laboratory of food chemistry

CE-MS analysis of oligosaccharides in complex matrices

Henk Schols Laboratory of Food Chemistry

Contents

� Prebiotics for a Healthy Colon

� Capillairy Electroforesis of oligosaccharides

� CE-Mass Spectrometry of oligosaccharides

� Analysis of prebiotics in food products

� Analysis of HMO/prebiotics after fermentation

� non-digestible food ingredient that selectively stimulates the growth of colonic bacteria, and thus benefits host health

Prebiotics

Influence of microflora activity on health

� Fermentation residues � Gases� Short Chain Fatty Acids

• drop pH

• positive effect by themselves

� Other residues => can be toxic

� Fermentations in the colon� Saccharolytic fermentation mostly in the proximal

part� Proteolytic fermentation mostly in the distal part

Influence of microflora composition on health

� Microflora = complex ecosystem with high heterogeneity� up to 1012 bacteria , more than 500 species

� Influence on health� Bacteria nature � Protective barrier role

Health effect of the predominant gut bacteria (Gibson et al,1995).

Complex mixture !

GOS: a commercial prebiotic preparation

Motivation for research :

analysis of GOS in complex matrices� Growing commercial interest in implementation of

functional carbohydrates in complex food products (e.g. dairy, juices)� Legislation – present as promised?� Reliable analysis in complex food products necessary

� Prebiotics – Health claims� Do they indeed reach the colon?� Are they indeed (selectively) fermented in the colon?� Any (relevant) degradation products formed?

� Dietary fibre - Novel oligosaccharides � Digestion and fermentation� In vitro vs in vivo fermentation

Capillary Electrophoresis with LIF-detection

� Most positive structures elute first

� Neutral structures elute with Electro \

Osmotic Flow (EOF)

� Negative structures can elute,

depending on the EOF

� Polyvinyl alcohol-coated capillary� Coating is reducing the charge of the wall

� Reversed polarity� Negative charge of APTS

+-

Fluorescence

DetectorCapillary

Electrode

(High voltage)

Laser

+ -+ + + + +++ ++ ++ ++ +++ + + + +++ ++ ++ ++ +++ + + + +++ ++ ++ ++ +++ + + + +++ ++ ++ ++ +++ + + + +++ ++ ++ ++ ++

+ + + + +++ ++ ++ ++ +++ + + + +++ ++ ++ ++ +++ + + + +++ ++ ++ ++ +++ + + + +++ ++ ++ ++ +++ + + + +++ ++ ++ ++ ++EOF

Labelling with APTS

� Not every sugar molecule is charged� Labeling with APTS

• To give charge to a sugar molecule

• To increase signal (fluorescent)

• To allow quantification on mole basis

� Laser Induced Fluorescence (LIF)� Detection based on fluorescence� Laser induces emission (520 nm)

OH

OH

RO

O

OH

O3S

O3S SO3

NH2

O3S

SO3NH

SO3

OH

OH

RO

OH

CH2

+

APTSCarbohydrate Labeled Carbohydrate

NaBH3CN

AcOH/ TEMP

OH

OH

RO

O

OH

O3S

O3S SO3

NH2

O3S

SO3NH

SO3

OH

OH

RO

OH

CH2

+OH

OH

RO

O

OH

O3S

O3S SO3

NH2

O3S

SO3NH

SO3

OH

OH

RO

OH

CH2

OH

OH

RO

O

OH

O3S

O3S SO3

NH2

O3S

SO3NH

SO3

OH

OH

RO

OH

CH2

OH

OH

RO

O

OH

O3S

O3S SO3

NH2

O3S

SO3NH

SO3

OH

OH

RO

OH

CH2

+

APTSCarbohydrate Labeled Carbohydrate

NaBH3CN

AcOH/ TEMP

Based on

Chen, et al (1998)

Separation of monomeric sugars using CE

� Separation depends on CE parameters

APTS side

products

Xylose

RhamnoseGlucose

Galactose

ArabinoseMannose

M a lto se

RF

U

min3 4 5 6

G1

G3

*

G4 G5

G6G7

G8G9 G10 G12 G14 G16 G18

APTS

*APTS G1

C2

C3C4

C5

C6C7

3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

LIF

inte

nsit

y

3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

LIF

inte

nsit

y APTS

X1

*

X2X3 X4

X5X6

APTS-(ββββ-1,4-xylo-oligosaccharides)

APTS-(ββββ-1,4-gluco-oligosaccharides)

APTS-(αααα-1,4-gluco-oligosaccharides)

C8

CE-LIF of different oligosaccharides

Migration behavior not as straightforward as expected

Quantification of oligosaccharides using CE-LIF

� Reproducibility

� Mole-based detection� Good results for of DP 3-5 of Cello-&

Malto dextrines

� Quantification possible� also if no standards available

� Elution behavior unpredictable

Cellotriose

0

10

20

30

40

50

60

0 10 20 30 40 50Labeled amount of carbohydrate (nmol)

4.42 4.63 4.75 4.88 5.00 5.13 5.25 5.38 5.50 5.63 5.75 5.88 6.00 6.13 6.25 6.38 6.50 6.63 6.75 6.92

-0.50

-0.25

-0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

min

321

DP3 DP4 DP5

CE-LIF

1 nmol per Maltodextrin

Capillary Zone Electrophoresis coupled with Mass spectrometry

Consequences of CE-MS coupling

� Find good interface

� Adapt buffer (volatile, lower pH)

� Adapt capillary (fused silica instead of coated silica)

� Longer capillary (to connect to MS; partially without cooling)

� (No) detection at end of capillary

LIF Detection in CE-MS analysis

LIF detector ‘in’ CE� Detection ‘in middle’ of capillary � Capillary cooling only tìll detector� Interpretation rather difficult

External LIF detector picometrix� Detection rather close to MS

interface � Capillary cooling up to MS� Interpretation more straight forward

MS / Base-Peak signal

Picometrics external LIF signalMaltose

Maltose

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0-100

0

100

200

300

400

500

600

700

800

900

1,000 test #13 MDnew2_080711 Channel_AmV

min

RT: 0.00 - 60.00

0 5 10 15 20 25 30 35 40 45 50 55 60Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lati

ve A

bu

nda

nce

37.54530.81

39.17584.86

40.85638.91

35.53476.70

42.46692.91

31.10368.66

45.77800.96

48.82909.03

50.27963.01

51.791017.09

22.16456.22

25.84390.9220.98

456.1417.21

387.2657.97

387.238.40

387.251.83

387.26

NL:5.11E5

Base Peak F: Full ms

MS 080711-

MD2_080711141741

CE-LIF versus CE-MS patterns of maltodextrines

external LIF detector

Iontrap MS (LTQ)

Conclusions CE & CE-MS

� CE provides high resolution in the separation of oligosaccharides

� Fluorescence label increases sensitivity and enables quantification

� Migration behaviour oligosaccharides is determined by sugar (linkage) composition and substitution pattern

� Mass spectrometry is quite helpful in structure elucidation of oligosaccharides

� Mass fragmentation mechanisms of oligosaccharides rather complex and not yet fully understood

Contents






Challenges of complex food matrixes

� Prebiotic oligosaccharides represented in very low amounts (0.2 – 2%) in food product

� Abundance of monomers and dimers

� High levels of Maltodextrins may disturb detection of prebiotic oligosaccharides

Model substrates used for analysis

Extraction and analysis schemeHot-water Extraction GOS (AOAC 2001.02)

(0.2 µm) Filtration

Derivatisation & Analysis

(CE-LIF)

Solid Phase Extraction (SPE)

- graphitized carbon columns

- Mono -/ Dimers removed by 2 – 5 % ACN

- Oligomers eluted by 25 % ACN in 0.05 % TFA

Amyloglucosidase-incubation

For Maltodextrin containing samples

CE-LIF analysis of GOS in fruit juice:

before and after SPE extraction (2% ACN)

3.6 4.0 4.4 4.8 5.2 5.6 6.0 6.4 6.8 7.2

2 - 27-02-08-r epeat+Annemi eke #18 [1 peak manuall y assig ned] Cb-0- 2LI

F in

tens

ity

migration time (min)

before SPE- extraction

mon

omer

s

AP

TS

Galacto-oligosaccharides

Cb-0- 2

21

ISTD

extracted

initial

after SPE- extraction

ISTD

DP3 DP4 DP5 DP6

Fructose and sucrose present (non-reducing sugars) will not be labeled

CE-LIF: infant-milkpowder containing Maltodextrins

after SPE extraction: with/without AMG incubation

Both samples extracted by SPE

Due to high Maltodextrin concentration, AMG-incubation necessary before SPE extraction

selective removal of Maltodextrins, GOS structures recovered

4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

1 - spe191207 #10 [modi fied by al bre003, 2 peaks manuall y assigned] frisospe2/200 LIF ___C hannel_1

LIF

inte

nsity


after SPE-extraction without AMG incubationafter SPE-extraction with AMG incubation

5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00-0.60

-0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

6.50

7.00

7.79

1 - 28 -02 -08 extr acts #2 [2 p eaks man ually a ssign ed] Friso -MDr em ove LIF_ __Ch an nel_ 12 - 27 -02 -08 -re peat +Ann emie ke # 14 [1 pea k ma nually assig ned, no rmaliz ed] LIF_ __Ch an nel_ 1

RFU

min

2

1

GOS original

Conclusions for extraction of oligosaccharides from food matrixes

� Extraction and analysis method seems to be promising and successful for GOS containing food matrices� Disturbing maltodextrin can be selectively removed by Amyloglucosidase

– pretreatment� SPE extraction eliminates the abundant amounts of mono- and dimers � CE-LIF results in a clear separation in a short analysis time

� Method for FOS and Inulin not yet applicable � APTS-label for CE-LIF needs an aldehyde group for binding to the

molecule

Contents






Glucose GluNAc Galactose Neu5Ac Fucose

Human Milk Oligosaccharides (HMOS) versa GOS

HMOs complex – but all have a Lactose core unit

GOS is transglycosylation product from Lactose

-> majority of GOS has also a Lactose core unit

-> also beneficial effects on gut flora expected

Experimental set-up for screening baby feces on oligosaccharides

Fluorescent Derivatization & Analysis

(CE-LIF(off-line), CE-MSn, CE-LIF(online)-MSn)

Baby-fecespreterm-born: breast-fed, GOS-fed, control

Breast-milk(not according to feces)

Purification/ Extraction0.2 µm-filtration

Moro et al (2005)

Purification/ Extractionremoval of fat/proteins

Stahl et al (1994)

Purification/Upconcentration- SPE (graphitized carbon columns)- Stepwise elution: water -> 2 % ACN ->

40 % ACN in 0.05 % TFA

Baby diet study

Fecal samples issuing from preterm-born babies…

1) …fed with infant formula supplied with prebiotic GOS (7)group 1: formula containing 5 g/L GOS (5)group 2: formula containing 0.8 g/L GOS (2)

2) … fed with infant formula not containing any prebiotic oligosaccharides (term born) (1)

3) … fed with mother milk (3)

Compared to…

1) …GOS initially present in infant formula

2) …initial mother milk

CE-LIF: Babies fed with GOS-formula

4.50 5.00 5.50 6.00 6.50 7.00

4 - 27-02-08-r epeat+Annemi eke #16 [1 peak manuall y assig ned]

4

3

2

1

LIF

inte

nsity


Mal

tose

ISTD

feces blank

B3

GOS reference

B17

Lact

ose

4.50 5.00 5.50 6.00 6.50 7.00

4

3

2

1

LIF

inte

nsity


Mal

tose

ISTD

feces blank

B14

GOS reference

B25

Lact

ose

Group 1:

Two babies fed with high dose GOS - formula

Group 2:

Two babies fed with low dose GOS - formula

Structures present in feces, dependent on baby and GOS-concentration in formula!

CE-LIF(-MS) of baby feces

4.00 5.00 6.00 7.00 8.00

5-0

LIF

inte

nsity

migration time

4.00 5.00 6.00 7.00 8.00

MMA 10x

LIF

inte

nsity

migration time

baby fed with GOS-containing formula breastfed baby

GOS reference

baby feces extract

mothermilk reference

baby feces extract

**

*

*

*

NHexAc-(Hex)2

Galacto-oligosaccharides

FL

LNF

PLN

FP

IILN

FP

IIILN

DF

H

Lact

ose

Lact

ose

Human milk oligosaccharides

FL: Fuc-Gal-Glc; LNFP: Fuc-Gal-GlcNAc-Gal-Glc, LNDFH: Fuc-Gal-GlcNAc-(Fuc)-Gal-Glc

* Maltose Internal Standard

Challenges of CE-LIF-MSn in HMO analysis

� Structure HMOs very complex� not all standards available� new structures formed during intestinal passage

� Some HMO-isomers separated by CE-LIF� same masses � MS can differentiate between several structures

� MSn experiments for structural characterisation !

Lacto-N-fucopentaose: 4 out of 5 isomers possible to separate with CE-LIF

β1,6β1,4

α1,3

β1,4

LNFP Y

β1,3 β1,4β1,3α1,2LNFP I

β1,3β1,3 β1,4

α1,4LNFP II

β1,4β1,3 β1,4

α1,3LNFP III

β1,3 β1,3β1,4

α1,3LNFP V

LIF

inte

nsity

Time (min)3.00 4.00 5.00 6.00 7.00 8.00 9.00

12

6

5

94

3

7

10

*

12815

13

14

19

17

2021

22

23

2527

29

31

3033

26

37

3839

4143

44 4546

47

2

6

3

*

18

16

15

19

17

22 23

3229

30

3328

4041

1 101112 20 25

24

34

35

36

39 454243

44 46 47

32

B

Abreast milk

fecal extractbreastfed baby

ISTD

+

?

?

Glucose GluNAc Galactose Neu5Ac Fucose

Abundant “simple structure” (Glu-Gal-Glu-Gal) not present any more in fecal extractSialidase and Fucosidase activity during intestinal passageLactase activity during intestinal passagePredominance of LNFP structures in fecal extract“unknown”peaks present: unknown Lacto-fucopentaose & unknown Lacto-fucotetraose-Isomer

1.5. CE-LIF mother milk and fecal extract mother milk-fed – introducing CE-MS data

Change in peak ratios

LNFP and LNT-Isomer analysis in fecal extracts by CE-LIF-MSn

Lacto-N-Fucopentaose Isomers

known for breast milk

New Lacto-N-Fucopentaose Isomer

in fecal extracts of breastfed babies

as identified by CE-MSn

β1,6

β1,4

α1,3

β1,4

β1,3

β1,3

F - LNH II

β1,6

β1,4

α1,3

β1,4

LNFP Y

Gastrointestinal

formation

of LNFP Y

Gastrointestinal

formation

of LnNT Y

New Lacto-N-Tetraose Isomer

in fecal extracts of breastfed babies

as identified by CE-MSn

Lacto-N-Fucotetraose Isomers

known for breast milk

β1,3 β1,4β1,3α1,2

LNFP I

β1,3β1,3 β1,4

α1,4

LNFP II

β1,4β1,3 β1,4

α1,3

LNFP III

β1,3 β1,3

β1,4

α1,3

LNFP V β1,3 β1,4β1,3

LNT

β1,3 β1,4β1,4

LnNT

β1,6β1,4β1,4

LnNT Y

Conclusions for HMOs/GOS extracted from baby feces

� Peak profile fecal extract different from mother milk

� Intestinal degradation of simple structures

� Predominant “leftover structures“ (resulting from degradation)

� Intestinal lactase-/sialidase-activity

� De-fucosylation of di-fucosyl-HMOseasier than mono-fucosyl HMOs(accumulation)

� Unknown peaks identified by CE-LIF-MSn: LNFP Y, LNT -> result of intestinal degradation

� Peak profile fecal extract different from GOS formula

� Peak profiles according to time of birth ?

� Accumulation of DP4

� Mucus derived oligosaccharides (other flora developed than breastfed baby)

Formula-fed babiesBreast-fed babies

Thanks for your attention!Acknowledgement

Simone AlbrechtEdwin Bakx Walter HeijnisMirjam Kabel

laboratory of food chemistry

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