Qualitative Profile and Quantitative Determination of Flavonoids from Crocus Sativus L. Petals by LC-MS/MS

Abstract

From the methanolic extract of Crocus sativus petals nine known flavonoids have been isolated and identified, including glycosidic derivatives of quercetin and kaempferol as major compounds (1-2), and their methoxylated and acetylated derivatives. Additionally, LC-ESI-MS qualitative and LC-ESI-MS/MS quantitative studies of the major compounds of the methanolic extract were performed. The high content of glycosylated flavonoids could give value to C. sativus petals, which are a waste product in the production of the spice saffron.

Full text

Qualitative Profile and Quantitative Determination of
Flavonoids from Crocus sativus L. Petals by LC-MS/MS
Paola Montoroa, Carlo I. G. Tuberosob, Mariateresa Maldinia, Paolo Cabrasb and Cosimo Pizzaa
aDipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo,
84084 Fisciano (SA), Italy
bDipartimento di Tossicologia, Università di Cagliari, via Ospedale 72, 09124 Cagliari, Italy
pmontoro@unisa.it
Received: June 10th, 2008; Accepted: October 16th, 2008
From the methanolic extract of Crocus sativus petals nine known flavonoids have been isolated and identified, including
glycosidic derivatives of quercetin and kaempferol as major compounds (1-2), and their methoxylated and acetylated
derivatives. Additionally, LC-ESI-MS qualitative and LC-ESI-MS/MS quantitative studies of the major compounds of the
methanolic extract were performed. The high content of glycosylated flavonoids could give value to C. sativus petals, which
are a waste product in the production of the spice saffron.
Keywords: Crocus sativus, LC-ESI-MS, LC-MS/MS, quercetin, kaempferol.
Saffron, the dried stigmas of Crocus sativus L.
(Iridaceae), is a very expensive spice, and is used as a
herbal medicine, for food coloring and as a flavoring
agent in different parts of the world [1a]. Saffron
originally grew in India, Iran, Europe and other
countries, and it has been successfully cultivated in
different countries, including Europe. The most
important European production areas are Sardinia
and Abbruzzo (Italy), Castile-la Mancha (Spain) and
western Macedonia (Greece). For saffron, the flowers
are cultivated to produce the stigmas. After
harvesting, the flowers are subjected to a delicate
treatment which will give the saffron spice. This
procedure is performed the same day of harvest. One
of the most traditional procedures is the separation of
petals from stigmas. A large amount of petals is
discarded for obtaining a small amount of stigmas.
Earlier investigation reported the isolation of
carotenoids, crocins, monoterpenoids and flavonoids
from the stigma, leaves, petals and pollen of C.
sativus [1b,1c].
Considering the large amount of petals that are waste
products in this production procedure, we have
undertaken a study to recover chemical compounds
from this matrix. Only one previous paper concerning
petals is reported in the literature, oriented towards
the biological activity of some new phenols isolated
from this part of the flowers [2a].
Flavonoids are polyphenolic compounds with
antioxidant properties [2b,2c]. Several studies have
shown that a high intake of flavonoids has been
correlated to a decrease in heart disease; in addition,
biological effects of this class of compounds have
been described in several in vivo and in vitro studies
[3a-3d]. These compounds are largely used for
chemotaxonomic surveys of plant genera and
families because of their almost ubiquitous presence
in vascular plants and of their structural variety.
A phytochemical study was undertaken with the aim
of identifying and determining quantitatively the
major compounds in the petals. In this study
flavonoid compounds were isolated; the major
compounds were glycosidic derivatives of quercetin
and kaempferol, including their methoxylated and
acetylated derivatives (1-9).
The study provided a method to define the flavonoids
fingerprint by LC-ESI- IT MS/MS (liquid chromato-
graphy electrospray tandem mass spectrometry with
ion trap analyser), with full scan acquisition in data
dependent scan mode, and a method to quantify
the content of the major compounds by LC-ESI- TQ
NPC Natural Product Communications 2008
Vol. 3
No. 12
2013 - 2016

2014 Natural Product Communications Vol. 3 (12) 2008 Montoro et al.
MS/MS (liquid chromatography electrospray tandem
mass spectrometry with triple quadrupole analyser)
by using a MRM (multiple reaction monitoring)
mode. The quantitative method, performed by using
internal and external standards, was validated in
agreement with EMEA note guidance on validation
of analytical methods [4].
By using tandem in time mass spectrometry it was
possible to reveal the compounds on the basis of their
specific fragmentation. The specific fragmentation
pattern was used for developing the selective MS/MS
method for the two major compounds (1, 2). The use
of tandem mass spectrometry in quantification of
secondary metabolites from plants has led to
sensitive, selective and robust methods for quality
control and standardisation of plant extracts [5a-5c].
Phytochemical investigation of the methanolic extract
of C. sativus led to the isolation of flavonoid
compounds 1-9. The compounds, identified by
comparing their NMR data with those reported
in the literature, were quercetin-3,7-di-O-β-D-
glucopyranoside (1) [6], kaempferol-3,7-di-O-β-D-
glucopyranoside (2) [2a], isorhamnetin-3,7-di-O-β-D-
glucopyranoside (3) [7], kaempferol-3-O-β-D-
glucopyranoside (4) [2a], quercetin-3-O-β-D-
glucopyranoside (5) [8], isorhamnetin-3-O-β-D-
glucopyranoside (6) [9], kaempferol-7-O-β-D-
glucopyranoside (7) [2a], kaempferol-3-O-β-D-(2-O-
β-D-glucosyl) glucopyranoside (8) [2a], and
kaempferol-3-O-β-D-(2-O-β-D-6-O-acetylglucosyl)
glucopyranoside (9) [2a]. The high content of
glycosylated flavonoids could give value to C.
sativus petals, which are a waste product in the
production of saffron spice.
In order to realise a qualitative analysis for the
flavonoid derivatives in C. sativus extracts, MS
experiments were performed by using an LC-MS
system equipped with an ESI source and an Ion Trap
analyser. Positive ion electrospray LC/MS analysis,
total ion current (TIC) profile and reconstructed
ion chromatograms (RICs) of extract are shown in
Figure 1. Flavonoid derivatives were identified by
comparing retention times and m/z values in the
total ion current chromatogram with those of the
selected standards, obtained in the isolation step.
Reconstructed ion chromatograms were obtained
for each value of m/z observed for the standard
compounds (m/z 627, 1; m/z 611, 2; m/z 641, 3; m/z
449, 4, 7; m/z 465, 5; m/z 479, 6; and m/z 653, 9) in
order to improve the separation and identification of
single compounds. The chromatographic profile
obtained in Total Ion Current revealed two very
major compounds, respectively 1 and 2, in C. sativus
extracts, whereas the other compounds were present
in lesser amounts. Quantitative analysis was focused
on compounds 1 and 2, which could potentially be
recovered from discarded petals as economic
secondary products.
In order to obtain an accurate quantitative
determination of compounds 1-2, a quantitative LC-
MS/MS method was developed. Since the sugar loss
is the most representative fragmentation for
glycosidic flavonoids, ESI-MS/MS analyses were
recorded for the two major compounds by using an
LC-MS equipped with an ESI source and a triple
quadrupole analyser. Analyses were performed by
direct introduction and both the spectra showed the
characteristic fragment resulting from sugar loss.
Thus an MRM method was developed. Transition
from the specific pseudomolecular ion [M+H]+ of
each compound to the corresponding aglycon ion
[A+H]+ was selected to monitor the flavone
glycosides in C. sativus using as internal standard
(I.S.), rutin (m/z 611) (I.S.).
Compound 1: precursor ion m/z 627.0, product ion
m/z 303.0, collision energy 30%; compound 2:
precursor ion m/z 611.0, product ion m/z 287.0,
collision energy 30%; I.S. precursor ion m/z 611.0,
product ion m/z 303.0, collision energy 30%.
The MRM analyses of C. sativus methanolic extract,
spiked with I.S., contained the peaks corresponding
to the compounds under investigation, with
appreciable intensity for quantitative purposes.
Validation of the method was realised in agreement
with EMEA note guidance on validation of analytical
methods [4].
Validation of the LC/MS/MS method included intra
and inter-day precision and accuracy studies on three
days. Accuracy and precisions were calculated by
analysing five samples of each extract (MeOH and
water). Standard deviations calculated in this assay
were < 7% for the two compounds under
investigation. Specificity is usually reported as the
non interference with other substances detected in the
region of interest; the present method, developed by
using a characteristic fragmentation of flavone
glycosides 1, 2, was specific with no other peak
interfering in the MS/MS detection mode.

Flavonoids from Crocus sativus petals Natural Product Communications Vol. 3 (12) 2008 2015
Figure 1. HPLC-MS qualitative analysis
Table 1: Quantitative results and quantification data.
n mg/g
MeOH ext.
mg/g
water ext.
mg/g
petals
Calibration
equation
r2
1 41.8±6.2 8.1±0.2 27.6±6.0 y = 0.183x-0.74 0.998
2 31.1±4.7 5.5±0.1 20.2±4.6 y = 0.094x-0.39 0.997
The calibration graphs, obtained by plotting area ratio
between external and internal standards versus the
known concentration of each compound, were linear
in the range of 1-100 μg mL-1 for all compounds.
Correlation values (r2) are reported in Table 1.
Five aliquots of methanol and water extracts,
respectively, obtained from C. sativus were analysed
in order to quantify the flavonoid contents. Table 1
reports the quantitative data for compounds 1-2,
regression of calibration curves, and quantitative
values. Quantitative analyses results confirmed that
waste petals of C. sativus can represent an interesting
source of such phenolic compounds, with respect to
the high content showed.
Experimental
Reagent and standards: Standards of pure
compounds 1-2 were isolated in our laboratory and
their structures were elucidated by NMR
spectroscopy (Bruker DRX-600). Each standard was
dissolved in methanol. HPLC grade methanol
(MeOH), acetonitrile (ACN) and trifluoroacetic acid
(TFA) were purchased from Merck (Merck KGaA,
Darmstadt, Germany). HPLC grade water (18mΩ)
was prepared using a Millipore Milli-Q purification
system (Millipore Corp., Bedford, MA). The reagents
used for the extractions, of analytical grade, were
purchased from Carlo Erba (Rodano, Italy). Column
chromatography was performed over Sephadex LH-
20 (Pharmacia, Uppsala, Sweden).
Equipment: Semi-preparative HPLC was performed
using an Agilent 1100 series chromatograph,
equipped with a G-1312 binary pump, a G-1328A
rheodyne injector and a G-1365B multiple wave
detector. The column was an RP C18 column
μ-bondapak 300 mm x 7.6 mm (Waters, Milford,
MA). HPLC–ESI-MS analysis was performed using a
Thermo Finnigan Spectra System HPLC coupled
with an LCQ Deca ion trap. Chromatography was
performed on an RP C18 column Symmetry Shield
(Waters, Milford, MA). HPLC–ES-MS/MS for
quantitative analysis was performed on a 1100 HPLC
system (Agilent, Palo Alto, CA) coupled with a triple
quadrupole instrument [API2000 (Applied
Biosystems, Foster City, CA, USA)]. The instrument
was used in the tandem MS mode, with multiple
reaction monitoring (MRM).
LC-ESI-MS and LC-ESI-MS/MS analysis: The
mass spectrometer was operated in the positive ion
mode under the following conditions: capillary
voltage 3 V, spray voltage 5 kV, tube lens offset 40
V, capillary temperature 260°C, and sheath gas
(nitrogen) flow rate 60 arbitrary units. Data were
acquired in the MS1 scanning mode with scan ranges
of 200 – 1000 m/z: the maximum injection time was
50 ms, and the number of microscans was 3. In order
to tune the LCQ for flavonoids, the voltages on the
lenses were optimised using the TunePlus function of
the Xcalibur software in the positive ion mode whilst
infusing a standard solution of quercetin (1 μg mL-1
in methanol) at a flow rate of 3 μL min-1. For
qualitative LC-ESI-MS analysis, a gradient elution
was performed on a RP C18 column Symmetry
shield (Waters, Milford, MA), 2mm x 150 mm, by
using a mobile phase A represented by water
acidified with trifluoroacetic acid (0.05%) and a
mobile phase B represented by water: acetonitrile
50:50 acidified with trifluoroacetic acid (0.05%). The
gradient started from 20% of eluent B, to achieve the
33% of solvent B in 18 min. After another 12 min the
percentage of B became 40%, and remained at this
value for 10 min, then became 50%. The flow (250
μL min-1) generated by chromatographic separation
was directly injected into the electrospray ion source.
MS were acquired and elaborated using the software
provided by the manufacturer.
For quantitative LC-ESI-MS/MS a gradient elution
was performed by using a mobile phase A
represented by water acidified with trifluoroacetic
acid (0.05%) and a mobile phase B represented
by acetonitrile acidified with trifluoroacetic acid
3
6
5
4
9
7
10
10
10
10
10
10
0
5
0
5
0
5
0
5
0
5
1.13 26.45
21.9
3.22 8.65 12.8 17.18 37.830.73 36.09
25.55
1.13
21.1
0.99 21.1
1.13 21.9
20.29
1.13 26.4
1.16
Base Peak
ESI Full ms
[ 200.00-1000.00]
m/z= 640.50-641.50
m/z= 478.50-479.50
m/z= 464.50-465.50
m/z= 448.50-449.50
0510 15 20 25 30 35
Time (min)
0
5
1.16
26.3
m/z= 652.50-653.50
19.5
10
10
0
5
0
5
21.9
20.32
1.13 26.45
m/z= 626.50-627.50
m/z= 610.50-611.50
1
2

2016 Natural Product Communications Vol. 3 (12) 2008 Montoro et al.
(0.05%). The gradient started from 5% of eluent B,
remained isocratic for 5 minutes, to achieve the 80%
of solvent B in 15 min. The flow (250 μL min-1)
generated by chromatographic separation was
directly injected into the electrospray ion source.
The mass spectrometer was operated in the positive
ion mode under the following conditions:
declustering potential 200 eV, focusing potential
155 eV, entrance potential 10 eV, collision energy
30 eV, and collision cell exit potential 15 eV, ion
spray voltage 5000, temperature 250°C. The
instrument was used in the tandem MS mode with
multiple reaction monitoring (MRM). For all
flavonoids analyzed, the selected fragmentation
reaction was the loss of the glycoside moiety.
Plant material: Petals of C. sativus, discarded by
production companies of saffron spice, were
collected in Sardinia (Italy) in November 2004.
Extraction and isolation: Dried and powdered petals
(23 g) of Crocus sativus were extracted for 3 days,
3 times, at room temperature with methanol to give
9.7 g of crude methanolic extract. This was extracted
for one day with water. The filtered extract was
lyophilized to give 3 g of crude extract. Part of the
methanolic extract (3.7 g) was fractionated initially
on a 100 cm×5.0 cm Sephadex LH-20 column, using
CH3OH as mobile phase, and 56 fractions (10 mL
each) were obtained. Fractions 28-29 (24.5 mg) (a),
19-20 (250 mg) (b), 31-33 (31.4 mg) (c) and 36-40
(40.4 mg) (d) were chromatographed by HPLC-UV.
The mobile phase was a linear gradient of
water/acetonitrile (50:50) with trifluoroacetic acid
0.1% (solvent B) in water acidified with
trifluoroacetic acid 0.1% (solvent A), at a flow rate of
2.000 mL min-1. From sample a, compounds 1 (3.3
mg, tR=26.8), 2 (2.9 mg, tR=29.7) and 3 (1 mg,
tR=28.7) were obtained; from sample c, compounds 4
(1.6 mg, tR=36.4) and 6 (0.9 mg, tR=37.8); and from
sample d, compounds 5 (0.6 mg, tR=32) and 7 (1.1
mg, tR=36.9) using the following gradient: 0 min,
10% B, 0-5 min, 10-20% B, 5-25 min, 20-40% B;
25-40 min, 40% B; 40-50 min, 40-70% B, 50-60 min,
70-100% B.
From sample b, compounds 8 (5.6 mg, tR=29.8) and 9
(1.2 mg, tR=37.9) were obtained using the following
gradient: 0 min, 20% B; 0-18 min, 20-33% B; 18-30
min, 33-40% B; 30-40 min, 40% B; 40-45 min,
40-85% B; and 45-55 min, 85-100%.
References
[1] (a) Fernandez, JA. (2004) Biology, biotechnology and biomedicine of saffron. Recent Research Developments in Plant Science, 2
127-159; (b) Rios JL, Recio MC, Giner RM, Manets S. (1996) An update review of saffron and its active constituents. Phytotheapy
Research, 10, 189-193; (c) Xi L, Qian Z. (2006) Pharmacological properties of crocetin and crocin (digentiobiosyl esters of
crocetin) from saffron, Natural Product Communications, 1, 65-75.
[2] (a) Li CY, Lee EJ, Wu TS. (2004) Antityrosinase principles and constituents of the petals of Crocus sativus. Journal of Natural
Products, 67, 437-440; (b) Rice-Evans CA, Miller NJ, Paganga G. (1997) Antioxidant properties of phenolic compounds. Trends in
Plant Science, 2, 152-159.
[3] (a) Cao G, Sofic E, Prior RL. (1997) Antioxidant and pro-oxidant behavior of flavonoids: structure-activity relationships. Free
Radical Biology and Medicine, 22, 749-760; (b) Rice-Evans CA, Miller NJ, Panaga G. (1996) Structure-antioxidant activity
relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20, 933-956; (c) Lien EJ, Ren S, Bui H, Wang
R. (1999) Quantitative structure-activity relationship analysis of phenolic antioxidants. Free Radical Biology and Medicine, 26,
285-294; (d) Montoro P, Braca A, Pizza C, De Tommasi N. (2005) Structure-antioxidant activity relationships of flavonoids
isolated from different plant species. Food Chemistry, 92, 349-355.
[4] ICH Q2B, International Conference on Harmonisation, London, 1995.
[5] (a) Li X, Xiong Z, Ying X, Cui L, Zhu W, Li F. (2006) A rapid ultra-performance liquid chromatography-electrospray ionization
tandem mass spectrometric method for the qualitative and quantitative analysis of the constituents of the flower of Trollius
ledibouri Reichb. Analytica Chimica Acta, 580, 170-180; (b) Benavides A, Montoro P, Bassarello C, Piacente S, Pizza C. (2006)
Catechin derivatives in Jatropha macrantha stems: Characterisation and LC/ESI/MS/MS quali-quantitative analysis. Journal of
Pharmaceutical and Biomedical Analysis, 40, 639-647; (c) Montoro P, Tuberoso CIG, Perrone A, Piacente S, Cabras P, Pizza C.
(2006) Characterisation by liquid chromatography -electrospray tandem mass spectrometry of anthocyanins in extracts of Myrtus
communis L. berries used for the preparation of myrtle liqueur. Journal of Chromatography A, 1112, 232-240.
[6] Merfort I, Wendisch D. (1992) New flavonoid glycosides from Arnicae flos DAB 9. Planta Medica, 58, 355-357.
[7] Grouiller A, Pacheco H. (1967) Flavonoid compounds. VI. Nuclear magnetic resonance spectra of some O-glucosylflavonals,
their aglycons and three synthetic mono- and di- O –glucosylflavanones. Bulletin de la Societe Chimique de France, 6, 1938-1943.
[8] Nawwar MAM, El-Mousallamy AMD, Barakat HH. (1989) Quercetin 3-glycosides from the leaves of Solanum nigrum.
Phytochemistry, 28, 1755-1757.
[9] Senatore F, D'Agostino M, Dini I. (2000) Flavonoid glycosides of Barbarea vulgaris L. (Brassicaceae). Journal of Agricultural
and Food Chemistry, 48, 2659-2662.