Characterization and quantification of hydroxycinnamate derivatives in Stevia rebaudiana leaves by LC-MSn

Article abstract of DOI:10.1021/jf202185m

Stevia rebaudiana leaves are used as a zero-calorie natural sweetener in a variety of food products in Asian countries, especially in Japan. In this study, the hydroxycinnamate derivatives of S. rebaudiana have been investigated qualitatively and quantita

Full text of DOI:10.1021/jf202185m

ARTICLE
pubs.acs.org/JAFC
Characterization and Quantification of Hydroxycinnamate Derivatives
in Stevia rebaudiana Leaves by LC-MSn†
Hande Karak€ose, Rakesh Jaiswal, and Nikolai Kuhnert*
School of Engineering and Science, Chemistry, Jacobs University Bremen, 28759 Bremen, Germany
S Supporting Information
b
ABSTRACT: Stevia rebaudiana leaves are used as a zero-calorie natural sweetener in a variety of food products in Asian countries,
especially in Japan. In this study, the hydroxycinnamate derivatives of S. rebaudiana have been investigated qualitatively and
quantitatively by LC-MSn. Twenty-four hydroxycinnamic acid derivatives of quinic and shikimic acid were detected, and 19 of them
were successfully characterized to regioisomeric levels; 23 are reported for the first time from this source. These comprise three
monocaffeoylquinic acids (Mr 354), seven dicaffeoylquinic acids (Mr 516), one p-coumaroylquinic acid (Mr 338), one feruloylquinic
acid (Mr 368), two caffeoyl-feruloylquinic acids (Mr 530), three caffeoylshikimic acids (Mr 336), and two tricaffeoylquinic acids
(Mr 678). Cis isomers of di- and tricaffeoylquinic acids were observed as well. Three tricaffeoylquinic acids identified in stevia leaves
are reported for the first time in nature. These phenolic compounds identified in stevia might affect the organoleptic properties and
add additional beneficial health effects to stevia-based products.
KEYWORDS: Stevia rebaudiana, chlorogenic acids, hydroxycinnamic acids, caffeoylquinic acids, caffeoylshikimic acids, tandem
mass spectrometry
’ INTRODUCTION
has been shown to be highly adaptable to cultivation in many
other parts of the world. S. rebaudiana occurs naturally on acid
soils of pH 4ꢀ5 but will also grow on soils with pH levels of
6.5ꢀ7.5, making it an interesting alternative to plants cultivated
on poor soils such as tobacco.7
Stevia rebaudiana is a plant belonging to the Asteraceae family
of plants, which is native to Brazil and Paraguay. Due to the
natural sweetness of its leaves, S. rebaudiana has caught attention
in scientific and industrial fields to act as a natural zero-calorie
sweetener in many applications in the food industry. The leaves
contain ent-kaurene glycosides, comprising stevioside, rebaudio-
sides A, B, C, D, E, and F, and dulcoside A. All of these diterpene
glycosides comprise a steviol backbone structure; they differ only
in the glucose moiety at positions C13 and C19 (Figure 1).
Stevioside is the main sweet-tasting glycoside in stevia and was
reported to be 250ꢀ300 times sweeter than sucrose.1 Rebaudio-
side A is the second most abundant ent-kaurene and sweetest
compound in stevia; its sweetness is 400 times greater than that
of sucrose, and it has more pleasant taste and is more water-
soluble than stevioside.2 The amounts of diterpene glycosides
may vary depending on the growth conditions of stevia; however,
stevioside accounts for 4ꢀ13% (w/w) and rebaudioside A
accounts for 2ꢀ4% (w/w),3 the other glycosides being present
in lower concentrations.
The principal advantage of stevia metabolites is that they are
natural, nonsynthetic products. Stevia leaves can be used in their
natural state (fresh or dried form), due to their high sweetening
intensity. Only small quantities are needed in comparison to white
sugar to achieve comparable sweetness. The primary use of stevia
is as a commercial sweetener; it is used in a wide range of products
such as soft drinks, ice cream, chocolate, yogurt, and baked and
cooked foods. Stevia products also have beneficial uses in various
consumer care products such as toothpaste or mouthwashes.4,5
Stevia may also be used for obesity, diabetics, dental caries, and
therapeutic effects such as hypoglycemic activity.6
In addition to diterpene glycosides, a number of secondary
plant metabolites have been identified from S. rebaudina including
labdane-type diterpenes, triterpenoids and steroids, flavonoids,
and oil components. From S. rebaudiana, 10 labdane-type diter-
penoids were identified, including austroinulin, isoaustroinulin,6
and sterebins (AꢀH).8,9 A triterpenoid, lupeol 3-palmitate, was
also separated from stevia.10 As plant sterols, β-sitosterol, stigmas-
terol, and campesterol were identified from S. rebaudiana.11
Plant phenols are a large and diverse group of compounds
including hydroxycinnamates, tannins, flavonoids, stilbenes, cou-
marins, lignans, and lignins.12 Chlorogenic acids (CGAs) are the
mostcommonhydroxycinnamate derivatives observedintheplant
kingdom. By definition, they are a large family of esters formed
between quinic acid and one to four residues of certain trans-
hydroxycinnamic acids, most commonly caffeic, p-coumaric, and
ferulic; sinapic and dimethoxycinnamic acids also occur, and in
some plant species various aliphatic acids may replace one or more
of the trans-cinnamic acid residues.13 CGAs are involved in
biological functions in plants such as defense against pathogens
and resistance to diseases. CGAs also participate in enzyme-
catalyzed browning reactions that may adversely affect the color,
flavor, and nutritional quality of dietary sources.14
Several pharmacological activities of CGAs including antiox-
idant activity, the ability to increase hepatic glucose utilization,15,16
Received: June 1, 2011
Revised:
July 15, 2011
The majority of the annual stevia production of an estimated
4000 t is produced in China and South America. The stevia crop
Accepted: August 2, 2011
Published: August 02, 2011
r
2011 American Chemical Society
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Figure 1. Structures and numberings of caffeoylquinic acids.
Figure 2. Base peak chromatogram of Stevia rebaudiana extract using ion trap MS in negative ion mode. For numbering, see Table 1.
inhibitionofthe HIV-1integrase,17,18 antispasmodicactivity,19 and
inhibition of the mutagenicity of carcinogenic compounds20
have been revealed by in vitro, in vivo, and human intervention
studies so far. CGAs and their metabolites display additionally
highly favorable pharmacokinetic properties.21ꢀ23 Because the
polyphenols in stevia might affect the organoleptic properties of
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Figure 3. Extracted ion chromatograms (EIC) of m/z 515 in negative ion mode (A) before and (B) after UV irradiation.
Figure 4. Structures and numbering of tricaffeoylquinic acids.
stevia-based product and could add additional health benefits to
the product, the objective of the present study was to profile the
phenolic content of S. rebaudina leaves with a particular emphasis
on hydroxycinnamate derivatives.
UV Irradiation. The prepared sample of stevia leaf extract (1 mL)
was placed in a photoreactor (LuzchemLZC -4 V, Ottawa, Canada)
under a shortwave UV lamp and irradiated at 245 nm for 40 min.
LC-MSn. The LC equipment (Agilent 1100 series, Bremen,
Germany) comprised a binary pump, an autosampler with a 100 μL
loop, and a diode array detector with a light-pipe flow cell (recording at
320 and 254 nm and scanning from 200 to 600 nm). This was interfaced
with an ion-trap mass spectrometer fitted with an ESI source (Bruker
Daltonics HCT Ultra, Bremen, Germany) operating in Auto-MSn mode
to obtain fragment ions m/z. As necessary, MS2, MS3, and MS4
fragment-targeted experiments were performed to focus only on com-
pounds producing a parent ion at m/z 335.1, 337.1, 367.1, 529.2, or
677.3. Tandem mass spectra were acquired in Auto-MSn mode (smart
fragmentation) using a ramping of the collision energy. Maximum
fragmentation amplitude was set to 1 V, starting at 30% and ending at
200%. MS operating conditions (negative mode) had been optimized
using 5-caffeoylquinic acid28 with a capillary temperature of 365 °C, a
dry gas flow rate of 10 L/min, and a nebulizer pressure of 50 psi.
’ MATERIALS AND METHODS
The chlorogenic acids, 3-caffeoylquinic acid, 4-caffeoylquinic acid,
5-caffeoylquinic acid (chlorogenic acid), 3,4-dicaffeoylquinic acid, 3,5-
dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid, were purchased from
PhytoLab (Vestenbergsgreuth, Germany). All other chemicals were
purchased from Sigma-Aldrich (Bremen, Germany). Stevia leaves were
purchased from a market in Bremen, Germany.
Sample Preparation. Two grams of S. rebaudiana leaves was
immersed in liquid nitrogen, ground in a hammer mill, and extracted first
with 150 mL of chloroform in a Soxhlet apparatus (Buchi B-811
extraction system) for 2 h and then with 150 mL of methanol for
another 2 h. Solvents were removed from the methanolic extract in
vacuo, and extracts were stored at ꢀ20 °C until required.
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Figure 5. Tandem mass spectra of 1,3,5-triCQA in negative ion mode.
Figure 6. Tandem mass spectra of 3,4,5-triCQA in negative ion mode.
High-resolution LC-MS was carried out using the same HPLC
equipped with a MicroTOF Focus mass spectrometer (Bruker
Daltonics) fitted with an ESI source, and internal calibration was
achieved with 10 mL of 0.1 mol/L sodium formate solution injected
through a six-port valve prior to each chromatographic run. Calibration
was carried out using the enhanced quadratic calibration mode.
HPLC. Separation was achieved on a 150 ꢁ 3 mm i.d. column
containing diphenyl 5 μm with a 4 ꢁ 3 mm i.d. guard column of the same
material (Varian, Darmstadt, Germany). Solvent A was water/formic
acid (1000 + 0.05 v/v), and solvent B was methanol. Solvents were
delivered at a total flow rate of 0.5 mL/min. The gradient profile was
from 10 to 70% B linearly in 60 min followed by 10 min isocratic and a
return to 10% B at 80 and 10 min isocratic to re-equilibrate.
Calibration Curve of Standard Compounds. Stock solutions
of the standard compounds were prepared in methanol. A series of
standard solutions was injected (5 μL) into the LC-MS system. The
areas of the peaks of each standard from UV chromatograms were used
to make the respective standard curves.
Synthesis of the Mixture of Regioisomers of Tricaffeoyl-
quinic Acids. To a solution of quinic acid (96 mg, 0.5 mmol) and
DMAP (16 mg, 0.12 mmol) in CH2Cl2 (10 mL) were added triethy-
lamine (4 mL) and 3,4-diacetylcaffeic acid chloride (423 mg, 1.5 mmol)
at room temperature. The reaction mixture was stirred for 6 h and
acidified with 2 mol/L HCl (pH ≈1) and then stirred for an additional
3 h to remove the acetyl protecting groups. The layers were separated,
and the aqueous phase was re-extracted with CH2Cl2 (1 ꢁ 20 mL) and
EtOAc (2 ꢁ 20 mL). The combined organic layers were dried over
Na2SO4 and filtered, and the solvents were removed in vacuo. The
resulting esters were analyzed by HPLC-MS.
’ RESULTS AND DISCUSSION
Methanol extracts of stevia dry leaves were directly used for
LC-MS analysis. Efficient separation and resolution were
achieved with diphenyl packing and acetonitrile/water as solvent
in the HPLC method. Negative ion mode was used for all MS
measurements. The HPLC method used here constitutes a
variation of methods employed previously,24 with variations
required to achieve sufficient separation of triacyl chlorogenic
acids and ent-kaurene glycosides. In comparison to isolation of
CGAs from green coffee beans, no removal of proteins/peptides
by Carrez reagent was necessary.13,24
All data for chlorogenic acids and diterpene glycosides pre-
sented in this paper use the IUPAC numbering system,32 and
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Table 1. Tandem Mass Spectral Data of Hydroxycinnamates in Stevia rebaudiana Leaf Extract
MS2
MS3
MS4
secondary peaks
secondary peaks
secondary peaks
m/z base
base
base
no.
compd
3-CQA
(neg) peak m/z int m/z int m/z int peak m/z int m/z int m/z int peak m/z int m/z int m/z int
1
2
3
4
5
6
7
8
9
353.0 190.7 178.8 49 134.9
353.0 190.7
8
126.8 172.8 37 85.2 55 110.8 65 188.8 174.6 64 134.4 84
126.8 172.7 49 85.1 61 110.8 21 108.8
8 93.0 110.8 62 154.7 24
5-CQA
4-CQA
353.0 172.7 178.8 60 190.6 14 134.8
515.1 353.0 190.8
515.1 353.0 335.0 12 172.8 17
3,5-diCQA
3,4-diCQA
4,5-diCQA
8
190.7 178.8 49 134.9
172.8 178.8 67 190.8 57 134.8 10 93.0 110.8 41 83.2
172.8 17 172.8 178.6 52 190.8 28 135.0 93.0 110.8 89 83.0 18
7
126.8 93.0 98 85.2 62 172.6 48
6
515.1 353.0 299.0
3
254.9
7
7
a cis-3,5-diCQA 515.1 353.0 190.8 10
a cis-4,5-diCQA 515.1 353.0 172.8 13
190.7 178.8 50 172.8 11 134.9 10 85.0 126.8 85 93.0 53 172.7 36
172.8 178.6 66 190.6 35 134.9 11 93.0 110.9 39 83.0 10
172.7 178.8 66 190.8 59 134.9 12 93.0 110.9 23 83.0 19
cis-4,5-diCQA 515.1 353.0 172.7
7
10 a cis-4,5-diCQA 515.1 353.0 172.8 12
172.7 178.6 76 190.6 70 134.8 18 93.0 110.8 39 83.0
126.8 172.7 42 108.8 44 92.8 32
6
11 5-p-CoQA
12 3F,5CQA
13 C,FQA
14 4C,5FQA
15 5-CSA
337.1 190.7 162.8
6
529.1 367.0 353.0 12 192.7
7
178.6
2
192.7 172.6 13 133.7 14
178.7 160.8 73 134.8 85
133.7 126.6 16
134.7
529.1 367.1 349.0
529.1 353.0 254.8
7
5
178.7 10
172.7 17
172.7 178.6 65 190.6 24 134.7 11 93.0 110.8 32 59.4 52
335.1 178.7 172.8 11 134.8 20
335.1 178.7 160.6 82 134.8 51
134.7
16 4-CSA
134.7
17 3-CSA
335.1 178.7 160.8
367.1 190.7 172.8
4
3
134.8 42
134.8
18 5-FQA
85.0 126.8 85 172.6 28
19 FQA
367.1 178.7 190.8 33 160.8 12 134.8 72 134.7 106.8
5
20 FQA
367.2 176.8
161.6 130.8 57
21 3,4,5-triCQA
22 1,3,5-triCQA
23 triCQA
24 triCQA
677.1 515.1 353.0 20
677.1 515.1 353.0 16
677.1 515.0 353.0 15
677.1 515.1 353.0 20
353.0 335.0 16 299.0
353.0 335.0 15 254.9
353.0 172.7 38 254.8
353.0 172.7 22 254.8
4
5
4
4
172.8 30 172.7 178.6 58 190.6 42 134.8 16
172.7 28 190.8 178.6 72 172.6 90 136.7 10
172.7 178.8 60 190.6 33 134.7 14
172.8 178.6 87 190.8 90 134.7 15
structures are presented in Figure 1. Peak assignments of CGAs
have been made on the basis of structure diagnostic hierarchical
keys previously developed,24ꢀ26 supported by means of their
parent ion, UV spectra, and retention times relative to 5-CQA
using validated methods in our laboratory.24,27 More sensitive
and more selective fragment-targeted MSn experiments were
used for quantitatively minor components. The base peak
chromatogram of stevia extract is shown in Figure 2. Abbrevia-
tions and numbering are given in Figure 1. Stevia extract was
analyzed by LC-MSn in the negative ion mode using an ESI ion-
trap mass spectrometer, allowing assignments of compounds to
regioisomeric level, and also by high-resolution mass spectro-
metry using ESI-TOF in negative ion mode connected to LC.
With the guidance of previous studies from Clifford,24ꢀ29 three
CQAs (1ꢀ3), seven di-CQAs (4ꢀ10), three FQAs (18ꢀ20),
one p-CoQA (11), three CFQAs (12ꢀ14), three CSAs
(15ꢀ17), and four tri-CQAs (21ꢀ24) were located in the
chromatogram. For all compounds the high-resolution mass data
were in good agreement with the theoretical molecular formulas,
with a mass error of below 5 ppm, confirming the elemental
compositions of all compounds investigated.
Figure 7. Extracted ion chromatograms (EIC) of m/z 677 in negative
ion mode (A) before and (B) after UV irradiation.
Reliable characterization of diterpene glycosides content in
stevia is crucial. Because the structures of single glycosides are
very similar, they have very similar retention times in LC and
therefore result in overlapping of peaks in the chromatogram.
In this paper, the general profile of diterpene glycosides in
S. rebaudiana is given. Characterization of the compounds was
achieved by ion-trap mass spectrometry with SIM, and confirmation
of elemental composition was provided by ESI-TOF measure-
ments (see the Supporting Information).
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Table 2. Quantities of Mono- and Di-CQAs in S. rebaudiana Leaves
compd
concn range
calibration curve
correl coeff
calcd amount (μg/g)
3-CQA
1 μg/mLꢀ1 mg/mL
Y = 4.457x ꢀ 460.04
Y = 17.719x ꢀ 2361.90
Y = 13.288x ꢀ 1223.00
Y = 5.3176x ꢀ 529.76
Y = 14.789x ꢀ 1401.40
Y = 16.251x ꢀ 697.77
0.99
0.99
0.99
0.99
0.99
0.99
35.5
44.3
70.3
145.6
28.6
37.2
5-CQA
1 μg/mLꢀ3 mg/mL
4-CQA
0.07 μg/mLꢀ1 mg/mL
0.09 μg/mLꢀ1 mg/mL
0.07 μg/mLꢀ1 mg/mL
0.03 μg/mLꢀ04 mg/mL
3,5-diCQA
3,4-diCQA
4,5-diCQA
Characterization of Caffeoylquinic Acids (Mr 354) and
Dicaffeoylquinic acids (Mr 516). Three peaks were detected
at m/z 353.1 and assigned using the hierarchial keys previously
developed24 as well-known 3-CQA, 5-CQA, and 4-CQA. Three
dicaffeoylquinic acid isomers were identified by their parent ion
m/z 515.2 and were assigned as 3,5-diCQA, 3,4-diCQA, and 4,5-
diCQA using the hierarchial keys.24,26 Three further peaks
present as minor components showed fragmentation patterns
similar to that of 4,5-diCQA. We have recently reported on cis
isomers of chlorogenic acids present in plant tissue exposed to
UV light, which have formed in a photochemical transꢀcis
isomerization reaction.30 To confirm if the remaining three peaks
correspond to cis isomers, the extract was irradiated with UV
light at 245 nm for 40 min. After irradiation, a significant increase
in the intensities of two peaks (9 and 10 in Figure 3) was
observed, if compared to their corresponding trans isomers from
the original plant extract. In addition, a significant increase was
observed in the intensity of cis-3,5-diCQA (7 in Figure 3) peak
accompanied by a decrease of the 3,4-diCQA (5 in Figure 3)
peak. This finding suggests that under the chromatographic
conditions employed the cis isomer is coeluting with 3,4-diCQA
(Figure 3).
regioisomers show m/z 178 (caffeic acid fragment) in their MS2
spectra. 4-CQA shows an intense characteristic fragment ion at
m/z 160, which is absent in the MS2 spectra of 3-CSA and 5-CSA.
Characterization of Tricaffeoylquinic Acid (Mr 678). Four
triacyl CQA isomers (Figure 4) were detected in the stevia
extract at 677 for tricaffeoyls in neg. mode and confirmed as
tricaffeoyl derivatives by targeted MS4 experiments. Assignment
of regiochemistry was assisted by an independent synthesis of a
mixture of all four possible regioisomers of tricaffeoylquinic
acids. The chromatogram of the mixture of all theoretically pos-
sible four regioisomers of tricaffeoylquinic acid obtained through
synthesis showed two well-resolved peaks with retention times
and MS data identical to those present in the stevia extract along
with an intense broad peak in a retention time range where the
two remaining isomers in the stevia extract were observed (see
the Supporting Information). Detailed studies of the tandem
mass spectra at various retention times within the broad peak
suggest that this broad peak must correspond to two distinct
unresolved regioisomers of tricaffeoylquinic acid. Comparison
of the chromatogram of the synthetic mixture with the extract
allowed unambiguous assignment of the two regioisomers in the
extract by identity of the fragmentation pattern compared to the
synthetic mixture. Identification of 1,3,5-triCQA in the extract
was followed automatically due to the absence of an MS4 base
peak at m/z 173 corresponding to a dehydrated MS2 base peak of
the quinic moiety characteristic of 4-acylated isomers. The MS4
base peak at m/z ∼191 and a secondary peak at m/z 178 (72% of
base peak) suggest the 3,5-disubstitution pattern (Figure 5).
3,4,5-triCQA was identified by comparison to material described
previously.36,37 (Figure 6)
The two remaining peaks might be cis isomers of 3,4,5-triCQA
and 1,3,5-triCQA, or they can correspond to either 1,4,5-tri-
CQA and 1,3,4-tri-CQA or any of their cis isomers (see Table 1).
However, current information does not allow us to discriminate
unambiguously between these regioisomers at the moment. To
probe whether cis isomers were present, the extract was again
irradiated with UV light, and after chromatographic analysis, a
significant increase in the intensity of the peaks of 4-acylated
isomers was observed (Figure 7). Otherwise, the experiment was
inconclusive. It is worth noting that in theory for each tricaffeoyl
derivative eight stereoisomers with various transꢀcis stereoche-
mistries are possible, thus increasing the total number of isomeric
tricaffeoylquinic acids to 32. Given the identity of MS data and
the absence of characteristic shoulders in the UV spectra
characteristic for cis-caffeoyl derivatives, we tentatively assign
the two remaining isomers as 1,4,5-tri-CQA and 1,3,4-tri-
CQA. Only 3,4,5-tri-CQA has been previously reported in
nature, whereas the remaining isomers are reported here for
the first time.
On the basis of increased intensity after UV irradiation and
fragmentation pattern, three additional cis isomers were ob-
served for 4,5-diCQA. One of these isomers was assigned as
cis-4,5-diCQA (9), and two of them were assigned as cisꢀtrans
(a cis) isomer, but the distinction between 4-cis,5-trans-diCQA
and 4-trans-5-cis-diCQA was not possible (8 and 10).
Characterization of Feruloylquinic Acid (Mr 368), p-Cou-
maroylquinic Acid (Mr 338), and Caffeoylferuloylquinic Acid
(Mr 530). Only one peak was detected at m/z 337.1, which was
identified as 5-p-CoQA according to its fragmentation pattern.
Three peaks were detected at m/z 367, and one of them was
identified as 5-FQA; the other two peaks could not be assigned
due to their uncommon fragmentation pattern.
A targeted MS3 experiment at m/z 529.2 ([M ꢀ H+]ꢀ)
applied to the extract located three peaks, and two of them were
identified as 3F,5CQA and 4C,5FQA on the basis of their
characteristic fragmentations in MS2 and MS3 spectra. The
assigments are achieved using the hierarchial keys previously
developed, and mass spectra published previously are not pre-
sented here.24,31
Characterization of Caffeoylshikimic Acids (Mr 336). Caf-
feoylshikimic acids (CSA) have been reported in date palms,
sweet basil, and carrot,32ꢀ35 and they have been characterized to
regioisomeric level in yerba matꢀe leaves by tandem mass spectra
previously.36 This class of compounds is reported here for the
first time from the Asteraceae family of plants. A targeted MS3
experiment at m/z 335.1 ([M ꢀ H+]ꢀ) applied to the extract
located three peaks, and they were identified by their fragmenta-
tion patterns as 5-CSA, 4-CSA, and 3-CSA (15ꢀ17).36 All three
Quantification of Caffeoylquinic Acids. Following the qua-
litative profiling of chlorogenic acids in S. rebaudiana, we decided
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Journal of Agricultural and Food Chemistry
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to quantify the levels of selected compounds. Chlorogenic acid
standard solutions were analyzed by LC-MS using the same
chromatographic method as used for stevia leaf extracts. For six
selected monoacyl- and diacylquinic acids, calibration curves
were obtained using six-point calibration from the UV chroma-
togram recorded at 320 nm. The individual amounts calculated
for mono- and dicaffeoylquinic acids are listed in Table 2, which
also lists the correlation coefficient of linear regression for each
standard sample and the concentration range.
Among the monocaffeoylquinic acids, 4-CQA was found to be
the most abundant compound, and among all CQAs 3,5-diCQA
was found to be the most abundant compound. The total
chlorogenic acid amount determined here is around 370 μg/g
of dry leaf.
In this study we profiled the chlorogenic acids in S. rebaudiana
employing LC-MSn and LC-TOF techniques. A total of 24
chlorogenic acids were detected in S. rebaudiana leaves, with
23 compounds described for the first time from this source. Tri-
CQAs were reported for the first time from S. rebaudiana with
three regioisomers found for the first time in nature. CSAs were
characterized for the first time from a plant belonging to the
Astareceae family by using tandem mass spectrometry. Quanti-
fication of selected mono- and di-CQAs was achieved by using
the UV chromatogram with total chlorogenic acid levels found to
be 370 μg/g of dry leaf.
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S
Supporting Information. Additional EIC of triacyl
b
CGAs, MS2 + MS3 data of all compounds mentioned in the text,
table of high-resolution MS-TOF data for compounds identified,
and structures of ent-kaurene terpenes. This material is free of
charge via the Internet at http://pubs.acs.org
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 49 421 200 3120. Fax: 49 421 200 3229. E-mail:
n.kuhnert@jacobs-university.de.
Funding Sources
Financial support from the European Union (project DIVAS) is
gratefully acknowledged.
’ ACKNOWLEDGMENT
We acknowledge the technical assistance of Anja M€uller.
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’ DEDICATION
†This paper is dedicated to Prof. M. N. Clifford on the occasion of
his 65th birthday.
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