MGDG, DGDG), ceramide (Cer), cerebroside,
cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine
(PC), sphingomyelin (SPH), lysoPE, and lysoPC (see Note 1).
3. The internal standard mixture for GC-MS analyses of fatty
acids and sterols consists of heptadecanoic acid (C17:0) in the
form of free fatty acid (FFA) and triacylglycerol (TAG, triheptadecanoate). The samples are spiked with the standards before
extraction at concentration levels of 10–30 μg FFA and
50–70 μg TAG. For identification, commercial fatty acid
methyl ester (FAME) and FFA mixtures can be used.
4. Extraction solvents: chloroform/methanol (2:1, v/v), dichloromethane (DCM), petroleum ether bp. 40–60
C, hexane, and
15 mM saline solution (NaCl).
5. Derivatization reagents: sodium methoxide in methanol
(NaOMe, 0.5 M), sodium hydrogen sulfate (NaHSO 4 , 15%), Nmethyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and trimethylchlorosilane (TMCS).
2.2 UPLC-ELSD
System
1. Acquity
® H-class Ultra Performance LC (UPLC) with a quaternary solvent manager (Waters Corporation, Wexford,
Ireland).
2. Evaporative
light-scattering
detector
(ELSD)
(Waters
Corporation).
3. LC column: Spherisorb Silica column (2.1 Â 100 mm, 3 μm,
narrow bore) (Waters Corporation).
4. Solvent A: iso-octane (2,2,4-trimethylpentane) and tetrahydrofuran (99:1, v/v) (see Note 2).
UPLC-ELSD Analysis of Algal Lipid Classes and Derivatization of Bound and. . .
225