laboratory of food chemistry
TRANSCRIPT
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
� 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
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
� 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
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
� 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
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
migration time (min)
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
� 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
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
migration time (min)
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
migration time (min)
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