Bioscience, Biotechnology, and Biochemistry

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Development of LC-MS/MS analysis of cyclic

dipeptides and its application to tea extract

Kenji Yamamoto, Miki Hayashi, Yuka Murakami, Yoko Araki, Yuuki Otsuka,
Takehiro Kashiwagi, Tomoko Shimamura & Hiroyuki Ukeda

To cite this article: Kenji Yamamoto, Miki Hayashi, Yuka Murakami, Yoko Araki, Yuuki Otsuka,
Takehiro Kashiwagi, Tomoko Shimamura & Hiroyuki Ukeda (2016) Development of LC-MS/MS
analysis of cyclic dipeptides and its application to tea extract, Bioscience, Biotechnology, and
Biochemistry, 80:1, 172-177, DOI: 10.1080/09168451.2015.1075865

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Bioscience, Biotechnology, and Biochemistry, 2016
Vol. 80, No. 1, 172–177
Development of LC-MS/MS analysis of cyclic dipeptides and its application to
tea extract
Kenji Yamamoto1,2, Miki Hayashi1, Yuka Murakami3, Yoko Araki1, Yuuki Otsuka1,
Takehiro Kashiwagi1, Tomoko Shimamura1,* and Hiroyuki Ukeda1
1
Faculty of Agriculture, Kochi University, Nankoku, Japan; 2Innovation Development Department, Suntory Global
Innovation Center Ltd., Osaka, Japan; 3Quality Assurance Division, Suntory Business Expert Ltd., Osaka, Japan
Received May 19, 2015; accepted July 9, 2015
http://dx.doi.org/10.1080/09168451.2015.1075865
2,5-Diketopiperazines (DKPs), also called cyclic
dipeptides, have been known to occur in various
foods. Recently, DKPs have attracted attentions as
bioactive components. There were some reports on
analytical methods for DKPs, but the number of
analyzed DKPs was only a part of all DKPs and the
quantitative performance was not studied in detail.
In this study, we selected 31 kinds of DKPs and
developed a quantitative and simultaneous analyti-
cal method using LC-MS/MS. This method was
applied to DKPs determination in Pu-erh tea, post-
fermentation tea, and 18 kinds of DKPs were deter-
mined at concentration of 0.0017–0.11 ppm. As a
result of spiked test, it was concluded that the
developed method using LC-MS/MS was useful for
estimating DKPs concentration in tea.
Key words: 2,5-diketopiperazines; DKPs; cyclic
dipeptide; LC-MS/MS; Pu-erh tea
2,5-Diketopiperazines (DKPs), cyclic dipeptides,
formed from the N-terminal amino acid residues of a
linear peptide or protein (Fig. 1) have been known to
occur in various foods, especially in heated and fer-
mented foods. For example, DKPs were found in
heated foods such as roasted coffee,1) cocoa,2) roasted
malt3), and chicken essence,4) and in fermented foods
such as beer,5) distillation residue of awamori6), and
aged sake.7) DKPs were also found in hydrolyzate of
protein such as whey protein8) and some beverages.9)
Thus, DKPs can be recognized as common compounds
in foods.
So far, DKPs were generally known as a taste com-
pound. For example, some DKPs found in roasted cof-
fee, roasted malt and cocoa contributed to bitterness. It
was also reported that cyclo(-Val-Gly) had bitterness,
while a linear dipeptide Val-Gly had no bitterness.10)
Recently, DKPs have been attracted attentions as a
functional component. Takaya et al. reported that some
DKPs found in a distillation residue of awamori
showed antioxidant activity.6) Tsuruoka et al. reported
*Corresponding author. Email: tomokos@kochi-u.ac.jp
© 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
that cyclo(-Phe-Phe) found in chicken essence acted as
a dual inhibitor of the serotonin transporter and
acetylcolinesterase.11) Cyclo(-His-Pro) was reported to
have biological effects such as food intake inhibition
and body weight reduction in rats.12,13) These reports
indicated that DKPs have possibilities to influence on
human biological regulation.
To progress research on functionalities of DKPs, a
simultaneous analytical method is required. Although
the simultaneous analytical methods of DKPs in
chicken essence4) and cocoa14) were reported, the num-
ber of DKPs which were already analyzed was only a
part of all DKPs. And in the previous report, MS
condition was optimized, but the quantitative perfor-
mance was not studied using calibration curves. There-
fore, we tried to develop the simultaneous analytical
method for 31 kinds of DKPs which confirm the
quantitative performance and repeatability. Among 31
kinds of DKP, cyclo(-Gly-His) (1), cyclo(-Ala-His) (2),
cyclo(-Ser-Ser) (3), cyclo(-Asp-Asp) (5), cyclo(-Glu-
Gly) (6), cyclo(-Ala-Gln) (7), cyclo(-His-Pro) (8), cyclo
(-Ser-Tyr) (12), cyclo(-His-Phe) (14), cyclo(-Gly-Trp)
(22), cyclo(-Met-Met) (24), cyclo(-Trp-Tyr) (26), cyclo
(-Leu-Trp) (27), and cyclo(-Phe-Trp) (30), are firstly
reported for simultaneous analytical method in this
study.
Materials and methods
Chemicals and materials. In this study, unless
otherwise mentioned, amino acids composing DKP
were all L-forms. The DKPs used were listed in Table 1.
In this study, cyclo(-D-Ala-L-Pro) (13) was used
because cyclo(-L-Ala-L-Pro) was not available as a
commercial reagent. Cyclo(-Glu-Gly) (6) and cyclo
(-Pro-Pro) (15) were purchased from Peptide Institute
Inc. (Osaka, Japan). Other DKPs were purchased from
Bachem AG (Bubendorf, Switzerland). Water was puri-
fied with a Milli-Q system. Formic acid, acetonitrile,
dimethyl sulfoxide (DMSO), and methanol were pur-
chased from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan). A 0.45-µm RC-membrane filter was
 LC-MS/MS analysis of cyclic dipeptides 173
H R2
O NH R2 O H2N
N O
R1 H
H H +
H O H R3
O N O
N HN R1
H H
H
H R3
DKP
N-terminal peptide

Fig. 1. DKP formation mechanism and chemical structure. (R1, R2, R3: Amino acid residue).

purchased from Sartorius stedim (Göttingen, Germany). LS-MS/MS analysis of DKPs. Mass spectra were
Pu-erh tea leaf, which was produced in Yunnan recorded by means of a tandem mass spectrometry
Province in 2011, was purchased from TAETEA (ripe (ACQUITY TQD, Waters, Massachusetts, USA) cou-
tea 7592, Yunnan, China). pled to an ultra performance liquid chromatography
system (ACQUITY UPLC system, Waters) equipped
with an Atlantis T3 column (100 Å, 3 μm,
Preparation of DKP standard solution. As shown 150 × 2.1 mm i.d., Waters) connected to an Atlantis T3
in Table 1, Cyclo(-Ser-Ser) (3) and cyclo(-Asp-Asp) (5) Sentry Guard Cartridge (100 Å, 3 μm, 10 × 2.1 mm i.d,
were dissolved with water. Cyclo(-Gly-Gly) (4), cyclo Waters). The eluent system was composed of water
(-Glu-Gly) (6), cyclo(-Ser-Tyr) (12), cyclo(-Asp-Phe) contained 0.1% (v/v) formic acid (A) and acetonitrile
(21), cyclo(-Met-Met) (24), and cyclo(-Phe-Phe) (31) (B). The flow rate was 0.2 mL/mL. Gradient system
were dissolved with DMSO. Cyclo(-Pro-Thr) (11), was performed as follows: 2% of eluent (B) at 0 min,
cyclo(-Pro-Pro) (15), cyclo(-Gly-Leu) (16), cyclo(-Val- 5% of the eluent (B) at 5 min, 50% of eluent (B) at
Pro) (19), cyclo(-Leu-Pro) (23), cyclo(-Phe-Pro) (25), 25 min, 98% of eluent (B) at 30–35 min. The mass
cyclo(-Leu-Trp) (27), and cyclo(-Leu-Leu) (28) were spectrometer was operated in the positive electrospray
dissolved with acetonitrile. Other DKPs were dissolved ionization mode (ESI+) with a capillary voltage of
with methanol. Using these solutions, we made a stan- 3 kV. The source temperature was 150 °C, and the des-
dard solution which contains all DKPs at 200 mg/L. olvation temperature was 400 °C, respectively. The
This standard solution was used for making standard collision cell was operated at a collision gas (argon).
curve and spiked test. Multiple reaction monitoring (MRM) was carried out
by setting [M + 1]+ as a precursor ion (m/z) and frag-
ment ion showing a satisfactory detection sensitivity as
Table 1. DKPs list in this study.
a product ion. Cone energy (V) and collision energy
No. DKP Mw Solvent (eV) were optimized for each DKP.
1 Cyclo(-Gly-His) 194.19 Methanol
2 Cyclo(-Ala-His) 208.22 Methanol
Sample preparation of Pu-erh tea. Two grams of
3 Cyclo(-Ser-Ser) 174.16 Water
4 Cyclo(-Gly-Gly) 114.10 DMSO Pu-erh tea leaves was milled, and then it was extracted
5 Cyclo(-Asp-Asp) 230.17 Water with 200 g of hot water (100 °C) for 10 min. After fil-
6 Cyclo(-Glu-Gly) 186.17 DMSO tration with a membrane filter (pore size: 0.45 μm) pur-
7 Cyclo(-Ala-Gln) 199.21 Methanol chased from Sartorius stedim (Göttingen, Germany), it
8 Cyclo(-His-Pro) 234.25 Methanol was used as an analytical sample (10 mg D.W. eq./mL)
9 Cyclo(-Ala-Ala) 142.16 Methanol for LC-MS/MS analysis.
10 Cyclo(-Gly-Pro) 154.17 Methanol
11 Cyclo(-Pro-Thr) 198.22 Acetonitrile
12 Cyclo(-Ser-Tyr) 250.25 DMSO
13 Cyclo(-D-Ala-Pro) 168.20 Methanol LC-MS/MS analysis of Pu-erh tea and spiked test.
14 Cyclo(-His-Phe) 284.31 Methanol LC-MS/MS system was used to quantify the DKPs in
15 Cyclo(-Pro-Pro) 194.23 Acetonitrile Pu-erh tea. The analytical conditions were the same as
16 cyclo(-Gly-Leu) 170.21 Acetonitrile ‘LS-MS/MS analysis of DKPs’. In this study, detection
17 Cyclo(-Phe-Ser) 234.25 Methanol limit and determination limit of each DKP were deter-
18 Cyclo(-Pro-Tyr) 260.29 Methanol mined based on the signal-to-noise ratio, 3:1 and 10:1,
19 Cyclo(-Val-Pro) 196.25 Acetonitrile
20 Cyclo(-Gly-Phe) 204.23 Methanol
respectively. In addition, the performance of our devel-
21 Cyclo(-Asp-Phe) 262.27 DMSO oped method was assessed by spiked test. Briefly, 31
22 Cyclo(-Gly-Trp) 243.26 Methanol kinds of DKP standards at final concentrations of 0.01
23 Cyclo(-Leu-Pro) 210.27 Acetonitrile or 0.1 ppm were added to Pu-erh tea. For DKP stan-
24 Cyclo(-Met-Met) 262.39 DMSO dards, detection limits were over 0.006 ppm, 0.1 ppm
25 Cyclo(-Phe-Pro) 244.29 Acetonitrile of DKP was spiked at the final concentration. For DKP
26 Cyclo(-Trp-Tyr) 349.38 Methanol
standards, detection limit were <0.006 ppm, 0.01 ppm
27 Cyclo(-Leu-Trp) 299.37 Acetonitrile
28 Cyclo(-Leu-Leu) 226.32 Acetonitrile
of DKP was spiked at the final concentration. Using
29 Cyclo(-Leu-Phe) 260.33 Methanol the results of spiked test, recovery (%) and RSD (%)
30 Cyclo(-Phe-Trp) 333.38 Methanol were calculated (n = 3). Recovery rate (%) was calcu-
31 Cyclo(-Phe-Phe) 294.35 DMSO lated by the following equation:
174 K. Yamamoto et al.

Recovery rate ð%Þ ¼ fdetected value ðppmÞ= predicted value ðppmÞg  100

DKPs,4,14) MS condition was optimized, but the

Results and discussion
Optimization of LS-MS/MS analysis of DKPs
In this study, we attempted to establish simultaneous
analytical method based on LC-MS/MS by selecting 31
kinds of DKPs which were available as commercial
reagents. MRM conditions were optimized individually
for each DKP (Table 2). Table 2 summarizes precursor
ions and fragment ions of each DKP along with cone
voltage and collision energy. A Waters Atlantis T3 col-
umn, a reversed-phase column, was used and the gradi-
ent condition of eluent was examined. As a result,
component peaks of 31 kinds of DKPs were success-
fully separated and detected within 35 min as shown in
Fig. 2.
Table 3 shows the summary of retention time, RSD
(%) for triplicates, and the linearity of calibration
curves of each DKP. RSD (%) was calculated at the
concentration of 0.1 ppm of DKP. The range of calibra-
tion curves was 0–0.5 ppm (9 points of 0, 0.0025,
0.005, 0.001, 0.025, 0.05, 0.1, 0.2, and 0.5 ppm). The
results showed satisfactory repeatability with RSD of
10% or below in all DKPs. The linearity of calibration
curves was 0.992 or above in all cases.
Summarizing the above results, we developed an
analytical LC-MS/MS method that is capable of
simultaneously quantifying 31 kinds of DKPs with
satisfactory repeatability and linearity. In the previous
report of the simultaneous analytical methods of
quantitative performance was not studied using calibra-
tion curves. So, this is the first report for trying to
develop an analytical method of DKPs which confirm
the quantitative performance and repeatability.
Determination of DKP in Pu-erh tea
Using the developed LC-MS/MS method, next, we
attempted to determine DKP concentration in a food.
As described above, DKP is known to be widely con-
tained in fermented food. Therefore, in this study, the
extract of Pu-erh tea, post-fermented tea, was used as
an analytical sample. Pu-erh tea, which is produced
mainly in China, is divided into two groups: raw
Pu-erh which is produced through a long process of
microbial fermentation, and riped Pu-erh which under-
goes compulsory fermentation by maintaining warm
and humid environment. In this study, the latter, riped
Pu-erh, was used for the experiment.
Fig. 3 shows MRM chromatogram of Pu-erh tea
extract. Table 4 shows the detection limit, determina-
tion limit, and concentration of each component con-
tained in the Pu-erh tea extract. Among 31 types of
DKPs, the detection limit of 28 DKPs was 0.01 ppm or
below and the determination limit of 30 DKPs was
0.1 ppm or below. Although the optimization of
parameters such as cone voltage and collision energy,
in the case of cyclo(-Gly-His) (1), cyclo(-Glu-Gly) (6),

Table 2. MS/MS condition for DKPs.

No. Precursor ion (m/z) Product ion (m/z) Cone energy (V) Collision energy (eV)
1 195 82 30 30
2 209 110 30 30
3 175 60 28 22
4 115 58 30 30
5 231 195 22 16
6 187 84 22 20
7 200 84 30 30
8 235 110 30 30
9 143 44 30 14
10 155 70 40 22
11 199 70 30 30
12 251 136 30 30
13 169 70 34 18
14 285 110 30 30
15 195 70 30 30
16 171 86 30 14
17 235 120 30 30
18 261 136 30 30
19 197 70 30 30
20 205 120 30 30
21 263 91 30 30
22 244 130 30 30
23 211 70 30 30
24 263 167 30 30
25 245 120 30 30
26 350 130 30 30
27 300 130 30 30
28 227 72 30 30
29 261 120 30 30
30 334 130 30 30
31 295 120 30 30
 LC-MS/MS analysis of cyclic dipeptides 175

Retention time (min)

Fig. 2. MRM chromatogram of the DKPs. Numbers of peak top corresponded to the DKPs shown in Table 1.

Table 3. Retention time, RSD, linearity of DKPs. not available as reagent. According to the previous
report on cyclo(-D-Ala-L-Pro) and cyclo(-L-Ala-L-
No. Retention time (min) RSD (%) (n = 3) Linearity
Pro),14) fragment ions of these two DKPs showed
1 2.0 4.4 1.000 identical tendency in terms of intensity. In addition, we
2 2.2 2.3 1.000 also confirmed that peaks of cyclo(-D-Ala-L-Pro) and
3 2.4 2.5 0.999 cyclo(-L-Ala-L-Pro) appeared at the same retention time
4 2.5 5.7 0.999
5 3.4 8.8 0.992
in this analytical method (data not shown). Considering
6 3.8 4.3 0.995 the fact that DKPs with L-amino acids were detected as
7 4.2 3.7 0.995 major components from food, it can be considered that
8 4.5 7.7 1.000 most of cyclo(-Ala-Pro) detected in this study was cor-
9 5.9 6.0 0.999 responding to the concentration of cyclo(-L-Ala-L-Pro).
10 7.1 0.72 0.999 To consider stereoisomers such as diastereomer and
11 8.2 3.1 1.000
enantiomer, it is necessary to select appropriate col-
12 9.0 3.0 1.000
13 9.2 3.7 0.999
umns such as chiral column and optimize eluents. The
14 10.7 2.8 0.998 quantitative determination of stereoisomers remains to
15 11.0 6.3 1.000 be developed.
16 12.2 1.5 0.999 Tea is considered to be one of the three favorite
17 12.3 4.4 0.999 beverages in the world along with coffee and cocoa.
18 12.9 4.1 1.000 The research on components such as catechin and caf-
19 13.0 6.2 1.000
feine has been advanced and their numerous health
20 13.1 4.2 1.000
21 14.0 5.9 0.999 effects have been elucidated. However, the health effect
22 14.2 3.5 1.000 of tea is diverse and some of the causal components
23 16.1 2.9 1.000 still remain unknown. The present study is the first
24 16.4 6.3 0.999 report which focused on DKPs contained in microbial
25 17.4 0.78 1.000 fermented tea. Considering the fact that DKPs poten-
26 17.9 7.6 0.999 tially have various effects, it is highly likely that DKPs
27 20.1 3.2 1.000
28 20.4 2.3 1.000
play a partial role in the health effect of microbial fer-
29 20.8 2.6 1.000 mented tea. There are a variety of types in microbial
30 21.7 3.3 1.000 fermented tea and various micro-organisms are
31 22.0 1.4 1.000 involved in manufacturing process. We plan to investi-
gate the association of DKPs with the types of micro-
bial fermented tea, related process, micro-organisms,
and cyclo(-Gly-Leu) (16), the detection limit was and their effects.
slightly higher, 0.02–0.05 ppm. The reason of this high
detection limit was caused by baseline noise. So, when
higher detection sensitivity is required, we had better to Result of spiked test
select another analytical instrument and method. To confirm the applicability of developed method for
In the extract of Pu-erh tea, 18 kinds of DKPs were tea analysis, the spiked test of DKPs was conducted.
confirmed to be present and their concentration was in DKP standards were added into Pu-eah tea extracts
the range 0.0017–0.11 ppm. Cyclo(-Ala-Pro) was con- with the final concentration of 0.01 or 0.1 ppm, and
tained at the highest concentration among the targeted calculated recovery rate (%) and RSD (%). Table 4
DKPs. As described above, in this study, cyclo(-D-Ala- summarizes the results. As for the recovery rate, 21
L-Pro) was used to prepare calibration curve and calcu- kinds of DKP were within the range between 70 and
late the concentration because cyclo(-L-Ala-L-Pro) was 120% of the predicted value, indicating satisfactory
176 K. Yamamoto et al.

Retention time (min)

Fig. 3. MRM chromatogram of the DKPs in Pu-erh tea extract.

Table 4. Analytical result and recovery of DKPs in Pu-erh tea.

No. Detection limit (ppm) Determination limit (ppm) concentration (ppm) Recovery (%) RSD (%) (n = 3)
*
1 0.02 0.06 n.d. 48 6.0
2 0.0003 0.001 n.d. 54 14
3 0.009 0.03 n.d. 77* 8.8
4 0.006 0.02 n.d. 54* 5.4
5 0.002 0.004 n.d. 55 5.7
6 0.02 0.05 n.d. 141* 8.5
7 0.0007 0.003 n.d. 121 6.5
8 0.00007 0.0003 0.055 126 4.8
9 0.0002 0.0006 0.0044 113 15
10 0.0002 0.0007 0.025 117 0.93
11 0.006 0.02 0.083 120* 6.8
12 0.0005 0.002 n.d. 125 15
13 0.004 0.02 0.11 108 6.8
14 0.00003 0.0001 0.0048 181 3.8
15 0.003 0.008 0.050 120 4.9
16 0.05 0.2 n.d. 106* 14
17 0.0002 0.0004 0.00080 84 9.8
18 0.0003 0.001 0.011 93 9.5
19 0.003 0.01 0.026 104 5.7
20 0.0004 0.002 n.d. 76 15
21 0.0002 0.0005 0.0017 80 6.1
22 0.0002 0.0005 n.d. 90 15
23 0.003 0.01 0.061 104 3.5
24 0.0005 0.002 n.d. 106 1.0
25 0.0002 0.0005 0.028 110 1.8
26 0.00005 0.0002 0.0025 93 5.6
27 0.00004 0.0002 0.0074 100 2.4
28 0.0003 0.0008 n.d. 127 7.3
29 0.00007 0.0003 0.0053 96 1.7
30 0.00004 0.0002 0.0063 102 8.1
31 0.00003 0.0001 0.0035 88 2.0
*Recovery data spiked 0.1 ppm of DKP standard.

agreement between the measured values and predicted
value.15) In contrast, the recovery of 10 kinds of DKPs
exceeded the range between 70 and 120% of the pre-
dicted values. Among them, overlaps with Pu-eah tea
extracts-originated broad peaks likely influenced the
results of DKPs with shorter retention time including
cyclo(-Gly-His) (1), cyclo(-Ala-His) (2), cyclo(-Gly-
Gly) (4), cyclo(-Asp-Asp) (5), cyclo(-Glu-Gly) (6),
cyclo(-Ala-Gln) (7), and cyclo(-His-Pro) (8). In addi-
tion, matrix effect was suggested to decrease the recov-
ery rate in the following compounds: cyclo(-Ser-Tyr)
(12), cyclo(-His-Phe) (14), and cyclo(Leu-Leu) (28). As
for RSD (%), all 31 kinds of DKPs were within the
range 0.93–15%, confirming satisfactory repeatability.
Based on the above results, although there are some
DKPs for their recovery rate to be improved, we suc-
cessfully established simultaneous analysis of 31 kinds
of DKPs to a practical level. The present method could
be applied to DKP analysis in various foods and to
greatly advance researches concerning DKP in some
foods in the near future.
Conclusion
In this study, we attempted to establish the simulta-
neous analysis method of 31 kinds of DKPs using LC-
MS/MS. Analytical conditions including cone voltage
 LC-MS/MS analysis of cyclic dipeptides
and collision energy have been examined, and success-
fully resolved, and detected 31 kinds of DKPs could be
determined within 35 min. When the developed method
was applied to Pu-erh tea and 18 kinds of DKPs were
confirmed to be present at the range 0.0017–0.11 ppm.
As a result of the spiked test of DKPs into Pu-erh tea,
21 kinds of DKPs showed satisfactory recovery rate,
within the range between 70 and 120% of the predicted
values. In addition, RSD (%) for triplicates was in the
range 0.93–15%.
Author contributions
K.Y. and Y.M. designed the study. M.H., Y.A., and
Y.O. carried out the experiments. K.Y. shared responsi-
bility for the writing of manuscript with T.S. and T.K.
And, H.U. provided valuable advice and project
management. All authors read and approved the final
manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
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