Degradation of tetrahydro-β-carbolines in the presence of nitrite: HPLC-MS analysis of the reaction products

Article abstract of DOI:10.1021/jf010363y

Motivated by the identification of numerous novel tetrahydro-β-carboline-carboxylic acids in food samples, we studied the reactions of tetrahydro-β-carbolines in the presence of nitrosating agents. The anticipated formation of nitroso derivatives from unsubstituted tetrahydro-β-carbolines, and from tetrahydro-β-carboline-3-carboxylic acids was indicated by HPLC-MS/MS analysis and validated by the characteristic product ion spectra of the respective nitroso compounds. In addition, oxidative decarboxylation resulted in formation of the corresponding dihydro-β-carbolines, and in the generation of the β-carbolines harman or norharman. Subsequently, we studied the reactivity of tetrahydro-β-carboline-1-carboxylic acids derived from the Pictet-Spengler condensation of indole amines with α-oxo acids. Again, in the presence of nitrosating agents the rapid disappearance of the starting material was obvious, but no nitroso derivatives could be observed. Instead, further HPLC-MS/MS studies demonstrated that dihydro-β-carbolines were the major products of tetrahydro-β-carboline-1-carboxylic acids. Finally, we demonstrated that freshly isolated nitroso-precursors spontaneously decomposed to yield harman alkaloids. In conclusion, we revealed that nitroso-tetrahydro-β-carbolines can represent intermediates involved in the generation of β-carbolines, and we established a novel pathway for the formation of harman alkaloids from nutritional tetrahydro-β-carbolines.

Full text of DOI:10.1021/jf010363y

J. Agric. Food Chem. 2001, 49, 5993−5998
5993
Degr a d a tion of Tetr a h yd r o-â-ca r bolin es in th e P r esen ce of Nitr ite:
HP LC-MS An a lysis of th e Rea ction P r od u cts
Stefanie Diem, Birgit Gutsche, and Markus Herderich*
Institut fu¨r Pharmazie und Lebensmittelchemie, Universita¨t Wu¨rzburg,
Am Hubland, 97074 Wu¨rzburg, Germany
Motivated by the identification of numerous novel tetrahydro-â-carboline-carboxylic acids in food
samples, we studied the reactions of tetrahydro-â-carbolines in the presence of nitrosating agents.
The anticipated formation of nitroso derivatives from unsubstituted tetrahydro-â-carbolines, and
from tetrahydro-â-carboline-3-carboxylic acids was indicated by HPLC-MS/MS analysis and
validated by the characteristic product ion spectra of the respective nitroso compounds. In addition,
oxidative decarboxylation resulted in formation of the corresponding dihydro-â-carbolines, and in
the generation of the â-carbolines harman or norharman. Subsequently, we studied the reactivity
of tetrahydro-â-carboline-1-carboxylic acids derived from the Pictet-Spengler condensation of indole
amines with R-oxo acids. Again, in the presence of nitrosating agents the rapid disappearance of
the starting material was obvious, but no nitroso derivatives could be observed. Instead, further
HPLC-MS/MS studies demonstrated that dihydro-â-carbolines were the major products of tetrahy-
dro-â-carboline-1-carboxylic acids. Finally, we demonstrated that freshly isolated nitroso-precursors
spontaneously decomposed to yield harman alkaloids. In conclusion, we revealed that nitroso-
tetrahydro-â-carbolines can represent intermediates involved in the generation of â-carbolines, and
we established a novel pathway for the formation of harman alkaloids from nutritional tetrahydro-
â-carbolines.
Keyw or d s: Tetrahydro-â-carbolines; nitroso compounds; harman alkaloids; HPLC-MS; tandem
mass spectrometry
INTRODUCTION
ences therein), but only the occurrence of 2-nitroso-
tetrahydro-â-carboline-3-carboxylic acid 9 in cured meat
products and of 1-methyl-2-nitroso-tetrahydro-â-carbo-
line-3-carboxylic acid 10a /b in soy sauce has been
reported (18-20).
In the presence of dietary nitrite sources, N-nitrosa-
tion of amines, amides, and nitrogen-containing hetero-
cycles is a common reaction, and most of the resulting
nitroso derivatives have been found to be mutagenic
substances (3). The DNA alkylating intermediates caus-
ing these genotoxic effects are formed either by spon-
taneous decomposition or after metabolic activation of
the respective nitroso compounds. In addition, N-nitroso
indoles can exert genotoxicity mediated by an alterna-
tive mechanism that relies on the transfer of the nitroso
group to DNA bases (4). Besides numerous indole
amines, tetrahydro-â-carbolines have been demon-
strated to exert mutagenic effects after nitrosation (5-
9). Yet, the identity of the mutagenic products derived
from reaction of tetrahydro-â-carbolines with nitrite
remains to be established (9-11).
Nitrosation of tetrahydro-â-carbolines under acidic
conditions with aqueous sodium nitrite is likely to yield
N2-nitroso-tetrahydro-â-carbolines. In addition, the re-
versible reaction at indole nitrogen N-9 has been
described (12-13). According to model reactions (14-
16), one can expect the presence of N2-nitroso-tetrahy-
dro-â-carbolines in nitrite-treated food samples as well
as the endogenous formation of such metabolites in the
digestive system. Rather surprisingly, tetrahydro-â-
carbolines are common food constituents (17 and refer-
Motivated by the identification of novel tetrahydro-
â-carbolines in food samples (1, 2), we examined the
reaction of various tetrahydro-â-carbolines 1-8 (Figure
1) with nitrosating agents. The initial focus of this study
was to confirm the predicted formation of nitroso-
tetrahydro-â-carbolines from the ubiquitously occurring,
but rather reactive, tetrahydro-â-carbolines 5-8. Con-
sequently, we planned to develop an analytical method
for the structure-specific detection of labile nitroso-
compounds by means of HPLC coupled with tandem
mass spectrometry (HPLC-MS/MS) analysis. In addi-
tion, we aimed to characterize the major products
obtained by the nitrosation of tetrahydro-â-carbolines
and intended to study the mechanistic aspects of the
nitrite-induced degradation reaction.
MATERIALS AND METHODS
Ca u tion . Nitroso-tetrahydro-â-carbolines and related nit-
rosation products are potential mutagens and should be
handled with extreme caution.
Ap p a r a tu s. HPLC analysis was performed with a binary
high-pressure gradient HPLC system (Knauer, Berlin, Ger-
many) equipped with analytical pumpheads, a dynamic mixing
chamber (200 µL), and a Rheodyne 7125 injector with a 20-µL
sample loop. For chromatographic separation a Eurospher
100-C18 column (250 mm × 4 mm i.d., 5 µm particle size;
Knauer, Berlin, Germany) was applied. UV detection was
achieved with an HP photodiode array detector 1040A (Hewlett-
* Corresponding author. Present address: The Australian
Wine Research Institute, Waite Road, Urrbrae, SA 5064,
Australia. Fax +61-8-83036601; e-mail markus.herderich@
awri.com.au.
10.1021/jf010363y CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/29/2001

5994 J. Agric. Food Chem., Vol. 49, No. 12, 2001
Diem et al.
F igu r e 3. HPLC-MS analysis of products obtained by the
nitrosation of 0.1 mg/mL tetrahydro-â-carboline 2a . A-C, Mass
chromatograms of the precursor 2a (m/z 231, 3B) and the
products harman 18 (m/z 183, 3A) and nitroso-tetrahydro-â-
carboline 10a (m/z 260, 3C); D, total ion chromatogram.
passage through an UV detector (270 nm, Knauer, Berlin,
Germany), a Peek micro-splitter valve (Upchurch Scientific,
Oak Harbor, WA) was applied to divide the eluate. Identity of
nitroso-tetrahydro-â-carboline was confirmed by ESI-MS/MS
(flow rate 20 µL/min, 40 psi N2 as sheath gas), and the
remaining 90% of the eluate containing the purified nitroso-
tetrahydro-â-carbolines was collected, concentrated to dryness
in a gentle stream of N2, redissolved in 40 µL of water, and
immediately reanalyzed by HPLC-MS using a binary gradi-
ent. Solvent A was 0.05% TFA in water (v/v), and solvent B
was acetonitrile. HPLC was programmed as follows: pressur-
izing with 50% B, equilibration time 10 min at 10% solvent
B, and linear gradient elution: 0 min, 10% B; 20 min, 50% B;
21 min, 100% B; and 27 min, 100% B.
F igu r e 1. Tetrahydro-â-carbolines under study.
Rea gen ts. Water and acetonitrile (both of HPLC gradient
grade), and trifluoroacetic acid (spectroscopic grade), were from
Merck (Darmstadt, Germany). Norharman 17 and harman 18
(purity >99%) were supplied by Aldrich and Fluka (Deisen-
hofen, Germany), respectively. Synthesis, isolation, and spec-
troscopic characterization of tetrahydro-â-carbolines 1-8 in
Figure 1 have been described previously (1, 2).
Nitr osa tion of Tetr a h yd r o-â-ca r bolin es. A 0.3-mg ali-
quot of each of the tetrahydro-â-carbolines 1-8 was dissolved
in 1 mL of water; we added 0.1 mL of nitrosation reagent (1
M aqueous solution of NaNO2, KNO2, or nitrite pickling salt)
plus 0.2 mL of ethanol and adjusted the mixture to pH 3 with
0.1 M HCl (11). For isolation of nitroso-tetrahydro-â-carbolines
10a and 12 we applied 3 mg of 2a or 4; for the initial
nitrosation experiment (Figure 3) we utilized 0.1 mg of
tetrahydro-â-carboline 2a . The reaction vials were incubated
in the dark at 37 °C for up to 1 h. Subsequently, the nitrosation
products were analyzed immediately without any further
cleanup. As control samples, 0.3 mg of tetrahydro-â-carbolines
1-8 in 1.1 mL of water and 0.2 mL of ethanol were treated
accordingly.
Ch a r a ct er iza t ion of Nit r osa t ion P r od u ct s: Nit r oso-
tetr a h yd r o-â-ca r bolin es 9-12. 2-Nitroso-tetrahydro-â-car-
boline-3-carboxylic acid 9 was detectable only at the very
beginning of the nitrosation reaction (incubation time less than
5 min); it decomposed during extended incubation times as
well as during isolation. ESI-MS (heated capillary 180 °C): m/z
246 [M + H]+, 287 [M + MeCN + H]+, 216, 257; ESI-MS/MS
product ions (precursor ion m/z 287, collision energy 10 eV):
m/z 246, 216 [M - NO + H]+, 200.
1-Methyl-2-nitroso-tetrahydro-â-carboline-3-carboxylic acid
10a /b decomposed during isolation. ESI-MS (heated capillary
240 °C): m/z 260 [M + H]+, 301 [M + MeCN + H]+, 214, 230,
271. ESI-MS/MS product ions (precursor ion m/z 260, 12 eV):
m/z 230 [M - NO + H]+, 214, 186, 168, 156, 142, 130.
2-Nitroso-tetrahydro-â-carboline 11 was detectable only at
the very beginning of the nitrosation reaction (incubation time
less than 5 min); it decomposed during extended incubation
times as well as during isolation. ESI-MS (heated capillary
200 °C): m/z 202, 243, 284 ([M + n × MeCN + H]+, n ) 0-2),
172, 213, 254; ESI-MS/MS product ions (precursor ion m/z 202,
10 eV): m/z 172 [M - NO + H]+, 143.
F igu r e 2. Dihydro-â-carbolines under study.
Packard, Waldbronn, Germany). Sampling interval was 640
ms for 1 scan from 190 to 400 nm. For data evaluation
chromatograms were obtained at 220 and 280 nm for com-
pounds 1-12 (Figure 1), at 300 nm for norharman 17 and
harman 18, and at 354 nm for dihydro-â-carbolines 13-16
(Figure 2). Solvent A was 0.05% trifluoroacetic acid (TFA) in
water (v/v), and solvent B was acetonitrile. For linear gradient
elution HPLC was programmed as follows: 0 min, 10% B; 20
min, 30% B; 22 min, 100% B; 27 min, 100% B. The flow rate
was 1 mL/min. Quantification was achieved with the help of
external calibration using reference substances dissolved in
water at concentrations from 5 µg/mL to 80 µg/mL.
HPLC-electrospray ionization-MS/MS analysis was per-
formed utilizing a TSQ 7000 tandem mass spectrometer
system equipped with an electrospray ionization (ESI) inter-
face (Finnigan MAT, Bremen, Germany) as has been described
previously (1, 2, 21). Chromatographic separation for ESI-MS/
MS experiments was performed on a Eurospher 100-C18
column (100 × 2.0 mm i.d., 5 µm particle size; Knauer, Berlin,
Germany) using a binary gradient. Solvent A was 0.05% TFA
in water (v/v), and solvent B was acetonitrile. The HPLC was
programmed as follows: pressurizing with 50% B, equilibra-
tion time 10 min at 10% solvent B, and linear gradient
elution: 0 min, 10% B; 20 min, 30% B; 30 min, 100% B; and
35 min, 100% B. The flow rate was 200 µL/min and the
injection volume was 5 µL. For the pneumatically assisted
electrospray ionization spray the capillary voltage was set to
3.5 kV and the temperature of the heated capillary was 180-
240 °C. Positive ions were detected with a total scan duration
of 1.0 s for a single spectrum (mass range m/z 150 to m/z 500).
MS/MS experiments such as product ion scans (mass range
m/z 50 to m/z 500) were performed at a collision gas pressure
of 2.0 mTorr Ar (0.26 Pa Ar) with a total scan duration of 3.0
s for a single spectrum.
For HPLC-ESI-MS with postcolumn splitting a 20-µL
sample was injected. After chromatographic separation and

Degradation of Tetrahydro-â-carbolines
J. Agric. Food Chem., Vol. 49, No. 12, 2001 5995
1-Methyl-2-nitroso-tetrahydro-â-carboline 12 decomposed
during isolation. ESI-MS (heated capillary 220 °C): m/z 216
[M + H]+, 257 [M + MeCN + H]+, 186, 227; ESI-MS/MS
product ions (precursor ion m/z 216, collision energy 16 eV):
m/z 186 [M - NO + H]+, 170, 156, 142.
the specific RDA fragmentation of the tetrahydropyrido
moiety (1, 2). To establish an analytical method for the
detection of nitroso-tetrahydro-â-carbolines we initially
studied the products obtained by the nitrosation of
1-methyl-tetrahydro-â-carboline-3-carboxylic acid 2a /b
(18, 19). As demonstrated by the mass chromatograms
derived from the HPLC-MS analysis after 1 h of
incubation time (Figure 3), the reaction of the major
diastereomer cis-2a with NaNO2 resulted in the com-
plete degradation of the tetrahydro-â-carboline precur-
sor (Figure 3B; m/z 231). As expected, formation of a
product showing the molecular ion of the corresponding
nitroso derivate could be detected by HPLC-MS (Figure
3C; m/z 260). In addition, a compound with the molec-
ular ion [M + H]+ at m/z 183 was generated from 2a
(Figure 3A). Subsequently, identity of this major deg-
radation product as harman 18 was established with
the help of an authentic reference compound that
showed identical retention time, UV spectra, and prod-
uct ions. Comparable results were obtained when we
studied the nitrosation of diastereomer 2b or varied the
nitrite source for the model experiments.
The ESI mass spectra of 10a shown in Figure 4
demonstrated the extraordinarily labile character of this
nitroso-tetrahydro-â-carboline. Together with the mo-
lecular ion m/z 260, additional cluster ions [M + MeCN
+ H]+ at m/z 301 could be detected and demonstrated
that the ionization conditions were obviously rather
gentle. Still, decomposition of the nitroso compound
during the electrospray process resulted in the neutral
loss of NO and yielded ions m/z 230 and m/z 271 (Figure
4A). By reducing the temperature of the heated inlet
capillary to 220 °C we enhanced the intensity of the
molecular ion m/z 260, thus demonstrating that the
degradation of the nitroso-tetrahydro-â-carboline hap-
pened in the interface and not during the chromato-
graphic separation. By taking advantage of additional
skimmer CID at 10 eV we successfully suppressed the
formation of cluster ions [M + MeCN +H]+ (Figure 4B).
Nitroso-tetrahydro-â-carboline 11 was even more un-
stable than 10a and could be detected only within the
first minutes of the nitrosation reaction. To record
molecular ions [M + H]+ at m/z 202 of 11, the temper-
ature of the heated inlet capillary was further reduced
to 200 °C. Yet, it was obvious that besides extensive
formation of cluster ions [M + n × MeCN + H]+ m/z
243 and 284, significant loss of NO yielded ions m/z 172,
213, and 254 (Figure 4C). Comparable decomposition
reactions of labile nitroso compounds have already been
observed during ESI-MS analysis of N-nitroso-peptides
and S-nitroso-conjugates (23-25).
Ch a r a cter iza tion of Nitr osa tion P r od u cts: 3,4-Dih y-
d r o-â-ca r bolin es 13-16 (F igu r e 2). 3,4-Dihydro-â-carboline-
3-carboxylic acid 13 decomposed during isolation. ESI-MS: m/z
215 [M + H]+; ESI-MS/MS product ions (precursor ion m/z 215,
17 eV): m/z 197 [M - H2O + H]+, 169 [M - HCOOH + H]+.
1-Methyl-3,4-dihydro-â-carboline-3-carboxylic acid 14 could
be isolated and purified as follows. Nitrosation products
obtained from 9 mg of 2a /b were concentrated to dryness by
lyophilization. The residue was extracted with 4 mL of MeOH,
diluted with water, filtered, and concentrated again. Final
purification by HPLC yielded 2 mg (22%) of 14. ESI-MS: m/z
229 [M + H]+. ESI-MS/MS product ions (precursor ion m/z 229,
21 eV): m/z 211 [M - H2O + H]+, 188 [M - CH3CN + H]+,
183, 146. 1H NMR (400 MHz, CD3OD): δ 1.84 (s, 3H, CH3),
3.26 (dd, 1H, H4ax), 3.42 (dd, 1H, H4eq), 4.24 (dd, 1H, H3), 6.99
(dd, 1H, H6), 7.08 (dd, 1H, H7), 7.26 (d, 1H, H8), 7.53 (d, 1H,
H5), J 3,4ax ) 9 Hz, J 3,4eq ) 5 Hz, J 4ax,4eq ) 14 Hz, J 5,6 ) 8 Hz,
J 6,7 ) 7 Hz, J 7,8 ) 8 Hz.
3,4-Dihydro-â-carboline 15 decomposed during isolation.
ESI-MS: m/z 171 [M + H]+. ESI-MS/MS product ions (precur-
sor ion m/z 171, 24 eV): m/z 154 [M - NH3 + H]+, 144 [M -
HCN + H]+, 118 [indole + H]+.
1-Methyl-3,4-dihydro-â-carboline 16 could be isolated and
purified as follows. Nitrosation products obtained from 9 mg
of 4 were concentrated to dryness by lyophilization. The
residue was extracted with 4 mL of MeOH, diluted with water,
filtered , and concentrated again. Final purification by HPLC
yielded 6 mg (66%) of 16. ESI-MS: m/z 185 [M + H]+. ESI-
MS/MS product ions (precursor ion m/z 185, 21 eV): m/z 168
[M - NH3 + H]+, 144 [M - CH3CN + H]+. 1H NMR (400 MHz,
CD3OD): δ 2.03 (s, 3H, CH3), 2.74 (m, 2H, H4), 3.97 (m, 2H,
H3), 7.20 (dd, 1H, H6), 7.46 (dd, 1H, H7), 7.53 (d, 1H, H8),
7.73 (d, 1H, H5), J 5,6 ) 8 Hz, J 6,7 ) 7 Hz, J 7,8 ) 9 Hz.
Id en tifica tion of Nitr osa tion P r od u cts: â-Ca r bolin es
17 a n d 18. â-Carboline (norharman) 17 was available as an
authentic reference compound and could be identified as
follows. ESI-MS: m/z 169 [M + H]+. ESI-MS/MS product ions
(precursor ion m/z 169, 31 eV): m/z 142 [M - HCN + H]+,
115 [M - C3H4N + H]+.
1-Methyl-â-carboline (harman) 18 was available as an
authentic reference compound and could be identified as
follows. ESI-MS: m/z 183 [M + H]+. ESI-MS/MS product ions
(precursor ion m/z 183, 31 eV): m/z 168, 115 [M - C4H6N +
H]+.
The extraordinarily labile character of the nitroso-
tetrahydro-â-carbolines was obvious from the ESI mass
spectra. Accordingly, all attempts to isolate and con-
centrate the compounds for further identification by
NMR spectroscopy resulted in the degradation of the
nitroso derivatives. Yet, the product ion spectra of all
compounds under study could be reproduced without
observation of significant alterations. Consequently, the
structural characterization of the nitrosation products
relied on the thorough interpretation of the MS/MS
experiments which are discussed now in detail.
Besides characteristic retention times and molecular
ions, all nitroso-tetrahydro-â-carbolines showed the
indicative loss of NO [M + H - 30]+ in the ESI mass
spectra and in the product ion spectra (Figure 5). As
was established before, tetrahydro-â-carboline-carbox-
RESULTS AND DISCUSSION
Nitr osa tion of Tetr a h yd r o-â-ca r bolin es: An a ly-
sis of th e P r od u cts by HP LC-ESI-MS/MS. Previous
studies on the analysis of â-carbolines in food samples
had revealed that alkaloids such as 1-8 were effectively
ionized by the electrospray process yielding exclusively
protonated molecule ions in the positive mode. Conse-
quently, all tetrahydro-â-carbolines could be identified
unambiguously by means of HPLC-MS/MS with the
help of their characteristic product ion spectra showing

5996 J. Agric. Food Chem., Vol. 49, No. 12, 2001
Diem et al.
F igu r e 5. Product ion spectrum of nitroso-tetrahydro-â-
carboline 10a (collision energy 12 eV).
of the typical loss of NO. In addition, 12 was also
characterized by elimination of H2NNO, whereas 11
showed the RDA product ion m/z 143 ([M - NO -
HNCH2 + H]+, intensity <10%). Taken together, the
MS/MS experiments suggested the formation of nitroso-
tetrahydro-â-carbolines 9-12 which were most likely
modified at the tetrahydropyrido nitrogen. It should be
noted that syn- and anti-isomers of these nitroso com-
pounds coeluted (26) and could not be differentiated by
HPLC-MS.
Analyzing the nitrosation reactions with tryptophan-
derived tetrahydro-â-carboline-1,3-dicarboxylic acids
5-6 or tryptamine-related tetrahydro-â-carboline-1-
carboxylic acids 7-8, we could not detect any of the
respective nitroso-tetrahydro-â-carbolines by means of
HPLC-MS/MS. Instead, the dihydro-â-carbolines 13-
16 in Figure 2 represented the major degradation
products of all tetrahydro-â-carbolines having a carboxy
group at C1. Pyruvate-derived 1-methyl-dihydro-â-car-
bolines 14 and 16 were more stable than the Pictet-
Spengler products of glyoxylic acid 13 and 15. Conse-
quently, we could isolate 14 and 16 and collect further
evidence by NMR spectroscopy to support the proposed
structures. The proton NMR data were consistent with
previously published results (22, 27). According to the
missing H1, combined with the signals and typical
coupling constants of H3 and the methylene group at
C4, the double bonds in 14 and 16 could be unambigu-
ously located at C1-N2. Besides elimination of NH3, the
product ion spectra of both 1-methyl-3,4-dihydro-â-
carbolines 14 and 16 showed fragments which were
formed by the neutral loss of acetonitrile and proved to
be characteristic for the methyl substituent. In analogy,
diagnostic ions such as the loss of HCN implied the
structure of labile 3,4-dihydro-â-carboline 15. Accord-
ingly, the unstable 3,4-dihydro-â-carboline-3-carboxylic
acid 13 was tentatively identified with the help of its
UV absorbance, molecular ion [M + H]+ at m/z 215, and
product ion m/z 169 ([M - HCOOH + H]+), which
confirmed the presence of the carboxyl group. Hence,
we established that oxidative decarboxylation of tet-
rahydro-â-carboline-1-carboxylic acids 5-8 yielded the
corresponding 3,4-dihydro-â-carbolines 13-16. In ad-
dition, HPLC-MS/MS analysis demonstrated formation
of 13-16 as minor products following the reaction of
tetrahydro-â-carbolines 2-4 in the presence of nitrite.
Nitr osa tion of Tetr a h yd r o-â-ca r bolin es: Mech a -
n istic Con sid er a tion s. Although thermal or electro-
chemical breakdown of tetrahydro-â-carbolines has been
reported before (28, 29), our experiments (which are
F igu r e 4. ESI mass spectra of nitroso-tetrahydro-â-carbolines
10a (4A, heated cap. 240 °C; 4B, heated cap. 220 °C plus
skimmer CID 10 eV) and 11 (4C, heated cap. 200 °C).
ylic acids can be identified unambiguously by the
specific RDA cleavage of the tetrahydropyrido moiety
(1, 2). Consequently, the absence of these prominent ions
[M + H - 73]+ at m/z 173 (9) and 187 (10a /b) dem-
onstrated the modification of the tetrahydropyrido
nitrogen of tetrahydro-â-carboline-carboxylic acids 1 and
2a /b which was indicative for N2-nitroso derivatives 9
and 10a /b. In contrast, 1-methyl-2-nitroso-tetrahydro-
â-carboline-carboxylic acids 10a /b yielded fragments at
m/z 186 from neutral loss of CO2 and NO. Product ions
m/z 214 and 168 in Figure 5 were formed by consecutive
loss of formic acid (or H2O and CO) from the carboxy
moiety and H2NNO (or H2O and N2). This demonstrated
that 10a /b most likely were N-nitroso-carboxylic acids
and did not represent nitrite anhydrides such as 1-meth-
yl-tetrahydro-â-carboline-CO2NO. In addition, perform-
ing our nitrosation reactions in the presence of ethanol
further reduced the risk of obtaining nitrite esters.
Product ion spectra of both nitroso-tetrahydro-â-carbo-
lines 11 and 12 revealed base peaks formed as a result

Degradation of Tetrahydro-â-carbolines
J. Agric. Food Chem., Vol. 49, No. 12, 2001 5997
Ta ble 1. Nitr osa tion of Tetr a h yd r o-â-ca r bolin es
precursora
products
1 (55%)
2 (8%)
9 (1%); 13 (n.d.); 17 (44%)
10 (28%); 14 (10%); 18 (54%)
11 (6%); 15 (25%); 17 (8%)
12 (3%); 16 (10%); 18 (1%)
13 (88%); 17 (2%)
14 (78%); 18 (6%)
15 (82%); 17 (n.d.)
16 (96%); 18 (n.d.)
3 (61%)
4 (86%)
5 (10%)
6 (16%)
7 (18%)
8 (4%)
a
2, 5, 6, and 10 as mixture of diastereomers.
F igu r e 6. HPLC-MS analysis of products obtained by
spontaneous decomposition of nitroso-tetrahydro-â-carboline
10a . A-C, Mass chromatograms of the educt 10a (m/z 260,
6C) and the products harman 18 (m/z 183, 6A) and tetrahydro-
â-carboline 2a (m/z 231, 6B); D, total ion chromatogram.
summarized in Table 1) demonstrated for the first time
the facile degradation of tetrahydro-â-carbolines in the
presence of commonly used nitrosation agents. More
important, these reactions established a novel chemical
pathway for the formation of bioactive harman alkaloids
from indole amines via their respective nitroso deriva-
tives. Obviously, tetrahydro-â-carboline-3-carboxylic ac-
ids 1 and 2a /b represented the most effective progeni-
tors of norharman 17 or harman 18, and yielded nitroso-
tetrahydro-â-carbolines and dihydro-â-carbolines as side
products. In contrast, isomeric tetrahydro-â-carboline-
1-carboxylic acids 7 and 8 were as reactive, but yielded
exclusively dihydro-â-carbolines 15 and 16 by oxidative
decarboxylation. As important, neither the correspond-
ing nitroso-tetrahydro-â-carbolines nor formation of
harmanes could be observed following the reaction of
tetrahydro-â-carboline-1-carboxylic acids 7 and 8 with
nitrite. Obviously, direct oxidation of dihydro-â-carbo-
lines 15 and 16 was not involved in formation of 17 or
18. As consequence, one pathway to the harman alka-
loids did proceed via two consecutive oxidative decar-
boxylation reactions of tetrahydro-â-carboline-1,3-di-
carboxylic acids 5 and 6 with dihydro-â-carboline-3-
carboxylic acids as likely intermediates (29).
Comparing the influence of substituents on the reac-
tivity of tetrahydro-â-carbolines, the data in Table 1
demonstrate that 1-carboxylic acids were more reactive
than the 3-carboxylic acids, whereas tetrahydro-â-
carbolines 3 and 4 were the most inert educts. Likewise,
the methyl group at position 1 stabilized both nitroso-
tetrahydro-â-carbolines 10 and 12 as well as the dihy-
dro-carbolines 14 and 16. Analyzing the reactivity of
tetrahydro-â-carbolines under study, compounds 1-4
yielded product patterns which were clearly different
from results obtained for 1-carboxylic acid derivatives
5-8. Particularly, the formation of nitroso-tetrahydro-
â-carbolines 9-12 from tetrahydro-â-carbolines 1-4
was obvious, which was paralleled with higher yields
of harman alkaloids. In addition, reactions of tetrahy-
dro-â-carbolines 3 and 4 demonstrated that, indepen-
dent from the oxidative decarboxylation mechanism we
discussed above, a second pathway significantly con-
tributed to the generation of harmanes. Consequently,
we adressed the question whether nitroso-tetrahydro-
â-carbolines 9-12 were involved in the formation of
harman 18 and norharman 17.
a stream of nitrogen. Instantaneous HPLC-MS analy-
sis of the purified nitroso-compound 10a revealed almost
complete degradation of this precursor together with
formation of harman 18 as the major product (Figure
6). The same results were obtained following the isola-
tion of nitroso-tetrahydro-â-carboline 12. The presence
of the educt 2a (or 4, respectively), as demonstrated by
Figure 6B, indicates that the degradation of nitroso-
tetrahydro-â-carbolines 10a and 12 can be initiated by
the loss of the NO group and may proceed via consecu-
tive radical chain reactions.
In conclusion, we revealed that the nitroso-tetrahy-
dro-â-carbolines can represent intermediates involved
in the generation of â-carbolines and established a novel
pathway for the formation of harman from nutritional
tetrahydro-â-carbolines in the presence of nitrite. Yet,
it has to be acknowledged that analysis of food samples
has revealed rather small concentrations of harman
alkaloids while tetrahydro-â-carbolines were almost
ubiquitously present in significantly higher amounts (17
and references therein). Consequently, further research
should address the identification of technological factors
or chemical compounds, such as antioxidative treat-
ments or naturally occurring radical traps, that could
provide extended protection from the undesirable for-
mation of harman alkaloids in the presence of nitrite.
LITERATURE CITED
(1) Gutsche, B.; Herderich, M. HPLC-MS/MS profiling of
tryptophan-derived alkaloids in food: Identification of
tetrahydro-â-carbolinedicarboxylic acids. J . Agric. Food
Chem. 1997, 45, 2458-2462.
(2) Gutsche, B.; Herderich, M. HPLC-MS/MS identification
of tryptophan-derived tetrahydro-â-carboline derivatives
in food. Fresenius’ J . Anal. Chem. 1998, 360, 836-839.
(3) Bartsch, H. N-Nitroso compounds and human cancer:
where do we stand? IARC Sci. Publ. 1991, 105, 1-10.
(4) Lucas, L. T.; Gatehouse, D.; Shuker, D. E. G. Efficient
nitroso group transfer from N-nitrosoindoles to nucle-
otides and 2′-deoxyguanosine at physiological pH. A new
pathway for N-nitrosocompounds to exert genotoxicity.
J . Biol. Chem. 1999, 274, 18319-18326.
(5) Wakabayashi, K.; Nagao, M.; Sugimura, T. Mutagens
and carcinogens produced by the reaction of environ-
mental aromatic compounds with nitrite. Cancer Surv.
1989, 8, 385-399.
(6) Ohta, T.; Isa, M.; Suzuki, Y.; Yamahata, N.; Suzuki, S.;
Kurechi, T. Formation of mutagens from tryptophan by
the reaction with nitrite. Biochem. Biophys. Res. Com-
mun. 1981, 100, 52-57.
P oten tia l of Nitr oso-tetr a h yd r o-â-ca r bolin es To
Act a s P r ecu r sor s of Ha r m a n Alk a loid s. To confirm
that labile nitroso-precursors were intermediates of
harman alkaloid synthesis, we isolated nitroso-tetrahy-
dro-â-carbolines 10a and 12 from the reaction mixtures
by HPLC-MS analysis with postcolumn splitting. A
minor part of the eluate was directly analyzed by on-
line ESI-MS/MS and confirmed the identity of the
individual nitroso-tetrahydro-â-carbolines while most of
the fractionated material was gently concentrated with
(7) Gatehouse, D.; Wedd, D. The bacterial mutagenicity of
three naturally occurring indoles after reaction with
nitrous acid. Mutat. Res. 1983, 124, 35-51.

5998 J. Agric. Food Chem., Vol. 49, No. 12, 2001
Diem et al.
(8) Ochiai, M.; Wakabayashi, K.; Sugimura, T.; Nagao, M.
Mutagenicities of indole and 30 derivatives after nitrite
treatment. Mutat. Res. 1986, 172, 189-197.
(9) Valin, N.; Haybron, D.; Groves, L.; Mower, H. F. The
nitrosation of alcohol-induced metabolites produces mu-
tagenic substances. Mutat. Res. 1985, 158, 159-168.
(10) Lin, K.-J .; Wu, S.-S.; Chen, J .-T. Mutagenicities of
nitrosated carboline derivatives. Proc. Natl. Sci. Counc.
Rep. China B 1986, 10, 280-286.
(11) Higashimoto, M.; Yamamoto, T.; Kinouchi, T.; Matsu-
moto, H.; Ohnishi, Y. Mutagenicity of 1-methyl-1,2,3,4-
tetrahydro-â-carboline-3-carboxylic acid treated with
nitrite in the presence of alcohols. Mutat. Res. 1996, 367,
43-49.
(12) Nakai, H.; Cassens, R. G.; Greaser, M. L.; Woolford, G.
Significance of the reaction of nitrite with tryptophan.
J . Food Sci. 1978, 43, 1857-1860.
(13) Lynch, S. C.; Gru¨nwedel, D. W.; Russell, G. F. Mutagenic
activity of a nitrosated early Maillard product: DNA
synthesis (DNA Repair) induced in HeLa S3 carcinoma
cells by nitrosated 1-(N-L-tryptophan)-1-deoxy-D-fruc-
tose. Food Chem. Toxicol. 1983, 21, 551-556.
(14) Salvi, N. A.; Choughuley, A. S. U. Nitrosation of some
food-related tetrahydro-â-carboline-3-carboxylic acids. J .
Sci. Food Agric. 1990, 52, 537-545.
(15) Salvi, N. A.; Choughuley, A. S. U. Studies in â-carbo-
lines: Nitrosation of some â-carboline derivatives. Ind.
J . Chem. 1990, 29B, 986-988.
(16) Salvi, N. A.; Choughuley, A. S. U.; Shetty, T. K.;
Bhattacharya, R. K. Nitrosation and mutagenicity of
tetrahydro-â-carboline derivatives. Toxicol. Environ.
Chem. 1992, 36, 47-56.
(17) Herderich, M.; Gutsche, B. Tryptophan derived bioactive
compounds in food. Food Rev. Int. 1997, 13, 103-135.
(18) Sen, N. P.; Seaman, S. W.; Baddoo, P. A.; Weber, D.;
Malis, G. Analytical methods for the determination and
mass spectrometric confirmation of 1-methyl-2-nitroso-
1,2,3,4-tetrahydro-â-carboline-3-carboxylic acid and 2-ni-
troso-1,2,3,4-tetrahydro-â-carboline-3-carboxylic acid in
foods. Food Addit. Contam. 1991, 8, 275-290.
(19) Sen, N. P.; Seaman, S. W.; Lau, B. P.-Y.; Weber, D.;
Lewis, D. Determination and occurrence of various
tetrahydro-â-carboline-3-carboxylic acids and the cor-
responding N-nitroso compounds in foods and alcoholic
beverages. Food Chem. 1995, 54, 327-337.
(21) Diem, S.; Herderich, M. Reaction of tryptophan with
carbohydrates: Identification and quantitative deter-
mination of novel â-carboline alkaloids in food. J . Agric.
Food Chem. 2001, 49, 2486-2492.
(22) Letellier, S.; Fleury, B.; Torreilles, J .; Previerro, A.
Derivatives of 3,4-dihydro-â-carboline 3-carboxylic acid
from C-terminal tryptophan-containing peptides. Syn-
thesis and properties. J . Heterocycl. Chem. 1988, 25,
1265-1270.
(23) Mirza, U. A.; Chait, B. T.; Lander, H. M. Monitoring
reactions of nitric oxide with peptides and proteins by
electrospray ionization-mass spectrometry. J . Biol. Chem.
1995, 270, 17185-17188.
(24) Zhang, Y.-Y.; Xu, A.-M.; Nomen, M.; Walsh, M.; Keaney,
J . F.; Loscalzo, J . Nitrosation of tryptophan residue(s)
in serum albumin and model dipeptides. Biochemical
characterization and bioactivity. J . Biol. Chem. 1996,
271, 14271-14279.
(25) Tsikas, D.; Raida, M.; Sandmann, J .; Rossa, S.; Forss-
mann, W.-G.; Fro¨lich, J . C. Electrospray ionization mass
spectrometry of low-molecular-mass S-nitroso com-
pounds and their thiols. J . Chromatogr. B 2000, 742,
99-108.
(26) Cheng, C. F.; Tsang, C. W. Separation of N-nitrosoamino
acids by C18 reversed-phase ion-pair high-performance
liquid chromatography and compatible detection by
electrospray ionization mass spectrometry. J . Chro-
matogr. A 1999, 849, 389-402.
(27) Hua, D. H.; Bharathi, N.; Panangadan, J . A. K.; Tsujim-
oto, A. Stereoselective additions of chiral R-sulfinyl
ketimine anions to ene esters. Asymmetric syntheses of
indolo[2,3-R]quinolizidine and yohimban alkaloids. J .
Org. Chem. 1991, 56, 6998-7007.
(28) Herraiz, T. Tetrahydro-â-carboline-3-carboxylic acid
compounds in fish and meat: possible precursors of co-
mutagenic â-carbolines norharman and harman in
cooked foods. Food Addit. Contam. 2000, 17, 859-866.
(29) Bobbitt, J . M.; Willis, J . P. Electrochemistry of natural
products. 7. Oxidative decarboxylation of some tetrahy-
dro-â-carbolinecarboxylic acids. J . Org. Chem. 1980, 45,
1978-1984.
(20) Wakabayashi, K.; Nagao, M.; Ochiai, M.; Tsuda, M.;
Yamaizumi, Z.; Saito, H.; Sugimura, T. Presence of
1-methyl-1,2,3,4-tetrahydro-â-carboline-3-carboxylic ac-
ids and tyramine as precursors of mutagens in soy sauce
after nitrite treatment. IARC Sci. Publ. 1984, 57, 17-
24.
Received for review March 16, 2001. Revised manuscript
received August 19, 2001. Accepted September 20, 2001. This
study was supported by the Deutsche Forschungsgemeinschaft
(DFG), Bonn, Germany (Grants He 2599/1-3 and /1-5).
J F010363Y