Chemical and Physical Aspects of Acrylamide Formation in Low Moisture … · · 2006-08-18Chemical and Physical Aspects of Acrylamide Formation in Low Moisture Systems 227th ACS

Nestlé Research Center

Chemical and Physical Aspects of AcrylamideFormation in Low Moisture Systems

227th ACS Meeting on Acrylamide, Anaheim, March 30th, 2004

I. Blank, T. Davidek, T. Goldmann, P. Pollien,F. Robert, R. Stadler, G. Vuataz

Nestlé Research Center2004-02-27 NRC/FCI - IBk2

Stadler et al.Nature (2002)

O NH2

NH2 COOH

O

NH2

N

OH

R

COOH OH

H

NH2

O

0

2000

4000

6000

8000

10000

-10.0 0.0 10.0 20.0 30.0 40.0 50.0 60.0

Time [min]

HS

conc

. [pp

bv]

Potato 170°C x 50Asn / Fructose 150°CAsn / Glucose 150°C x 5

Pollien et al.Anal. Chem. (2003)

m/z = 72

H3O+

PTR-MS

Stadler et al.Chem. Res. Tox. (2003) R

NH2

COOHRSugar

Recent work on acrylamide formation published from our laboratories


Chemistry Physics

Physical state:molecular mobility,

melting point, Tg

DSC, TGA,microscopy

Melting behavior,phase diagram

Wateractivity

Chemical reactivity:Aldehydes, ketones

Quantification,synthesis

GC-MS, HPLC, HRMS, NMR

Acrylamideformation

PTR-MS

Objective: Study chemical & physicalaspects of acrylamide formation


Acrylamide formation from asparagine: Fragmented information, hypotheses

Stadler, 2002

N

OH

Y

COOH OH

H

NH2

O

NH2

O

Yaylayan, 2003

XN

O

Y

H H

N

OH

Y

OOHNH2

O

N

O

OH

Y

O

H

X

N

OH

Y

H

X

+

NH2

O

Mottram, 2002

N

OH

Y

OOHNH2

O

NH2

O

NH2

O O

Zyzak, 2003

N

OH

Y

H

X

+

N

OH

Y

H

X

+

N

OH

Y

OOHNH2

O

NH2

O

NH2

O NH2

m/z 251


Quantitative results obtained withpotential precursors of acrylamide

Acrylamide (mmol/mol Asn)

R''

O

R'

O

Asn 0.1-0.3

OH

O

NH2

0.2

N

OH

R

COOH OH

H

O

NH22.4

N

O

R

COOH

H

O

NH20.1

NH2

COOHO

NH2

Glc 0.4

Pyrolysis: 180 °C, 5 min


OH NH2

O

NH2

O

- H2O

0.00.51.01.52.02.53.03.54.0

120 140 160 180 200 220

Temperature (°C)

Acr

ylam

ide

(mm

ol/m

ol)

3-Hydroxypropanamide

Pyrolysis: 5 min

Fructose/asparagine (1:1)


0.0

0.5

1.0

1.5

2.0

2.5

80 100 120 140 160 180 200 220

Temperature (°C)

Acr

ylam

ide

(mm

ol/m

ol)

A

O H

N HH OHO

OH

O H

C O N H 2OH C O 2 H

C ON H 2

t= 60 min

0.00.20.40.60.81.01.21.4

0 10 20 30 40 50 60 70

Time (min)

Acr

ylam

ide

(mm

ol/m

ol)

T= 100 °C

B


Pyrolysis of decarboxylated Amadori compounds

Precursor Products

OH

OH

OH

O

NH

OHH

HOO

Hofmann-type β-elimination of quaternary ammonium compounds: R1

NR3

H

R2

+

OH -

R1R2 + NR3 + H2O

Styrene Strecker aldehyde

ΔT

OH

OH

OH

O

NH

OH O HO

Strecker aldehyde

ΔT

XN

O

Y

H H

X


Fructose / phenylalanine(1 : 1)

NH2

COOH Sugar

200-300 μmol/mol Phe

180 °C 15 min

TIC

0

500000

1000000

1500000

10 12 14 16 18 20 22 24 26 28 30min

Styrene

17.74

m/z 104

0

50000

100000

150000

200000

10 12 14 16 18 20 22 24 26 28 30min

Styrene

17.76

m/z 107

0

50000

100000

150000

200000

10 12 14 16 18 20 22 24 26 28 30min

α,β,β -2H3-Styrene17.73

H2

H2

H2

TIC

0

500000

1000000

1500000

10 12 14 16 18 20 22 24 26 28 30min

Styrene

17.74

TIC

0

500000

1000000

1500000

10 12 14 16 18 20 22 24 26 28 30min

Styrene

17.74

m/z 104

0

50000

100000

150000

200000

10 12 14 16 18 20 22 24 26 28 30min

Styrene

17.76

m/z 104

0

50000

100000

150000

200000

10 12 14 16 18 20 22 24 26 28 30min

Styrene

17.76

m/z 107

0

50000

100000

150000

200000

10 12 14 16 18 20 22 24 26 28 30min


H2

H2

H2

m/z 107

0

50000

100000

150000

200000

10 12 14 16 18 20 22 24 26 28 30min


H2

H2

H2


XN

OH

Y

COOH OH

H

XNH2

COOHO

OH

Y+

N-Glycosyl conjugateAmino acid Carbonyl

Decarboxylated Amadori compound

XN

O

Y

H H

- H2O

Schiff base

XN

OH

Y

OOH

Azomethine ylide

N

OH

Y

H

X

+

II

X

Vinylogous compound

Oxazolidin-5-one

N

O

OH

Y

O

H

X

- CO2

Schiff base betaine

XN

OH

Y

OO

H

+

-

- CO2

XN

O

Y

COOH

H

Amadori compound

I


XN

OH

Y

Imine 2

1,2-Prototropy of the 1,3-dipole to azomethine ylides 1 and 2

Isomerization of the decarboxylated Schiff base

(Grigg et al., 1988; Rizzi, 1970)

Azomethine ylide 1

XN

OH

Y

H

+ XN

OH

Y

H+

XN

OH

Y

H

+

Azomethine ylide 21,3-dipole

XN

OH

Y

Imine 1

XN

O

Y

HH

DecaboxylatedAmadori compound


Strecker aldehyde

Imine 2

N

O

R

NH2

O

IIIa

O

NH2

O

H2O

NH2

O

R

+

NH2

COOH

O

NH2O

O

R+

- H2O

Schiff base

Azomethine ylide

Asparagine Dicarbonyl

- CO2

N

O

R

H

NH2

O

+

N

O

R

OO

H

NH2

O

-

+

Imine 1

N

O

R

H

NH2

O IIIb

NH2

NH2

O

O

RO

+

H2O

3-Aminopropionamide

NH2

O

Acrylamide

- NH3

Time Acetol Diacetyl

5 4.6 0.320 2.9 0.5

(min) AA (mmol/mol)

α-Hydroxycarbonyls: Highest conversion yields of acrylamide from asparagine so far.

X

O

O

H

O

OH

Pyrolysis (180 °C)

Asn +


02468

1012141618

80 100 120 140 160 180

Temperature (°C)

AA

(mm

ol/m

ol A

sn)

0

1

2

3

4

5

20 50 100 200

Acr

ylam

ide

(mm

ol/m

ol A

sn)

Glc/Asn

0Water (μL)

Fru/AsnFructose is more reactive than glucose in generating acrylamide from asparagine. Moisture does influence acrylamide formation.

Effect of (i) reaction time and temperature(ii) type of sugar and moisture

Reaction time and temperatureare covariant parameters.High amounts of acrylamideformed at 120-160 °C,depending on reaction time.

60 min5 minGlc/Asn


Acrylamide release from sugar/Asn•H2O (measuring m/z 72 by PTR-MS)

Fructose / Asn·H2O

Galactose / Asn·H2O

Glucose / Asn·H2O

Fructose generates more acrylamide and at a lower temperature

Tem

pera

ture

[°C

]

Type of reducing sugar: Differences in acrylamide yields in headspace

0

20

40

60

80

100

120

140

160

0 20 40 60 80 100 120

Time [min]

Mas

s flo

w a

cryl

amid

e re

lese

ad [u

mol

/mol

Asn

/ m

in]

30

50

70

90

110

130

150

170

190


Fructose/Asn·H2O

35°C 163°C152°C134°C

Glucose/Asn·H2O

152°C 173°C 185°C35°C

Type of reducing sugar:Differences in browning reaction


Fructose/Asn•H2O (1)Galactose/Asn•H2O (2)Glucose/Asn•H2O (3)

Result: Fructose generates more acrylamide than glucose.

ΑΑ

(μm

ol/m

ol A

sn)

2000

(1) (2) (3)

Conclusion: Chemical reactivity of the sugar is not proportional to the acrylamide amounts generated under low moisture conditions !

Acrylamide generated from crystalline sugar/Asn•H2O (pyrolysis at 180 °C for 5 min)

Hypothesis: Molecular mobility plays a key role in acrylamide formation by solid-state Maillard reactions.

Type of reducing sugar:Differences in overall acrylamide yields


DSC of different sugar/Asn•H2O mixtures (open reaction system, 5 °C/min)

Acrylamide formation depends on melting point of the sugars

Molecular mobility is keyin acrylamide formation

-1.6-1.4-1.2-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.01.21.4

40 60 80 100 120 140 160 180Temperature [°C]

Hea

t flo

w [W

/g]

Glucose

Fructose

Galactose

Crystallisation water release

pureAsparagine

monohydrate


0

5000

10000

15000

20000

25000

30000

35000

0 10 20 30 40 50 60

Time (min)

Acr

ylam

ide

(um

ol/m

ol A

sn)

galactose/Asnglucose/Asnfructose/Asn

High molecular mobility in non-aqueous liquid systems:Chemical reactivity of the sugar is the major driver in acrylamide formation

Kinetics of acrylamide formation in DMSO at 150 °C


Summary / Conclusions

Major chemical pathway of acrylamide formation from asparagine has been established, with α-hydroxy carbonyls as the most reactive species.

N

O

Y

HH

O

NH2

Reaction time and temperature are covariant parameters in acrylamide formation from asparagine.

0

4

8

12

16

20

80 100 120 140 160 180T [°C]

AA

[mm

ol/m

ol]

60 min5 minGlc/Asn

Nature of sugar and molecular mobility are important parameters of acrylamide formation from asparagine under low moisture conditions.

-1.6-1.2-0.8-0.40.00.40.81.2

60 80 100 120 140 160 180

FruGlc

T [°C]

DSC (Sugar/Asn)

-1.6-1.2-0.8-0.40.00.40.81.2

60 80 100 120 140 160 180

FruGlc

T [°C]

DSC (Sugar/Asn)


Acknowledgments

ChemistryT. Davidek, S. Devaud, P. Pollien, F. Robert, F. Saucy Synthesis, mechanismsOn-line monitoring (PTR-MS)Quantification (HPLC, GC-MS)

T. Goldmann, S. Riediker, R. Stadler, A. Studer, N. Varga Quantification (LC-MS/MS)Method development, mechanisms

PhysicsM.-I. Alonso, I. Bauwens, G. Vuataz Analytics (DSC), crystalline and amorphous systemsPhase diagram, TG, aW

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Chemical and Physical Aspects of Acrylamide Formation in Low Moisture … · · 2006-08-18Chemical and Physical Aspects of Acrylamide Formation in Low Moisture Systems 227th ACS