Pyrolysis/GC/MS Analysis of N-(1-Deoxy-D-fructos-1-yl)-L-phenylalanine: Identification of Novel Pyridine and Naphthalene Derivatives

Article abstract of DOI:10.1021/jf950418u

Pyrolysis/GC/MS has been employed to analyze phenylalanine specific products formed during Maillard reaction. Phenylalanine Amadori product and different model systems containing phenylalanine and glucose, ribose, or glycerladehyde were studied. Ribbon py

Full text of DOI:10.1021/jf950418u

J. Agric. Food Chem. 1996, 44, 223
−229
223
P yr olysis/GC/MS An a lysis of N-(1-Deoxy-D-fr u ctos-1-yl)-L-p h en yl-
a la n in e: Id en tifica tion of Novel P yr id in e a n d Na p h th a len e
Der iva tives
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
Pyrolysis/GC/MS has been employed to analyze phenylalanine specific products formed during
Maillard reaction. Phenylalanine Amadori product and different model systems containing
phenylalanine and glucose, ribose, or glycerladehyde were studied. Ribbon pyrolysis was used to
study the effect of temperature (150, 200, 250 °C) on the efficiency of formation of initial pyrolysis
products from phenylalanine and Amadori phenylalanine. Quartz tube pyrolysis was used at 250
1
3
°
C to enhance the secondary reactions. To address specific mechanistic questions, [1- C]glucose
was used. These studies revealed the formation of pyridine and naphthalene derivatives such as
3
2
,5-diphenylpyridine, 1(2)-naphthaleneamine, N-methyl-1(2)-aminonaphthalene, 1-aminoanthracene,
′-phenylpyrrolo[4,5-a]dihydronaphthalene, 1(2)-(N-phenethyl)naphthaleneamine, and 1(2)-(N-phen-
ethyl-N-methyl)naphthaleneamine. The precursors for pyridine and naphthalene derivatives were
verified by GC/MS identification of the target compounds in the reaction mixtures of the postulated
precursors.
Keyw or d s: Amadori; decomposition mechanisms; Maillard reaction; 13C-labeled glucose; phenyl-
alanine; pyridine and naphthalene derivatives
INTRODUCTION
thylpyridine and 3-phenylpyridine. In a subsequent
study, Kunert-Kirchhoff and Baltes (1990) identified 58
phenylalanine specific products from the reaction of an
equimolar mixture of glucose and phenylalanine at 120,
Pyrolysis coupled with gas chromatography/mass
spectrometry (Py/GC/MS) has been demonstrated to be
a fast and convenient technique for the analysis of
Maillard reaction products arising from the Amadori
intermediate, especially with amino acids that are stable
enough under pyrolysis conditions to react with the
sugar rather than decompose (Huyghues-Despointes et
al., 1994). Py/GC/MS analysis reduces reaction char-
acterization to microscale level, which enables the
efficient use of isotopically enriched compounds for
mechanistic studies (Huyghues-Despointes and Yay-
layan, 1995). In addition, different sample introduction
techniques can reveal primary and secondary pyrolysis
products. For example, quartz tube pyrolysis promotes
bimolecular interactions, and ribbon pyrolysis produces
initial degradation products (Huyghues-Despointes et
al., 1994).
1
50, and 180 °C, among them, 3-(2′-furyl)-2-phenyl-2-
propenales, phenylhydroxy ketones, benzylpyrazines,
phenethylpyrazines, and phenethylamides, in addition
to 2-[5′-(hydroxymethyl)-2′-formylpyrrol-1′-yl]-3-phenyl-
propionic acid lactone. Sugimara et al. (1982) found
mutagenic activity in phenylalanine pyrolysate and
identified a pyridine derivative, 2-amino-5-phenylpyri-
dine, arising from phenylalanine as mutagenic. In this
paper, we report the effect of temperature on the
products arising from pyrolysis of phenylalanine and its
Amadori product, especially naphthalene derivatives,
that have not been reported in phenylalanine model
systems. The increased utilization of Maillard reaction
mixtures to impart roasted flavors to microwaveable,
vegetarian, and extruded foods has prompted regulatory
agencies to focus their attention on their chemical
composition (Manley, 1994).
Phenylalanine and its corresponding Amadori com-
pound (ARPP) thermally degrade at temperatures above
1
50 °C (Kato et al., 1971; Westphal et al., 1988). Kato
et al. (1971) identified, in the volatiles generated from
heating of phenylalanine at 300 °C, several alkyl-
substituted benzene derivatives, aromatic amines, biben-
zyl, stilbene, acetaldehyde, and aromatic aldehydes.
Papadopoulou and Ames (1994) extracted and identified
two nonvolatile products, N-(2-phenethyl)-3,4-diphen-
ylpyrrole and N-(2-phenethyl)-3,4-diphenyl-3-pyrroline-
MATERIALS AND METHODS
All reagents and chemicals were purchased from Aldrich
Chemical Co. (Milwaukee, WI). The synthesis of Amadori
phenylalanine was performed according to the procedure of
Sosnovsky et al. (1993).
P yr olysis/GC/MS An a lysis. A Hewlett-Packard GC/mass
selective detector (5890 GC/5971B MSD) interfaced to a CDS
Pyroprobe 2000 unit was used for the Py/GC/MS analysis. Two
modes of sample introduction were used: (a) 1 mg equivalent
of sample dissolved in deionized water was applied to the
platinum filament with a total heating time (THT) of 20s (the
water was evaporated using a nitrogen stream) or (b) 1-2 mg
of solid samples was introduced inside a quartz tube (0.3 mm
thickness), plugged with quartz wool, and inserted inside the
coil probe with a THT of 20 s. The GC column flow rate was
2
,5-dione, from phenylalanine heated at 210 °C in
paraffin oil. According to Baltes and Mevissen (1988),
under roasting conditions phenylalanine with a 6-fold
excess of glucose produces 155 volatile components
including furans, pyrones, pyrazines, pyrroles, aromatic
compounds, and, in addition to parent pyridine, 4-me-
*
Author to whom correspondence should be addres-
0
.8 mL/min for a split ratio of 92:1 and a septum purge of 3
sed. [telephone (514) 398-7918; fax (514) 398-7977;
e-mail czd7@MusicA.McGill.CA].
mL/min. The pyroprobe interface temperature was set at 150,
200, or 250 °C depending on the analysis, and the Pyroprobe
0021-8561/96/1444-0223$12.00/0
© 1996 American Chemical Society

224 J. Agric. Food Chem., Vol. 44, No. 1, 1996
Keyhani and Yaylayan
Ta ble 1. P r od u cts Id en tified d u r in g P yr olysis of P h en yla la n in e Mod el System s, Usin g Qu a r tz Tu be a t 250 °C for 20 s
MW
Phea
ARPPa
ARPP/Phea
Glu/Phea
Rib/Phea
Gly/Phea
furans
2
2
2
2
2
1
5
3
,2′-methylene-bis(5-methyl)furan
-ethylfuran
-furancarboxaldehyde
,5-dimethylfuran
-furanmethanol
-(2-furanyl)ethanone
-methyl-2-furancarboxaldehyde
-phenylfuran
176
96
96
96
98
110
110
144
-b
-
-
-
-
-
-
-
-
+
-
-
+
+
+
+
-
-
-
-
-
-
-
+
+b
-
+
+
+
+
+
+
-
-
+
+
+
+
-
+
-
-
-
+
-
-
+
+
pyrazines
3
2
,4-dimethylpyrrolo[1,2-a]pyrazine
,6-dimethylpyrazine
146
108
122
94
-
-
-
-
-
-
+
+
-
-
-
-
-
-
-
+
-
-
-
-
+
+
+
-
trimethylpyrazine
methylpyrazine
pyrroles
-methyl-1H-pyrrole
2
81
109
147
-
-
-
+
+
-
-
-
-
-
-
-
-
-
+
-
-
-
1
1
-(1H-pyrrol-2-yl)ethanone
-(2-furanylmethyl)-1H-pyrrole
aromatics
1
1
,1′-(1,2-ethanediyl)bis(benzene)
-propynylbenzene
182
116
107
121
117
131
120
163
150
104
149
122
206
92
+
+
+
+
+
+
-
-
-
+
-
-
+
+
+
+
-
-
-
-
+
+
+
-
+
+
+
+
+
+
+
+
-
-
+
+
-
-
-
-
+
+
+
+
+
+
+
+
+
-
-
-
-
-
+
+
-
+
+
+
+
+
+
+
-
-
-
-
-
-
+
+
+
+
-
+
+
+
+
+
-
+
-
-
-
-
-
+
-
-
+
+
-
+
+
+
benzenemethanamine
phenethylamine
benzeneacetonitrile
benzenepropanitrile
phenylacetaldehyde
N-(2-phenethyl)acetamide
benzenepropionic acid
styrene
N-(2-phenethyl)formamide
benzeneethanol
trans,trans-1,4-diphenyl-1,3-butadiene
methylbenzene
ethylbenzene
1
pyridines
106
194
,1-diphenylpropene
3
4
3
-phenylpyridine
-(phenylmethyl)pyridine
,5-diphenylpyridine (X)
155
169
231
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
+
naphthalenes
2
1
′-phenylpyrollo[4,5-a]-3,4-dihydronaphthalene
(2)-naphthaleneamine
245
143
157
193
247
261
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
-
-
+
+
-
-
+
-
+
+
N-methylnaphthaleneamine
aminoanthracene
N-phenethyl-1(2)-naphthaleneamine (Y)
N-phenethyl-N-methyl-1(2)-naphthaleneamine (Z)
miscellaneous compounds
1
,2-dimethyl-1H-imidazole
96
255
144
60
-
-
-
-
-
-
+
+
+
+
-
-
-
-
-
-
+
+
+
-
-
-
-
-
+
+
-
-
-
+
lactone*
pyranone*
acetic acid
1
-hydroxy-2-propanone
74
a
Phe, phenylalanine; Glu, glucose; Gly, glyceraldehyde; Rib, ribose; ARPP, Amadori phenylalanine. b +, present; -, absent. c Lactone,
2
4
-[5′-(hydroxymethyl)-2′-formylpyrrol-1′-yl]-3-phenylpropionic acid lactone. d Pyranone, 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-
-one.
was set at the desired temperature at a heating rate of 50 °C/
ms. 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 1494 V. The mass
range analyzed was 35-350 amu. The column was a fused
silica DB-5 column (30 m length × 0.25 mm i.d. × 25 µm film
thickness; Supelco, Inc.). The column initial temperature was
RESULTS AND DISCUSSION
To investigate the mechanism of formation of phenyl-
alanine specific products during Maillard reaction,
phenylalanine, Amadori phenylalanine (ARPP), and
equimolar mixtures of glucose/phenylalanine, ribose/
phenylalanine, glyceraldehyde/phenylalanine, and Ama-
dori phenylalanine in the presence of phenylalanine
were pyrolyzed using Py/GC/MS as described under
Materials and Methods. Ribbon pyrolysis was used to
study the effect of temperature (150, 200, 250 °C) on
the efficiency of formation of initial pyrolysis products
from phenylalanine and Amadori phenylalanine. Quartz
tube pyrolysis was used at 250 °C to enhance the
secondary reaction products. The products identified
in all of the model systems studied using the quartz tube
at 250 °C are listed in Table 1. Thermal degradation
of phenylalanine alone at temperatures above 150 °C
-
5 °C for 3 min and was increased to 50 °C at a rate of 30
C/min; immediately the temperature was further increased
°
to 270 °C at a rate of 8 °C/min and kept at 270 °C for 5 min.
Products that were not found in the mass spectral libraries
were identified by comparison with literature mass spectral
data or by generating the products from their proposed
precursors and comparing mass spectra and chromatographic
retention times.
Con fir m ation of th e P r ecu r sor s of P yr idin e an d Naph -
th a len e Der iva tives. Equimolar concentrations of the pos-
tulated reactants were mixed with or without a solvent (ether)
and heated at 130 °C for 15 h and analyzed by GC/MS. The
details of each reaction are elaborated in the text.

Novel Pyridine and Naphthalene Derivatives
J. Agric. Food Chem., Vol. 44, No. 1, 1996 225
Ta ble 2. Ma ss Sp ectr om etr ic Da ta
3
-phenylfuran
145 (12), 144 (100), 116 (11), 115 (86), 89 (14), 87 (3), 86 (3), 63 (12), 62 (5)
46 (10), 145 (100), 144 (38), 117 (10), 116 (82), 115 (36), 90 (10), 89 (10), 64 (7), 63 (10),
3 (12), 62 (5)a
1
6
3
3
,5-diphenylpyridine (X)
,4-diphenylpyridine
232 (19), 231 (100), 230 (24), 203 (5), 202 (12), 115 (3), 102 (10), 101 (6), 77 (4), 76 (6)
232 (19), 231 (100), 230 (70), 216 (14), 202 (21), 115 (9), 114 (9), 102 (8), 101 (13),
8
8 (8), 76 (7)
232 (18), 231 (100), 230 (61), 228 (6), 204 (4), 203 (3), 202 (9), 154 (6), 127 (10), 116 (4),
14 (8), 102 (20), 101 (7), 77 (15), 76 (10)
170 (13), 169 (98), 168 (100), 167 (38), 166 (6), 155 (3), 154 (18), 143 (1), 142 (8), 141 (13),
39 (9), 115 (20), 63 (10), 62 (3)
71 (11), 170 (84), 169 (100), 168 (53), 167 (13), 166 (1), 155 (14), 154 (7), 143 (5), 142 (11),
41 (21), 140 (8), 139 (7), 115 (19), 63 (9), 62 (4)a
156 (12), 155 (100), 154 (49), 128 (11), 127 (13), 102 (10), 77 (5), 51 (7)
57 (4), 156 (26), 155 (100), 154 (46), 128 (12), 127 (13), 102 (11), 77 (10), 51 (12)
2
4
,6-diphenylpyridine
1
-(phenylmethyl)pyridine
1
1
1
3
2
-phenylpyridine
1
-[5′-(hydroxymethyl)-2′-formylpyrrol-1′-yl]-
256 (11), 255 (27), 211 (4), 210 (5), 193 (2), 182 (2), 180 (2), 167 (2), 148 (5),
3
-phenylpropionic acid lactone
147 (5), 131 (11),120 (9), 108 (12), 104 (5), 103 (4), 92 (11), 91 (100), 78 (7), 77 (7), 63 (5),
5
1 (8)
N-(phenethyl)-1(2)-naphthaleneamine (Y)
248 (7), 247 (31), 157 (10), 156 (100), 143 (9), 128 (6), 127 (3), 78 (9), 77 (4)
N-(phenethyl)-N-methyl-1(2)-aminonaphthalene (Z) 262 (7), 261 (34), 171 (13), 170 (100),157 (3), 155 (3), 156 (3), 128 (7), 92 (4)
N-(phenylmethylene)phenethylamine
210 (1), 209 (5), 208 (3), 132 (7), 119 (9), 118 (100), 117 (6), 103 (4), 92 (7), 91 (95), 90 (6),
9 (7), 78 (4), 77 (11), 65 (11), 63 (4), 51 (7)
8
a
With [1-13C]glucose.
A. P yr id in e Der iva tives. Pyridines as a group
have exhibited mutagenic activity based on the Ames
and Williams tests (Sasaki et al., 1987). Two pyridine
derivatives, 3-phenylpyridine and 4-(phenylmethyl)-
pyridine, were identified in the model systems on the
basis of a spectral library search. 3-Phenylpyridine has
also been identified by Baltes and Mevissen (1988). In
addition, one of the major chromatographic peaks
designated X (molecular ion at m/z 231) in the pyrogram
of ARPP (see Figure 1) showed close similarity with 2,6-
diphenyl- and 3,4-diphenylpyridine mass spectra (see
Table 2). However, one of the common major peaks (m/z
F igu r e 1. Pyrogram of phenylalanine Amadori product
generated from the ribbon probe at 250 °C for 20 s. Pyranone
2
30) differed in relative intensity when compared with
the known diphenylpyridine spectra, indicating a dif-
ferent isomer that was tentatively assigned to 3,5-
diphenylpyridine structure. The two pyridine deriva-
tives, 3-phenylpyridine and 4-(phenylmethyl)pyridine,
and compound X were formed during pyrolysis of ARPP
on the ribbon probe at 250 °C, while at 150 °C only
compound X was formed (Table 3) and it increased in
intensity with increasing temperature (Table 4). Py-
rolysis of phenylalanine on the ribbon probe produced
compound X at all temperatures studied, whereas the
other two pyridine derivatives were formed only at 250
)
2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one.
leads to the formation of components with benzyl and
phenyl residues as characteristic structural features
(
Kato et al., 1971). Py/GC/MS of phenylalanine on the
ribbon probe and in the quartz tube yields phenethyl-
amine as the main degradation product. No thermal
degradation products of phenylalanine were observed
below 200 °C. However, ARPP degraded at 150 °C and
exhibited a more complex product profile. The main
reaction products were phenylacetaldehyde (Strecker
aldehyde), styrene, 2,3-dihydro-3,5-dihydroxy-6-methyl-
°
C. These observations indicate that the precursors of
compound X are readily formed from phenylalanine
compared to the precursors of the two other pyridine
derivatives.
4
(H)-pyran-4-one (pyranone), and three unknown com-
pounds designated X, Y, and Z (see Figure 1). In
addition, pyrazines were identified only in the ARPP
model systems. The identification of the Strecker
aldehyde and the pyrazines in the glucose/phenylalanine
model system and the similarity of the pyrograms of
ARPP and the glucose/phenylalanine mixture are in-
dicative of the efficiency of Amadori rearrangement
under pyrolysis conditions similar to the proline/glucose
system (Huyghues-Despointes et al., 1994). Formation
of phenylacetaldehyde from phenylalanine has been
reported at trace levels (Kato et al., 1971). Due to its
acidic R-hydrogens, phenylacetaldehyde is capable of
condensation reactions with other aldehydes such as
glycoladehyde to form 3-phenylfuran, as reported by
Baltes et al. (1988), and was also detected in our model
systems containing glucose, ribose, and glyceraldehyde.
Proposed Mechanism of Formation of 3,5-Diphenylpy-
ridine (Compound X) and 3-Phenylpyridine. Phenethyl-
amine can be formed by the decarboxylation of phenyl-
alanine. Phenylacetaldehyde, on the other hand, can
be formed through Strecker degradation, although it has
been detected in trace amounts in the absence of sugars
(Kato et al., 1971). These two compounds are known
to react to form an imine which rearranges into enamine
due to conjugation with the benzene ring (Figure 2). The
corresponding benzaldehyde adduct [N-(phenylmethyl-
ene)phenethylamine] was detected in the heated (10
min) methanolic solution of ARPP in 10% water (Table
2). If the above enamine reacts with another mole of
phenylacetaldehyde as an N-nucleophile, it eventually
leads to the formation of N-(2-phenethyl)-2,4-diphen-
ylpyrrole (Papadopoulou and Ames, 1994). However,
enamines can act as C-nucleophiles and undergo con-
densation reactions with other carbonyls such as phe-
nylacetaldehyde or acetaldehyde to produce 1 (Figure
2). Intermediate 1 can lose a styrene molecule as shown
in Figure 2 to produce the triene 2, which can undergo
1
3
Labeling experiments with [1- C]glucose indicated that
0% of the glycoaldehyde moiety in 3-phenylfuran
originates from C1-C2 of the sugar (see Table 2). 2-[5′-
Hydroxymethyl)-2′-formylpyrrol-1′-yl)-3-phenylpropi-
onic acid lactone reported by Kunert-Kirchhoff et al.
1990) was similarly observed in the model systems
containing glucose as the carbohydrate source.
7
(
(

226 J. Agric. Food Chem., Vol. 44, No. 1, 1996
Keyhani and Yaylayan
Ta ble 3. Effect of Tem p er a tu r e on th e F or m a tion of P yr olysis P r od u cts fr om P h en yla la n in e Am a d or i Com p ou n d Usin g
th e Ribbon P r obe
system
MW
150 °C
200 °C
250 °C
aromatics
benzaldehyde
trans,trans-1,4-diphenyl-1,3-butadiene
methylbenzene
ethylbenzene
styrene
106
206
92
106
104
150
122
194
120
-a
-
-
-
-
-
-
+
+
-
+
+
+
+
+
+
+
+
+a
+
+
+
+
+
+
+
+
benzenepropionic acid
benzeneethanol
1
,1-diphenylpropene
phenylacetaldehyde
furans
-furanmethanol
2
98
110
110
144
-
-
-
-
-
+
+
+
+
+
+
+
1
5
3
-(2-furanyl)ethanone
-methyl-2-furancarboxaldehyde
-phenylfuran
pyridines
4
3
3
-(phenylmethyl)pyridine
-phenylpyridine
,5-diphenylpyridine (X)
169
155
231
-
-
+
-
+
+
+
+
+
naphthalenes
2
′-phenylpyrollo[4,5-a]-3,4-dihydronaphthalene
245
193
247
261
143
157
-
-
-
-
+
+
-
+
+
+
+
+
+
+
+
+
+
+
anthracenamine
N-phenylethyl-1(2)-naphthaleneamine (Y)
N-phenylethyl-N-methyl-1(2)-naphthaleneamine Z)
1
(2)-naphthaleneamine
N-methyl-1(2)-naphthaleneamine
miscellaneous compounds
acetic acid
60
74
144
255
-
-
+
+
+
+
+
+
+
+
+
+
2
-hydroxypropanone
b
pyranone
lactone
c
a
, present; -, absent. b 2,3-Dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one. c 2-[5′-(hydroxymethyl)-2′-formylpyrrol-1′-yl]-3-phe-
+
nylpropionic acid lactone.
Ta ble 4. Effect of Tem p er a tu r e on th e Efficien cy of F or m a tion (× 1011) in Ar ea /Mole of Molecu la r Ion s of P yr id in e a n d
Na p h th a len e Der iva tives a n d Th eir P r ecu r sor s Gen er a ted fr om Ribbon P yr olysis for 20 s of P h en yla la n in e a n d Am a d or i
P h en yla la n in e
1
50 °C
ARPPa
200 °C
ARPP
250 °C
ARPP
compound
MW
Phea
Phe
Phe
phenylacetaldehyde
phenethylamine
benzenemethanamine
benzaldehyde
benzeneethanol
N-methyl-1(2)-naphthaleneamine
120
121
107
106
122
157
143
233
247
261
155
169
231
0
0
0
0
tr
0
0
0
0
0
3
0
0
0
0
tr
2
0
0
0
0
0
9
0
6
0
0
0
0
0
0
0
0
0
0
17
18
0
0
0
135
0
0
0
0
0
0
tr
0
25
0
0
tr
tr
tr
9
0
10
5
4
2
b
0
tr
tr
7
0
4
2
2
0
41
1
1
1
1
3
4
3
(2)-naphthaleneamine
-(N-benzyl)naphthaleneamine
(2)-(N-phenethyl)naphthaleneamine (Y)
(2)-(N-phenethyl-N-methyl)naphthaleneamine (Z)
-phenylpyridine
-(phenylmethyl)pyridine
,5-diphenylpyridine (X)
0
0
tr
tr
0.5
9
113
a
Phe, phenylalanine; ARPP, Amadori phenylalanine; systems containing two components are equimolar. b tr, trace.
thermally allowed electrocyclic ring closure to produce
either 3,5-diphenylpyridine or 3-phenylpyridine after an
aromatization step. To test the validity of this assump-
tion, equimolar concentrations of phenylacetaldehyde
and phenethylamine were dissolved in ether solution
and mixed at room temperature for 15 h. The solution
was injected through the pyrolysis interface and also
through the injection port and analyzed by GC/MS
under the same conditions as mentioned under Materi-
als and Methods. The resulting chromatogram had a
peak at the same retention time with a mass spectrum
identical to that of compound X. When ARPP was
dissolved in methanol and heated for 1 h at 90 °C and
analyzed by GC/MS, 3,5-diphenylpyridine was observed
as the predominant reaction product in the pyrogram.
Proposed Mechanism of Formation of 4-Substituted
Pyridine Derivatives. The 4-substituted pyridine de-
rivatives observed in the model systems can be envis-
aged to be formed by a mechanism similar to that
depicted in Figure 2. Replacing phenylacetaldehyde
with acetaldehyde, which can be formed from phenyl-
alanine in trace amounts (Kato et al., 1971) and more
efficiently from sugars, a similar condensation product
to that shown in Figure 2 can be formed as shown in
Figure 3. The enamine (3) formed can react with either
phenylacetaldehyde or any other aldehyde such as
formaldehyde or acetaldehyde to form a triene (4) after
losing a benzene molecule. The phenylacetaldehyde
adduct, for example, can form 4-(phenylmethyl)pyridine
after an electrocyclic ring closure and aromatization
steps. When an equimolar mixture of acetaldehyde and
phenethylamine was stirred at room temperature for
15 h and subsequently heated with phenylacetaldehyde
and injected through the pyrolysis interface and also

Novel Pyridine and Naphthalene Derivatives
J. Agric. Food Chem., Vol. 44, No. 1, 1996 227
F igu r e 2. Proposed mechanism of formation of 3- and/or
5
-substituted pyridine derivatives.
F igu r e 4. (A) Mass spectrum of compound Z, tentatively
assigned the structure of N-methyl-N-phenethyl-1(2)-amino-
naphthalene. (B) Mass spectrum of compound Y, tentatively
assigned the structure of N-phenethyl-1(2)-aminonaphthalene.
In addition to the above naphthalene derivatives, two
peaks in the chromatogram of various model systems
containing phenylalanine and sugar or ARPP were
observed, one at a retention time of 31.5 min with a
parent ion at m/z 247 (compound Y) and the other at
3
1.7 min with a parent ion at m/z 261 (compound Z).
Both compounds exhibited characteristic fragmentations
of a naphthalene derivative (see Figure 4 and Table 2)
such as a fragment ion at m/z 128 (parent naphthalene)
and the presence of multiply charged ions. These two
compounds were produced at trace levels in phenylal-
anine pyrolysis products. On the ribbon probe phenyl-
alanine produced only compound Y (m/z 247) at 250 °C,
whereas in the quartz tube both compounds were
produced at 250 °C (Tables 4 and 5). On the basis of
their molecular weights and mass spectral fragmenta-
tion patterns (Table 2 and Figure 4), naphthalene
derivatives containing phenethyl groups were postu-
lated for the structures of the two unknown compounds
as shown in Figure 4. In general, the number of
naphthalene derivatives and the efficiency of their
formation (area/mole) increased for phenylalanine when
pyrolysis was conducted in the quartz tube at 250 °C
F igu r e 3. Proposed mechanism of formation of 4-substituted
pyridine derivatives.
through the injection port and analyzed by GC/MS
under the same conditions as the pyrolysis of ARPP, the
resulting chromatogram had a peak at the same reten-
tion time with a mass spectrum identical to that of
4
-(phenylmethyl)pyridine. In addition, experiments
1
3
with [1- C]glucose (Table 2) show a large percentage
of label incorporation in 4-(phenylmethyl)pyridine, indi-
cating involvement of an acetaldehyde fragment coming
from the sugar moiety, whereas 3,5-diphenylpyridine
did not show any label incorporation.
(
Table 4). At 250 °C the naphthalene derivatives
B. Na p h th a len e Der iva tives. Naphthalene de-
rivatives are suspected carcinogens (Orzechowski, 1992).
Aminoanthracene is a recognized promutagen (Phillip-
son, 1983). Thermal degradation of ARPP at 250 °C,
both in the quartz tube and on the ribbon probe,
resulted in the formation of several naphthalene deriva-
tives such as 1(2)-naphthaleneamine, N-methyl-1(2)-
aminonaphthalene, 1-aminoanthracene, and 2′-phe-
nylpyrrolo[4,5-a]dihydronaphthalene (Tables 1 and 3).
At 200 °C on the ribbon probe only the pyrrolo derivative
did not form, and at 150 °C only naphthaleneamine and
its N-methyl derivative were formed from the ARPP.
On the other hand, phenylalanine alone on the ribbon
probe did not produce any of the naphthalene deriva-
tives mentioned above. However, in the quartz tube at
produced by ARPP and phenylalanine in the quartz tube
were similar; however, the efficiency of their formation
increased by 8-fold on the basis of area/mole in the case
of the Amadori compound in the presence of phenyl-
alanine.
Proposed Mechanism of Formation of N-Substituted
1- and 2-Aminonaphthalene Derivatives. N-Substituted
1- or 2-aminonaphthalene derivatives could be formed
from corresponding aminonaphthalenes by nucleophilic
substitution reaction with proper electrophiles. Proto-
nated benzeneethanol could be one such reactant.
When commercially available 2-naphthaleneamine and
benzeneethanol were mixed in a reaction vial in the
presence of trace amounts of phenylalanine as the
proton source and then incubated at 130 °C for 15 h,
2
50 °C all, except the pyrrolo derivative, were formed.

228 J. Agric. Food Chem., Vol. 44, No. 1, 1996
Keyhani and Yaylayan
F igu r e 5. Proposed mechanisms of formation of N-substituted 1- and 2-aminonaphthalenes.
Ta ble 5. Efficien cy of F or m a tion (× 1011) in Ar ea /Mole of Molecu la r Ion s of P yr id in e a n d Na p h th a len e Der iva tives a n d
Th eir P r ecu r sor s Gen er a ted fr om P yr olysis a t 250 °C for 20 s in th e Qu a r tz Tu be of Mod el System s Con ta in in g
P h en yla la n in e
compound
MW
Phea
Glu/Phea
ARPPa
ARPP/Phea
phenylacetadehyde
phenethylamine
benzenemethanamine
benzaldehyde
benzeneethanol
N-methyl-1(2)-naphthaleneamine
120
121
107
106
122
157
143
233
247
261
155
169
231
0
187
tr
0
0
tr
3
tr
4
34
0
0
0
3
tr
8
tr
17
9
27
0
0
0
4
tr
4
0
6
4
0
340
tr
0
6
tr
7
0
35
20
6
4
156
a
1
1
1
1
3
4
3
(2)-naphthaleneamine
-(N-benzyl)naphthaleneamine
(2)-(N-phenethyl)naphthaleneamine (Y)
(2)-(N-phenethyl-N-methyl)naphthaleneamine (Z)
-phenylpyridine
-(phenylmethyl)pyridine
,5-diphenylpyridine (X)
3
tr
0.5
14
3
1
30
4
3
88
a
Phe, phenylalanine; Glu, glucose; ARPP Amadori phenylalanine; systems containing two components are equimolar. b tr, trace.
the GC/MS analysis of the reaction mixture yielded a
compound with a parent ion of m/z 247 and with the
same mass spectrum and retention time as compound
Y. 1-Aminonaphthalenes and N-substituted 1-amino-
naphthalenes could also be formed in phenylalanine
model systems by the aldol condensation of phenylac-
etaldehyde and acetaldehyde as illustrated in Figure 5.
The resulting R,â-unsaturated aldehyde (5) can react
with any primary amine to produce an R,â-unsaturated
imine (6), and this adduct can undergo an intramolecu-
lar electrophilic aromatic substitution reaction (EAS)
and can aromatize to produce the target compounds. An
equimolar mixture of phenylacetaldehyde and acetal-
dehyde was refluxed for 30 min, at the end of which
time phenethylamine was added and refluxing was
continued for an additional 15 min. Analysis of the
mixture indicated the presence of compound Y. Alter-
natively, phenylacetaldehyde can condense with glyco-
aldehyde to produce N-substituted 2-aminonaphthalene
derivatives. The initial R,â-dihydroxyaldehyde inter-
mediate (7) can undergo intramolecular electrophilic
aromatic substitution reaction (EAS) and after dehydra-
tion can produce hydroxy â-tetralone [3,4-dihydro-3-
hydroxy-2(1H)-naphthalenone] (8), which can react with
different primary and secondary amines to form N-
substituted 2-aminonaphthalene derivatives. When
phenylacetaldehyde was reacted with glycoladehyde
dimer for 15 h at 120 °C, the reaction mixture contained
3-phenylfuran as the major product. Subsequently,
further reaction with phenethylamine in the presence
of a catalytic amount of phenylalanine produced com-
pound Y (m/z 247). The aminonaphthalene derivatives,
therefore, could be a mixture of 1- and/or 2-substituted
isomers.

Novel Pyridine and Naphthalene Derivatives
J. Agric. Food Chem., Vol. 44, No. 1, 1996 229
Con clu sion . Thermal degradation of phenylalanine
containing model systems produced naphthalene and
pyridine derivatives, some of which have already been
classified as mutagenic (Orzechowski, 1992). Pyridines
as a group are heterocyclic compounds which produce
positive results with the Ames and Williams test (Sasaki
et al., 1987). Py/GC/MS was demonstrated to be a
useful and rapid technique that can be employed in
understanding the degradation pathways of Amadori
compounds. The use of isotopically enriched compounds
to determine reaction mechanisms is facilitated since
Py/GC/MS analyses can be conducted on a microscale.
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2
1
-naphthylamine in isolated rat hepatocyes. Carcinogenesis
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