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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1000000

1500000

2000000

2500000

3000000

Time

Response_

Signal: Mix-HFBD-10.D\FID1A.CH

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500000

1000000

1500000

2000000

2500000

3000000

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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600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

1900000

2000000

2100000

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”