A new method for the quantification of N-(ldeoxy-D-huctas-l-yl)-glycine (DFG) was developed based on isotope dilution fast atom bombardment tandem mass spectrometry using &apos;3C-laheUed DFG as an internal standard. This method, which requires neither derivatization nor clean-up of the samples, was used to study the degradation of DFG under different conditions (time and pH). It was found that the decomposition of DFG at 90°C was favoured at pH 7 compared to pH 6. The higher stability of DFG at pH 6 was due to 1,2-enolization leading to the relatively stable 3deoxglucoson

Andreas A Staempg

Imre Blank

Laurent B Fay

Re&amp

CiteSeerX

BIOLOGICAL MASS SPECTROMETRY, VOL. 23, 612-646 (1994) 
Study on the Decomposition of the Amadori 
Compound N-( l-Deoxy-D-fructos-l-yl)-glycine in 
Model Systems : Quantification by Fast Atom 
Bombardment Tandem Mass Spectrometry 
Andreas A. StaempG, Imre Blank, Re& Fumesux and Laurent B. Fay 
Nestec Ltd, Nest16 Research Centre, Vers-chez-les-Blanc, PO Box 44, lo00 Lausanne 26, Switzerland 
A new method for the quantification of N-(ldeoxy-D-huctas-l-yl)-glycine (DFG) was developed based on isotope 
dilution fast atom bombardment tandem mass spectrometry using '3C-laheUed DFG as an internal standard. This 
method, which requires neither derivatization nor clean-up of the samples, was used to study the degradation of 
DFG under different conditions (time and pH). It was found that the decomposition of DFG at 90°C was favoured 
at pH 7 compared to pH 6. The higher stability of DFG at pH 6 was due to 1,2-enolization leading to the relatively 
stable 3deoxglucosone 
INTRODUCTION 
Amadori compounds are N-(1-deoxyketos-1-yl)-amine 
derivatives formed in the course of the non-enzymatic 
browning (Maillard) reaction.' They are key interme- 
diates of the Maillard reaction and responsible for 
changes in colour, flavour and nutritive value of 
They have been found, for example, in malt 
and brewed product~,4.~ dried vegetables,6 soya sauce' 
and cocoa.8 
The degradation pathways of Amadori compounds 
have been widely discussed.' They decompose (Scheme 
1)' via the enediol or the aminoenol intermediate and 
form, by 2,3-enolization and l,Zenolization, the reac- 
tion products l-deoxy-2,3-hexodiulose (1-deoxyosone) 
and 3-deoxy-2-hexosulose (3-deoxyosone), respec- 
tively." These reactive a-dicarbonyls show a tendency 
to further enolization, dehydration and carbon fission 
and are thus capable of forming polymeric colourants 
and volatile compounds. 
Several methods have been reported for the quanti- 
tative analysis of Amadori compounds," including 
high-performance liquid chromatography (HPLC)6 and 
capillary gas chromatography/mass spectrometry (GC/ 
MS).4 The latter involves oximation of the isolated 
Amadori compounds followed by silylation, thus theo- 
retically resulting in two peaks (syn and anti isomers) 
for each Amadori compound. In order to simplify the 
isolation procedure, to avoid any derivatization step 
and to maintain high selectivity and sensitivity of the 
mass spectrometer, we have developed a new, accurate 
method based on isotope dilution fast atom bombard- 
ment tandem mass spectrometry (FAB MS/MS). 
Isotope dilution with FAB mass spectrometry provided 
a high dynamic range.I2 MS/MS gave a compromise 
between improved selectivity and decreased sensitivity. 
The aim of our work was to study the decomposition 
of N-( 1 -deoxy-~-fructOs-l-yl)-glycine (DFG) in model 
CCC 1052-9306/94/100642-05 
0 1994 by John Wiley & Sons, Ltd. 
systems. The newly developed quantification method for 
DFG based on isotope dilution FAB MS/MS is report- 
ed here in detail, as is the synthesis of the 13C-labelled 
DFG used as internal standard. 
EXPERIMENTAL 
Sythesis of N-(Ideoxy-D-fructos-l-y1)-(1-' 'C)glycine 
The target compound was synthesized according to the 
l i t e r a t~ re . ' ~ . ' ~  A mixture of 50 g (278 mmol) of D-(+)- 
HzC-N€-R 
C-OH 
C ~ H  2,3-cnolisation 
I 
I I  
I 
I 
I 
P 
HC-OH 
HC-OH 
HzC-OH 
\b 
CH3 
I 
c=o 
I 
c=o 
I 
HC-OH 
I 
HC-OH 
I 
HzC-OH 
1 -dmxyglucosone 
H2C-NH-R HC-NH-R 
I1 I 
c=o 
I I 
I I 
I I 
C 4 H  
H ~ H  1.2-cnolisation HOZH __ 
7
H C 4 H  
HC-OH 
HC-OH 
HC-OH 
I 
H2C4H 
DFG 
I 
H2C-OH 
I 
\L 
HC=O 
I 
c=o 
I 
CH2 
HC-OH 
I 
I 
HC-OH 
HzC-OH 
3-deoxyglucmone 
Scheme 1. Decomposition pathway of the Amadori compound 
N-(1 -deoxy-o-fructos-1 -yl)-glycine (DFG) according to Led1 and 
Schleicher.' R = CH,COOH. 
Received 31 March 1994 
Accepted 30 June 1994 

DECOMPOSITION OF N-(I-DEOXY-D-FRUCTOS-~-YL)-GLYCINE 643 
glucose '(Merck, Switzerland) and 8 g (42 mmol) of 
sodium pyrosulphite (Siegfried, Switzerland) in 7.5 ml of 
water was heated to 95°C. After stabilization of the 
temperature 5 g (66 mmol) of (1-13C)-glycine (Tracer 
Tech. Inc., Sommerville, Massachusetts) were added 
step by step. The temperature was then held for 1 h at 
95 "C before cooling. The orange-brown reaction 
mixture was diluted with aqueous ethanol to a final 
volume of 360 ml containing 61% ethanol. The whole 
mixture was then transferred to a cationic ion-exchange 
column (Amberlite IR 120 H + ,  Rohm & Haas). The 
remaining glucose was washed out with ethanol-water 
(7: 3, v/v) and water, then the labelled DFG was eluted 
with 0.2 N ammonia (15 ml I - '  25% ammonia, Merck, 
Switerland). The elution was monitored by thin-layer 
chromatography (SiO, HPTLC, Merck) using butanol- 
water acetic acid (2: 1 : 1, v/v) as mobile phase. The 
solvent was evaporated and the residue recrystallized in 
ethanol-water (7:3 , v/v). The yield was 2.3 g (9.7 
mmol; 15% theor.) of white N-(1-deoxy-D-fructos-l-yl)- _ .  
(i-13C)-glycine. 
Non-labelled DFG was synthesized using the same 
reaction scheme. 
The purity of the labelled and the non-labelled DFG 
was checked by nuclear magnetic resonance (NMR) and 
elementary analysis: for ("C)DFG, C 40.76% (40.76%), 
H 6.33% (6.35%), N 5.84% (5.88%); for non-labelled 
DFG, C 40.30% (40.51%), H 6.34% (6.37%), N 5.94% 
(5.94%). 
Stock solutions of the labelled and the non-labelled 
DFG were prepared in water prior to use. 
!+ample preparation for kinetic studies 
The decomposition of DFG was studied a t  pH 6 and 
pH 7. For each series of experiments, 1 M solutions of 
DFG were prepared by dissolving 1.19 g of DFG in 5 
ml of distilled water. The pH of the solution was 6. To 
perform the reaction under neutral conditions, the pH 
was adjusted with 6 N NaOH prior to heating. The 
samples were then heated for 15, 30, 60 or 120 min at  
90°C. The kinetics were performed at  constant pH 
using a pH-stat device (Metrohm, Switzerland). About 
0-1 ml 6 N NaOH was automaticalfy added dropwise 
to the mixture during the experiments to maintain the 
pH at its initial value. The reaction mixture was imme- 
diately cooled down to room temperature and stored at 
-20°C before further treatment. 
Sample preparation for isotope dilution FAB MS/MS 
The reaction vials were vigorously shaken on a Vortex 
and centrifuged to separate possible precipitated 
material. Then, 50 pl of the sample were added to 1 ml 
water. Fifty microlitres of the diluted solution were 
added to 50 pI of ("C)DFG stock solution (2 mg ml-*) 
and 1 1.11 of it was mixed directly on the probe tip with 
approximately 1 pI of a thioglycerol-glycerol (1 : 1) 
matrix. 
The calibration curve was prepared by mixing 50 p1 
of various concentrations of (l 2C)DFG (ranging from 
0.02 mg m1-l to 8 mg ml-') with 50 p1 of (I3C)DFG 
stock solution. Aliquots (1 pl) were directly placed on 
the probe tip and mixed with approxiamtely 1 pI of the 
matrix. 
Mass spectrometry 
All experiments were performed on a Finnigan TSQ700 
triple-quadrupole mass spectrometer (Finnigan MAT, 
Bremen, Germany) equipped with an Ion Tech saddle- 
field atom gun operated at  10 kV and 0.2 mA using 
xenon as gas. The mass spectrometer was run under full 
data control (ICIS 11). 
For the isotope dilution experiments the triple quad- 
rupole was set to fragment ion scan mode. With the first 
quadrupole, the protonated molecular ion of either 
DFG (m/z  238) or labelled (13C)DFG (m/z 239) was 
selected. At the same time the third quadrupole was set 
to scan the masses 220 Da and 221 Da respectively, rep- 
resenting the loss of one water molecule from the 
[M + H] + ion. The offset of the collision cell was set to 
10 eV (laboratory reference). Argon was the collision 
gas at a pressure of 1.6 mTorr. The two mass-analysing 
quadrupoles scanned alternatively for 0.1 s the two sets 
of corresponding masses. Both quadrupoles were tuned 
for unit mass resolution. 
RESULTS AND DISCUSSION 
Fast atom bombardment tandem mass spectrometry of 
DFG 
FAB mass spectrometry produces from DFG the com- 
monly observed protonated molecular ion with little 
fragmentation. The protonated molecular ion for DFG 
is 238 Da and for '3C-labelled DFG is 239 Da. 
Figure 1 shows the FAB mass spectra of the labelled 
and the non-labelled DFG. In addition to the molecular 
ion, the losses of one or two water molecules is 
observed leading to the ions at 221/220 Da and 203/202 
Da, respectively. 
The CID spectrum of the protonated molecular ion 
leads to similar spectra showing enhanced fragment 
ions [M + H - H,0]+ and [M + H - 2H,O]+, and 
an additional fragment corresponding to protonated 
glycine at  m/z 77 and 76 for the labelled and the 
unlabelled DFG, respectively. This low-mass fragment 
I ion was of too low intensity to be used for the quantifi- 
cation. 
The acquisition of a fragment ion instead of the 
protonated molecule itself enhances the selectivity and 
therefore decreases the probability of interference 
through matrix ions or ions from other compounds in 
the mixture. 
The use of thioglycerol in the matrix and the presence 
of sodium ions from the stabilization of the pH during 
the reaction process could form a sodium adduct of two 
matrix molecules at m/z 239 as a possible interfering 
ion. This ion also loses one water molecule, producing a 
fragment ion of the mass of 221 Da. However, thiogly- 
cero1 easily undergoes oxidation to the disulphide, 
644 
’I‘ 
A. A. STAEMPFLI, I. BLANK, R. FUMEAUX A N D  L. B. FAY 
y = - 5.6037 x2+ 9.0661 x + 0.M35 
I I E+ 06 
1001 
P 3 
*: glycerol 
m l z  
6.19 
m l z  
Figure 1. FAB mass spectra of the non-labelled and the “C-labelled DFG showing the two neutral losses of a water moiety from the 
protonated molecular ion. 
which forms an ion at  m/z 215. Its sodium adduct 
appears at m/z 237. Therefore no interfering ions were 
observed. 
The calibration curve (Fig. 2) was prepared by adding 
various amounts of DFG to a constant known amount 
of the 13C-labelled DFG and, thereafter, by measuring 
the ratio of the signal at m/z 220 to the signal a t  m/z 
221. The results show that the calibration line rep- 
resents a second-order curve. This is due to the natural 
I3C isotope peak of the unlabelled DFG (9.8% at mass 
221) which coincides with the acquired mass of the 
internal standard.” This second-order calibration curve 
was used to quantify DFG in model reactions. 
The goodness of fit (accuracy) of the measured values 
with the theoretical values was checked as follows. The 
theoretical I3C enrichment of the labelled DFG was 
calculated for each calibration point, knowing the 
. 
E 80 P 
DECOMPOSITION OF Nil-DEOXY-o-FRUCTOS-I-YL)-GLYCINE 615 
y=O.9727x+O.9438 
0.99977 r"/Z - -  r2=0.99954; r =  
amount of labelled and unlabelled DFG in the mixture. 
The I3C enrichments were expressed in mole per cent 
excess (MPE) and were calculated according to the 
formula: 
[Qi/(Qi + QJI x 100 
where Q1 = quantity of (I3C)DFG in the calibration 
point; and Q, =quantity of ("C)DFG in the cali- 
bration point. 
Furthermore, with the measured ratio of the inten- 
sities of the masses 221/220, we calculated the measured 
"C enrichment :Is 
URS - RoM(1 + Rs - Roll x 100 
where R, = ratio 221/220 in each calibration point; and 
R ,  = ratio 221/220 measured with pure unlabelled 
DFG. 
Finally, we plotted theoretical 13C enrichment versus 
measured ''C enrichment (Fig. 3). This curve displays a 
slope of 0.9727, which slightly deviates from the theo- 
retical value of 1.oooO. This difference is due to the iso- 
topic enrichment of the starting (l-'3C)glycine (99 at.% 
excess) used for the preparation of (I3C)DFG and due 
to the chemical purity of the synthesized DFG iso- 
topomers (99% each). The correlation coeficent of the 
curve was calculated as 0.99977. These experiments 
E 1 O L  l 
0 20 40 60 80 100 
Theoretical l 3  C enrichment (MPE) 
Figure 3. Determination of the accuracy of the measurements. 
The theoretical "C enrichments were calculated from the known 
amounts of ("C)DFG and ('*C)DFG mixed together. The mea- 
sured "C enrichments were obtained from the ratio of the mea- 
sured masses 220 to 221. Each point represents the mean of three 
determinations. 
demonstrate that the measured results are in good 
agreement with the theoretical values. 
The repeatability of the method was checked with 
several samples containing the same amount of DFG. 
Each sample was diluted separately. The results are 
listed in Table 1, where the ratio 220/221 is the ratio of 
the observed fragment ion and represents the mean of 
two measurements. 
This rapid method using the labelled DFG as internal 
standard works well for the quantification of DFG. 
Other Amadori compounds vary in their hydropho- 
bicity and therefore their presence at  the surface of the 
matrix would change in a non-linear manner. For each 
target compound the corresponding labelled Amadori 
compound therefore has to be synthesized and the 
experiment has to be repeated. 
It must be pointed out that this FAB MS/MS method 
does not tolerate too strong salt concentrations or 
buffer solutions. Preliminary experiments showed that 
high concentrations of alkali metals favour the forma- 
tion of the single- or multi-metalated molecular species 
which lowers the accuracy of quantification. Buffers, on 
the other hand, lead to a complete suppression of all the 
signals, and thus prevenf any quantification. 
Kinetics of the degradation of DFG 
The method was applied to different model reaction 
mixtures to determine the kinetic properties of the deg- 
radation of DFG. Figure 4 represents the decomposi- 
tion rate of DFG in an unbuffered aqueous solution at 
90°C over a period of 2 h a t  pH 6 (m) and pH 7 (O), 
kept constant during the whole reaction. 
Continuous decomposition of DFG was observed in 
all samples analysed. The decomposition rate was 
Table 1. Repeatability of the method after mea- 
suring twice several samples containing 
the same amount of DFG 
Variation Ratio of the Standard 
ions 2201221 deviation cwRicient 1%) 
Sample A 2.96 0.03 1 .oo 
Sample B 2.84 0.03 0.91 
Sample C 2.88 0.01 0.35 
Sample D 2.80 0.01 0.52 
c 
0.20 -- 
0.00 i P 
0 20 40 60 80 IO0 I20 
Time [min] 
Figure 4. Kinetics of the degradation of DFG at pH 6 (m) and pH 7 (0). 
646 A. A. STAEMPFLI. 1. BLANK, R. FUMEAUX AND L. B. FAY t 
strongly affected by pH. DFG decomposed at pH 6 
moderately (B) and more than 50% was still unreacted 
after 2 h heating. The rate of decomposition was con- 
siderably increased a t  pH 7. Thus, more than 50% was 
already degraded after 1 h reaction (0). The lower 
decomposition rate of DFG a t  pH 6 compared to pH 7 
may be explained by the type of enolization favoured 
and the stability of the resulting deoxyosones. At pH 6, 
DFG mainly decomposes via 12enolization forming 
relatively stable 3-deoxyglucosone, whereas a t  pH 7 the 
2,3-enolization pathway dominates, thus leading to the 
very unstable I-deoxyglucosone. 
According to Anet," the pH of the reaction medium 
is one important parameter which controls the decom- 
position pathway of Amadori compounds. 1,2-Enoliza- 
tion is promoted under acidic conditions, resulting in 
3-deoxyosones, whereas neutral and alkaline conditions 
favour 2,3-enolization, forming 1-deoxyosones. This 
effect has been explained by the degree of protonation 
of the nitrogen atom.". Led1 and co-workers have 
recently confirmed the predominance of I-deoxyosones 
at  pH 7.16 Significantly higher amounts of 3-deoxy- 
osones were found at  pH 5 compared to  pH 7. A more 
rapid fragmentation of these reactive ct-dicarhonyls was 
observed with increasing pH. So far, 1-deoxyosones 
could only be detected as quinoxaline 
which is an indication for their high instability, whereas 
3-deoxyosones have long been isolated in pure form.18 
In conclusion, these experiments show that FAB 
MS/MS is a straightforward method to study mechanis- 
tic aspects of the degradation of DFG in model systems. 
These results will contribute to our basic knowledge on 
the Maillard reaction. To continue this work, we will 
link kinetic experiments with studies on the formation 
of volatiles which will allow us to evaluate the role of 
Amadori compounds in generating flavours. 
Acknowledgements 
The authors are grateful to Drs I. Horman and G. Philippossian for 
helpful discussions, Dr D. Welti for N M R  measurements of the 
labelled and the non-labelled DFG and to S. Devaud for her expert 
technical assistance. 
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1. F. Led1 and E. Schleicher. Angew. Chem. Int. Ed. Engl. 29, 
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2. J. Mouron,'Pmg. FoodNutr.Sci. 5, 5 (1981). 
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Study on the Decomposition of the Amadori Compound N-( l-Deoxy-D-fructos-l-yl)-glycine in Model Systems : Quantification by Fast Atom Bombardment Tandem Mass Spectrometry

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Study on the Decomposition of the Amadori Compound N-( l-Deoxy-D-fructos-l-yl)-glycine in Model Systems : Quantification by Fast Atom Bombardment Tandem Mass Spectrometry

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