Glycine Specific Novel Maillard Reaction Products: 5-Hydroxy-1,3-dimethyl-2(1H)-quinoxalinone and Related Compounds

Article abstract of DOI:10.1021/jf960528g

A major product of the reaction of D-glucose with excess glycine was detected by Py/GC/MS analysis and subsequently synthesized and isolated using focused microwave irradiation at atmospheric pressure conditions. Spectroscopic analyses by NMR, FTIR, MS, and UV in conjunction with labeling studies have indicated the unknown compound to be 5-hydroxy-1,3-dimethyl-2(1H)-quinoxalinone. Analysis of the mass spectra of the unknown compound obtained by reacting separately labeled [¹³C]-D-glucoses with unlabeled glycines and reacting separately labeled [¹⁵N]- and [¹³C]glycines with unlabeled D-glucoses indicated the incorporation of 10 carbon atoms (six from sugar, one C-1 atom of glycine, and three C-2 atoms of glycine) and two nitrogens. In addition, other derivatives of quinoxalinone formed in D-glucose/glycine and D-glucose/L-alanine mixtures were also tentatively identified based on similar amino acid incorporation patterns and mass spectroscopic analysis.

Full text of DOI:10.1021/jf960528g

J. Agric. Food Chem. 1997, 45, 697
−702
697
Glycin e Sp ecific Novel Ma illa r d Rea ction P r od u cts:
5-Hyd r oxy-1,3-d im eth yl-2(1H)-qu in oxa lin on e a n d Rela ted
Com p ou n d s
Anahita Keyhani and Varoujan A. Yaylayan*
Department of Food Science and Agricultural Chemistry, McGill University, 21,111 Lakeshore,
Ste. Anne de Bellevue, Quebec, Canada H9X 3V9
A major product of the reaction of D-glucose with excess glycine was detected by Py/GC/MS analysis
and subsequently synthesized and isolated using focused microwave irradiation at atmospheric
pressure conditions. Spectroscopic analyses by NMR, FTIR, MS, and UV in conjunction with labeling
studies have indicated the unknown compound to be 5-hydroxy-1,3-dimethyl-2(1H)-quinoxalinone.
Analysis of the mass spectra of the unknown compound obtained by reacting separately labeled
[13C]-D-glucoses with unlabeled glycines and reacting separately labeled [15N]- and [13C]glycines
with unlabeled D-glucoses indicated the incorporation of 10 carbon atoms (six from sugar, one C-1
atom of glycine, and three C-2 atoms of glycine) and two nitrogens. In addition, other derivatives
of quinoxalinone formed in D-glucose/glycine and D-glucose/L-alanine mixtures were also tentatively
identified based on similar amino acid incorporation patterns and mass spectroscopic analysis.
Keyw or d s: Amadori; decomposition mechanisms; Maillard reaction; 13C-labeled D-glucose; [15N]-
and [13C]glycine and L-alanine; pyrazinones; quinoxalinone; Py/ GC/ MS; microwave-assisted synthesis
INTRODUCTION
glycine/D-glucose model systems can generate also fused
benzopyrazinone derivatives (quinoxalinones) in addi-
tion to alkyl-substituted pyrazinones, by a related
mechanism. Alkyl- and tetrahydroquinoxaline deriva-
tives have been identified in different food products such
as brewed coffee (Sasaki et al., 1987), pork liver (Mus-
sinan and Walradt, 1974), and roasted filberts (Kinlin
et al., 1972). Imidazoquinoxalines, specific products of
a creatinine/glucose/glycine mixture, have been identi-
fied in fried beef (Nagao et al., 1983).
In this paper, we report the isolation and mechanism
of formation of 5-hydroxy-1,3-dimethyl-2(1H)-quinox-
alinone in a glycine/D-glucose model system. Details of
the microwave-assisted synthesis (Yaylayan et al., 1997)
and the spectroscopic characterization (Yaylayan et al.,
1996) have been reported elsewhere.
The complexity of the Maillard reaction mixtures
precludes their complete analysis through classical
organic chemistry and requires the use of isotopically
labeled starting materials to establish the origin and
fate of multitudes of reactive intermediates that form
during the process. Identification of these intermediates
can help to predict the formation of certain end products
and eventually can lead to the classification of Maillard
reaction into its underlying elementary processes. Such
approaches have been successfully applied on a micro-
scale level by the use of pyrolysis/gas chromatography/
mass spectrometry (Py/GC/MS) as an integrated reac-
tion, separation, and identification system to elucidate
the mechanism of formation of alkyl-substituted pyrazi-
nones from D-glucose/glycine model systems (Yaylayan
and Keyhani, 1996; Keyhani and Yaylayan, 1996a) and
pyridines and naphthalenes from D-glucose/L-phenyla-
lanine (Keyhani and Yaylayan, 1996b). However, many
of the major products formed and detected by GC/MS
in the D-glucose/glycine model system could not be
identified and required the production of analytically
pure samples for spectroscopic identification.
The ability of focused microwave irradiation under
atmospheric pressure conditions to generate and extract
chemical reaction products, using a two-stage microwave-
assisted process (MAP) (Pare´ et al., 1991, 1994), was
successfully applied (Yaylayan et al., 1997) to synthesize
and isolate some of the major unknown products formed
when D-glucose was pyrolyzed in the presence of excess
glycine. MAP has been applied successfully to various
liquid-phase and gas-phase extractions and is currently
used extensively as a sample preparation tool (Be`langer
et al., 1996). Spectroscopic analysis (Yaylayan et al.,
1996) of one of the major products that has been isolated
using the microwave extraction has indicated that
MATERIALS AND METHODS
All reagents, chemicals, [1-13C]-D-glucose (99% enriched),
[2-13C]-D-glucose (99% enriched), [6-13C]-D-glucose (99% en-
riched), and [15N]glycine (98% enriched) were purchased from
Aldrich Chemical Co. (Milwaukee, WI). [3-13C]-D-Glucose (99%
enriched), [4-13C]-D-glucose (99% enriched), [5-13C]-D-glucose
(99% enriched), [1-13C]glycine (99% enriched), [2-13C]glycine
(90% enriched), [1,2-13C]glycine (99% enriched), [3-13C]-DL-
alanine (99% enriched), and [1-13C]-L-alanine (99% enriched)
were purchased from ICON Services Inc. (Summit, NJ ). [15N]-
L-Alanine (99% enriched) was purchased from CIL, Inc.
(Andover, MA). [2-13C]-DL-Alanine (92.1% enriched) was pur-
chased from C/D/N Isotopes Inc. (Pointe-Claire, Quebec).
Precoated preparative silica gel plates were obtained from
Merck (Germany). The synthesis of Amadori glycine was
1
performed according to Sosnovsky et al. (1993). 13C- and H-
NMR spectra were recorded in CDCl3 at 300 MHz using TMS
as internal standard on a Varian Unity Inova 300 instrument.
Infrared spectra were recorded in CaF2 IR cells with a 25 µm
Teflon spacer, on a Nicolet 8210 Fourier-transform spectrom-
eter equipped with a deuterated triglycine sulfate (DTGS)
detector. The Soxwave100 focused microwave extraction
system was obtained from Prolabo (Fontenay-Sous-Bois,
France), operating at an emission frequency of 2450 MHz and
a 300 W full power, equipped with at 250 mL quartz vessel
and a Graham type refrigerant column (400 mm length).
* Author to whom correspondence should be ad-
dressed [tel, (514) 398-7918; fax, (514) 398-7977; e-mail,
czd7@MusicA.McGill.CA].
S0021-8561(96)00528-6 CCC: $14.00
© 1997 American Chemical Society

698 J. Agric. Food Chem., Vol. 45, No. 3, 1997
Keyhani and Yaylayan
Ta ble 1. Ma ss Sp ectr om etr ic Da ta of Qu in oxa lin on es a n d P yr a zin on es
5-hydroxy-1,3-dimethyl-2(1H)-quinoxalinone (from glycine/D-glucose)
191(11), 190(100), 162(32), 161(23), 134(18), 133(42), 119(23),
106(6), 93(12), 92(19), 56(27)
5-hydroxy-1,3,7-trimethyl-2(1H)-quinoxalinone (from glycine/D-glucose)
5-hydroxy-1-methyl-2(1H)-quinoxalinone (from glycine/D-glucose)
205(12), 204(100), 176(21), 175(36), 161(11), 148(17), 147(48),
133(11),92(14), 56(28)
177(10), 176(100), 148(30), 120(21), 119(23), 105(24), 92(11),
79(15), 51(15)
1,7-dimethylcyclopenta-2(1H)-pyrazinone (from glycine/D-glucose or
glycine/cyclotene)
165(11), 164(100), 149(34), 135(37), 121(21), 107(24), 106(26),
94(15), 92(10), 79(10), 66(9)
5-hydroxy-1-ethyl-3-methyl-2(1H)-quinoxalinone (from L-alanine/D-glucose)
205(13), 204(100), 176(34), 148(24), 147(30), 120(22), 119(14),
106(15), 92(17), 79(14), 65(13)
1-ethyl-3,7-dimethylcyclopenta-2(1H)-pyrazinone (from L-alanine/D-glucose or
L-alanine/cyclotene)
193(13), 192(100), 191(91), 177(69), 163(11), 149(10), 121(19),
120(47), 108(29), 106(20), 94(25)
P y/GC/MS An a lysis. A Hewlett-Packard GC/mass selec-
tive detector (5890 GC/5971B MSD) interfaced to a CDS
pyroprobe 2000 unit was used for the Py/GC/MS analysis. Solid
samples (1-4 mg) of amino acid/glucose in different ratios were
introduced inside a quartz tube (0.3 mm thickness), plugged
with quartz wool, and inserted inside the coil probe. The
pyroprobe was set at the desired temperature (250 °C) at a
heating rate of 50 °C/ms and with a THT (total heating time)
of 20 s. The GC column flow rate was 0.8 mL/min for a split
ratio of 92:1 and a septum purge of 3 mL/min. The pyroprobe
interface temperature was set at 250 °C. Capillary direct MS
interface temperature was 180 °C; ion source temperature was
280 °C. The ionization voltage was 70 eV, and the electron
multiplier was 1682 V. The mass range analyzed was 30-
300 amu. The column was a fused silica DB-5 column (30 m
length × 0.25 mm i.d. × 25 µm film thickness; Supelco, Inc.).
Unless otherwise specified, the column’s initial temperature
was -5 °C for 2 min and was increased to 50 °C at a rate of
30 °C/min; immediately the temperature was further increased
to 250 °C at a rate of 8 °C/min and kept at 250 °C for 5 min.
Products that were not found in the mass spectral libraries
were identified by generating them from their proposed
precursors and comparing mass spectra and chromatographic
retention times.
Micr owave-Assisted Syn th esis an d Extr action of 5-Hy-
dr oxy-1,3-dim eth yl-2(1H)-qu in oxalin on e. A D-glucose (1.00
g, 0.005 mol) and glycine (1.25 g, 0.016 mol) mixture was
transferred into the 250 mL quartz extraction vessel of the
Soxwave 100 microwave extraction system; 2 mL of water was
then added. The vessel was inserted inside the extraction
cavity and fitted with a condenser. The irradiation was carried
out in the following sequence at full power (300 W): 2 min
on, 30 s off, 2 min on, 30 s off, 2 min on, 30 s off, and 2 min on,
for a total of 8 min of irradiation. At the end of the irradiation
sequence a dark brown and dry slurry was obtained. The
extraction step was carried out with 40 mL of hexane using
the following sequence of irradiation: 40 s on, 30 s off, and 90
s on. The solvent was decanted, dried over sodium sulfate,
and evaporated under vacuum. The resulting oil was further
purified by thick layer chromatography on silica gel using ethyl
acetate as the solvent. The title compound was the only
fluorescent band visible under UV at 365 nm: νmax (ethyl ether)
(absorbance units) 350 nm (0.68), 279 (2.58), 207 (2.47); FTIR
(CDCl3) 3158 cm-1 (Ar-OH), 2979 (-CH3), 2894 (-CH3), 1668
(N-CdO), 1653 (CdN), 1600, 1560, and 1539 (ArH), 1469 and
1382 (CH3), 1095 (C-O); 1H-NMR (CDCl3) δ 7.49 (ArH, dd,
1H), 6.66 (ArH, dd, 1H), 6.51 (ArH, q, 1H), 2.62 (NdC-CH3,
s, 3H), 3.60 (N-CH3, s, 3H), 2.20 (OH, br, H/D exch); EIMS
m/z (rel intensity) 191 (12), 190 (100), 162 (29), 161 (22), 134
(20), 133 (51), 120 (6), 119 (28), 106 (7), 93 (14), 92 (22), 81
(7), 79 (5), 78 (6), 77 (11), 76 (5), 68 (5), 67 (5), 66 (9), 64 (14),
63 (5), 56 (44), 55 (5), 54 (5), 52 (7), 51 (23), 50 (9), 42 (12), 39
(14), 38 (5).
nes in these model systems are formed through such
addition reactions. These studies have also identified
a novel aldol type reaction between the C-2 atom of
glycine and R-keto aldehydes followed by deamination
and decarboxylation, resulting in the conversion of the
aldehyde moiety into a methyl ketone, such as the
conversion of pyruvaldehyde into 2,3-butanedione and
glyoxal into pyruvaldehyde. This property of glycine as
a methylating agent, during Maillard reaction, was also
demonstrated in the present study. In addition, it has
been observed that carbon atoms of sugar, especially
C-1, can also be incorporated to a certain extent, as
methyl groups, to form methylated quinoxalinones.
P r ed iction , Con fir m a tion , a n d Id en tifica tion of
P r od u cts Ar isin g fr om Mu ltip le Ad d ition s of Gly-
cin e d u r in g Ma illa r d Rea ction . Products arising
from the incorporation of more than one amino acid
moiety into a sugar fragment could be identified by a
method based on Py/GC/MS utilized as an integrated
reaction, separation, and identification system and by
the use of labeled sugars and amino acids as reactants
(Yaylayan and Keyhani, 1996). In this approach, sugars
are reacted in the pyrolysis probe with increasing
concentrations of the amino acid relative to the sugar;
consequently, chromatographic peaks arising from mul-
tiple additions of the amino acid will increase and thus
could be identified and further confirmed by reacting
the sugar with 15N- and 13C-labeled amino acids and
observing the incorporation of multiple labels into the
product by Py/GC/MS analysis. Performing such ex-
periments with D-glucose/glycine revealed the participa-
tion of three glycine molecules in the formation of alkyl-
substituted pyrazinones (Keyhani and Yaylayan, 1996a).
In addition, other chromatographic peaks (Table 1)
such as m/z 176, 190, and 204 also showed increased
intensity by the addition of excess amino acid. Experi-
ments performed with 13C-labeled D-glucoses (indepen-
dently labeled at each carbon atom) and 15N- and 13C-
labeled glycines indicated the incorporation of all six
carbon atoms of the sugar and two nitrogens, one C-1
and three C-2 atoms of glycine into m/z 190 (see Scheme
1 and Table 2). However, m/z 176 contained only two
C-2 atoms of glycine, and m/z 204 contained up to four
C-2 atoms of glycine (see Table 3). Comparison of their
mass spectra (see Figure 1) indicated that they are
structurally related compounds, differing only in the
number of methyl group substituents, arising mainly
from the C-2 atom of glycine. The chromatographic
peak related to m/z 190 was the most abundant followed
by m/z 204 and 176, respectively. When excess glycine
was reacted with synthetic Amadori glycine, the peak
due to m/z 190 was the most intense in the pyrogram.
RESULTS AND DISCUSSION
Multiple addition reactions of amino acids to sugar
dicarbonyl fragments have not been studied extensively,
despite their importance in elucidating cross-linking of
proteins. Model studies (Keyhani and Yaylayan, 1996a)
using [13C]-D-glucoses and a series of C2, C3, and C4
dicarbonyl compounds with labeled [15N]- or [13C]gly-
cines have indicated that methyl-substituted pyrazino-
In addition, due to the similarity of the glycine
substitution pattern to that of alkylpyrazinones (Key-
hani and Yaylayan, 1996a) and intense molecular ions
in their mass spectra, it was predicted that they should
possess aromatic pyrazinone structures and as such

Glycine Specific Maillard Products
J. Agric. Food Chem., Vol. 45, No. 3, 1997 699
Ta ble 3. P er cen t Distr ibu tion of Molecu la r Ion m /z 204
Gen er a ted fr om La beled D-Glu coses or Excess La beled
Glycin esa
Sch em e 1. Or igin of Ca r bon Atom s in Qu in oxa -
lin on es
m/z m/z m/z m/z m/z
204 205 206 207 208
D-Glucose/Excess Glycine
D-glucose/glycine
98
0
0
0
0
0
0
11
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
[1-13C]-D-glucose/glycine
[2-13C]-D-glucose/glycine
[3-13C]-D-glucose/glycine
[4-13C]-D-glucose/glycine
[5-13C]-D-glucose/glycine
[6-13C]-D-glucose/glycine
D-glucose/[1-13C]glycine
85 15
97
92
100
97
92
91
3
3
8
0
3
8
0
D-glucose/[2-13C]glycine (90% enriched)
D-glucose/[15N]glycine (98% enriched)
11 34 52
98
2
0
0
ARP Glycine/Excess Glycine
ARP glycine/[1-13C]glycine
67
0
0
33
0
0
0
0
0
ARP glycine/[2-13C]glycine (90% enriched)
ARP glycine/[15N]glycine (98% enriched)
42 41 17
97
0
3
a
Values are adjusted for natural abundance; compounds less
than 99% enriched are indicated. ARP ) Amadori rearrangement
product.
Ta ble 2. P er cen t Distr ibu tion of Molecu la r Ion m /z 190
Gen er a ted fr om La beled D-Glu coses or Excess La beled
Glycin esa
m/z m/z m/z m/z
190 191 192 193
D-Glucose/Excess Glycine
D-glucose/glycine
99
0
0
0
0
0
0
0
0
1
100
100
100
100
100
95
100
0
2
0
0
0
0
0
0
5
0
16
98
0
0
0
0
0
0
0
0
84
0
[1-13C]-D-glucose/glycine
[2-13C]-D-glucose/glycine
[3-13C]-D-glucose/glycine
[4-13C]-D-glucose/glycine
[5-13C]-D-glucose/glycine
[6-13C]-D-glucose/glycine
D-glucose/[1-13C]glycine
D-glucose/[2-13C]glycine (90% enriched)
D-glucose/[15N]glycine (98% enriched)
0
ARP Glycine/Excess Glycine
ARP glycine/[1-13C]glycine
63
64
35
37
28
52
0
8
15
0
0
0
ARP glycine/[2-13C]glycine (90% enriched)
ARP glycine/[15N]glycine (98% enriched)
a
Values are adjusted for natural abundance; compounds less
than 99% enriched are indicated. ARP ) Amadori rearrangement
product.
F igu r e 1. Mass spectra of selected quinoxalinone derivatives
formed in the D-glucose/glycine mixture.
could be extracted into nonpolar solvents such as
hexane. All attempts to extract these compounds from
heated D-glucose/glycine systems failed to produce enough
quantities for spectroscopic analysis; however, when the
synthesis and extraction by hexane were performed by
focused microwave irradiation under atmospheric pres-
sure conditions (Yaylayan et al., 1997), a simple mixture
was obtained containing the compound having the ion
m/z 190 as the major component. Further purification
by preparative chromatography yielded the pure com-
pound which was assigned the structure of 5-hydroxy-
1,3-dimethyl-2(1H)-quinoxalinone (Scheme 1), based on
spectroscopic analysis.
labeled [13C]-D-glucoses at each carbon atom with
unlabeled glycines and reacting separately labeled [15N]-
and [13C]glycines with unlabeled D-glucose revealed the
incorporation of 10 carbon atoms (six from sugar, one
C-1 atom of glycine, and three C-2 atoms of glycine) and
two nitrogens into the structure of m/z 190. This
combination of carbons and nitrogens can produce only
the following elemental formula: C10H10N2O2. Analysis
by 2-D NMR (COSY) and 1H-NMR confirmed the
presence of three mutually coupled aromatic protons,
one phenolic proton and two methyl groups. FTIR
analysis indicated the presence of phenolic, alkyl, car-
bonyl, and aromatic functional groups. The parent
2(1H)-quinoxalinone shows carbonyl-stretching absorp-
tion in the region of 1660-1690 cm-1 and UV maxima
Sp ect r oscop ic Ch a r a ct er iza t ion of 5-Hyd r oxy-
1,3-d im eth yl-2(1H)-qu in oxa lin on e. The mass spec-
tra of compound m/z 190 obtained by reacting separately

700 J. Agric. Food Chem., Vol. 45, No. 3, 1997
Keyhani and Yaylayan
Ta ble 4. In cor p or a tion of La bels in Selected Ma ss
Sch em e 2. Electr on Im p a ct F r a gm en ta tion P a th w a ys
of 5-Hyd r oxy-1,3-d im eth yl-2(1H)-qu in oxa lin on e, Ba sed
on La belin g Stu d ies
Sp ectr a l F r a gm en ts of 5-Hyd r oxy-1,3-d im eth yl-2(1H)-
qu in oxa lin on ea
m/z m/z m/z m/z m/z
190 162 161 133 56
D-Glucose/Excess Glycine
D-glucose/glycine
190 162 161 133 56
191 163 162 134 57
191 163 162 134 56
191 163 162 134 56
191 163 162 134 56
191 163 162 134 56
191 163 162 133 56
191 162 161 133 56
[1-13C]-D-glucose/glycine
[2-13C]-D-glucose/glycine
[3-13C]-D-glucose/glycine
[4-13C]-D-glucose/glycine
[5-13C]-D-glucose/glycine
[6-13C]-D-glucose/glycine
D-glucose/[1-13C]glycine
D-glucose/[2-13C]glycine (90% enriched) 193 165 164 136 58
D-glucose/[15N]glycine (98% enriched) 192 164 163 135 57
a
Compounds less than 99% enriched are indicated.
droxy-1,3-dimethyl-2(1H)-quinoxalinone formation di-
rectly from the Amadori product.
Mech a n ism of F or m a t ion of 5-H yd r oxy-1,3-d i-
m eth yl-2(1H)-qu in oxa lin on e. To confirm the above
assertion that 5-hydroxy-1,3-dimethyl-2(1H)-quinoxali-
none could be formed directly from the glycine Amadori
compound, 2-fold molar excess of [15N]glycine was
reacted with unlabeled glycine Amadori product (ARP),
and label distribution was analyzed for the parent ion
m/z 190 and fragment m/z 56. If the compound is
formed only through the 3-deoxyglucosone (3DG) path-
way, by reacting with 3 mol of free glycine, it is expected
to observe a high percentage of 15N incorporation into
the product due to the presence of excess [15N]glycine,
with the formation of ions at m/z 192 and 57. For
example, in the same reaction mixture, trimethylpyrazi-
none (m/z 138), which is formed by the reaction of 3 mol
of glycine with 2,3-butanedione (Keyhani and Yaylayan,
1996a), through a similar pathway, generates 60% of
doubly labeled parent ion (m/z 140), 30% singly labeled,
and 10% unlabeled. However according to Table 2, only
15% of the parent ion of 5-hydroxy-1,3-dimethyl-2(1H)-
quinoxalinone was doubly labeled (m/z 192, 3DG + 2 ×
[15N]glycine), 52% was singly labeled (m/z 191, ARP +
[15N]glycine or 3DG + [15N]glycine + glycine), and 35%
was unlabeled (m/z 190, ARP + glycine). In addition,
only 25% of m/z 56 was labeled (m/z 57), indicating 75%
of the 5-hydroxy-1,3-dimethyl-2(1H)-quinoxalinone was
formed by direct addition of glycine (either released from
ARP or added) to the Amadori product when it was
pyrolyzed in the presence of excess [15N]glycine.
The proposed mechanism of formation of 5-hydroxy-
1,3-dimethyl-2(1H)-quinoxalinone is illustrated in Scheme
3. The enol form of Amadori glycine undergoes a
â-elimination reaction to produce an R-keto imine
derivative which could be generated also from 3DG
through reaction with glycine. Reaction with a second
mole of glycine and subsequent dehydrations produce
a conjugated diimine which undergoes a dehydration
reaction between the two carboxylic acid groups of the
glycine to generate a cyclic anhydride. The cyclic
anhydride then undergoes an intramolecular cyclization
followed by decarboxylation to form a 6-substituted
N-methylpyrazinone. The latter can undergo an aldol
type intramolecular condensation and aromatization to
produce 5-hydroxy-1-methyl-2(1H)-quinoxalinone, which
has been detected in the same reaction mixture (Table
1). The imine functionality in 2(1H)-quinoxalinones is
known to react with carbon nucleophiles (Cheeseman
and Cookson, 1979); the C-2 carbon of glycine or acetic
acid generated from either glycine or glucose can
(H2O) at 343, 287, 254, and 228 nm (Cheeseman and
Cookson, 1979). These values are consistent with those
reported in Materials Methods. In addition, the parent
2(1H)-quinoxalinone fragments under electron impact
conditions by successive losses of carbon monoxide and
hydrogen cyanide from the molecular ion (Kovacik et
al., 1973). According to Scheme 2, the compound m/z
190 shows similar fragmentations. Since position C-3
is substituted with a methyl group, a loss of methyl
cyanide (41 amu) is observed rather than that of a
hydrogen cyanide.
One of the advantages of using labeled reactants is
that all the atoms of a product can be traced back to
their origin in the starting material. This fact facilitates
not only the elucidation of their mechanism of formation
but also the assignment of their mass spectral frag-
ments. The molecular ion m/z 190 (100%) loses carbon
monoxide (originating from C-1 of glycine) to produce
m/z 162 (29%), which in turn loses another carbon
monoxide (originating from C-6 of glucose) to produce
m/z 134 (20%), which either loses a methyl or methyl
cyanide group to produce m/z 119 (28%) and 93 (14%),
respectively. Ion at m/z 162 (29%) can also lose a
hydrogen atom to produce m/z 161 (22%), which in turn
can lose carbon monoxide to produce m/z 133 (51%) that
undergoes successive losses of methyl cyanide and a
methyl group to produce m/z 92 (22%) and 77 (11%)
successively, as illustrated in Scheme 2 (see also Table
4). Labeling experiments indicate that fragment m/z
56 (44%) incorporates one C-1 atom of D-glucose, one
nitrogen atom, and two C-2 atoms of glycine. The
importance of this fragment lies in the fact that it
establishes the connectivity of nitrogen atom to C-1
atom of D-glucose, indicating the possibility of 5-hy-

Glycine Specific Maillard Products
J. Agric. Food Chem., Vol. 45, No. 3, 1997 701
Sch em e 3. P r op osed Mech a n ism of F or m a tion of
5-Hyd r oxy-1,3-d im eth yl-2(1H)-qu in oxa lin on e fr om D-
Glu cose a n d Glycin e, Ba sed on La belin g Stu d ies
Sch em e 4. P r op osed Mech a n ism of F or m a tion of 5-
Hyd r oxy-1,3,7-tr im eth yl-2(1H)-qu in oxa lin on e fr om D-
Glu cose a n d Glycin e, Ba sed on La belin g Stu d ies
Ta ble 5. P er cen t Distr ibu tion of Molecu la r Ion m /z 204
Gen er a ted fr om La beled D-Glu coses or Excess La beled
L-Ala n in ea
m/z
m/z
m/z
D-glucose/L-alanine
D-glucose/L-alanine
204 205 206
98
0
0
0
0
0
0
12
1
0
2
98
98
98
100
100
100
88
18
5
0
2
2
2
0
0
0
0
[1-13C]-D-glucose/L-alanine
[2-13C]-D-glucose/L-alanine
[3-13C]-D-glucose/L-alanine
[4-13C]-D-glucose/L-alanine
[5-13C]-D-glucose/L-alanine
[6-13C]-D-glucose/L-alanine
D-glucose/[1-13C]-L-alanine
D-glucose/[2-13C]-DL-alanine (92% enriched)
D-glucose/[3-13C]-DL-alanine
D-glucose/[15N]-L-alanine
methylate (Keyhani and Yaylayan, 1996a) this position
to form 5-hydroxy-1,3-dimethyl-2(1H)-quinoxalinone.
Oth er Qu in oxa lin on e a n d P yr a zin on e Der iva -
tives Detected in Glycin e or L-Ala n in e Mod el
System s. Pyrazinones and quinoxalinones, in general,
are formed during Maillard reaction, by multiple addi-
tions of amino acids to an R-dicarbonyl compound. This
interaction can lead to the formation of different deriva-
tives, depending on the structure of the dicarbonyl
fragment. It has been demonstrated that pyruvalde-
hyde and 2,3-butanedione can generate alkyl-substitut-
ed pyrazinones, and Amadori products or 3-DG can
generate alkyl-substituted quinoxalinones. In addition,
the initially formed 5-hydroxy-1-methyl-2(1H)-quinox-
alinone can undergo methylation at the imine site to
produce 5-hydroxy-1,3-dimethyl-2(1H)-quinoxalinone,
which in turn can produce a 5-hydroxy-1,3,7-trimethyl-
2(1H)-quinoxalinone by methylation, through, for ex-
ample, Michael type addition, as proposed in Scheme
4. However, the position of the methylation site is not
confirmed. To demonstrate the generality of this reac-
tion with other amino acids containing alkyl side chains,
the L-alanine reaction was investigated, similar to that
of glycine, with variously labeled starting materials. The
main quinoxalinone structure, tentatively identified by
labeling studies, was 5-hydroxy-1-ethyl-3-methyl-2(1H)-
quinoxalinone (m/z 204, Tables 1 and 5), the equivalent
product to that of 5-hydroxy-1-methyl-2(1H)-quinoxali-
none from glycine (Scheme 1, m/z 176). It is worth
mentioning that when D-glucose was replaced with a
keto sugar, such as D-fructose or D-tagatose, the peaks
81
95
99
0
1
a
Values are adjusted for natural abundance; compounds less
than 99% enriched are indicated.
associated with quinoxalinone structures increased
significantly, in both glycine and L-alanine systems. This
observation could be related to the fact that Heyn’s
product is more reactive toward the second amino acid
attack, considering the carbonyl group being reacted is
an aldehyde.
Cyclic R-dicarbonyls, such as cyclotene, are also
known to be formed in Maillard systems. Since cyclo-
tene was detected in D-glucose/glycine and D-glucose/L-
alanine systems, its ability to undergo similar reactions
was also investigated. Commercial cyclotene was re-
acted in the pyrolysis probe, with excess glycine or
L-alanine in separate experiments, and the mass spectra
of the products formed were compared to that of glycine
or L-alanine reaction products with glucose (Table 1).
The main product formed in both systems had a mo-
lecular ion at m/z 188 which could arise from dimeriza-
tion of cyclotene followed by the loss of 2 mol of water.
However, the glycine/cyclotene reaction also produced

702 J. Agric. Food Chem., Vol. 45, No. 3, 1997
Keyhani and Yaylayan
Sch em e 5. P r op osed Mech a n ism of F or m a tion of Cy-
clop en ta -2(1H)-p yr a zin on es fr om D-Glu cose or Cy-
clop en ten e a n d Glycin e or L-Ala n in e, Ba sed on La -
belin g Stu d ies
LITERATURE CITED
Be´langer, J . M. R.; Li, K.; Fingas, M. F.; Lao, R. C.; Shu, Y.
Y.; Punt, M. M.; Pare´, J . R. J . Microwave-Assisted Process
(MAP®): A Survey of Applications to Sample Preparation.
Proc. 30th Microwave Power Symposium, International
Microwave Power Institute, 1996.
Cheeseman, G. W. H.; Cookson, R. F. Condensed pyrazines.
The chemistry of heterocyclic compounds; J ohn Wiley and
Sons: New York, 1979; Vol. 35, pp 78-119.
Keyhani, A.; Yaylayan, V. Elucidation of the mechanism of
pyrazinone formation in glycine model systems using labeled
sugars and amino acids. J . Agric. Food Chem. 1996a , 44,
2511-2516.
Keyhani, A.; Yaylayan, V. Pyrolysis/GC/MS analysis of N-(1-
deoxy-D-fructos-1-yl)-l-phenylalanine: Identification of novel
pyridine and naphthalene derivatives. J . Agric. Food Chem.
1996b, 44, 223-229.
Kinlin, T. E.; Muralidhara, R.; Pittet, A. O.; Sanderson, A.;
Walradt, J . P. Volatile components of roasted filberts. J .
Agric. Food Chem. 1972, 20, 1021-1028.
Kovacik, V.; Federonko, M.; J ezo, I. Effect of 2,3-substituents
on the mass spectrometric fragmentation of quinoxalinones.
Org. Mass. Spectrom. 1973, 7, 449-457.
Mussinan, C. J .; Walradt, J . P. Volatile constituents of
pressure cooked pork liver. J . Agric. Food Chem. 1974, 22,
827-831.
Nagao, M.; Sato, S.; Sugimura T. Mutagens produced by
heating foods. In The Maillard reaction in foods and
nutrition; Waller, G. R., Feather, M. S., Eds.; ACS Sympo-
sium Serries 215; American Chemical Society: Washington,
DC, 1983; pp 521-536.
Pare´, J . R. J .; Sigouin, M.; Lapointe J . U.S. Patent 5,002,784,
March 1991 (various international counterparts).
Pare´, J . R. J .; Be´langer, J . M. R.; Stafford, S. S. Microwave-
Assisted Process (MAP®): A New Tool for the Analytical
Laboratory. Trends Anal. Chem. 1994, 13 (4), 176-184.
Sasaki, Y.; Shibamoto, T.; Wei, C.; Fernando, S. Biological and
chemical studies on overheated brewed coffee. Food Chem
Toxicol. 1987, 25, 225-228.
Sosnovsky, G.; Gnewuch, C. T.; Ryoo, E.-S. In the search for
new anticancer drugs. XXV: Role of N-nitrosated Amadori
compounds derived from glucose-amino acid conjugates in
cancer promotion or inhibition. J . Pharm. Sci. 1993, 82,
649-656.
a product with a molecular ion at m/z 164 and the
cyclotene/L-alanine mixture produced a product with a
molecular ion at m/z 192. Both of these compounds were
also detected in the respective mixtures of amino acids
with D-glucose and exhibited similar retention times and
amino acid incorporation patterns as that of quinoxali-
nones and pyrazinones. Based on these findings, they
were tentatively assigned 1,7-dimethylcyclopenta-2(1H)-
pyrazinone and 1-ethyl-3,7-dimethylcyclopenta-2(1H)-
pyrazinone structures, respectively. Scheme 5 depicts
the proposed mechanism of formation of cyclopentapy-
razinones from glycine and L-alanine model systems.
CONCLUSION
Yaylayan, V.; Keyhani, A. Pyrolysis/GC/MS analysis of non-
volatile flavor precursors: Amadori compounds. In Contri-
bution of low and non-volatile materials to the flavor of foods;
Pickenhagen, W.; Ho, C.-T., Spanier, A. M., Eds.; Allured
Publishing Co.: Carol Stream, IL, 1996; pp 13-26.
Yaylayan, V.; Keyhani, A.; Pare, J . R. J .; Belanger, J . Micro-
wave-assisted synthesis and spectroscopic characterization
of 5-hydroxy-1,3-dimethyl-2(1H)-quinoxalinone. Spectros-
copy 1996, in press.
Quinoxalinones and pyrazinones are formed in Mail-
lard systems by multiple additions of amino acids to
different dicarbonyls; as such, they could be viewed as
in situ indicators of formation of dicarbonyl compounds
in Maillard model systems. Elucidiation of their mech-
anism of formation was possible through the use of
labeled sugars and amino acids utilizing Py/GC/MS.
Yaylayan, V.; Matni, G.; Pare, J . R. J .; Belanger, J . Microwave-
assisted synthesis and extraction of selected Maillard reac-
tion products. J . Agric. Food Chem. 1997, 45, 149-152.
ACKNOWLEDGMENT
Received for review J uly 12, 1996. Revised manuscript
received December 6, 1996. Accepted December 27, 1996.X
V.Y. acknowledges funding for this research by the
Natural Sciences and Engineering Research Council
(NSERC) of Canada.
X Abstract published in Advance ACS Abstracts, Feb-
ruary 15, 1997.