Formation of ester and amine derivatives of 5-O-caffeoylquinic acid in the process of its simulated extraction

Article abstract of DOI:10.1021/jf3029682

Chlorogenic acid (CQA), the ester of caffeic acid with quinic acid, supplied to human organisms mainly through coffee, tea, fruits, and vegetables, is one of the most studied polyphenols. It is potentially useful in pharmaceuticals, foodstuffs, feed additives, and cosmetics due to its recently discovered biomedical activity. This finding caused new interest in its properties, its isomers, and its natural occurrence. The presented study shows that 5-O-caffeoylquinic acid, during its buffered water extraction, not only undergoes such transformation as isomerization, water molecule addition, and hydrolysis but also reacts with buffer components forming its derivatives. The amount of each formed component depends on the heating time, buffer pH, and buffer type. Although the concentrations of these components are low, they can be mistakenly treated as a new component not previously found in the examined plant or can be a cause of erroneous quantitative estimations of plant composition.

Full text of DOI:10.1021/jf3029682

Article
pubs.acs.org/JAFC
Formation of Ester and Amine Derivatives of 5‑O‑Caffeoylquinic Acid
in the Process of Its Simulated Extraction
Andrzej L. Dawidowicz* and Rafal Typek
Faculty of Chemistry, Maria Curie Sklodowska University, 20-031 Lublin, Pl. Maria Curie Sklodowska 3, Poland
ABSTRACT: Chlorogenic acid (CQA), the ester of caffeic acid with quinic acid, supplied to human organisms mainly through
coffee, tea, fruits, and vegetables, is one of the most studied polyphenols. It is potentially useful in pharmaceuticals, foodstuffs,
feed additives, and cosmetics due to its recently discovered biomedical activity. This finding caused new interest in its properties,
its isomers, and its natural occurrence. The presented study shows that 5-O-caffeoylquinic acid, during its buffered water
extraction, not only undergoes such transformation as isomerization, water molecule addition, and hydrolysis but also reacts with
buffer components forming its derivatives. The amount of each formed component depends on the heating time, buffer pH, and
buffer type. Although the concentrations of these components are low, they can be mistakenly treated as a new component not
previously found in the examined plant or can be a cause of erroneous quantitative estimations of plant composition.
KEYWORDS: chlorogenic acid, citric 5-CQA derivatives, acetic 5-CQA derivatives, amine 5-CQA derivatives,
5-CQA buffered extraction
INTRODUCTION
MATERIALS AND METHODS
■
■
Reagents. Acetonitrile (HPLC), sodium phosphate, phosphoric acid,
ammonium chloride, ammonium hydroxide, sodium borate (borax), boric
acid, sodium citrate, citric acid, sodium acetate, and acetic acid (all of
analytical grade) were purchased from the Polish Chemical Plant POCh
(Gliwice, Poland); formic acid from Sigma Aldrich (Seelze, Germany);
and chlorogenic acid from Loba-Chemie Austranal Praparate (Austria).
Water was purified on the Milli-Q system from Millipore (Millipore,
Bedford, MA, USA).
Methods. The investigations of pH influence on the 5-O-caffeoylquinic
acid transformation process were performed by heating 5-O-caffeoylquinic
acid buffered water solution under reflux. Glass equipment composed of a
boiling flask (100 mL) and a small condenser was used for this purpose.
The following pH values were applied during the experiments:
phosphoric buffers of pH 4.0; 5.0; 6.0; 7.0; 8.0; and 9.0; boric buffers
of pH 6.0; 7.0; 8.0; and 9.0; citric buffers of pH 4.0; 5.0; 6.0; and 7.0;
acetic buffers of pH 4.0; 5.0; 6.0; and 7.0; and ammonia buffers of pH
6.0; 7.0; 8.0; and 9.0. The heated 5-O-caffeoylquinic acid solutions
contained 10 mg of 5-O-caffeoylquinic acid in 50 mL of buffer. Portions
of the acid were inserted to the already boiling buffer (∼100 °C). The
solvents were heated for 10 min or 1, 3, or 5 h.
HPLC Measurements. Chromatographic measurements were
performed using LC/ESI/IT/MS from Finnigan (LCQ Adventage
Max) equipped with the ion-trap mass spectrometric system (Thermo-
Electron Corporation, San Jose, CA, USA) and a diode array detector
from Finningan (Surveyer PDA Plus Detector). The column used was a
100 mm × 4.6 mm i.d., 3 μm, Gemini C18 (Phenomenex, Torrance, CA,
USA). Chromatographic separation was performed using gradient
elution in isothermic conditions (25 °C). Mobile phase A was 25 mM
formic acid in water; mobile phase B was 25 mM formic acid in
acetonitrile. The gradient program started at 5% B increasing to 35% for
30 min, next 35% B to 100% B for 5 min, followed by isocratic elution
(100% B) for 5 min. The total run time was 40 min at the mobile phase
flow rate 0.4 mL/min.
Chlorogenic acid, the ester of caffeic acid with quinic acid,
supplied to human organisms mainly by coffee, tea, fruits, and
vegetables,1,2 is one of the most studied polyphenols.3,4 As shown
in ref 5, the metabolic track of chlorogenic acid synthesis starts
with the deaminase of phenylalanine by the specific enzyme
forming trans-cinnamic acid, which is then transformed into the
caffeoyl moiety of chlorogenic acid.6,7 Antioxidant and anti-
carcinogenic properties of CQA have been well established in
numerous in in vitro studies.8−12 Chlorogenic acid in the human
body is metabolized to ferulic and isoferulic acids, m-coumaric
acid, and the derivatives of phenylpropionic, benzoic, and
hippuric acids.13 Coffee, a beverage rich in chlorogenic acids,
modifies gastrointestinal hormone secretion and glucose tolerance
in humans, although the mechanism has not been fully eluci-
dated.14,15 According to recent studies, attempts have been made
to use CQA as a UVA and UVB filter.16 These newly found
properties of CQA caused unabated interest in 5-CQA, its isomers,
and its natural occurrence.
According to previous reports,17 the heating of 5-CQA water
solution in the temperature range of 100−200 °C causes
chlorogenic acid isomerization and transformation. It has been
found that as many as 14 derivative compounds and reaction
products with water can be formed from 5-O-caffeoylquinic acid
by heating its water solution at different pH.17 The cited inves-
tigations were performed using phosphate buffers of different
pH. The question asked in the present study is about the
influence of buffer type on 5-CQA transformation during its
heating in buffered water solutions. It is a valid question as the
5-CQA molecule possesses free hydroxyl and carboxyl groups
able to react with different buffer components. The presented
results may be valuable for researchers examining plant materials
in which chlorogenic acid derivatives formed during extraction
can be mistaken for natural components of the examined plants.
Received: July 10, 2012
Revised: November 7, 2012
Accepted: November 25, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jf3029682 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Table 1. Names, Shortcuts, and Peak Numbers of Estimated 5-O-Caffeoylquinic Transformation Products Obtained in
Phosphorous Buffer
peak
a
name of compounds
shortcut
(QA)
number
(1S,3R,4S,5R)-1,3,4,5-tetrahydroxycyclohexanecarboxylic acid “quinic acid”
1
(1S,3R,4R,5R)-3-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]-1,4,5-trihydroxycyclohexanecarboxylic acid
(1S,3R,4R,5R)-3-[3-(3,4-dihydroxyphenyl)-3S-hydroxypropanoyl]-1,4,5-trihydroxycyclohexanecarboxylic acid
(1S,3R,4R,5R)-5-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]-1,4,5-trihydroxycyclohexanecarboxylic acid
(1S,3R,4R,5R)-5-[3-(3,4-dihydroxyphenyl)-3S-hydroxypropanoyl]-1,4,5-trihydroxycyclohexanecarboxylic acid
(1S,3R,4R,5R)-4-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]-1,4,5-trihydroxycyclohexanecarboxylic acid
(1S,3R,4R,5R)-4-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]-1,4,5-trihydroxycyclohexanecarboxylic acid
3,4-dihydroxybenzoic acid “protocatechuic acid”
(3-CQA-3R-OH)
(3-CQA-3S-OH)
(5-CQA-3R-OH)
(5-CQA-3S-OH)
(4-CQA-3R-OH)
(4-CQA-3S-OH)
(PA)
2 or 3
2 or 3
4 or 5
4 or 5
6 or 7
6 or 7
8
(1S,3R,4R,5R)-1-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-3,4,5-trihydroxycyclohexanecarboxylic acid
“1-O-caffeoylquinic acid”
(1-CQA)
9
(1S,3R,4R,5R)-3-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic acid
“3-O-caffeoylquinic acid”
(3-CQA)
(5-CQA)
(4-CQA)
10
11
12
(1S,3R,4R,5R)-5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,3,4-trihydroxycyclohexanecarboxylic acid
“5-O-caffeoylquinic acid”
(1S,3R,4R,5R)-4-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,3,5-trihydroxycyclohexanecarboxylic acid
“4-O-caffeoylquinic acid”
(E)-3-(3,4-dihydroxyphenyl)-2-propenoic acid “caffeic acid”
(Z)-3-(3,4-dihydroxyphenyl)-2-propenoic acid “cis-caffeic acid”
CA
13
14
15
cis-CA
cis-5-CQA
(1S,3R,4R,5R)-5-{[(2Z)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,3,4-trihydroxycyclohexanecarboxylic acid
“cis-5-O-caffeoylquinic acid”
a
See Figure 1.
Figure 1. Chromatograms of 5-CQA heated under reflux for 5 h in water solutions buffered at pH 7.0 by the following buffers: phosphoric (A); boric
(B); citric (C); and acetic (D), and buffered at pH 9.0 by ammonium buffer (E). Peak numbers correspond to compound numbers in Figure 2. The
sequence of diastereoisomeric components in the 2/3, 4/5, 6/7, 16/17, 21/22, 23/24, or 25/26 pairs are tentative as it was impossible to differentiate the
R/S components and the ortho-/para-position in the given pair.
B
dx.doi.org/10.1021/jf3029682 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Table 2. Time Periods and Ions Monitored in Buffered Solutions of 5-O-Caffeoylquinic Acid
Phosphoric Buffer
time range (min)
0−9
9−13.5
13.5−16
16−20
20−22
22−40
m/z
191
371
153
353
179
353
Boric Buffer
time range (min)
0−9
9−13.5
13.5−16
16−20
20−22
22−40
m/z
191
371
153
353
179
353
Citric Buffer
time range (min)
0−9
9−13.5
13.5−16
16−20
20−22
22−40
m/z
191
371
153
353
179
527
Acetic Buffer
time range (min)
0−9
191
9−13.5
371
13.5−16
153
16−20
353
20−22
179
22−23
353
23−40
395
m/z
Ammonia Buffer
13.5−14
time range (min)
0−4.5
4.5−9
9−13.5
14−16
16−20
20−22
22−40
m/z
191
352
371
153
353
353
179
353
Table 3. Names, Shortcuts, and Peak or Structure Numbers of Estimated 5-O-Caffeoylquinic Transformation Products
peak
number
structure
b
a
name of compounds
shortcut
number
(1S,3R,4R,5R)-3-[(4-carboxy-3R-hydroxybutanoyl)oxy]-5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4-
dihydroxycyclohexanecarboxylic acid
(5-CQA-3-1-3R-Cit) 16 or 17
1
(1S,3R,4R,5R)-3-[(4-carboxy-3S-hydroxybutanoyl)oxy]-5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4-
dihydroxycyclohexanecarboxylic acid
(5-CQA-3-1-3S-Cit) 16 or 17
2
(1S,3R,4R,5R)-3-hydroxypentanedioic-5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4-
(5-CQA-3-3-Cit)
(5-CQA-4-Ac)
18
3
dihydroxycyclohexanecarboxylic acid
(1S,3R,4R,5R)-4-acetyloxy-5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,3-
dihydroxycyclohexanecarboxylic acid
19
4
(1S,3R,4R,5R)-3-acetyloxy-5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4-
dihydroxycyclohexanecarboxylic acid
(5-CQA-3-Ac)
20
5
(1S,3R,4R,5R)-3-{[(2E)-3-(3-amine-4-hydroxyphenyl)prop-2-enoyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic
(3-CQA-m-NH2)
(3-CQA-p-NH2)
(5-CQA-m-NH2)
(5-CQA-p-NH2)
(4-CQA-m-NH2)
(4-CQA-p-NH2)
21 or 22
21 or 22
23 or 24
23 or 24
25 or 26
25 or 26
6
acid
(1S,3R,4R,5R)-3-{[(2E)-3-(4-amine-3-hydroxyphenyl)prop-2-enoyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic
acid
7
(1S,3R,4R,5R)-5-{[(2E)-3-(3-amine-4-hydroxyphenyl)prop-2-enoyl]oxy}-1,3,4-trihydroxycyclohexanecarboxylic
acid
8
(1S,3R,4R,5R)-5-{[(2E)-3-(4-amine-3-hydroxyphenyl)prop-2-enoyl]oxy}-1,3,4-trihydroxycyclohexanecarboxylic
acid
9
(1S,3R,4R,5R)-4-{[(2E)-3-(3-amine-4-hydroxyphenyl)prop-2-enoyl]oxy}-1,3,5-trihydroxycyclohexanecarboxylic
acid
10
11
(1S,3R,4R,5R)-4-{[(2E)-3-(4-amine-3-hydroxyphenyl)prop-2-enoyl]oxy}-1,3,5-trihydroxycyclohexanecarboxylic
acid
a
b
See Figure 1. See Figure 2.
In each run, PDA spectra in the range 200−600 nm and MS spectra in
the range of m/z100−1000 were collected continuously. SIM function
was used to better visualize the chromatographic separation and to
remove the signal connected with the buffer components. The time
periods and monitored ions are collected in Table 1.
Because of the lack of standards of esters and amine derivatives, the
amounts of these compound were calculated by relating their chro-
matographic responses to the calibration curve for 5-CQA.
RESULTS AND DISCUSSION
■
The column effluent was ionized by electrospray ionization (ESI).
The ESI needle potential was 4.5 kV in the negative ionization mode. To
identify the chlorogenic acid isomers and the chlorogenic acid trans-
formation products, the functions of secondary (MSn) ion fragmenta-
tion were applied. The collision energy was chosen individually for each
group of the examined compounds. The comparison of the obtained
results with the literature data18 was helpful for assignment of CQA
region-isomers and their derivatives. To identify 5-O-caffeoylquinic acid
derivatives in the examined samples, LC/ESI/TOF/MS analysis was
additionally performed. LC/ESI/TOF/MS analysis was carried out on
the Agilent (Agilent Technologies, Palo Alto, CA, USA) liquid
chromatography system. MS analysis was performed on the orthogonal
TOF/MS equipped with an electrospray interface (Agilent Technolo-
gies, Santa Clara, CA, USA). Negative mode using full scan mode in
TOF/MS analysis was applied, and the mass range was set at 100−600
Da. Chromatographic separation was performed using the same chro-
matographic column and the same gradient elution as those described
for the LC/ESI/IT/MS analysis.
Figure 1 presents the chromatograms of 5-CQA water solutions
buffered at pH 7.0 using phosphoric, boric, citrate, and acetate
buffers and the chromatogram of 5-CQA water solutions buf-
fered at pH 9.0 using ammonia buffer, all heated under reflux for
5 h. The exemplary samples imitate the chlorogenic acid extract
obtained for this compound during its hot buffered water
extraction at pH 7.0 and/or 9.0.
As in results from ref 17, the heating of 5-CQA water solution
in phosphoric buffer leads to the formation of 14 compounds,
which are the transformation and isomerization products of the
parent compound. The analysis of the results reported in this
article (the experiments were performed in analogous way as in
ref 17) shows that the same compounds appear in 5-CQA water
solutions buffered by different buffers; see Table 2.
A more detailed consideration of the obtained chromatograms
shows that additional compounds are formed. PDA and MSn data
C
dx.doi.org/10.1021/jf3029682 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Figure 2. Molecular structures of individual ester and amine derivatives formed during 5-CQA transformation in citric, acetic, and ammonium buffers.
1, (1S,3R,4R,5R)-3-[(4-carboxy-3R-hydroxybutanoyl)oxy]-5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4-dihydroxycyclohexanecarboxylic
acid; 2, (1S,3R,4R,5R)-3-[(4-carboxy-3S-hydroxybutanoyl)oxy]-5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4-dihydroxycyclohexanecar-
boxylic acid; 3, (1S,3R,4R,5R)-3-hydroxypentanedioic-5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4-dihydroxycyclohexanecarboxylic
acid; 4,(1S,3R,4R,5R)-4-acetyloxy-5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,3-dihydroxycyclohexanecarboxylic acid; 5, (1S,3R,4R,5R)-
3-acetyloxy-5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4-dihydroxycyclohexanecarboxylic acid; 6, (1S,3R,4R,5R)-3-{[(2E)-3-(3-amine-
4-hydroxyphenyl)prop-2-enoyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic acid; 7, (1S,3R,4R,5R)-3-{[(2E)-3-(4-amine-3-hydroxyphenyl)prop-
2-enoyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic acid; 8, (1S,3R,4R,5R)-5-{[(2E)-3-(3-amine-4-hydroxyphenyl)prop-2-enoyl]oxy}-1,3,4-trihy-
droxycyclohexanecarboxylic acid; 9, (1S,3R,4R,5R)-5-{[(2E)-3-(4-amine-3-hydroxyphenyl)prop-2-enoyl]oxy}-1,3,4-trihydroxycyclohexanecarboxylic
acid; 10, (1S,3R,4R,5R)-4-{[(2E)-3-(3-amine-4-hydroxyphenyl)prop-2-enoyl]oxy}-1,3,5-trihydroxycyclohexanecarboxylic acid; 11, (1S,3R,4R,5R)-
4-{[(2E)-3-(4-amine-3-hydroxyphenyl)prop-2-enoyl]oxy}-1,3,5-trihydroxycyclohexanecarboxylic acid.
indicate that these compounds can be identified as ester and
amine derivatives of CQA. In the case of citric buffer, three
additional derivatives are formed during 5-CQA transtormation
(peaks 16−18 in Figure 1C): peaks 16 and 17, diastereoizomeric
pair (1S,3R,4R,5R)-3-[(4-carboxy-3R/S-hydroxybutanoyl)oxy]-
5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4-dihy-
droxycyclohexanecarboxylic acid, (5-CQA-3-1-3R/S-Cit); peak
18, (1S,3R,4R,5R)-3-hydroxypentanedioic-5-{[(2E)-3-(3,4-
dihydroxyphenyl)prop-2-enoyl]oxy}-1,4-dihydroxycyclohexane-
carboxylic acid, (5-CQA-3-3-Cit).
In the case of acetic buffer, two additional derivatives are
formed (peaks 19 and 20 in Figure 1D): peak 19, (1S,3R,4R,5R)-
4-acetyloxy-5-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]-
oxy}-1,3-dihydroxycyclohexanecarboxylic acid, (5-CQA-4-Ac);
peak 20, (1S,3R,4R,5R)-3-acetyloxy-5-{[(2E)-3-(3,4-
dihydroxyphenyl)prop-2-enoyl]oxy}-1,4-dihydroxycyclohexane-
carboxylic acid, (5-CQA-3-Ac);
In the case of ammonium buffer, six additional derivatives
appear in the mixture after 5-CQA transformation (peaks 21−
26 in Figure 1E): peaks 21 and 22, (1S,3R,4R,5R)-3-{[(2E)-
3-(3/4-amine-4-hydroxyphenyl)prop-2-enoyl]oxy}-1,4,5-trihy-
droxycyclohexanecarboxylic acid, (3-CQA-m/p-NH2); peaks
23 and 24, (1S,3R,4R,5R)-5-{[(2E)-3-(3/4-amine-4-hydro-
xyphenyl)prop-2-enoyl]oxy}-1,3,4-trihydroxycyclohexanecar-
boxylicacid, (5-CQA-m/p-NH2);peaks 25 and 26, (1S,3R,4R,5R)-
4-{[(2E)-3-(3/4-amine-4-hydroxyphenyl)prop-2-enoyl]oxy}-
1,3,5-trihydroxycyclohexanecarboxylic acid, (4-CQA-m/p-NH2).
Such identification results from the following. The ion mole-
cular weights of the compounds corresponding to peaks 16−18
are the same and exceed the 5-CQA ion molecular weight by the
molecular weight of citric acid lessened by 18 Da (molecular
weight of water, a byproduct of the esterification reaction). The
performed MS2 does not allow one to identify the position of the
citric moiety substitution in 5-CQA citric derivatives. Because of
the very low concentration of these derivatives and their low
D
dx.doi.org/10.1021/jf3029682 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
stability in the ionization process, MS3 spectra could not be carried
out. The same relationship is observed in acetic esters of 5-CQA
(peaks 19 and 20); however, the lower number of acetic in relation
to citric 5-CQA derivatives is difficult to explain. Citric acid is able
to react with hydroxyl groups in positions 1-, 3-, and 4-, and with
the carboxylgroupof quinic moietydueto the presence of carboxyl
and hydroxyl groupsinits molecule. Because of steric hindrance, its
reaction with the 1- and 4- hydroxyl group and with the carboxyl
group of quinic moiety is less likely. It is more probable that the
3-hydroxyl group reacts with the side carboxyl group of citric acid
forming two diastereoisomers, and with the central carboxyl group
of citric acid forming the third ester 5-CQA derivative. The last
assumption is consistent with the obtained chromatograms (see
Figure 1C). Peaks 16 and 17 have a close retention and a similar
magnitude, which indicates a similarity of the chemical prop-
erties and amounts of corresponding 5-CQA citric derivatives
(diastereoisomeric pair).
In the case of acetic acid, due to a smaller size of its molecule in
relation to the citric acid molecule, the esterification of the 3- and
4-hydroxyl groups of quinic moiety is more probable; however,
the formation of the 5-CQA acetic derivative in position 3 seems
to be more likely. If so, the last derivative should correspond with
peak 19.
The esterification of hydroxyl groups in the aromatic ring of
5-CQA requires the application of acid anhydride or acid
chloride. Hence, the esterification of hydroxylic groups coupled
to the aromatic ring looks impossible in the applied experi-
mental conditions. Probably the employment of a very sensitive
LC-NMR system would be more helpful for an unequivocal
identification of citric and acetic moiety substitution in CQA.
The ion molecular weights of the compounds corresponding
to peaks 21−26 are also the same and lower by 1 Da than the
5-CQA ion molecular weight. The observed difference in
molecular weight of molecular ions indicates the substitution
of the −OH group (alcoholic or phenolic group) in the quinic
moiety or in the aromatic ring, or the substitution of −OH group
in the carboxylic group by the −NH2 group (the difference in the
molecular weight of these groups equals 1 Da). It results from the
MS2 analysis that these CQA derivatives decompose into two
ions −191 m/z, corresponding to the molecular weight of quinic
ion, and 178 m/z, which is lower by 1 Da than the molecular
weight of caffeic acid. Hence, the obtained results indicate the
substitution of −OH groups by −NH2 in the caffeic moiety.
There are some additional arguments showing the correctness
of this identification. Low temperature substitution in the aro-
matic ring is more privileged than in the aliphatic and/or cyclic
structure; however, it requires the application of a catalyst (Lewis
acid).19 The used ammonium buffer was contaminated with Fe3+
(5 ppm), which played the role of catalyst of the substitution
reaction during aromatic amine synthesis from phenolic sub-
strates. Moreover, hydroxyl groups in the caffeic moiety are in
ortho position. The ortho position of hydroxyl groups favor the
substitution of one of them. It is worth mentioning that a higher
temperature than that used in these experiments is required for
the substitution of the −OH group by −NH2 in the −COOH
group.
Table 4. Negative Ion MSn Data for Product Transformation
of 5-O-Caffeoylquinic Acid
MS1
MS2
parent
ion
base
peak
secondary peak
intensity
peak
number
m/z
m/z
m/z
(%)
compds
16
17
18
19
20
527.1
191.2
179.1
365.3
179.2
365.3
179.1
365.2
179.2
233.3
179.1
233.3
178.1
22.1
9.1
(5-CQA-3-1-3S-Cit) or
(5-CQA-3-1-3S-Cit)
527.2
527.1
395.3
395.4
191.2
191.2
191.2
191.1
18.2
11.2
31.2
27.2
19.1
31.8
11.3
39.8
67.4
(5-CQA-3-1-3R-Cit) or
(5-CQA-3-1-3R-Cit)
(5-CQA-3-3-Cit)
(5-CQA-4-Ac)
(5-CQA-3-Ac)
21
22
23
24
25
26
351.9
351.8
351.8
351.9
351.8
351.9
191.4
191.4
191.3
191.3
178.2
178.1
(3-CQA-p-NH2) or
(3-CQA-m-NH2)
178.2
178.1
178.1
191.2
191.3
73.2
5.3
(3-CQA-m-NH2) or
(3-CQA-p-NH2)
The formation of six amine CQA derivatives results from the
substitution of the meta or para −OH group by −NH2 in the
most abundant CQA isomers present in heated 5-CQA water
solution, 3-CQA, 4-CQA, and 5-CQA. From the results from the
chromatograms (see the exemplary chromatogram in Figure 1E),
three pairs of peaks differing in height can be distinguished
among the peaks representing six amine CQA derivatives.
Assuming that the amount of amine CQA derivatives is directly
(5-CQA-m-NH2) or
(5-CQA-p-NH2)
8.8
(5-CQA-m-NH2) or
(5-CQA-p-NH2)
32.1
24.2
(4-CQA-m-NH2) or
(4-CQA-p-NH2)
(4-CQA-m-NH2) or
(4-CQA-p-NH2)
Table 5. HRMS Data for Product Transformation of 5-O-Caffeoylquinic Acid
[M − H]− (Da)
error
peak number
elemental composition [M − H]−
experimental
calcd
(ppm)
(mDa)
compds
16
17
18
19
20
21
22
23
24
25
26
C22H23O15
C22H23O15
C22H23O15
C18H19O10
C18H19O10
C16H18O8N
C16H18O8N
C16H18O8N
C16H18O8N
C16H18O8N
C16H18O8N
527.1032
527.1039
527.1033
395.0981
395.0975
352.1030
352.1029
352.1037
352.1036
352.1039
352.1027
527.1037
527.1037
527.1037
395.0978
395.0978
352.1032
352.1032
352.1032
352.1032
352.1032
352.1032
0.9
0.4
0.8
0.8
0.8
0.6
0.9
1.4
1.1
2.0
1.4
−0.5
0.2
(5-CQA-3-1-3R-Cit) or (5-CQA-3-1-3S-Cit)
(5-CQA-3-1-3R-Cit) or (5-CQA-3-1-3S-Cit)
(5-CQA-3-3-Cit)
−0.4
0.3
(5-CQA-4-Ac)
−0.3
−0.2
−0.3
0.5
(5-CQA-3-Ac)
(3-CQA-m-NH2) or (3-CQA-p-NH2)
(3-CQA-m-NH2) or (3-CQA-p-NH2)
(5-CQA-m-NH2) or (5-CQA-p-NH2)
(5-CQA-m-NH2) or (5-CQA-p-NH2)
(4-CQA-m-NH2) or (4-CQA-p-NH2)
(4-CQA-m-NH2) or (4-CQA-p-NH2)
0.4
0.7
−0.5
E
dx.doi.org/10.1021/jf3029682 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
proportional to the amount of their precursors, the peak pairs
21/22, 23/24, and 25/26 correspond to the amine derivatives of
3-CQA, 5-CQA, and 4-CQA, respectively.
For better clarity, the names of all the identified new com-
pounds, their peaks and structure numbers, shortcuts, and chemical
structures are collected in Table 3 and graphically presented in
Figure 2. The MSn and HRMS data for the mentioned compounds
are collected in Tables 4 and 5.
The influence of pH and heating time on the amount of
individual ester and amine derivatives formed during 5-CQA
transformation and degradation in water solutions buffered by
different buffer types is presented in Figures 3−5.
Figure 4. Influence of pH on the amount of acetic esters formed during
the heating of 5-CQA water solution buffered by acetic buffer. Heating
time of buffered 5-CQA water solution: 10 min (A); 1 h (B); 3 h (C);
and 5 h (D).
Figure 3. Influence of pH on the amount of citric esters formed during
the heating of 5-CQA water solution buffered by citric buffer. Heating
time of buffered 5-CQA water solution: 10 min (A); 1 h (B); 3 h (C);
and 5 h (D).
Figure 3 illustrates the influence of pH increase versus the
amount of three citric esters of 5-CQA. As seen in the presented
plots, the increase of heating time causes a small increase in the
amount of the 5-CQA citric derivatives. Moreover, the elevated
hydrogen ion concentration favors the formation of these
derivatives, which agrees with literature. The same conclusion can
be made for the plots in Figure 4, which presents the influence of pH
increase on the amount of two acetic esters of 5-CQA. Quite a
different situation is observed in the case of amine CQA derivatives;
see Figure 5. The formation of CQA amines is favored in an alkali
environment. Moreover, there is no significant influence of heating
time on the amount of these derivatives, which can result from the
gradual thermal decomposition of ammonium buffer.
The presented study proves that 5-O-caffeoylquinic acid,
during its buffered water extraction, not only undergoes such
transformations as isomerization, water molecule addition,
hydrolysis and recombination of hydrolysis products but also
can react with buffer components forming esters and amine
derivatives, depending on buffer composition. The performed
experiments show that the amount of each forming component is
low and depends on the heating time, buffer pH, and buffer type.
Liquid extraction is applied most frequently today as a sample
preparation procedure for plant analysis. To increase extraction
Figure 5. Influence of pH on the amount of amine derivatives formed
during the heating of 5-CQA water solution buffered by ammonium
buffer. Heating time of buffered 5-CQA water solution: 10 min (A); 1 h
(B); 3 h (C); and 5 h (D).
efficiency, buffering extractants of different pH are frequently
used. The pH of the same plant extract can vary owing to the
contents of plant components differing in their acidic−basic
character, e.g., organic acids and basic, mineral, and organic
salts. In light of these facts and the presented results, it is
quite possible that the transformation and reaction products of
5-O-caffeoylquinic acid can be mistakenly treated as new components
F
dx.doi.org/10.1021/jf3029682 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
(16) Rivelli, D. P; Filho, C. A. H.; Almeida, R. L.; Ropke, C. D.; Sawada,
T. C. H.; Barros, S. B. M. Chlorogenic acid UVA−UVB photostability.
Photochem. Photobiol. 2010, 86, 1005−1007.
(17) Dawidowicz, A. L.; Typek, R. The influence of pH on the thermal
stability of 5-O-caffeoylquinic acids in aqueous solutions. Eur. Food Res.
Technol. 2011, 233, 223−232.
(18) Clifford, M. N.; Johnston, K. L.; Knight, S.; Kuhnert, N. A.
Hierarchical scheme for LC-MSn identification of chlorogenic acids. J.
Agric. Food Chem. 2003, 51, 2900−2911.
(19) McMurry, J. Organic Chemistry, 7th ed.; Brooks/Cole-Thomson:
Belmont, CA, 2007.
not previously found in the examined plant. The presence of
these products can also lead to erroneous quantitative estima-
tions of plant composition when some or all components
formed from 5-O-caffeoylquinic acid during its water or
buffered water extraction really exist in the examined plant
and the 5-O-caffeoylquinic acid transformation process only
adds to their amount. This is why the presented results are
important for researchers investigating plant metabolism and
looking for new plant components.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +48 81 537 55 45. Fax: +48 81 533 33 48. E-mail: dawid@
poczta.umcs.lublin.pl.
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Clifford, M. N.; Wu, W.; Kuhnert, N. The chlorogenic acids of
Hemerocallis. Food Chem. 2006, 95, 574−578.
(2) Wang, Z.; Clifford, M. N. Profiling the chlorogenic acids of sweet
potato (Ipomoea batatas) from China. Food Chem. 2008, 106, 147−152.
(3) Clifford, M. N. Chlorogenic acids and other cinnamates − nature,
occurrence, dietary burden, absorption and metabolism. J. Sci. Food
Agric. 2000, 80, 1033−1042.
(4) Nobuji, N.; Shin-ichi, K.; Hiroe, K.; Keiko, S.; Kiyoshi, K.;
Takahiko, M. Identification, quantitative determination, and antiox-
idative activities of chlorogenic acid isomers in prune (Prunus domestica
L.). J. Agric. Food Chem. 2000, 48, 5512−5516.
(5) Koukol, J.; Conn, E. E. The metabolism of aromatic compounds in
higher plants. IV. Purification and properties of the phenylalanine
deaminase of Hordeum vulgare. J. Biol. Chem. 1961, 236, 2692−2698.
(6) Hanson, K. R.; Zucker, M. The biosynthesis of chlorogenic acid and
related conjugates of the hydroxycinnamic acids. Chromatographic
separation and characterization. J. Biol. Chem. 1963, 238, 1105−11015.
(7) Levy, C. C.; Zucker, M. Cinnamyl and p-coumaryl esters as
intermediates in the biosynthesis of chlorogenic acid. J. Biol. Chem. 1960,
235, 2418−2425.
(8) Marinova, E. M.; Toneva, A.; Yanishlieva, N. Comparison of the
antioxidative properties of caffeic and chlorogenic acids. Food Chem.
2009, 114, 1498−1502.
(9) Niggeweg, R.; Michael, A. J.; Martin, C. Engineering plants with
increased levels of the antioxidant chlorogenic acid. Nat. Biotechnol.
2004, 22, 746−754.
(10) Rodriguez de Sotillo, D.; Hadley, M.; Wolf-Hall, C. Potato peel
extract a nonmutagenic antioxidant with potential antimicrobial activity.
J. Food Sci. 1998, 63, 907−911.
(11) Sawa, T.; Nakao, M.; Akaike, T.; Ono, K.; Maeda, H. Alkylperoxyl
radical-scavenging activity of various flavonoids and other phenolic
compounds: implications for the anti-tumor-promoter effect of
vegetables. J. Agric. Food Chem. 1999, 47, 397−402.
(12) Zhao, M.; Wang, H.; Yang, B.; Tao, H. Identification of
cyclodextrin inclusion complex of chlorogenic acid and its antimicrobial
activity. Food Chem. 2010, 120, 1138−1142.
́ ́
(13) Gonthier, M. P.; Verny, M. A.; Besson, C.; Remesy, C.; Scalbert,
A. Chlorogenic acid bioavailability largely depends on its metabolism by
the gut microflora in rats. J. Nutr. 2003, 133, 1853−1859.
(14) Welsch, C. A.; Lachance, P. A.; Wasserman, B. P. Dietary phenolic
compounds: Inhibition of Na+-dependent D-glucose uptake in rat
intestinal brush border membrane vesicles. Nat. Biotechnol. 1989, 119,
1698−1704.
(15) Johnston, K. L.; Clifford, M. N.; Morgan, L. M. Coffee acutely
modifies gastrointestinal hormone secretion and glucose tolerance in
humans: Glycemic effects of chlorogenic acid and caffeine. Am. J. Clin.
Nutr. 2003, 78, 728−733.
G
dx.doi.org/10.1021/jf3029682 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX