Abstract:Membrane lipids of photosynthetic organisms consist of glycerophospholipids and glyceroglycolipids. We investigated a method for the simultaneous quantitative analysis of neutral and acidic lipids using HPLC-ELSD, and quantified monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG). Ten complex lipid classes were separated with a binary gradient system consisting of chloroform and methanol-acetone-water-acetic acid (30:60:9:1, v/v/v/v) with 0.3% triethy… Show more
“…With these properties, HILIC is similar to silica gel chromatography. However, the chromatography solvents were different: in our method, we used a mixture of methanol, acetonitrile, and water, while ordinary silica gel chromatography for plant lipid analysis is often performed using chloroform (Sugawara and Miyazawa 1999; Malavolta et al 2004; Pacetti et al 2007; Yunoki et al 2009; Rocha et al 2010). Thus, lipid analysis by HILIC can reduce toxic halogenated solvent waste and is better suited for electrospray ionization, the most normal ionization mode of LC-MS.Fig.…”
Plants synthesize a wide range of hydrophobic compounds, generally known as lipids. Here, we report an application of liquid chromatography ion trap time-of-flight mass spectrometry (LC-IT-TOF-MS) for plant lipidomics. Using hydrophilic interaction chromatography (HILIC) for class separation, typical membrane lipids including glycerolipids, steryl glucosides and glucosylceramides, and hydrophobic plant secondary metabolites such as saponins were analyzed simultaneously. By this method, we annotated approximately 100 molecules from Arabidopsis thaliana. To demonstrate the application of this method to biological study, we analyzed Arabidopsis mutant trigalactosyldiacylglycerol3 (tgd3), which has a complex metabolic phenotype including the accumulation of unusual forms of galactolipids. Lipid profiling by LC-MS revealed that tgd3 accumulated an unusual form of digalactosyldiacylglycerol, annotated as Gal(β1 → 6)βGalDG. The compositional difference between normal and unusual forms of digalactosyldiacylglycerol was detected by this method. In addition, we analyzed well-known Arabidopsis mutants ats1-1, fad6-1, and fad7-2, which are also disrupted in lipid metabolic genes. Untargeted lipidome analysis coupled with multivariate analysis clearly discriminated the mutants and their distinctive metabolites. These results indicated that HILIC-MS is an efficient method for plant lipidomics.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-011-0318-z) contains supplementary material, which is available to authorized users.
“…With these properties, HILIC is similar to silica gel chromatography. However, the chromatography solvents were different: in our method, we used a mixture of methanol, acetonitrile, and water, while ordinary silica gel chromatography for plant lipid analysis is often performed using chloroform (Sugawara and Miyazawa 1999; Malavolta et al 2004; Pacetti et al 2007; Yunoki et al 2009; Rocha et al 2010). Thus, lipid analysis by HILIC can reduce toxic halogenated solvent waste and is better suited for electrospray ionization, the most normal ionization mode of LC-MS.Fig.…”
Plants synthesize a wide range of hydrophobic compounds, generally known as lipids. Here, we report an application of liquid chromatography ion trap time-of-flight mass spectrometry (LC-IT-TOF-MS) for plant lipidomics. Using hydrophilic interaction chromatography (HILIC) for class separation, typical membrane lipids including glycerolipids, steryl glucosides and glucosylceramides, and hydrophobic plant secondary metabolites such as saponins were analyzed simultaneously. By this method, we annotated approximately 100 molecules from Arabidopsis thaliana. To demonstrate the application of this method to biological study, we analyzed Arabidopsis mutant trigalactosyldiacylglycerol3 (tgd3), which has a complex metabolic phenotype including the accumulation of unusual forms of galactolipids. Lipid profiling by LC-MS revealed that tgd3 accumulated an unusual form of digalactosyldiacylglycerol, annotated as Gal(β1 → 6)βGalDG. The compositional difference between normal and unusual forms of digalactosyldiacylglycerol was detected by this method. In addition, we analyzed well-known Arabidopsis mutants ats1-1, fad6-1, and fad7-2, which are also disrupted in lipid metabolic genes. Untargeted lipidome analysis coupled with multivariate analysis clearly discriminated the mutants and their distinctive metabolites. These results indicated that HILIC-MS is an efficient method for plant lipidomics.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-011-0318-z) contains supplementary material, which is available to authorized users.
“…Seaweeds synthesize three major types of GL: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG) (Adhikari et al, 2006;Yunoki et al, 2009). …”
“…For example, nine TAG from Walnut ( Juglans regia L.) were separated and identified by HPLC–ELSD including trilinolein, dilinoleoyl-oleoyl-glycerol and dilinolenoyl-glycerol as the major TAG at 38, 19 and 18 %, respectively [7]. In addition, ten complex lipid classes were separated with a binary gradient system by HPLC–ELSD and glyceroglycolipids including monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG) were quantified simultaneously in different plants [8] and 17 neutral and polar lipid classes were separated by HPLC–ELSD using a monolithic silica phase and quantified in four different zooplankton [9]. A 15 min HPLC gradient with hexane based solvent and isopropanol based solvent is used to resolve carotenoids, TAG, DAG and free fatty acids, and the major membrane components: phosphophatidylcholine (PtdCho), MGDG and DGDG.…”
Triacylglycerol (TAG) analysis and quantification are commonly performed by first obtaining a purified TAG fraction from a total neutral lipid extract using thin-layer chromatography (TLC), and then analyzing the fatty acid composition of the purified TAG fraction by gas chromatography (GC). This process is time-consuming, labor intensive and is not suitable for analysis of small sample sizes or large numbers. A rapid and efficient method for monitoring oil accumulation in algae using high performance liquid chromatography for separation of all lipid classes combined with detection by evaporative light scattering (HPLC–ELSD) was developed and compared to the conventional TLC/GC method. TAG accumulation in two Chlamydomonas reinhardtii (21 gr and CC503) and three Chlorella strains (UTEX 1230, CS01 and UTEX 2229) grown under conditions of nitrogen depletion was measured. The TAG levels were found to be 3–6 % DW (Chlamydomonas strains) and 7–12 % DW (Chlorella strains) respectively by both HPLC–ELSD and TLC/GC methods. HPLC–ELSD resolved the major lipid classes such as carotenoids, TAG, diacylglycerol (DAG), free fatty acids, phospholipids, and galactolipids in a 15-min run. Quantitation of TAG content was based on comparison to calibration curves of trihexadecanoin (16:0 TAG) and trioctadecadienoin (18:2 TAG) and showed linearity from 0.2 to 10 μg. Algal TAG levels >0.5 μg/g DW were detectable by this method. Furthermore TAG content in Chlorella kessleri UTEX 2229 could be detected. TAG as well as DAG and TAG content were estimated at 1.6 % DW by HPLC–ELSD, while it was undetectable by TLC/GC method.
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