Quantitative analysis of N-phenylpropenoyl-L-amino acids in roasted coffee and cocoa powder by means of a stable isotope dilution assay

Article abstract of DOI:10.1021/jf053207q

Since recent reports on the role of N-phenylpropenoyl-L-amino acids as powerful antioxidants and key contributors to the astringent taste of cocoa nibs, there is an increasing interest in the concentrations of these phytochemicals in plant-derived foods.

Full text of DOI:10.1021/jf053207q

J. Agric. Food Chem. 2006, 54, 2859−2867 2859
Quantitative Analysis of N-Phenylpropenoyl- -amino Acids in
L
Roasted Coffee and Cocoa Powder by Means of a Stable
Isotope Dilution Assay
TIMO STARK,† HELENE JUSTUS,# AND THOMAS HOFMANN*,#
Deutsche Forschungsanstalt fu¨r Lebensmittelchemie, Lichtenbergstrasse 4, D-85748 Garching,
Germany, and Institut fu¨r Lebensmittelchemie, Universita¨t Mu¨nster, Corrensstrasse 45,
D-48149 Mu¨nster, Germany
Since recent reports on the role of N-phenylpropenoyl-L-amino acids as powerful antioxidants and
key contributors to the astringent taste of cocoa nibs, there is an increasing interest in the
concentrations of these phytochemicals in plant-derived foods. A versatile analytical method for the
accurate quantitative analysis of N-phenylpropenoyl-L-amino acids in plant-derived foods by means
of HPLC-MS/MS and synthetic stable isotope labeled N-phenylpropenoyl-L-amino acids as internal
standards was developed. By means of the developed stable isotope dilution assay (SIDA), showing
recovery rates of 95-102%, 14 N-phenylpropenoyl-L-amino acids were quantified for the first time in
cocoa and coffee samples. On the basis of the results of LC-MS/MS experiments as well as
cochromatography with the synthetic reference compounds N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-
tryptophan, N-[4′-hydroxy-(E)-cinnamoyl]-L-tryptophan, and N-[4′-hydroxy-3′-methoxy-(E)-cinnamoyl]-
L-tyrosine, respectively, were detected for the first time in cocoa powder, and (-)-N-[4′-hydroxy-(E)-
cinnamoyl]-L-tyrosine, (-)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-tyrosine, N-[4′-hydroxy-3′-methoxy-(E)-
cinnamoyl]-L-tyrosine, (+)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[4′-hydroxy-(E)-
cinnamoyl]-L-aspartic acid, N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-tryptophan, N-[4′-hydroxy-(E)-cinnamoyl]-
L-tryptophan, and N-[4′-hydroxy-3′-methoxy-(E)-cinnamoyl]-L-tryptophan, respectively, were detected
for the first time in coffee beverages.
KEYWORDS: N-Phenylpropenoyl-L-amino acids; taste; cocoa; coffee; stable isotope dilution assay; SIDA;
N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-aspartic acid
INTRODUCTION
cinnamoyl]-L-aspartic acid (5) and (+)-N-[4′-hydroxy-3′-meth-
oxy-(E)-cinnamoyl]-L-aspartic acid (9) have been earlier isolated
from cell suspension cultures of Arabidopsis thaliana (9) and
cell cultures of Beta Vulgaris (10), respectively. In addition,
(-)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-tyrosine (4) has been
previously identified in cocoa flowers (11), and together with
the tryptophan derivatives 11 (13), 12 (14), and 13 (15) as well
as the tyrosine conjugate 14 was detected in raw Robusta coffee
beans (12).
Recently, application of taste dilution analyses (1) revealed
besides procyanidins the N-phenylpropenoyl amino acids 1-10
(Figure 1) as the key contributors to the typical astringent taste
of cocoa beans as well as roasted cocoa nibs. Using the so-
called half-tongue test, human recognition thresholds for the
astringent and mouth-drying oral sensation were determined to
be between 26 and 220 µmol/L (water) depending on the amino
acid moiety (2, 3).
Although these N-phenylpropenoyl amino acids are biologi-
cally active as astringent chemosensates (2, 3), phytoalexins
(16), and plant antioxidants (7), suitable methodologies for the
rapid and accurate quantification of these unstable, highly polar
phytochemicals are still missing. As the use of stable isoto-
pomers of analytes is known to enable the correction of
compound discrimination during extraction, cleanup, chromato-
graphic separation, and MS detection, the purpose of the present
investigation was to develop a versatile and reliable stable
isotope dilution assay (SIDA) for the quantitative determination
of N-phenylpropenoyl amino acids in plant foods such as roasted
cocoa and coffee.
Besides reports of its taste activity, (-)-N-[3′,4′-dihydroxy-
(E)-cinnamoyl]-3-hydroxy-L-tyrosine (3), named clovamide, was
earlier identified in red clover (Trifolium pratense) (4-6), and
together with (-)-N-[4′-hydroxy-(E)-cinnamoyl]-L-tyrosine (8),
named deoxyclovamide, was earlier found as an antioxidant in
cocoa (7) and as a constituent of the bark of African blackwood
(Dalbergia melanoxylon) (8). Moreover, (+)-N-[4′-hydroxy-(E)-
* Author to whom correspondence should be addressed (telephone
+49-251-83-33-391; fax +49-251-83-33-396; e-mail thomas.hofmann@
uni-muenster.de).
† Deutsche Forschungsanstalt fu¨r Lebensmittelchemie.
# Institut fu¨r Lebensmittelchemie.
10.1021/jf053207q CCC: $33.50
© 2006 American Chemical Society
Published on Web 03/04/2006

2860 J. Agric. Food Chem., Vol. 54, No. 8, 2006
Stark et al.
Figure 1. Chemical structures of N-phenylpropenoyl-L-amino acids 1−14.
MATERIALS AND METHODS
mm) filled with a slurry of LiChroprep 25-40 µm RP-18 material
(Merck) in aqueous formic acid (0.1% in water; pH 2.5). Chromatog-
raphy was performed using aqueous formic acid (0.1% in water; pH
2.5) as the effluent, followed by aqueous formic acid (0.1% in water;
pH 2.5) containing increasing amounts of methanol. The target
compounds were detected by monitoring the effluent at 300 nm, and
the fractions containing these compounds were confirmed by RP-HPLC-
DAD and RP-HPLC-MS/MS. After the individual fractions had been
freeze-dried three times, the corresponding N-phenylpropenoyl-L-amino
acids were obtained as white, amorphous powders in high purities of
>99%.
N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-L-tryptophan, 11: 0.29 mmol;
29% yield; UVmax ) 221, 290, 323 nm; LC-MS (ESI+), m/z 367 (100%,
[M + 1]+), 733 (15%, [2M + 1]+), 389 (10%, [M + Na]+); 1H NMR
(400 MHz, DMSO-d6, COSY) δ 3.07 [dd, 1H, J ) 8.8, 14.4 Hz,
H-C(9a)], 3.23 [dd, 1H, J ) 5.0, 14.4 Hz, H-C(9b)], 4.62 [m, 1H, J
) 5.0, 8.8 Hz, H-C(10)], 6.44 [d, 1H, J ) 15.6 Hz, H-C(8′)], 6.75
[d, 1H, J ) 8.0 Hz, H-C(5′)], 6.83 [dd, 1H, J ) 2.0, 8.0 Hz, H-C(6′)],
6.94 [d, 1H, J ) 1.6 Hz, H-C(2′)], 6.97 [m, 1H, J ) 0.8 Hz, J ) 8.0
Hz, H-C(5)], 7.07 [m, 1H, J ) 0.8 Hz, J ) 8.0 Hz, H-C(6)], 7.16 [d,
1H, J ) 2.4 Hz, H-C(1)], 7.22 [d, 1H, J ) 15.6 Hz, H-C(7′)], 7.34
[d, 1H, J ) 8.0 Hz, H-C(7)], 7.56 [d, 1H, J ) 8.0 Hz, H-C(4)], 8.26
[d, 1H, J ) 7.6 Hz, H-N], 9.13 [s, 1OH, HO-C(3′)], 9.37 [s, 1OH,
HO-C(4′)], 10.84 [d, 1H, H-N(indole)], 12.66 [s, 1OH, HOO-C(11)];
13C NMR (100 MHz, DMSO-d6, HMQC, HMBC) δ 27.8 [C-9], 53.5
[C-10], 110.4 [C-2], 111.8 [C-7], 114.3 [C-2′], 116.1 [C-5′], 118.5 [C-4],
118.6 [C-8′], 118.9 [C-5], 121.0 [C-6′], 121.3 [C-6], 124.0 [C-1], 126.8
[C-1′], 127.7 [C-3], 136.4 [C-8], 140.0 [C-7′], 146.0 [C-3′], 147.8 [C-4′],
165.8 [C-9′], 174.2 [C-11].
Chemicals. The following compounds were obtained commer-
cially: Amberlyst 15 (H+, 20-50 mesh) and (dimethylamino)pyridine
(Fluka, Neu-Ulm, Germany); acetic anhydride, (E)-4-hydroxycinnamic
acid, thionyl chloride, and (E)-4-hydroxy-3-methoxycinnamic acid
(Sigma-Aldrich, Steinheim, Germany); (E)-3,4-dihydroxycinnamic acid,
potassium carbonate, l-aspartic acid, L-dopa, L-glutamic acid, L-tyrosine,
L-tryptophan, sodium sulfate, pyridine, and tetrahydrofuran (Merck,
Darmstadt, Germany); [2,3,3-2H]-L-aspartic acid, [2,5,6-2H]-L-dopa,
[2,3,5,6-2H]-L-tyrosine, and [1,4,5,6,7-2H]-L-tryptophan (Cambridge
Isotope Laboratories, Andover, MA). Solvents were of HPLC grade
(Merck). Deuterated solvents were obtained from Euriso-Top (Gif-Sur-
Yvette, France). Roasted and nonalkalized cocoa powder and roasted
coffee (Arabica Columbia) as well as decaffeinated coffee (70%
Arabica, 30% Robusta) were obtained from the food industry. (+)-N-
[3′,4′-Dihydroxy-(E)-cinnamoyl]-L-aspartic acid (1) (Figure 1), (-)-
N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-glutamic acid (2), (-)-N-[3′,4′-
dihydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine (3), (-)-N-[3′,4′-
dihydroxy-(E)-cinnamoyl]-L-tyrosine (4, Figure 1), (+)-N-[4′-hydroxy-
(E)-cinnamoyl]-L-aspartic acid (5), (-)-N-[4′-hydroxy-(E)-cinnamoyl]-
L-glutamic acid (6), (-)-N-[4′-hydroxy-(E)-cinnamoyl]-3-hydroxy-L-
tyrosine (7), (-)-N-[4′-hydroxy-(E)-cinnamoyl]-L-tyrosine (8), (+)-N-
[4′-hydroxy-3′-methoxy-(E)-cinnamoyl]-L-aspartic acid (9), and (+)-
N-[(E)-cinnamoyl]-L-aspartic acid (10) were synthesized following the
procedure reported recently (3).
Syntheses of N-Phenylpropenoyl-L-Amino Acids. Closely follow-
ing the synthetic route reported recently for the synthesis of N-
phenylpropenoyl-L-amino acids (3), N-[3′,4′-dihydroxy-(E)-cinnamoyl]-
L-tryptophan (11), N-[4′-hydroxy-(E)-cinnamoyl]-L-tryptophan (12),
N-[4′-hydroxy-3′-methoxy-(E)-cinnamoyl]-L-tryptophan (13), and N-[4′-
hydroxy-3′-methoxy-(E)-cinnamoyl]-L-tyrosine (14) were prepared
starting from (E)-caffeic acid, (E)-p-coumaric acid, or (E)-ferulic acid
and L-tryptophan or L-tyrosine, respectively. The target compounds were
then applied onto the top of a water-cooled glass column (40 × 140
N-[4′-Hydroxy-(E)-cinnamoyl]-L-tryptophan, 12: 0.41 mmol; 41%
yield; UVmax ) 219, 292, 307 nm; LC-MS (ESI+), m/z 351 (100%, [M
+ 1]+), 701 (24%, [2M + 1]+), 373 (22%, [M + Na]+), 723 (17%,
[2M + Na]+); 1H NMR (400 MHz, DMSO-d6, COSY) δ 3.05 [dd, 1H,
J ) 9.0, 14.8 Hz, H-C(9a)], 3.09 [dd, 1H, J ) 5.0, 14.8 Hz, H-C(9b)],

N-Phenylpropenoyl-L-amino Acis in Roasted Coffee and Cocoa Powder
J. Agric. Food Chem., Vol. 54, No. 8, 2006 2861
Figure 2. Synthetic sequence used for the preparation of N-[3′,4′ L-tryptophan (d5-11).
-dihydroxy-(E)-cinnamoyl]-[1,4,5,6,7-2H]-
4.62 [m, 1H, J ) 5.0, 9.0 Hz, H-C(10)], 6.52 [d, 1H, J ) 16.0 Hz,
H-C(8′)], 6.79 [d, 2H, J ) 8.4 Hz, H-C(3′, 5′)], 6.99 [m, 1H, J )
1.2, 8.0 Hz, H-C(5)], 7.05 [m, 1H, J ) 1.2, 8.0 Hz, H-C(6)], 7.16
[d, 1H, J ) 2.0 Hz, H-C(1)], 7.30 [d, 1H, J ) 16.0 Hz, H-C(7′)],
7.32 [d, 1H, J ) 8.4 Hz, H-C(7)], 7.38 [d, 2H, J ) 8.4 Hz, H-C(2′,
6′)], 7.56 [d, 1H, J ) 8.4 Hz, H-C(4)], 8.23 [d, 1H, J ) 7.6 Hz, H-N],
9.84 [s, 1OH, HO-C(4′)], 10.84 [d, 1H, J ) 1.6 Hz, H-N(indole)],
12.65 [s, 1OH, HOO-C(11)]; 13C NMR (100 MHz, DMSO-d6, HMQC,
HMBC) δ 27.6 [C-9], 53.5 [C-10], 110.3 [C-2], 111.7 [C-7], 116.0
[C-3′, 5′], 118.1 [C-4], 118.2 [C-8′], 118.3 [C-5], 121.4 [C-6], 124.0
[C-1], 126.3 [C-1′], 127.6 [C-3], 129.8 [C-2′, 6′], 136.5 [C-8], 139.1
[C-7′], 159.2 [C-4′], 165.9 [C-9′], 174.0 [C-11].
and the aqueous phase was acidified to pH 2.0 with aqueous
hydrochloric acid (2 mol/L). After extraction with a mixture of ethyl
acetate and tetrahydrofuran (3:1, v/v; 3 × 50 mL), the combined organic
extracts were dried over Na2SO4, filtered, and freed from solvent in a
vacuum affording the acetylated phenylpropenoic acid derivative. An
aliquot (1.0 mmol) of this product was mixed with thionyl chloride
(1.0 mmol) and was then heated under reflux until no further formation
of hydrogen chloride was observable. After cooling to room temper-
ature, the reaction mixture was dried under a stream of nitrogen, and
either [2,3,3-2H]-L-aspartic acid (for d3-1 and d3-5), [2,5,6-2H]-L-dopa
(for d3-3 and d3-7), [2,3,5,6-2H]-L-tyrosine (for d4-4 and d4-8), or
[1,4,5,6,7-2H]-L-tryptophan (for d5-11-d5-13) (each 1.0 mmol) dis-
solved in dry tetrahydrofuran (30 mL) was added. After this solution
had been stirred for up to 72 h at room temperature, the solvent was
evaporated in a vacuum, and the residue was dissolved in a solution of
K2CO3 (0.25 mol/L; 20 mL) in water/methanol (1:1; v/v) and stirred
at room temperature. After 20 min, Amberlyst 15 ion-exchange resin
was added with stirring until a pH value of 3.5 was reached. The resin
was then filtered off and washed with water (20 mL), and the filtrate
was freed from methanol in a vacuum and, finally, freeze-dried. The
residue was taken up in water/methanol (1:1, v/v; 3 mL) and applied
onto the top of a water-cooled glass column (40 × 140 mm) filled
with a slurry of LiChroprep 25-40 µm RP-18 material (Merck) in
aqueous formic acid (0.1% in water; pH 2.5). Chromatography was
performed using aqueous formic acid (0.1% in water; pH 2.5) as the
effluent, followed by aqueous formic acid (0.1% in water; pH 2.5)
containing increasing amounts of methanol. The target compounds were
detected by monitoring the effluent at 300 nm, and the fractions
containing these compounds were confirmed by RP-HPLC-DAD. After
the individual fractions had been freeze-dried three times, the corre-
sponding deuterium-labeled N-phenylpropenoyl-L-amino acids were
obtained as white, amorphous powders in high purities of >99%.
N-[4′-Hydroxy-3′-methoxy-(E)-cinnamoyl]-L-tryptophan, 13: 0.26
mmol; 26% yield; UVmax ) 229, 291, 322 nm; LC-MS (ESI+), m/z
381 (100%, [M + 1]+), 761 (39%, [2M + 1]+), 403 (35%, [M + Na]+),
1
783 (18%, [2M + Na]+); H NMR (400 MHz, DMSO-d6, COSY) δ
3.07 [dd, 1H, J ) 8.8, 14.8 Hz, H-C(9a)], 3.23 [dd, 1H, J ) 4.8, 14.8
Hz, H-C(9b)], 3.80 [s, 3H, H-C(10′)], 4.61 [m, 1H, J ) 4.8, 8.8 Hz,
H-C(10)], 6.56 [d, 1H, J ) 15.6 Hz, H-C(8′)], 6.78 [d, 1H, J ) 8.0
Hz, H-C(5′)], 6.97 [dd, 1H, J ) 2.0, 8.0 Hz, H-C(6′)], 6.99 [m, 1H,
H-C(5)], 7.06 [m, 1H, H-C(6)], 7.12 [d, 1H, J ) 2.0 Hz, H-C(2′)],
7.15 [d, 1H, J ) 2.4 Hz, H-C(1)], 7.30 [d, 1H, J ) 15.6 Hz, H-C(7′)],
7.33 [d, 1H, J ) 8.0 Hz, H-C(7)], 7.55 [d, 1H, J ) 7.6 Hz, H-C(4)],
8.16 [d, 1H, J ) 8.4 Hz, H-N], 9.42 [s, 1OH, HO-C(4′)], 10.83 [s,
1H, H-N(indole)], 12.69 [s, 1OH, HOO-C(11)]; 13C NMR (100 MHz,
DMSO-d6, HMQC, HMBC) δ 27.7 [C-9], 53.5 [C-10], 56.0 [C-10′],
110.3 [C-2], 111.2 [C-2′], 111.7 [C-7], 116.1 [C-5′], 118.6 [C-4], 118.7
[C-5], 119.2 [C-8′], 121.2 [C-6], 122.2 [C-6′], 124.0 [C-1], 126.6 [C-1′],
127.5 [C-3], 136.5 [C-8], 140.0 [C-7′], 147.8 [C-3′], 148.3 [C-4′], 165.7
[C-9′], 174.0 [C-11].
N-[4′-Hydroxy-3′-methoxy-(E)-cinnamoyl]-L-tyrosine, 14: 0.32 mmol;
32% yield; UVmax ) 221, 297, 322 nm; LC-MS (ESI+), m/z 358 (100%,
[M + 1]+), 715 (22%, [2M + 1]+), 380 (18%, [M + Na]+); 1H NMR
(400 MHz, DMSO-d6, COSY) δ 2.81 [dd, 1H, J ) 9.2, 14.0 Hz,
H-C(7a)], 2.99 [dd, 1H, J ) 4.8, 14.0 Hz, H-C(7b)], 3.80 [s, 3H,
H-C(10′)], 4.48 [m, 1H, J ) 4.8, 14.0 Hz, H-C(8)], 6.54 [d, 1H, J )
15.6 Hz, H-C(8′)], 6.66 [d, 2H, J ) 8.4 Hz, H-C(3, 5)], 6.79 [d, 1H,
J ) 8.0 Hz, H-C(5′)], 6.99 [dd, 1H, J ) 1.6, 8.0 Hz, H-C(6′)], 7.04
[d, 2H, J ) 8.4 Hz, H-C(2, 6)], 7.13 [d, 1H, J ) 1.6 Hz, H-C(2′)],
7.29 [d, 1H, J ) 15.6 Hz, H-C(7′)], 8.13 [d, 1H, J ) 8.0 Hz, H-N],
9.20 [s, 1OH, HO-C(4)], 9.44 [s, 1OH, HO-C(4′)], 12.70 [s, 1OH,
HOO-C(9)]; 13C NMR (100 MHz, DMSO-d6, HMQC, HMBC) δ 36.5
[C-7], 54.4 [C-8], 56.0 [C-10′], 111.2 [C-2′], 115.4 [C-3, 5], 116.0
[C-5′], 119.0 [C-8′′], 122.1 [C-6′], 126.6 [C-1′], 128.2 [C-1], 130.4
[C-2, 6], 140.0 [C-7′], 148.0 [C-3′], 148.8 [C-4′], 156.3 [C-4], 165.7
[C-9′], 173.7 [C-9].
Syntheses of 2H-Labeled N-Phenylpropenoyl-L-amino Acids. As
exemplified for the preparation of N-[3′,4′-dihydroxy-(E)-cinnamoyl]-
[1,4,5,6,7-2H]-L-tryptophan in Figure 2, acetic anhydride (25 mmol)
was added dropwise to a solution of (E)-caffeic acid, (E)-p-coumaric
acid, or (E)-ferulic acid (10 mmol each), respectively, in pyridine (5
mL) and (dimethylamino)pyridine (0.25 mmol) at 0 °C. The reaction
mixture was stirred for 1 h at 0 °C and then poured onto crushed ice,
(+)-N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-[2,3,3-2H]-L-aspartic acid,
d3-1: 0.28 mmol; 28% yield (Figure 3); UVmax ) 207, 285, 309 nm;
LC-MS (ESI+), m/z 619 (100%, [2M + Na]+), 321 (55%, [M + Na]+),
163 (52%, [M - 136]+), 933 (32%, [3M + K]+); 1H NMR (400 MHz,
DMSO-d6, COSY) δ 6.44 [d, 1H, J ) 15.7, H-C(8′)], 6.74 [d, 1H, J
) 8.2 Hz, H-C(5′)], 6.85 [dd, 1H, J ) 1.9, 8.2 Hz, H-C(6′)], 6.95
[d, 1H, J ) 1.9 Hz, H-C(2′)], 7.24 [d, 1H, J ) 15.7 Hz, H-C(7′)],
8.19 [s, 1H, H-N], 9.10 [s, 1OH, HO-C(3′)], 9.32 [s, 1OH, HO-
C(4′)].
(-)-N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-[2,5,6-2H]-3-hydroxy-L-ty-
rosine, d3-3: 0.25 mmol; 25% yield (Figure 3); UVmax ) 225, 255,
297 nm; LC-MS (ESI+), m/z 725 (100%, [2M + 1]+), 363 (45%, [M
+ 1]+) 163 (25%, [M - 200]+); 1H NMR (400 MHz, DMSO-d6,
COSY) δ 2.73 [dd, 1H, J ) 9.1, 13.9 Hz, H-C(7a)], 2.91 [dd, 1H, J
) 5.0, 13.9 Hz, H-C(7b)], 4.44 [m, 1H, H-C(8)], 6.41 [d, 1H, J )
15.7 Hz, H-C(8′)], 6.74 [d, 1H, J ) 8.1 Hz, H-C(5′)], 6.82 [dd, 1H,
J ) 2.0, 8.1 Hz, H-C(6′)], 6.94 [d, 1H, J ) 2.0 Hz, H-C(2′)], 7.19
[d, 1H, J ) 15.7 Hz, H-C(7′)], 8.15 [d, 1H, J ) 8.0, H-N], 8.63,
8.68, 9.08, 9.32 [4s, 4OH, HO-C], 12.56 [s, 1OH, HOOC(9)].
N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-[2,3,5,6-2H]-L-tyrosine, d4-4: 0.18
mmol; 18% yield (Figure 3); UVmax ) 216, 297, 323 nm; LC-MS

2862 J. Agric. Food Chem., Vol. 54, No. 8, 2006
Stark et al.
Figure 3. Structures of deuterated N-phenylpropenoyl-L-amino acids d3-1, d3-3, d4-4, d3-5, d3-7, d4-8, and d5-11−d5-13.
(ESI+), m/z 348 (100%, [M + 1]+), 370 (33%, [M + Na]+), 695 (12%,
(ESI+), m/z 332 (100%, [M + 1]+), 354 (38%, [M + Na]+), 663 (25%,
[2M + 1]+), 685 (18%, [2M + Na]+); 1H NMR (400 MHz, DMSO-d6,
COSY) δ 2.83 [dd, 1H, J ) 9.2, 14.0 Hz, H-C(7a)], 3.01 [dd, 1H, J
) 4.8, 14.0 Hz, H-C(7b)], 4.51 [m, 1H, J ) 4.8, 8.4, 8.8, 9.6, 13.2
Hz, H-C(8)], 6.52 [d, 1H, J ) 15.6 Hz, H-C(8′)], 6.82 [d, 1H, J )
8.8 Hz, H-C(3′, 5′)], 7.33 [d, 1H, J ) 15.6 Hz, H-C(7′)], 7.42 [d,
1H, J ) 8.4 Hz, H-C(2′, 6′)], 8.13 [d, 1H, J ) 8.4 Hz, H-N], 9.23
[s, 1OH, HO-C(4)], 9.89 [s, 1OH, HO-C(4′)], 12.72 [s, 1OH, HOOC-
(9)].
1
[2M + 1]+); H NMR (400 MHz, DMSO-d6, COSY) δ 2.83 [dd, 1H,
J ) 9.6, 14.0 Hz, H-C(7a)], 3.00 [dd, 1H, J ) 4.8, 14.0 Hz, H-C(7b)],
4.49 [m, 1H, J ) 4.8, 9.6, 14.0 Hz, H-C(8)], 6.52 [d, 1H, J ) 15.8
Hz, H-C(8′)], 6.78 [d, 1H, J ) 8.4 Hz, H-C(5′)], 6.97 [d, 1H, J )
2.0 Hz, H-C(2′)], 7.23 [d, 1H, J ) 15.8 Hz, H-C(7′)], 7.86 [dd, 1H,
J ) 2.0, 8.0 Hz, H-C(6′)], 8.24 [d, 1H, J ) 8.0 Hz, H-N], 9.17 [s,
1OH, HO-C(3′)], 9.23 [s, 1OH, HO-C(4)], 9.41 [s, 1OH, HO-C(4′)],
12.68 [s, 1OH, HOOC(9)].
(+)-N-[4′-Hydroxy-(E)-cinnamoyl]-[2,3,3-2H]-L-aspartic acid, d3-
5: 0.30 mmol; 30% yield (Figure 3); UVmax ) 207, 285, 309 nm; LC-
MS (ESI+), m/z 305 (100%, [M + Na]+), 587 (93%, [2M + Na]+),
603 (60%, [2M + K]+) 885 (40%, [3M + K]+), 147 (20%, [M -
N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-[1,4,5,6,7-2H]-L-tryptophan, d5-
11: 0.27 mmol; 27% yield (Figure 3); UVmax ) 221, 290, 323 nm;
LC-MS (ESI+), m/z 372 (100%, [M + 1]+), 394 (49%, [M + Na]+),
765 (33%, [2M + Na]+), 743 (28%, [2M + 1]+); 1H NMR (400 MHz,
DMSO-d6, COSY) δ 3.10 [dd, 1H, J ) 8.8, 14.4 Hz, H-C(9a)], 3.25
[dd, 1H, J ) 4.8, 14.4 Hz, H-C(9b)], 4.64 [m, 1H, J ) 4.8, 8.0, 13.2
Hz, H-C(10)], 6.47 [d, 1H, J ) 15.6 Hz, H-C(8′)], 6.78 [d, 1H, J )
8.0 Hz, H-C(5′)], 6.86 [dd, 1H, J ) 1.6, 8.0 Hz, H-C(6′)], 6.97 [d,
1H, J ) 1.6 Hz, H-C(2′)], 7.25 [d, 1H, J ) 15.6 Hz, H-C(7′)], 8.29
[d, 1H, J ) 8.0 Hz, H-N], 9.18 [s, 1OH, HO-C(3′)], 9.42 [s, 1OH,
HO-C(4′)], 10.80 [s, 1H, H-N(indole)], 12.70 [s, 1OH, HOOC(11)].
N-[4′-Hydroxy-(E)-cinnamoyl]-[1,4,5,6,7-2H]-L-tryptophan, d5-12:
0.43 mmol; 43% yield (Figure 3); UVmax ) 219, 292, 307 nm; LC-
MS (ESI+), m/z 356 (100%, [M + 1]+), 378 (32%, [M + Na]+), 711
1
136]+); H NMR (400 MHz, DMSO-d6, COSY) δ 6.52 [d, 1H, J )
15.7 Hz, H-C(8′)], 6.79 [d, 2H, J ) 8.6 Hz, H-C(3′, 5′)], 7.33 [d,
1H, J ) 15.7 Hz, H-C(7′)], 7.40 [d, 2H, J ) 8.6 Hz, H-C(2′, 6′)],
8.18 [s, 1H, H-N], 9.81 [s, 1OH, HO-C(4′)], 12.82 [s, 2OH, HOOC-
(1, 4)].
(-)-N-[4′-Hydroxy-(E)-cinnamoyl]-[2,5,6-2H]-3-hydroxy-L-
tyrosine, d3-7: 0.25 mmol; 25% yield (Figure 3); UVmax ) 225, 255,
297 nm; LC-MS (ESI+), m/z 1407 (100%, [4M + Na]+), 1077 (85%,
[3M + K]+), 731 (46%, [2M + K]+), 1423 (44%, [4M + K]+), 147
(23%, [M - 200]+), 347 (15%, [M + 1]+); 1H NMR (400 MHz,
DMSO-d6, COSY) δ 2.73 [dd, 1H, J ) 8.7, 13.8 Hz, H-C(7a)], 2.91
[dd, 1H, J ) 5.1, 13.8 Hz, H-C(7b)], 4.41 [m, 1H, H-C(8)], 6.49 [d,
1H, J ) 15.7 Hz, H-C(8′)], 6.78 [d, 2H, J ) 8.6 Hz, H-C(3′, 5′)],
7.27 [d, 1H, J ) 15.7 Hz, H-C(7′)], 7.38 [d, 2H, J ) 8.6 Hz, H-C(2′,
6′)], 8.06 [d, 1H, J ) 8.0 Hz, H-N], 8.62, 8.67, 9.82 [3s, 3OH, HO-
C].
1
(18%, [2M + 1]+); H NMR (400 MHz, DMSO-d6, COSY) δ 3.09
[dd, 1H, J ) 8.6, 14.6 Hz, H-C(9a)], 3.26 [dd, 1H, J ) 5.0, 14.6 Hz,
H-C(9b)], 4.64 [m, 1H, J ) 5.0, 8.6, 13.2 Hz, H-C(10)], 6.55 [d,
1H, J ) 16.0 Hz, H-C(8′)], 6.82 [d, 2H, J ) 8.4 Hz, H-C(3′, 5′)],
7.33 [d, 1H, J ) 16.0 Hz, H-C(7′)], 7.41 [d, 2H, J ) 8.4 Hz, H-C(2′,
6′)], 8.26 [d, 1H, J ) 7.6 Hz, H-N], 9.89 [s, 1OH, HO-C(4′)], 10.87
[s, 1H, H-N(indole)], 12.72 [s, 1OH, HOOC(11)].
N-[4′-Hydroxy-(E)-cinnamoyl]-[2,3,5,6-2H]-L-tyrosine, d4-8: 0.33
mmol; 33% yield (Figure 3); UVmax ) 224, 299, 309 nm; LC-MS
N-[4′-Hydroxy-3′-methoxy-(E)-cinnamoyl]-[1,4,5,6,7-2H]-L-tryp-

N-Phenylpropenoyl-L-amino Acis in Roasted Coffee and Cocoa Powder
J. Agric. Food Chem., Vol. 54, No. 8, 2006 2863
tophan, d5-13: 0.26 mmol; 26% yield (Figure 3); UVmax ) 229, 291,
322 nm; LC-MS (ESI+), m/z 386 (100%, [M + 1]+), 408 (38%, [M +
connected to the API 3200 LC-MS/MS (Applied Biosystems, Darm-
stadt, Germany) running in the positive electrospray ionization mode.
By means of the selected reaction monitoring (SRM) mode, the
individual N-phenylpropenoyl amino acids were analyzed using the
following mass transitions given in parentheses: 1 (m/z 296.3f163.0),
d3-1 (m/z 299.3f163.0), 2 (m/z 310.3f163.0), 3 (m/z 360.3f163.0),
d3-3 (m/z 363.3f163.0), 4 (m/z 344.3f163.0), d4-4 (m/z 348.3f163.0),
5 (m/z 280.3f147.0), d3-5 (m/z 283.3f147.0), 6 (m/z 294.3f147.0),
7 (m/z 344.3f147.0), d3-7 (m/z 347.3f147.0), 8 (m/z 328.3f147.0),
d4-8 (m/z 332.3f147.0), 9 (m/z 310.3f177.0), 10 (m/z 264.3f131.0),
11 (m/z 367.3f163.0), d5-11 (m/z 372.3f163.0), 12 (m/z 351.3f147.0),
d5-12 (m/z 356.3f147.0), 13 (m/z 381.3f177.0), d5-13 (m/z 386.3f
177.0), 14 (m/z 358.3f177.0).
1
Na]+), 771 (23%, [2M + 1]+), 793 (12%, [2M + Na]+); H NMR
(400 MHz, DMSO-d6, COSY) δ 3.11 [dd, 1H, J ) 8.6, 14.6 Hz,
H-C(9a)], 3.27 [dd, 1H, J ) 5.0, 14.6 Hz, H-C(9b)], 3.84 [s, 3H,
H-C(10′)], 4.65 [m, 1H, J ) 5.0, 8.0, 8.6, 13.2 Hz, H-C(10)], 6.59
[d, 1H, J ) 16.0 Hz, H-C(8′)], 6.82 [d, 1H, J ) 8.0 Hz, H-C(5′)],
7.03 [dd, 1H, J ) 1.6, 8.0 Hz, H-C(6′)], 7.15 [d, 1H, J ) 1.6 Hz,
H-C(2′)], 7.32 [d, 1H, J ) 16.0 Hz, H-C(7′)], 8.20 [d, 1H, J ) 8.0
Hz, H-N], 9.49 [s, 1OH, HO-C(4′)], 10.87 [s, 1H, H-N(indole)],
12.77 [s, 1OH, HOOC(11)].
Quantitative Analysis of N-Phenylpropenoyl Amino Acids in
Roasted Cocoa and Coffee. Roasted Cocoa. Cocoa powder (5.0 g)
was spiked with solutions (100 µL, each) of the labeled internal
standards d3-1, d3-3, d4-4, d3-5, d3-7, d4-8, d5-11, and d5-12 in methanol
(1.0 mg/mL), and the mixture was homogenized in a laboratory shaker
for 30 min. After equilibration, the cocoa powder was extracted with
n-pentane (5 × 30 mL) at room temperature for 30 min. The residual
cocoa material was then extracted five times with acetone/water (70:
30, v/v; 30 mL each) for 45 min at room temperature with stirring.
After centrifugation, the liquid layer was freed from acetone under
reduced pressure at 30 °C and then freeze-dried to give the acetone/
water extract. Aliquots (∼40 mg) of the acetone/water extract were
taken up in a methanol/water mixture (1:1, v/v; 10 mL), which was
acidified to pH 2.5 with formic acid. After membrane filtration, aliquots
(5-10 µL) were analyzed by means of LC-MS/MS, which was
equipped with a 150 × 2 mm i.d., 4 µm, RP-18 Synergi Fusion column
(Phenomenex) operated with a flow rate of 0.2 mL/min. Chromatog-
raphy was performed starting with a mixture (20:80, v/v) of methanol
and aqueous formic acid (0.1% in water; pH 2.5) for 5 min; the
methanol content was increased to 60% within 35 min, and, finally, to
100% within 5 min. The labeled compounds d3-1, d3-3, d4-4, d3-5,
d3-7, d4-8, and d5-11-d5-13 were used as the internal standards for
the quantitative analysis of the corresponding analytes. In addition,
compound 2 was quantified via d3-1, compounds 6, 9, and 10 were
quantified via d3-5, and compound 14 was quantified via d3-8 as the
internal standard. The amounts of the individual N-phenylpropenoyl
amino acids were calculated using the response factors (given in
parentheses) for the individual compounds 1 (1.00), 2 (0.80), 3 (0.92),
4 (0.94), 5 (0.99), 6 (0.85), 7 (0.86), 8 (0.99), 9 (0.86), 10 (0.95), 11
(0.95), 12 (0.96), 13 (0.91), and 14 (1.00), which had been determined
by analysis of solutions containing defined amounts of the internal
standards and the target compounds in five mass ratios from 0.2 to 5.
Roasted Coffee. Roast coffee beans were frozen in liquid nitrogen,
ground in a mill, and passed through a sieve (pore size ) 0.5 mm).
Coffee powder (5.4 g) was spiked with the labeled internal standards
d3-1, d4-4, d3-5, d4-8, and d5-11-d5-13 in methanol (1.0 mg/mL), and
the mixture was homogenized by means of a laboratory shaker for 30
min. After equilibration, the coffee powder was percolated with hot
water (110 mL) using a no. 4 cellulose filter (Melitta), and the brew
obtained (100 mL) was analyzed by means of LC-MS/MS as described
above.
Nuclear Magnetic Resonance Spectroscopy (NMR). 1H, 13C,
DEPT-135, COSY, HMQC, and HMBC measurements were performed
on an AMX-400 spectrometer (Bruker, Rheinstetten, Germany). Evalu-
ation of the experiments was carried out using 1D- and 2D-WIN-NMR
(version 6.1) as well as XWin-NMR software (version 3.5; Bruker).
Tetramethylsilane was used as the internal standard.
RESULTS AND DISCUSSION
Recent investigations revealed the N-phenylpropenoyl-L-
amino acids 1-10 (Figure 1) as the key compounds contributing
to the astringent taste of roasted cocoa nibs (2, 3). In addition,
the structurally related N-phenylpropenoyl-L-tryptophans 11-
13, as well as N-[4′-hydroxy-3′-methoxy-(E)-cinnamoyl]-L-
tyrosine (14), have been detected in coffee beans (12-15). To
investigate the natural occurrence of all these compounds in
roasted cocoa and coffee, reference compounds needed to be
synthesized for compounds 11-14 (Figure 1). Closely follow-
ing the procedure reported recently for the synthesis of the
N-phenylpropenoyl-L-amino acids 1-10 (3), compounds 11-
14 were prepared starting from (E)-caffeic acid, (E)-p-coumaric
acid, or (E)-ferulic acid and L-tryptophan or L-tyrosine, respec-
tively.
Identification of N-Phenylpropenoyl-L-Amino Acids in
Cocoa and Coffee. To identify the N-phenylpropenoyl-L-amino
acids 1-14 in cocoa and coffee, an aqueous acetone extract
isolated from cocoa powder as well as an aqueous brew prepared
from roasted ground coffee, respectively, was analyzed by RP-
HPLC coupled to an LC-MS/MS instrument running in the
positive electrospray ionization mode. By means of the SRM
mode, the mass transitions m/z 296.3f163.0 (1), m/z 310.3f
163.0 (2), m/z 360.3f163.0 (3), m/z 344.3f163.0 (4), m/z
280.3f147.0 (5), m/z 294.3f147.0 (6), m/z 344.3f147.0 (7),
m/z 328.3f147.0 (8), m/z 310.3f177.0 (9), m/z 264.3f131.0
(10), m/z 367.3f163.0 (11), m/z 351.3f147.0 (12), m/z
381.3f177.0 (13), and m/z 358.3f177.0 (14) were recorded
for the individual N-phenylpropenoyl-L-amino acids.
Recovery. Aliquots (5.0 g) of cocoa powder were spiked with known
amounts of a mixture of selected N-phenylpropenoyl amino acids (1-
9, 11, 12, 14) dissolved in methanol. After stirring and equilibration
for 30 min, the quantitative analysis of the N-phenylpropenoyl amino
acids was done as detailed above. As the basis for the calculation of
the recovery rate, the content of the N-phenylpropenoyl amino acids
in cocoa powder, which was not spiked with additional N-phenylpro-
penoyl amino acids (control), was determined as the mean of triplicates.
High-Performance Liquid Chromatography (HPLC). The HPLC
apparatus (Jasco, Gross-Umstadt, Germany) consisted of a PU 1580
HPLC pump system with a DG-1580-53 in-line degasser, an LG-1580-
02 low-pressure gradient unit, and an MD 1515 diode array detector
(DAD). Chromatographic separations were performed on stainless steel
columns packed with ODS-Hypersil, 5 µm (ThermoHypersil, Kleinos-
theim, Germany) in either an analytical (250 × 4.6 mm i.d., flow rate
) 0.8 mL/min) or a semipreparative scale (250 × 10 mm i.d., flow
rate ) 3.0 mL/min).
Analysis of the cocoa sample demonstrated for the first time
that, besides the previously reported compounds 1-10, also
N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-tryptophan (11), N-[4′-
hydroxy-(E)-cinnamoyl]-L-tryptophan (12), and N-[4′-hydroxy-
3′-methoxy-(E)-cinnamoyl]-L-tyrosine (14) are naturally occur-
ring in cocoa. These data were further confirmed by comparing
the mass spectra and retention times (RP-HPLC) with those
obtained for the synthetic reference compounds as well as by
cochromatography. In contrast, N-[4′-hydroxy-3′-methoxy-(E)-
cinnamoyl]-L-tryptophan (13) was not detectable in significant
amounts.
LC-MS/MS analysis of the coffee brew revealed besides the
previously reported compounds 4, 8, and 11-13, the L-aspartic
acid derivatives 1 and 5 as phytochemicals in coffee. These
data were further confirmed by comparing the mass spectra and
retention times (RP-HPLC) with those obtained for the synthetic
reference compounds as well as by cochromatography.
Liquid Chromatography-Mass Spectrometry (LC-MS/MS). LC-
MS/MS analysis was performed using an Agilent 1100 HPLC system

2864 J. Agric. Food Chem., Vol. 54, No. 8, 2006
Stark et al.
+
Figure 4. MS/MS (ESI ) spectrum of (A) N-[3
′
,4
′
-dihydroxy-(E)-cinnamoyl]-[1,4,5,6,7-H]-
L
-tryptophan (11) and (B) N-[3
′
,4
′
-dihydroxy-(E)-cinnamoyl]-
[1,4,5,6,7-2H]-
L-tryptophan (d5-11).
Synthesis of Labeled N-Phenylpropenoyl-L-Amino Acids.
To accurately determine the amounts of N-phenylpropenoyl-L-
amino acids in roasted coffee beverage and cocoa nibs, a SIDA
should be developed in the following. To achieve this, first,
corresponding labeled internal standards needed to be synthe-
sized.
Exemplified for N-[3′,4′-dihydroxy-(E)-cinnamoyl]-[1,4,5,6,7-
2H]-L-tryptophan (d5-11), the synthetic sequence used for the
preparation of deuterium-labeled N-phenylpropenoyl-L-amino
acids is outlined in Figure 2. (E)-Caffeic acid was acetylated
with acetic anhydride in pyridine and (dimethylamino)pyridine,
converted into the corresponding chloride using thionyl chloride,
and, finally, reacted with [1,4,5,6,7-2H]-L-tryptophan in dry
tetrahydrofuran. After K2CO3-catalyzed cleavage of the protect-
ing groups and Amberlite treatment, the title compound was
isolated by column chromatography on RP-18 material, followed
by a final HPLC purification yielding N-[3′,4′-dihydroxy-(E)-
cinnamoyl]-[1,4,5,6,7-2H]-L-tryptophan (d5-11) as a white,
amorphous powder in high purities of >99%. Following the
same procedure, the labeled N-phenylpropenoyl-L-amino acids
d3-1, d3-3, d4-4, d3-5, d3-7, d4-8, d5-12, and d5-13 were
synthesized (Figure 3). When compared to N-[3′,4′-dihydroxy-
(E)-cinnamoyl]-L-tryptophan (11), the MS/MS spectrum
of N-[3′,4′-dihydroxy-(E)-cinnamoyl]-[1,4,5,6,7-2H]-L-tryp-
tophan (d5-11) measured in the ESI+ mode revealed an increase
of the pseudomolecular ion by 5 units, thus reflecting the
incorporation of the five deuterium atoms in d5-11 (Figure 4).
In addition, the daugther ion m/z 163 corresponding to caffeic
acid was observed as the base peak. Similarily, the 4′-hydroxy-
(E)-cinnamoyl and 4′-hydroxy-3′-methoxy-(E)-cinnamoyl amino
acids showed the fragments m/z 147 and 177 for p-coumaric
acid and ferulic acid, respectively, as the corresponding base
ions.
In addition, the incorporation of the deuterium atoms into
1
the target molecules was confirmed by means of H NMR
spectroscopy. For example, N-[4′-hydroxy-3′-methoxy-(E)-
cinnamoyl]-L-tryptophan (13) showed 10 olefinic resonance
signals each integrating for one proton (Figure 5A). In

N-Phenylpropenoyl-L-amino Acis in Roasted Coffee and Cocoa Powder
J. Agric. Food Chem., Vol. 54, No. 8, 2006 2865
Figure 5. Excerpt of the 1H NMR spectra (400 MHz; DMSO-d6) of (A) N-[4
′-hydroxy-3′-methoxy-(E)-cinnamoyl]-L-tryptophan (13) and (B) N-[4′-hydroxy-
3
′
-methoxy-(E)-cinnamoyl]-[1,4,5,6,7-2H]-
L-tryptophan (d5-13).
1
comparison, the same segment of the H NMR spectrum of
N-[4′-hydroxy-3′-methoxy-(E)-cinnamoyl]-[1,4,5,6,7-2H]-L-tryp-
tophan (d5-13) showed only five signals, each integrating for
one proton as part of the ferulic acid moiety (Figure 5B). Taking
all of the NMR and MS data into account, it was proven that
no significant deuterium-protium exchange occurred during
their chemical synthesis.
peak area ratios in LC-MS/MS. Good linearity was found for
mass ratios ranging from 0.2 to 5.0.
For the quantitative analysis of the N-phenylpropenoyl-L-
amino acids by means of a SIDA, cocoa powder or ground
roasted coffee was dosed with defined amounts of d3-1, d3-3,
d4-4, d3-5, d3-7, d4-8, d5-11, d5-12, and d5-13 as the internal
standards, followed by homogenization and equilibration at room
temperature. After isolation of the N-phenylpropenoyl-L-amino
acids from cocoa or coffee by acetone/water extraction or hot
water percolation, respectively, mass chromatography was
performed by analytical RP-HPLC-MS/MS as shown in Figure
6.
To check the stability of the labeled compounds under
aqueous acidic conditions, the deuterated compounds were
individually incubated in H2O acidified with HCl to pH 4.0 for
4 h at room temperature and, after lyophylization, were then
1
analyzed by means of H NMR spectroscopy. However, the
aromatic protons H-C(1) and H-C(4)-H-C(7) were still not
observable for the labeled compound (data not shown), thus
demonstrating the lack of any significant D/H exchange under
workup conditions and confirming the suitability of the labeled
compounds as internal standards for the SIDA.
To check the accuracy of the analytical method, recovery
experiments were performed (Table 1). To achieve this,
synthetic reference material of the N-phenylpropenoyl-L-amino
acids 1-9, 11, 12, and 14 was added to cocoa powder in three
different concentrations prior to quantitative analysis, and the
amounts determined after cleanup were compared with those
found in the blank cocoa sample (control). The recovery rates,
calculated on the basis of the content of each N-phenylprope-
noyl-L-amino acid added to the cocoa powder prior to cleanup,
were found to range between 95 and 102% (Table 1). These
Development of a SIDA and Quantitation of N-Phenyl-
propenoyl-L-Amino Acids. To convert the measured ion
intensities into the mass ratios of labeled and nonlabeled
N-phenylpropenyl-L-amino acids, a graph was calculated from
calibration mixtures of known mass ratios and the corresponding

2866 J. Agric. Food Chem., Vol. 54, No. 8, 2006
Stark et al.
Table 1. Determination of the Recovery Rates for the Quantitative
Analysis of N-Phenylpropenoyl-L-amino Acids in Cocoa Powder
compd
no.
amount
concn calcd
g/g)
concn determined
g/g)
recovery
(%)
added (
µ
g/g)
(
µ
(µ
1
428.43
463.83
526.21
588.59
47.0
94.0
188.0
475.43
522.43
616.43
97.6
100.7
95.5
2
5.55
47.88
88.71
42.0
84.0
47.55
89.55
100.7
99.1
95.9
168.0
173.55
166.39
3
58.58
108.70
159.08
250.80
49.0
98.0
196.0
107.58
156.58
254.58
101.0
101.6
98.5
4
19.07
65.09
111.17
207.93
47.0
94.0
188.0
66.07
113.07
207.07
98.5
98.3
100.4
5
120.42
158.31
208.88
286.22
44.0
88.0
176.0
164.42
208.42
296.42
96.3
100.2
96.6
6
3.28
53.85
103.63
206.13
50.0
100.0
200.0
53.28
103.28
203.28
101.1
100.3
101.4
7
9.04
58.43
106.96
213.96
Figure 6. MS/MS chromatograms for the quantitative analysis of the
N-phenylpropenoyl amino acids 1, 3, 5, and 7 in cocoa powder via the
internal standards d3-1, d3-3, d3-5, and d3-7 by using the SRM mode.
50.0
100.0
200.0
59.04
109.04
209.04
99.0
98.1
102.4
8
55.37
108.04
163.44
271.31
Table 2. Concentrations of N-Phenylpropenoyl-L-amino Acids in Cocoa
Powder and Coffee
56.0
112.0
224.0
111.37
167.37
279.37
97.0
97.7
97.1
concna (mg/kg) in
coffeeb
9
18.8
62.24
111.64
201.25
compd
no.
46.0
92.0
184.0
64.80
110.8
202.80
96.1
100.8
99.2
cocoa nibs
decaffeinated coffeec
1
2
3
4
5
6
7
8
428.43
±
±
±
±
±
±
±
±
±
±
±
±
10.11d
0.04
nde
nd
0.10
0.01
nd
nd
0.01
nd
0.03
nd
nd
0.13
0.01
nd
nd
0.05
nd
5.55
58.58
19.07
120.42
3.28
0.32d
1.12d
0.54d
1.18d
0.91d
0.23d
0.62d
0.91d
0.51d
0.06d
0.1d
11
12
14
0.45
45.98
90.94
46.0
92.0
184.0
46.45
92.45
184.45
99.0
98.4
99.7
183.94
0.39
46.18
88.55
9.04
45.0
90.0
180.0
45.39
90.39
180.39
101.7
98.0
97.7
55.37
18.80
2.26
0.45
0.39
9
176.20
10
11
12
13
14
nd
nd
3.32
0.55
0.04
nd
3.11
0.32
0.03
nd
1.11
61.74
116.86
230.63
59.0
118.0
236.0
60.11
119.11
237.11
102.7
98.1
97.3
nd
1.11
±
0.04d
a Concentrations are given as the mean of triplicates. b Arabica Columbia.
c Decaffeinated coffee consisted of 70% Arabica and 30% Robusta. d Standard
deviation. e Not detectable.
data clearly demonstrate the developed SIDA as a reliable tool
enabling a rapid and accurate quantitative determination of
N-phenylpropenoyl-L-amino acid concentration in foods.
Next, the concentrations of N-phenylpropenoyl-L-amino acids
in roasted cocoa powder and a roasted decaffeinated coffee as
well as a roasted regular coffee were quantitatively determined.
As given in Table 2, independent of the coffee sample analyzed,
N-[3′,4′-dihydroxy-(E)-cinnamoyl)-L-tryptophan (11) was found
as the quantitatively predominating N-phenylpropenoyl-L-amino
acid, accounting for up to 84% of the total amino acid amides
in both of the coffee samples. In contrast, all of the other
N-phenylpropenoyl-L-amino acids were present in significantly
smaller amounts ranging between 0.01 and 0.55 mg/kg (Table
2). It is interesting to note that the Arabica Columbia showed
higher concentrations for N-phenylpropenoyl-L-tryptophan and
aspartic acid, respectively, whereas in the decaffeinated coffee
comparatively higher amounts of N-phenylpropenoyl-tyrosines
were detectable (Table 2).
In comparison to the coffee samples, by far the highest
concentrations of 428.43 and 120.42 mg/kg in cocoa were found
for N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-aspartic acid (1) and
N-[4′-hydroxy-(E)-cinnamoyl]-L-aspartic acid (5). With some-
what lower amounts of 55.37 and 58.58 mg/kg N-[3′,4′-
dihydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine (3) and N-[4′-
hydroxy-(E)-cinnamoyl]-L-tyrosine (8), both of which have been
reported earlier as cocoa antioxidants (7), were determined in
cocoa, whereas all of the other N-phenylpropenoyl-L-amino acids

N-Phenylpropenoyl-L-amino Acis in Roasted Coffee and Cocoa Powder
J. Agric. Food Chem., Vol. 54, No. 8, 2006 2867
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were present in amounts below 19.07 mg/kg. In particular, the
tryptophan derivates were found as trace constituents in cocoa
ranging from 0.39 to 0.45 mg/kg (Table 2).
In summary, a versatile analytical method enabling an
accurate quantitative analysis of biologically active N-phenyl-
propenoyl-L-amino acids in foods such as cocoa and coffee by
means of SIDA using RP-HPLC-MS/MS was developed. This
sophisticated and reliable quantification method is the scientific
basis to evaluate the contribution of these phytochemicals to
the astringent taste of foods and natural plant materials such as
coffee and cocoa and offers the possibility to bridge the gap
between the antioxidant activity of cocoa and coffee products
and their amounts of individual N-phenylpropenoyl-L-amino
acids.
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Received for review December 22, 2005. Revised manuscript received
February 10, 2006. Accepted February 10, 2006. This research project
was supported by a grant from FEI (Forschungskreis der Erna1hr-
ungsindustrie e.V., Bonn, Germany) the AiF (Arbeitsgemeinschaft
industrieller Forschung), and the German Ministry of Economics
(Project 13235 N) as well as by a grant from the Fonds der Chemischen
Industrie (FCI).
JF053207Q