Alkylpyridiniums. 1. Formation in model systems via thermal degradation of trigonelline.

Article abstract of DOI:10.1021/jf011234k

Trigonelline is a well-known precursor of flavor/aroma compounds in coffee and undergoes significant degradation during roasting. This study investigates the major nonvolatile products that are procured after trigonelline has been subjected to mild pyrolysis conditions (220-250 degrees C) under atmospheric pressure. Various salt forms of trigonelline were also prepared and the thermally produced nonvolatiles analyzed by thin layer chromatography, liquid chromatography-electrospray ionization tandem mass spectrometry, and (1)H and (13)C nuclear magnetic resonance. Results revealed the decarboxylated derivative 1-methylpyridinium as a major product of certain salts, the formation of which is positively correlated to temperature from 220 to 245 degrees C. Moreover, trigonelline hydrochloride afforded far greater amounts of 1-methylpyridinium compared to the monohydrate over the temperature range studied. Investigations into other potential quaternary amine products of trigonelline also indicate nucleophilic substitution reactions that lead to dialkylpyridiniums, albeit at concentration levels approximately 100-fold lower than those recorded for 1-methylpyridinium.

Full text of DOI:10.1021/jf011234k

1192 J. Agric. Food Chem. 2002, 50, 1192−1199
Alkylpyridiniums. 1. Formation in Model Systems via Thermal
Degradation of Trigonelline
RICHARD H. STADLER,* NATALIA VARGA, J O¨ RG HAU, FRANCIA ARCE VERA, AND
DIETER H. WELTI
Nestl e´ Research Center, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland
Trigonelline is a well-known precursor of flavor/aroma compounds in coffee and undergoes significant
degradation during roasting. This study investigates the major nonvolatile products that are procured
after trigonelline has been subjected to mild pyrolysis conditions (220-250 °C) under atmospheric
pressure. Various salt forms of trigonelline were also prepared and the thermally produced nonvolatiles
analyzed by thin layer chromatography, liquid chromatography-electrospray ionization tandem mass
1
13
spectrometry, and H and C nuclear magnetic resonance. Results revealed the decarboxylated
derivative 1-methylpyridinium as a major product of certain salts, the formation of which is positively
correlated to temperature from 220 to 245 °C. Moreover, trigonelline hydrochloride afforded far greater
amounts of 1-methylpyridinium compared to the monohydrate over the temperature range studied.
Investigations into other potential quaternary amine products of trigonelline also indicate nucleophilic
substitution reactions that lead to dialkylpyridiniums, albeit at concentration levels ∼100-fold lower
than those recorded for 1-methylpyridinium.
KEYWORDS: Trigonelline; nicotinic acid; 1-methylpyridinium; 1,4-dimethylpyridinium; alkylpyridinium;
liquid chromatography-mass spectrometry (LC-MS); nuclear magnetic resonance spectroscopy (NMR);
model system studies; pyrolysis
INTRODUCTION
The alkaloid trigonelline (1-methylnicotinic acid) (Figure 1)
is fairly widely distributed in the plant kingdom, and its presence
has been corroborated in a number of higher plant species such
as the Lamiaceae and Asteraceae (1) and also in marine shellfish
(2). Particularly high levels of trigonelline are found in green
coffee beans, with typical levels reported for the various Coffea
species and varieties ranging from 0.3 to 1.3% on a dry matter
basis (3). The importance of trigonelline, as a precursor of flavor
and aroma compounds as well as beneficial nutritional factors,
is well documented in the literature (4, 5).
The products of trigonelline resulting from thermal treatment
were already addressed more than a half-century ago (6), which
is not surprising given the abundance of the compound in green
and roasted coffees. These initial studies demonstrated the
formation of nicotinic acid when trigonelline is heated in a
sealed tube. In fact, trigonelline decomposes via two major
routes under typical temperatures as encountered during coffee
roasting (220-250 °C), that is, (1) decarboxylation and methyl
rearrangement to afford pyridines and (2) N-demethylation to
give nicotinic acid (6).
Figure 1. Chemical structures of trigonelline, 1, and nicotinic acid, 2.
degradation and formed during roasting (6). Moreover, endog-
enous nicotinic acid in the green coffee bean is present at low
concentrations (8-17 mg/kg) but is significantly augmented due
to N-demethylation of the pyridine moiety of the progenitor,
which is initiated at temperatures >180 °C (5).
There exist to date only very few mechanistic studies
addressing the breakdown and conversion of trigonelline under
mild pyrolysis conditions. The pathway of pyridine formation
has been investigated in thermally treated trigonelline hydrate,
a study that was based on deuterium exchange in alkylpyridines
(8). Furthermore, in this model, nicotinic acid and pyridine were
identified as the major reaction products, reaching 6.1 and 5.3%
yields, respectively. An earlier study by Viani and Horman
suggested the presence of relatively high amounts of methyl-
nicotinoate, N-methylnicotinamide, and N,N-dimethylnicotina-
mide formed under their pyrolytic conditions (4), in contrast to
only traces or low levels (<1%) of the same compounds
measured in the more recent study (8). These discrepancies in
levels of key reaction products may be due to the conditions of
The consumption of 3.5 standard cups of coffee per day
contributes up to one-third of the minimum dietary requirement
for an adult of nicotinic acid (7), a key product of trigonelline
*
Corresponding author (telephone +41 21 785 83 60; fax +41 21 785
8
5 53; e-mail richard.stadler@rdls.nestle.com).
1
0.1021/jf011234k CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/02/2002

Alkylpyridinium Formation via Thermal Degradation of Trigonelline
J. Agric. Food Chem., Vol. 50, No. 5, 2002 1193
pyrolysis, for example, heating times, purity of the trigonelline
salt, and quantification techniques used.
with methanol containing 20% (v/v) formic acid; the eluate was
concentrated in vacuo at 40 °C, and complete dryness was attained by
subsequent lyophilization. This fraction (24 mg) was subjected to
Despite these reports, there is clearly a lack of fundamental
studies particularly with regard to the nonvolatile reaction
products. This prompted us to characterize the major nonvolatile
products of thermally treated trigonelline and to determine the
impact of the nature of the trigonelline salt on the decomposition
products formed as a function of both time and temperature. In
the work presented here, liquid chromatography coupled to
electrospray ionization tandem mass spectroscopy (LC-ESI-MS/
MS) is mainly used to identify and quantify the major
nonvolatile reaction products in a model system.
HRMS and NMR analysis: HRMS, calcd mass molecular ion )
1
9
4.0656, measured mass molecular ion ) 94.0647; H NMR 4.380 ppm
(
8
s, 3 H, N-CH
3
), 8.031 ppm (“t” sl. br., Jav ∼ 6.9 Hz, 2 H, 3,5-H),
.516 ppm (“t”, J ∼ 7.8 Hz, 1 H, 4-H), 8.770 ppm (d, J ∼ 5.8 Hz, 2
13
H, 2,6-H); C NMR 50.94 ppm (q, broadened, N-CH
3
), 130.77 ppm
1
(
d, 3,5-CH), 147.86 ppm (d 1:1:1, JC-N ∼ 8.6 Hz, 2,6-CH), 148.10
ppm (d, 4-CH).
High-Resolution Mass Spectrometry. High-resolution measure-
ments were performed on a Finnigan MAT 8430 double-focusing mass
spectrometer working in the electron ionization mode at 70 eV. The
samples were directly introduced in the source heated at 180 °C. The
resolution power was 5000, and perfluorokerosene was used as reference
compound.
MATERIALS AND METHODS
Materials and Reagents. Trigonelline hydrochloride and nicotinic
acid were purchased from Sigma (Buchs, Switzerland). Pyridine
LC-ESI-MS/MS. A Micromass Quattro-LC (Micromass, Man-
chester, U.K.) quadrupole mass spectrometer was used in this study,
equipped with a ZSpray electrospray ion source and coupled to a Waters
2690 Alliance separation module. HPLC separations were performed
by ion exchange chromatography on a GromSiL SCX column (5 µm,
50 × 2 mm i.d.), injecting 5 µL of sample. All runs were performed
under isocratic conditions at a flow rate of 0.3 mL/min and using
methanol/water 1:1 (v/v) containing a final concentration of 50 mmol/L
ammonium acetate (pH ∼6.8, not adjusted). The column was at room
temperature.
(
(
anhydrous, 99.8%), 1,4-dimethylpridinium iodide, and iodomethane
99.5%) were from Aldrich (Buchs, Switzerland). Anion exchange resin
-
AG 1-X8, 100-200 mesh, OH form, was from Bio-Rad, and Sil G/UV
TLC plates from Macherey and Nagel. Silica gel plates 60F254 (1 mm)
were from Merck. Iodoplatinate was purchased from Fluka. All other
reagents and solvents were of analytical grade purity. D
3-trimethylsilyltetradeuteriopropionic acid sodium salt) were purchased
from Dr. Glaser AG, Basel, Switzerland.
2
O and TSP
(
Instrument control and data processing were performed using
MassLynx NT software, version 3.4 (Micromass). Operating parameters
were as follows: positive ion mode, needle voltage typically set to
Synthesis of 1-Methylpyridinium Iodide. Pyridine (2.5 g, 31.6
mmol) was added to ∼10 mL of MeCN (dry, J. T. Baker), and methyl
iodide (5 g, 35.23 mmol) was added dropwise. The mixture was kept
for 30 min at room temperature and heated for 10 min at 50 °C. The
oily yellow mixture was then kept for 16 h at room temperature.
Placement on ice resulted in immediate precipitation of the iodide salt.
The precipitate was washed with MeCN (cold), dried in a desiccator
under vacuum, and afforded 6.55 g (94% yield) of solid material (slight
yellow color): mp 116-117 °C [lit. 116-117 °C (9)]; high-resolution
mass spectrometry (HRMS): calcd mass molecular ion ) 94.0656,
2
.83 kV, cone voltage 32 V, and RF lens 0.15 V. The source block
and desolvation temperatures were set at 140 and 400 °C, respectively.
Nebulizer and desolvation gas flows were set to 90 and 710 L of N /h,
respectively. The ion energy of the first and second quadrupole was
.8 and 1.0 V, respectively. All data were acquired at a collision energy
2
0
of 24 eV using argon as collision gas at a pressure of 0.25 Pa (1.9
mTorr). The dwell time was 0.2 s, the interchannel delay 0.03 s, and
the mass span 0.1 Da. At least two single reaction monitoring (SRM)
transitions were chosen for each compound as follows:
1
measured mass molecular ion ) 94.0656; H NMR 4.436 ppm (s, 3 H,
N-CH
3
), 8.090 ppm (“t” sl. br., Jav ∼ 6.9 Hz, 2 H, 3,5-H), 8.570 ppm
(
“t”, J ∼ 7.8 Hz, 1 H, 4-H), 8.827 ppm (d, J ∼ 5.8 Hz, 2 H, 2,6-H);
1
3
1-methylpyridinium, m/z 94 f 79*, 94 f 78, 94 f 67
C NMR 51.15 ppm (q, N-CH
3
), 130.88 ppm (d, 3,5-CH), 147.96
ppm (d 1:1:1, 2,6-CH), 148.19 ppm (d, 4-CH).
1,4-dimethylpyridinium, m/z 108 f 93*, 108 f 92, 108 f 65
Standard Pyrolysis Procedures. Typically, 50 mg of trigonelline,
as either the hydrate, hydrochloride, or hydrogen sulfate salt (salts
prepared by passage of trigonelline, 500 mg, through a column packed
with 4 g of anion exchange resin, AG 1-X8), was heated on a heating
module (Brouwer) in a tightly closed vacuum hydrolysis tube. After
the predefined heating period, the tubes were allowed to reach room
temperature and the residue was taken up in a small volume (2 × 2
mL) of 50% methanol/water (v/v). The tubes were sonicated to assist
dissolution and the final volume made up to 5 mL with the same solvent.
The samples were stored at -20 °C and diluted 500-fold with water
prior to analysis by LC-ESI-MS/MS.
nicotinic acid, m/z 124 f 80, 124 f 78*
trigonelline, m/z 138 f 94, 138 f 92, 138 f 78*
For quantification purposes, mass transitions marked with an asterisk
(*) were used.
NMR Measurements. The NMR samples were prepared by dis-
solution in 0.7 mL of 99.95% deuterated water (D O) and transferring
2
the solution into a Wilmad 528-PP NMR tube with an outer diameter
of 5 mm. No shift standard was added, and shift referencing was done
by comparison with a 0.75% solution of 3-trimethylsilyltetradeuterio-
Large Scale Pyrolysis and Isolation of 1-Methylpyridinium
Chloride. Trigonelline hydrochloride (500 mg, 3.64 mmol) was
weighed in a B u¨ chi round-bottom flask and 500 µL of water added at
room temperature. The slurry was dried in a B u¨ chi vacuum oven at 75
propionic acid sodium salt (TSP) in D O measured under the same
2
experimental conditions. All NMR spectra were measured on a Bruker
DPX-360 spectrometer equipped with a 5 mm quadrinuclear (QNP)
probehead at ∼23 °C in a laboratory with regulated room temperature
1
°
C and then heated to 220 °C within ∼1.5 min. The compound was
((0.5 °C). H NMR spectra were acquired at 360.13 MHz, using a
pyrolyzed at this temperature for a further 15 min. The residue was
dissolved in 5 mL of water and applied to two C18 disposable cartridges
spectral width of 19.95 ppm, number of scans usually 64, 64K data
points, acquisition period of 4.56 s, and a relaxation delay of 10 s. The
1
3
(M&N, 1 g), preconditioned consecutively with 5 mL each of methanol,
pulse duration was 8 µs, corresponding to ∼67° pulse angle. C NMR
spectra were acquired at 90.56 MHz, using a level switched waltz16
proton decoupling pulse program with full decoupling during the 1.507
s acquisition period and 2 dB power level reduction during the 10 s
relaxation delay. The spectral width was 240.04 ppm with 64K data
points free induction decay (FID) size, and the pulse width 4 µs, also
corresponding to ∼67° pulse angle. Exponential line broadening of 0.05
Hz for proton spectra and 0.5 Hz for carbon spectra was applied before
Fourier transformation. To confirm the assignments, a two-dimensional
heteronuclear correlation experiment optimized for a 145 Hz one-bond
proton-carbon coupling was done for one of the methylpyridinium
salt samples, using a spectral width of 130.21 ppm for 8K data points
water, and 10 mM HCl. Each 2.5 mL of the reaction product was
charged onto one column and eluted with 2 bed-volumes of water. The
extract was concentrated in vacuo at 35 °C to remove excess solvent
and lyophilized, affording in total 324 mg of water soluble material.
This fraction was then taken up in a small volume of methanol and
acetonitrile added and left overnight at 5 °C. The crystals that formed
were separated by filtration (identified as unreacted trigonelline by
TLC), and the filtrate concentrated in vacuo at 40 °C. The residue was
taken up in ethanol, acidified with concentrated HCl, and chromato-
graphed by TLC on silica gel sheets in ethyl acetate/methyl ethyl ketone/
formic acid/water (5:3:2:1). 1-Methylpyridinium (R ) 0.3) was eluted
f

1194 J. Agric. Food Chem., Vol. 50, No. 5, 2002
Stadler et al.
in the carbon dimension and 6.0 ppm for 256 data points in the proton
dimension. An acquisition period of 0.347 s and a relaxation delay of
triplet was ca. 1:1.9:1. This does not represent a “classical” 1:2:1
triplet, as would be caused by coupling with two equivalent
protons, but it is in fact a quadrupolar 1:1:1 pattern ( JC-N g
4
.2 s were used. Sine square filtering was applied before two-
1
dimensional Fourier transformation and the spectrum displayed and
plotted in the magnitude mode. Quotes indicate approximate description
of multiplets, sl. br. stands for slightly broadened, etc.
8
.3 Hz), where the outer lines contribute to the intensity of the
center line because of the significant line width. This pseudo-
triplet structure was also present on the signals of the methyl
but was not resolved, because the coupling constant is smaller.
Quantitation. Standards were prepared in Millipore-grade water
from a stock solution (0.2 mg/mL). External calibration curves (five-
point) were established in the concentration range from 200 to 2000
pg/µL, and analytes not within the given range were adequately diluted
1
13
The H NMR, C NMR, and HRMS data thus confirm
-methylpyridinium ion as a major product of trigonelline
1
2
with water and re-injected. For all standard curves, r > 0.99 and the
breakdown in this model system.
amount of the compounds in the samples was calculated from the
respective linear regression equation.
Quantification of the Major Nonvolatile Transformation
Products of Trigonelline Hydrochloride. LC-ESI-MS/MS. A
major challenge at the onset of this study was to achieve a
separation of the starting material and the major pyrolysis
products, particularly 1-methylpyridinium ion, in one single
analytical run. Thus, different LC stationary phases/columns
were assessed, ranging from C18 to various cation exchange
resins. Unfortunately, the quaternary base could not be retained
on the C18 columns tested but showed retention on a strong
cation exchange column, under isocratic conditions with 50 mM
ammonium acetate (pH not adjusted) in methanol (50%, v/v).
Our first approach was to use conventional LC coupled to UV
detection, but this revealed impurities in most fractions at the
retention time of 1-methylpyridinium. Therefore, to achieve
quantification of the alkylpyridiniums and nicotinic acid in a
single run, separation/detection was done by LC-ESI-MS/MS.
RESULTS
Identification of 1-Methylpyridinium as a Major Product
of Trigonelline Pyrolysis. Earlier reports on the thermal
degradation of trigonelline have revealed nicotinic acid and
nicotinamide, as well as their O- and N-methyl analogues, as
reaction products (4, 8). Thus, a first step in this study was to
identify the major nonvolatile products formed during pyrolysis
of trigonelline hydrochloride at temperatures ranging from 220
to 250 °C. The crude pyrolysates in methanol were subjected
to TLC on silica gel sheets in a number of different solvent
systems, spraying the sheets with iodoplatinate reagent. A
prominent compound that did not correspond in its Rf value to
the commonly known trigonelline breakdown/conversion prod-
ucts was detected in all pyrolysates, at various relative amounts
over the whole temperature range. This compound reacted as
an intense dark blue spot and migrated in a relatively acidic
solvent (ethyl acetate/methyl ethyl ketone/formic acid/water, 5:3:
Under positive ESI conditions, 1-methylpyridinium and 1,4-
+
dimethylpyridinium show abundant M ions without fragmenta-
tion. Because N-alkylpyridiniums are already charged in solu-
tion, they are amenable to ionization directly from the liquid
phase. The daughter ion spectra obtained upon dissociation of
2
:1), with an Rf at ∼0.3.
+
the M ion of 1-methylpyridinium and 1,4-dimethylpyridinium
Compounds with a quaternary amine function show intense
are portrayed in parts A and B of Figure 2, respectively. Details
of fragmentation for the compounds studied here have been
reported previously (10), and only salient features for the
alkylpyridiniums will be mentioned. The main fragmentation
in positive ESI-MS of 1-methylpyridinium is the loss of the
coloration with iodoplatinate reagent, indicating that this major
reaction product has retained the N-methyl group. Large-scale
pyrolysis was conducted with more starting material (0.5 g of
trigonelline) and the extract purified by TLC in the same solvent
system as described above, eluting the compound with 20%
formic acid in methanol. Preliminary mass spectral analysis by
•+
+
methyl group to afford m/z 79 [C5H5N] and m/z 78 [C5H4N] .
Ring opening of the pyridinium moiety with retention of the
direct infusion ESI-MS/MS in the positive mode suggested a
+
+
N-methyl group affords the fragment m/z 67 [C5H7] . A
1
-methylpyridinium salt, with a peak at m/z 94 [M ], corre-
characteristic transition for the dialkylpyridinium is the [M -
sponding to the molecular formula C6H8N. This approach also
enabled the detection of additional azaheterocycles under certain
reaction conditions, identified as dimethylpyridinium salts,
substituted with a methyl group at either the 2-, 3-, or 4-position
of the pyridinium moiety. However, due to the paucity of
material and relatively low concentrations of the dialkyl-
pyridiniums, no attempts were made to isolate and further
identify the compound(s) by NMR techniques.
+
1
[
]
ion at m/z 107. The fragmentation pattern also shows
+
C6H6N] at m/z 92 (loss of a methyl group) and loss of HCN
to afford m/z 65 [C5H5] .
+
In this study, ESI-MS/MS was the method of choice to
quantify the aforementioned compounds, with emphasis on
chromatographic separation of the quaternary amine base
1-methylpyridinium salt. Potential ion suppression by coelution
of matrix constituents was largely avoided due to the strong
dilution of the fractions, and thus quantification was done by
establishing external calibration curves. All analyses were done
using SRM and choosing three transitions for each compound
of interest for certainty of the analyte (i.e., responses in all three
transitions and ion ratios comparable to the standards). Typical
SRM traces detecting the aforementioned two nonvolatile
products nicotinic acid and 1-methylpyridinium of pyrolyzed
trigonelline (240 °C) are illustrated in Figure 3.
Thorough NMR analysis of the pyrolysis product was done
1
13
in D2O. The H and C data of synthetic 1-methylpyridinium
iodide were compared with methylpyridinium isolated from the
pyrolysate. Both compounds show the classical AA′BB′C spin
system of a pyridine, where the AA′ signals are slightly
broadened due to the neighboring quadrupolar 14N atom, and
an N-methyl signal, which is also slightly broadened. The shifts
of the signals are very similar for the synthetic and the pyrolysis
products, the only significant difference observed being the
much broader water signal (HDO, ∼4.81 ppm) in the methyl-
pyridinium isolated from the pyrolysate, probably due to a
different pH of the latter.
Impact of Temperature. A series of experiments were
designed to follow the formation of 1-methylpyridinium and
nicotinic acid (a major N-demethylated product) at different
temperatures and over time (Figure 4).
Analogous findings are also reflected in the carbon-13 spectra.
Here, the signals of the proton-decoupled 2,6-CH carbons
showed a resolved triplet structure due to the one-bond coupling
Three independent determinations were conducted with
trigonelline hydrochloride as the starting material, which was
heated under atmospheric conditions in tightly closed hydrolysis
1
4
with the quadrupolar N nucleus. The intensity ratio of the

Alkylpyridinium Formation via Thermal Degradation of Trigonelline
J. Agric. Food Chem., Vol. 50, No. 5, 2002 1195
treatment, the residue was dissolved in 50% methanol/water (v/
v) and a strongly diluted aliquot analyzed by LC-ESI-MS using
the conditions described. The amount of 1-methylpyridinium
formed during thermal treatment of trigonelline is strictly
dependent on temperature, increasing from 220 to 245 °C and
then decreasing suddenly at 250 °C (Figure 4).
Interestingly, the maximum amount of the decarboxylated
product was always measured at 245 °C (five independent
experiments conducted over the same temperature range), with
2
excellent correlations between independent experiments (r >
0
.99). An individual plot of temperature versus natural log
2
amount of 1-methylpyridium formed shows a correlation of r
0.975 up to 245 °C. However, the rate equation(s) leading to
-methylpyridinium may be rather complex, and kinetics would
)
1
have to take into account methyl rearrangements and radical-
based mechanisms (4). In this context, methyl groups attached
to quaternary nitrogens are potential alkylating agents, and thus
we included in our analyses the determination of 1,4-dimethyl-
pyridinium, which could be procured by nucleophilic displace-
ment (11). Notably, the transitions detected could originate from
a number of methyl substitution patterns, that is, 1,2-, 1,3-, and/
or 1,4-dimethylpyridinium. Under the LC conditions employed,
no separation of the aforementioned structural isomers was
possible. All three compounds, synthesized either in our
laboratory or obtained commercially, showed identical daughter
ion spectra (data not shown).
Dimethylpyridinium could be detected in most of the samples
based on LC-ESI-MS/MS (using three SRM transitions, m/z 108
f 93, m/z 108 f 92, and m/z 108 f 65, and comparable ion
ratios) and eluted from the LC column ∼0.5 min later than
1
-methylpyridinium. Even though the yield of the dialkyls in
the pyrolysis experiments was in all cases significantly lower
than the monoalkyl derivative (by a factor of ∼100), it
nevertheless was detected at various levels in many of the
samples and showed a positive correlation to the amount of
1
-methylpyridinium formed in this model system (Figure 5).
Figure 2. ESI-MS/MS daughter ion spectra of (A) 1-methylpyridinium cation
A route relatively well described in reports on the fate of
and (B) 1,4-dimethylpyridinium cation.
trigonelline during pyrolysis or during the roasting of coffee is
N-demethylation of the progenitor to afford nicotinic acid, which
is positively correlated to the degree of roasting and does not
decompose per se at elevated temperatures (5, 6, 12). Using
tubes over the temperature range of 220-250 °C, in 5 °C
increments, with a heating period of 15 min. After thermal
Figure 3. LC-ESI-MS/MS chromatogram of pyrolyzed trigonelline hydrogen chloride, using transitions (SRM) for nicotinic acid (A, B, t
-methylpyridinium (C−E, t ) 6.6 min) as described in the text.
r
) 0.6 min.) and
1
r

1196 J. Agric. Food Chem., Vol. 50, No. 5, 2002
Stadler et al.
Table 1. Formation of 1-Methylpyridinium from Trigonelline
Hydrochloride Heated at 220, 230, and 240 °C as a Function of Time,
a
Determined by LC-ESI-MS/MS and Expressed as Percent Conversion
b,c
on a Molar Basis
pyrolysis
time (min)
220 °C
230 °C
240 °C
5
0.64 ± 0.1
1.14 ± 0.14
1.77 ± 0.16
3.08 ± 0.53
3.18 ± 0.13
2.18 ± 0.06
1.77 ± 0.61
2.53 ± 0.8
6.63 ± 0.67
18.79 ± 1.55
4.56 ± 0.74
7.12 ± 1.8
1
0
1.33 ± 0.05
1.63 ± 0.27
1.77 ± 0.28
1.29 ± 0.28
1.08 ± 0.21
2
0
3
0
4
5
60
9.15 ± 1.79
a
Transition m/z 94 f 79. b Based on the assumption that upon 100%
conversion, 0.287 mmol of trigonelline may afford 0.287 mmol of 1-methylpyridinium
determined as the free cation). c Entries are averages ± SD of n ) 3 independent
(
determinations.
Figure 4. Formation of nicotinic acid (dashed line) and 1-methylpyridinium
solid line) from trigonelline hydrogen chloride as a function of temperature,
(
Table 2. Formation of Nicotinic Acid from Trigonelline Hydrochloride
Heated at 220, 230, and 240 °C as a Function of Time, Determined
expressed as percent conversion on a molar basis. Pyrolysis time ) 15
min. Entries are averages of three independent determinations, depicting
a
by LC-ESI-MS/MS and Expressed as Percent Conversion on a Molar
±
SD.
b,c
Basis
pyrolysis
time (min)
220 °C
230 °C
240 °C
5
0.35 ± 0.13
1.56 ± 0.27
4.34 ± 0.77
6.86 ± 0.73
9.3 ± 1.16
1.47 ± 0.27
5.1 ± 0.96
12.75 ± 0.83
14.26 ± 1.0
15.27 ± 0.35
17.08 ± 0.83
5.26 ± 0.72
14.1 ± 0.31
16.85 ± 2.74
11.36 ± 0.46
10.0 ± 0.88
6.7 ± 1.46
1
0
2
0
3
0
4
5
60
10.85 ± 0.73
a
Taking transition m/z 124 f 78. b Based on the assumption that upon 100%
conversion, 0.287 mmol of trigonelline may afford 0.287 mmol of nicotinic acid.
c
Entries are averages ± SD of n ) 3 independent determinations.
could be recovered after 1 h of thermolysis at 220 and 230 °C,
respectively, demonstrating that trigonelline is relatively stable
over an extended time. Trigonelline decomposition was ac-
celerated at relatively higher temperatures (240 °C), with 94%
of the starting material decomposed after 45 min. Thus,
trigonelline breakdown is markedly increased at temperatures
of g240 °C, as already observed in the experiments over
different temperature ranges.
Figure 5. Plot of the ratio of the amounts of the quaternary amine reaction
products 1-methylpyridinium/dimethylpyridinium, obtained after pyrolysis
of trigonelline hydrochloride at 240 °C over various time periods ranging
from 5 to 45 min.
the SRM trace m/z 124 f 78, nicotinic acid could be quantified
in all experiments even though it showed no retention on the
SCX column under the conditions used (tr ) ∼0.6 min). Three
independent experiments with trigonelline hydrochloride showed
a maximum conversion to nicotinic acid at 235-240 °C, with
a prominent decrease above this temperature (Figure 4). This
indicates that N-demethylation is favored at slightly lower
temperatures versus decarboxylation, probably due to the
quaternary nitrogen that favors methyl transfer reactions, to yield
methyl nicotinoate, for example. Furthermore, the decomposition
of trigonelline over the temperature range 220-240 °C showed
only a relatively slight decline (from 46.6 to 37.5% of
trigonelline remaining, expressed on a molar basis) in the
temperature range 220-240 °C, with prominent degradation of
the starting material measurable at temperatures >240 °C (5-
The generation of 1-methylpyridinium and nicotinic acid
under conditions as described above over time and temperature
is shown in Tables 1 and 2, respectively. The formation of the
quaternary base reaches a maximum after 20 min over all three
temperature ranges, positively correlated to the temperature of
pyrolysis as illustrated in the temperature-time-dependent
measurements. At the highest temperature in the series (240
°C), 1-methylpyridinium reached 18% conversion in an experi-
ment with three independent determinations, showing a steep
incline up to 20 min (Table 1). This is also reflected by the
data of Figure 4, which shows a maximum decarboxylation at
245 °C to yield close to 15% 1-methylpyridinium on a molar
basis (note, in this case the heating time was 15 min). Prolonged
heating at 240 °C (>30 min) resulted in a significant decrease
in 1-methylpyridinium levels, probably due to further decom-
position and/or interaction with other thermolysis products. In
contrast to the temperature-time pattern observed for 1-meth-
ylpyridinium, nicotinic acid formation follows a clear positive
correlation at 220 and 230 °C over the measured time period
of 1 h (Table 2).
7% of starting material remaining at 245 °C), which is in accord
with data published in the literature (12).
Formation of Major Reaction Products oVer Time. In all of
the experiments described above, a fixed time point, that is, 15
min, was chosen over a variable temperature range. To
determine the formation of the key reaction products as a
function of time, trigonelline hydrochloride was heated at 230,
2
3
40, and 250 °C at six different time points, that is, 5, 10, 20,
0, 45, and 60 min (all data are averages of three independent
Reaction rate plots [ln C100/(C100 - Ct) versus time, where
C100 ) moles of nicotinic acid at 100% conversion, and Ct
represents the amount formed at time t] were calculated over
determinations). Moreover, 41 and 38% of the starting material

Alkylpyridinium Formation via Thermal Degradation of Trigonelline
J. Agric. Food Chem., Vol. 50, No. 5, 2002 1197
Scheme 1. Proposed Reaction Pathways Leading to the Formation of
Alkylpyridiniums
Figure 6. Yield of 1-methylpyridinium from trigonelline salts (hydrate,
hydrogen sulfate) as a function of temperature, expressed as percent
conversion on a molar basis. Pyrolysis time ) 15 min. All entries are
averages of duplicate determinations.
2
the first 20 min and showed good correlation (r ) 0.9997 and
rates to nicotinic acid of the hydrate and sulfate salts are
comparatively decreased, versus the hydrochloride (see Figure
0
.9988 at 230 and 220 °C, respectively). The rate ∆C/∆t
determined from the slope was ≈3-fold higher at 230 °C
compared to that at 220 °C. The kinetics of nicotinic acid
formation do not, however, continue to show linearity after 20
min with a significant decrease particularly at 240 °C, indicating
that at this temperature other reaction routes may be preferred
over simple demethylation of trigonelline or its O-methyl ester
methyl nicotinoate.
Impact of the Trigonelline Salt on the Reaction Products
Formed during Thermal Treatment of Trigonelline. Tri-
gonelline hydrochloride showed a very distinct pattern of
degradation over the temperature range studied, and the question
addressed in this section is the potential impact of various salts
on the degradation pathway of this compound. To our knowl-
edge, none of the published studies on thermal degradation
products of trigonelline take into account the possible ionic
interaction with salts and how these could affect the product
profile (4, 8).
Commercially available trigonelline hydrochloride was con-
verted to the hydrate by passage over an anion exchange column.
Trigonelline hydrate was then subjected to thermal treatment
over the same temperature range as described for the hydro-
chloride, and the reaction products 1-methylpyridinium and
nicotinic acid were quantified by LC-ESI-MS.
Trigonelline hydrate revealed far less stability over the
temperature range studied than the corresponding hydrochloride,
with only ∼3% of the starting material after a 15 min heating
period at 220 °C. In addition, there was no further loss of
trigonelline hydrate at relatively higher temperatures of 250 °C,
which suggests that most of the hydrate was degraded already
at relatively low temperatures. Advanced degradation/polym-
erization was reflected by a tarry black residue with a “sticky”
consistency that was difficult to dissolve in organic solvents
even after 30 min of sonification.
4
), over the same temperature range, yielding on a molar basis
approximately 1% and 3.5% nicotinic acid at 235 and 245 °C,
respectively.
DISCUSSION
This is the first report on the identification and quantification
of the quaternary base 1-methylpyridinium as a major reaction
product of trigonelline subjected to pyrolytic conditions. This
quaternary base is formed by decarboxylation and, as demon-
strated in this study, represents an important stable product/
intermediate in the thermal reaction pathway(s) of trigonelline.
Two major parameters have a pronounced impact on the
degradation route and formation of 1-methylpyridinium, namely,
the temperature and the nature of the trigonelline salt.
The failure to detect 1-methylpyridinium in previous model
system studies (4, 8) may in fact be due to employment of
trigonelline hydrate, which shows a degradation pattern that
differs from the hydrogen chloride salt. A familiar reaction of
1-methylpyridiniums is ring opening by reaction with nucleo-
philes, such as hydroxide ions, primary amines, and carbanions
(13). The more basic hydrate may be present in the pseudobase
form as 2-hydroxy-1-methyl-1,2-dihydropyridine, the open-chain
tautomer (Scheme 1, route A), which could undergo hydrolysis
to afford degradation products such as short-chain aldehydes
and methylamine (8, 9). In contrast, the replacement of the
hydrate with hydrochloride or hydrogen sulfate counterions
appears to confer resistance to the pyridine nucleus, possibly
by electronically stabilizing the quaternary amine up to certain
temperatures to afford 1-methylpyridinium as a major reaction
product.
The formation of alkylpyridiniums such as 1,2-, 1,3-, and/or
1,4-dimethylpyridinium in this model system suggests that
1-methylpyridinium per se may further react with nucleophiles
present in the reaction mixture, e.g., substituted pyridines.
Thermally induced methyl rearrangements of quaternary salts
such as 1-methylpyridine can procure a range of volatile
products in the presence of a nucleophile such as pyridine, in
particular good yields of 2- and 4-methylpyridine (30.1 and
34.6%, respectively), and minor formation of 3-methylpyridine
(5.8%) and ethylpyridines (1.4%) (14, 15) (Scheme 1, route
B). Alkylpyridines thus act as nucleophiles and participate in
methyl displacement reactions with 1-methylpyridinium (Scheme
1, route C) to afford pyridine and the new disubstituted
Salient differences were observed upon comparison of the
yields of 1-methylpyridinium (Figure 6). Exchange of hydrogen
chloride with hydrogen sulfate by passage of trigonelline through
an anion exchange column shows accelerated decomposition
to form 1-methylpyridinium at temperatures >225 °C. Also
characteristic in this case is a clear increase of 1-methylpyri-
dinium in the range 240-250 °C, analogous to the tendency
observed for trigonelline hydrogen chloride (Figure 4), albeit
with lower yield of the quaternary base.
The nature of the salt also has a pronounced impact on the
formation of nicotinic acid. We observed that the conversion

1198 J. Agric. Food Chem., Vol. 50, No. 5, 2002
Stadler et al.
dialkypyridinium products (9). The latter may in principle react
even further in a reverse “demethylation” reaction with available
nucleophiles. In particular, the 2-substituted 1-methylpyridini-
ums show greater rate constants of demethylation, due to the
impact of steric effects (16).
ACKNOWLEDGMENT
We thank M. Blanc for critically reading the manuscript and
for valuable comments.
Depending on the nature of the salt, the various reactions
described above may be driven to more defined “homogeneous”
products as in the case of the hydrochloride or, as observed for
trigonelline hydrate, toward pyridine ring-opening reactions that
yield more or less undefined polymeric material that is difficult
to isolate and characterize (Scheme 1, route A).
LITERATURE CITED
(
1) Bonham, C. C.; Wood, K. V.; Yang, W.-J.; Nadolska-Orczyk,
A.; Samaras, Y.; Gage, D. A.; Poupart, J.; Burnet, M.; Hanson,
A. D.; Rhodes, D. Identification of quaternary ammonium and
tertiary sulfonium compounds by plasma desorption mass
spectrometry. J. Mass Spectrom. 1995, 30, 1187-1194.
(2) Taguchi, H. Biosynthesis and metabolism of trigonelline, and
physiological action of the compound. Vitamins 1988, 62, 549-
The second major thermal degradation route of trigonelline
involves N-demethylation to provide nicotinic acid, the forma-
tion of which is positively correlated to the roasting degree of
coffee (7) and corroborates the model described here. Nicotinic
acid can be generated by hydrolysis of methyl nicotinoate (8)
and then remains as a stable reaction product, resisting degrada-
tion to pyridine even at temperatures of ∼230 °C (6). Interest-
ingly, a significant impact of the salt form of trigonelline on
the formation of the acid was also observed in this study, that
is, far lower yields with trigonelline hydrate or hydrogen sulfate
than with the hydrochloride. This indicates that methyl migration
may be favored by chloride as the counterion, with a better yield
of the progenitor methyl nicotinoate. Considering that trigonel-
line, pK ) 2.92 (17), is present in its zwitterionic form, then
the low yield of nicotinic acid in trigonelline hydrogen sulfate
suggests rapid decarboxylation to afford a zwitterionic carbanion
intermediate (18), which may hydrolyze or yield substituted
alkylpyridines (8).
5
57.
(3) Stennert, A.; Maier, H. G. Trigonelline in coffee. Z. Lebensm.
Unters. Forsch. 1994, 199, 198-200.
(4) Viani, R.; Horman, I. Thermal behavior of trigonelline. J. Food
Sci. 1974, 39, 1216-1217.
(
5) Taguchi, H.; Sakaguchi, M.; Shimabayashi, Y. Trigonelline
content in coffee beans and the thermal conversion of trigonelline
into nicotinic acid during the roasting of coffee beans. Agric.
Biol. Chem. 1985, 49, 3467-3471.
(
6) Hughes, E. B.; Smith, R. F. The nicotinic acid content of coffee.
J. Soc. Chem. Ind. 1946, 65, 284-286.
(
7) Teply, L. J.; Prier, R. F. Nutritional evaluation of coffee including
niacin bioassay. J. Agric. Food Chem. 1957, 5, 375-377.
(8) Rizzi, G. P.; Sanders, R. A. Mechanism of pyridine formation
from trigonelline under coffee roasting conditions. In FlaVour
Science, Recent DeVelopments; Taylor, A. J., Mottram, D. S.,
Eds.; Royal Society of Chemistry: Cambridge, U.K., 1996.
(9) Kosower, E. M. Additions to pyridinium rings. I. 1-Methylpy-
ridinium iodide. J. Am. Chem. Soc. 1955, 77, 3883-3885.
A number of model pyrolysis experiments have been de-
scribed recently on trigonelline to investigate its potential
mutagenic (19) or antimutagenic (20) effects in bacterial
mutation assays. The results of such in vitro tests are in general
difficult to interpret and extrapolate to in vivo conditions, which
has been highlighted (21). Hence, some of these pyrolytic model
studies may not be comparable because the nature of the salt
of trigonelline is not documented or considered of importance.
However, as shown in this work, the counterion plays a major
role in defining the reaction route under mild pyrolytic condi-
tions. Thus, an explanation for the contradictory findings of anti-
and promutagenic activities of the trigonelline pyrolysis products
may in fact be due to the usage of different salt forms of the
quaternary amine and thus a significantly different product/
activity profile.
(10) Hau, J.; Stadler, R.; Jenny, T.; Fay, L. B. Tandem mass
spectrometric accurate mass performance of time-of-flight and
Fourier transform ion cyclotron resonance mass spectrometry:
a case study with pyridine derivatives. Rapid Commun. Mass
Spectrom. 2001, 15, 1840-1848.
11) Katritzky, A. R.; Banerji, A.; El-Osta, B. S.; Parker, I. R.;
Ramsden, C. A. J. Chem. Soc., Perkin Trans. 2 1979, 5, 690-
693.
(
(12) Casal, S.; Oliveira, M. B.; Ferreira, M. A. HPLC/diode-array
applied to the thermal degradation of trigonelline, nicotinic acid
and caffeine in coffee. Food Chem. 2000, 68, 481-485.
(
13) Barton, D.; Ollis, W. D. ComprehensiVe Organic Chemistry. The
Synthesis and Reactions of Organic Compounds. Vol. 4. Het-
erocyclic Compounds; Sammes, P. G., Ed.; Permagon Press:
Oxford, U.K., 1979.
(14) Chumakov, Y. I.; Novikova, V. F. Alkylation and aralkylation
of pyridine during the thermal decomposition of quaternary
pyridinium salts by the Ladenburg-Chichibabin method. Chem.
Abstr. 1968, 69, 43743m.
At this stage, it is important to address the relevance of these
model studies in the food product, that is, coffee. Depending
on the nature of the trigonelline salt in the coffee bean, the
degradation route may indeed favor the formation of 1-methyl-
pyridinium, which will undoubtedly have a direct and indirect
impact on other physicochemical properties such as flavor and
aroma. In relation to the former, trigonelline per se is not a
dominant contributor to roasted coffee bitterness (22), and
alkylpyridiniums may indeed be responsible for a part of
perceived bitterness in the brew. Furthermore, this compound
was recently mentioned as a constituent of roasted coffee (23),
but this observation is based only on global proton NMR
investigations of the whole coffee brew, and no attempts were
made to isolate and characterize the compound.
(
15) Digiovanna, C. V.; Cislak, P. J.; Cislak, G. N. Some aspects of
the direct alkylation of pyridine and methyl pyridines. ACS Symp.
Ser. 1977, No. 55, 397-407.
(16) Berg, U.; Gallo, R.; Metzger, J. Demethylations of quaternary
pyridinium salts by a soft nucleophile, triphenylphosphine.
Electronic and steric accelerations. J. Org. Chem. 1976, 41,
2
621-2624.
(17) Niazi, M. S. K.; Mollin, J. Dossociation constants of some amino
2
acid and pyridinecarboxylic acids in ethanol-H O mixtures. Bull.
Chem. Soc. Jpn. 1987, 60, 2605-2610.
(18) March, J. AdVanced Organic Chemistry. Reactions, Mechanisms,
and Structure, 4th ed.; Wiley: New York, 1992; p 564.
(19) Wu, X.; Skog, K.; J a¨ gerstad, M. Trigonelline, a naturally
occurring constituent of green coffee beans behind the mutagenic
activity of roasted coffee? Mutat. Res. 1997, 391, 171-177.
Investigations into the impact of coffee roasting on 1-methyl-
pyridinium and dialkylpyridinium formation have recently been
completed and are the subject of a following paper (24).
(
20) Nguyen, V. C.; Araki, Y.; Jinnouchi, T.; Murai, H. On the
desmutagenicity of coffee. Proceedings of the 16th International
Colloquium on Coffee, Kyoto, Japn; 1995; pp 88-93.

Alkylpyridinium Formation via Thermal Degradation of Trigonelline
J. Agric. Food Chem., Vol. 50, No. 5, 2002 1199
(
(
(
21) Stadler, R. H.; Turesky, R. J.; M u¨ ller, O.; Markovic, J.; Leong-
Morgenthaler, P.-M. The inhibitory effects of coffee on radical-
mediated oxidation and mutagenicity. Mut. Res. 1994, 308, 177-
(24) Stadler, R. H.; Varga, N.; Milo, C.; Schilter, B.; Arce Vera, F.;
Welti, D. H. Alkylpyridiniums. 2. Isolation and quantification
in roasted and ground coffees. J. Agric. Food Chem. 2002, 50,
1
90.
1
200-1206.
22) Belitz, H. D. Geschmacksaktive Substanzen in Kaffee. Proceed-
ings of the 7th International Scientific Colloquium on Coffee,
Hamburg, Germany; 1975; Association Scientifique International
du Caf e´ : Bremen, Germany, 1976; pp 242-252.
Received for review September 24, 2001. Revised manuscript received
November 26, 2001. Accepted November 26, 2001.
23) Bosco, M.; Toffanin, R.; de Palo, D.; Zatti, L.; Segre, A. High-
1
resolution H NMR investigation of coffee. J. Sci. Food Agric.
1
999, 79, 869-878.
JF011234K