Analysis of monogalactosyldiacylglycerol(MGDG) and digalactosyldiacylglycerol (DGDG)
as heptafluorobutyrate derivatives of their deacylated products by gas-liquid
chromatography
Eduard V. Nekrasov, Mikhail Vyssotski
INTRODUCTION
Structure and occurrence of MGDG and DGDG
1,2-diacyl-[β-D-galactopyranosyl-(1′→3)]-sn-glycerol (monogalactosyldiacylglycerol, MGDG)
1,2-diacyl-[α-D-galactopyranosyl-(1′→6′)]-β-D-galactopyranosyl-(1′→3)-sn-glycerol (digalactosyldiacylglycerol, DGDG)
Occurrence: photosynthetic organisms (plants, cyanobacteria)
Cell localization: membranes of chloroplasts and other forms of plastids (plants), thylakoids of cyanobacteria
Abundance: major polar lipids in photosynthetic tissues
O
CH2OH
OH
H
H
OH H
OH
O
H
CH2H
CHOCO
CH2OCO
OCH2OH
OH
H
H
OH H
OH
H
O
H
OCH2
OH
H
H
OH H
OH
O
H
CH2H
CHOC
O
CH2OCO
INTRODUCTIONPractical applications
Pharmacological activity• MGDG and MGMG from spinach: growth inhibitor of human
gastric cancer cells via inhibition of mammalian DNA polymerases• MGDG and DGDG from higher plants: antiinflammatory activity
Beneficial effects on the intestinal environment
Potential industrial application as natural detergents due to the hydrophilic property of the carbohydrate polar head groups
INTRODUCTIONAnalysis of glycolipids
Khotimchenko (2005) Chem. Natur. Comp. 41, 285-288.
Overlapping of compounds; separation of molecular species within a lipid class
No calibration is requiredGC analysis of fatty acid methyl esters of glycolipids scraped from TLC plate
Parrish et al. (1996) J. Chromat. A 741, 91-97.
One-dimensional; limitation of material loads; see also above
Fast analysisflame-ionisation detector (Iatroscan)
Heinz (1996) Advances in Lipid Methodology –Three. The Oily Press Ltd. 211-332.
Calibration for individual compounds; overlapping of compounds; separation of molecular species within a lipid class
Fast analysisdensitometry after charring or specific staining
Roughan & Batt (1968) Anal. Biochem. 22, 74-88.
Laborious, time-consumingEasy to calibratecolorimetry (phenol-sulfuric acid, anthrone)
Complex mixture can be separated; no expensive equipment is required
Thin-layer chromatography coupled with:
ReferenceDisadvantagesAdvantagesMethod of separation and analysis
INTRODUCTIONAnalysis of glycolipids (cont.)
ReferenceDisadvantagesAdvantagesMethod of separation and analysis
Williams et al. (1975) Anal. Biochem. 66, 110-122.Kojima et al. (1990) Biochem. Cell Biol. 68, 59-64.
Introduction of additional steps (glycolipid hydrolysis and derivatization); fatty acid composition of glycolipid is required for precise calculation of its content in the mixture.
A less complicated picture of compounds in complex mixtures due to preliminary separation of glycolipid backbones; no complication with molecular species of glycolipid.
Gas-liquid chromatography of different derivatives of glycolipid backbones: trimethylsilyl ethers, methyl, acetyl, trifluoroacetylderivatives.
Christie & Urwin (1995) J. High Resol. Chromatogr.18, 97-100.
Calibration for individual compounds
Evaporative light-scattering detection (HPLC-ELSD)
Heinz (1996) Advances in Lipid Methodology – Three. The Oily Press Ltd. 211-332.
Limited to unsaturated molecular species of lipids; calibration for individual molecular species
UV detection
Christie & Urwin (1995) J. High Resol. Chromatogr.18, 97-100. Yunoki et al. (2009) Lipids 44, 77-83.
Up to tertiary gradient elution can be required for effective separation; recovery of glycolipids depends on column phase.
Complex mixture can be separated; automatic and fast analysis
High-performance liquid chromatography with:
Fluorinated derivatives
• Formation in the presence of contaminants (salts etc.)• Weak interaction with non-polar liquid phases resulting
in elution at relatively low temperature• Stability with time• Have been succesfully used for GC analysis of amino
acids, monosaccharides, sialic acids, fatty acids
Objectives
• Method development for analysis of glycolipid backbones as fluorinated derivatives
• Selection of an internal standard for quantitative analysis
• Comparison of derivatization of standard oligosaccharides with trifluoroacetic and heptafluorobutyric anhydrides
• Analysis of glycolipid content in plant lipid extracts
General procedure
OCH2OH
HOH
H OH
H
H
OH
O
H
CH2
C HOH
CH2OH
OCH2
HO
H O
H
H
O
O
H
F7C3C
CC3F7
CC3F7
O
O
O
CH2
CHO
CH2O
CC3F7
O
CC3F7
O
OF7C3CO
Total lipid extract, fraction or an individual component
Alkaline hydrolysis
CH3NH2
Biphasic partitioning O-Acylationwith a
fluorinated anhydride
GC analysis
Monogalactosylglycerol (MGG) N-methyl fatty acid amides (MFAA)
Organic phase:MFAA
Aqueous phase:MGG
Exsiccation
Heptafluorobutyrate derivative of MGG
OCH2OH
HOH
H OH
H
H
OH
O
H
CH2
CHO
CH2O
O
O
CH3NHCO
CH3NHCO
Alkaline hydrolysis
Salts of fatty acid are formed which may be difficult to remove from the aqueous phase
Salts are formed under neutralization
N-methyl fatty acid amides are formed which not suitable for further analysis of fatty acid composition
Disadvantage
Bergelson (1980) Lipid Biochemical Preparations. Elsevier. P. 253.
Sodium hydroxide
Dawson (1976) Lipid Chromatographic Analysis. Vol. 1. Marcel Dekker, INC. PP. 151-153.
Methyl esters of fatty acids are formed which can be used for further analysis of fatty acid composition
Sodium methoxide
Clarke & Dawson (1981) Biochem. J. 195, 301-306.
The excess of the reagent can be easily removed
Monomethylamine(CH3NH2)
ReferenceAdvantageReagent
General disadvantage of alkaline hydrolysis: does not attack O-alkyl ether or O-alkenyl groups of lipids
Biphasic partitioning
AP OP AP OP Init AP OP AP OP
I II III IV
TLC of total lipids from spinach leaves after alkaline hydrolysis (CH3NH2) and biphasic partitioning in different solvent systems. Detection with the anthrone reagent.
AP – aqueous phase
OP – organic phase
Init – initial lipid extract
SQDG - sulfoquinovosyldiacylglycerol
Solvent systems for biphasic partitioning:
I: chloroform – butanol – water*
II: butanol – petroleum ether – ethyl acetate
III: hexane – methanol – water + chloroform
IV: hexane – toluene – water + chloroform
*The preferable solvent system with higher recovery of glycolipid backbone and least contamination with pigments
MGDG
DGDG
SQDG
Deacylated lipid backbones
Selection of an internal standard
Presence of the compounds should be first tested in the material under investigation; esters of sucrose may present in the lipid extracts from some plants (Solanaceaefamily).Cellobiitol has a retention time close to one of sucrose; for their separation a slower temperature program can be applied
Similar in structure to the glycolipid backbones to be analyzed; produce a single peak in the chromatogram.
Sucrose and cellobiitolwere in the area free from contaminants when plant lipid extracts were analyzed.
Non-reducing sugars and polyol alcohols:Disaccharides (sucrose, trehalose)
Cellobiitol (4-O-ß-D-glucopyranosyl-D-glucitol)
Produce several peaks in the chromatogram; monosaccharides have too small retention times compared to MGG and DGG
Reducing sugars (monosaccharides, lactose)
DisadvantageAdvantageHeptafluorobutyrate
derivatives of compounds
Comparison of derivatization of different oligosaccharides with trifluoroacetic (TFAA) and heptafluorobutyric (HFBA) anhydrides
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3000000
Time
Response_
Signal: Mix-HFBD-10.D\FID1A.CH
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Time
Response_
Signal: MIX-TFAD-10.D\FID1A.CH (*)
O-Heptafluorobutyrate derivatives (HFBD)
O-Trifluoroacetatederivatives (TFAD)
Suc
Suc
Raffinose
Raffinose Stachyose
StachyoseOctadecanol
Octadecanol
HFBD have a higher FID response compared to TFAD (~40%)
Treatment with HFBA has a “discriminating”effect on a FID response of hydrophobic compounds (octadecanol, stearic acid methyl ester) Capillary GC column: SolGel-1ms™, 30 m x 0.25 mm x 0.25 μm (SGE,
Australia). Temperature program: 150-270°C, 4°C/min.
Optimization: solubility of fluorinated derivatives
FID response of the heptafluorobutyrate derivatives of oligosaccharides in the presence of a fluorinated
solvent or reagent
0
20
40
60
80
100
120
1 2 3 4Content of fluorinated solvents
Rel
ativ
e FI
D re
spon
se,
% o
f suc
rose
Sucrose (disaccharide)
Raffinose (trisaccharide)
Stachyose (tetrasaccharide)
No 20% (v/v) HFIP 16% (v/v) HFBA 40% (v/v) HFBA
HFIP – hexafluoro-2-propanol
HFBA – heptafluorobutyric anhydride
TFAA – trifluoroacetic anhydride
FID response of the trifluoroacetate derivatives of oligosaccharides in the presence of a fluorinated
solvent or reagent
0
20
40
60
80
100
120
1 2 3 4Content of fluorinated solvents
Rel
ativ
e FI
D re
spon
se,
% o
f suc
rose
Sucrose
Raffinose
Stachyose
No 20% (v/v) HFIP 9% (v/v) TFAA 23% (v/v) TFAA
Comparison of HFBD and TFAD
O-Heptafluorobutyrate derivatives (HFBD):• Higher FID response as compared to TFAD (~40%)• Better chromatographic resolution at a higher temperature program• Better stability in solution• Different response for oligosaccharides with different chain length• “Discriminating" effect on FID response of hydrophobic compounds
O-Trifluoroacetate derivatives (TFAD):• Similar response for oligosaccharides with different chain length • Lower FID response as compared to HFBD• Peaks of sucrose and monogalactosylglycerol (MGG),
digalactosylglycerol (DGG) and sulfoquinovosylglycerol (SQG) overlapped so a slower temperature program should be applied
Analysis of glycolipid content in plant lipid extracts: spinach leaves
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Time
Response_
Signal: Spinach-TL-HFBA-16.D\FID1A.CH
2.56
6.66
8.81
14.73
15.39
Suc
MGG
DGG
SQG
Content of glycolipids in spinach leaves was determined using their avarage molecular weights calculated on the basis of their fatty acid composition available in literature (Yamauchi et al. (1982) Agric. Biol. Chem. 46, 2847-2849) : MW(MGDG)=762.4, MW(DGDG)=932.6
Comparison of GLC and HPLC-ELSD
05
101520
2530354045
MGDG DGDG
Con
tent
, % (w
/w) o
f tot
al li
pids GLC as HFBD
HPLC-ELSD*
*Yunoki et al. (2009) Lipids 44, 77-83.
Analysis of glycolipid content in plant lipid extracts: pumpkin fruit
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1400000
1500000
1600000
1700000
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Time
Response_
Signal: Pump-HFBD.D\FID1A.CH
6.67
8.81
14.53
15.40
16.25
21.37
25.26
Suc
MGG
DGG
all-ß-DGG ?
TGG0
2
4
6
8
10
12
14
MGDG DGDG TGDG TeGDG
Con
tent
, % (w
/w) o
f tot
al li
pids
TeGG
Content of glycolipids in pumpkin fruit was determined using their avarage molecular weights calculated on the basis of MS analysis of a glycolipid fraction: MW(MGDG)=774.1, MW(DGDG)=935.3, MW(TGDG)=1100.7, MW(TeGDG)=1262.7 (A. MacKenzie (2009) personal communication)
TGDG – trigalactosyldiacylglycerol
TeGDG – tetragalactosyldiacylglycerol
Conclusions• Both the heptafluorobutyrate and trifluoroacetate
derivatives of glycosylglycerols can be used for identification and quantitative analysis of glycolipids
• HFBD have advantages over TFAD if complete dissolving of HFBD is achieved
• GC analysis of MGDG and DGDG as HFBD or TFAD can be extended to their isomers and higher homologues (TGDG, TeGDG).
Acknowledgments
Dr. A. MacKenzie and Dr. Y. Lu (IRL) for help with MS analysis
This work was supported by the New Zealand Foundation for Research, Science and Technology grant C08X0709 “High value lipids”