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Oriental Journal of Chemistry

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0.225 number of documents published in that journal. The
equal'. SJR is a measure of scienti c in uence of chart shows the evolution of the average number of
0.15 0.6
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0.075
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2009
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2011
c in uence
2013
of the2015
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Total Cites Self-Cites Cites per document Year Value

Cites / Doc. (4 years) 2008
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800 Cites / Doc. (4 years) 2009 0.096
Evolution of the total number of citations and journal's
self-citations received by a journal's published Cites / Doc. (4 years) 2010 0.183
documents during the three previous years. Cites / Doc. (4 years) 2011 0.167
0
Cites / Doc. (4 years) 2012 0.428
400
Journal Self-citation is de ned as the number of citation
from a journal citing article to articles published by the Cites / Doc. (4 years) 2013 0.498
2008 2010 2012 2014 2016 2018
same journal.
Cites / Doc. (4 years) 2014 0.456
Cites / Doc.
Cites / Doc.(4
(4years)
years) 2015 0.463
0 Cites / Doc. (3 years)
Cites / Doc. (4 years) 2016 0.607
Cites
2008 Year
2010 Value
2012 2014 2016 2018 Cites
Cites / Doc.(4
/ Doc. (2years)
years) 2017 0.608
S lf Cit 2008 0
External Cites per Doc Cites per Doc % International Collaboration

Evolution of the number of total citation per document

0.8 International Collaboration accounts for the articles that
21
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14
0.4
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self-citations from the total number of citations received
0 0
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2008 2010 2012 2014 2016 2018 2008
2008 1.60 2010 2012 2014 2016 2018
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 ISSN: 0970-020 X
ORIENTAL JOURNAL OF CHEMISTRY CODEN: OJCHEG
An International Open Access, Peer Reviewed Research Journal
2019, Vol. 35, No.(5):
Pg. 1579-1583
www.orientjchem.org

Rapid Identification of Anthocyanin in Ficus aurata Fruits by

Liquid Chromatography-Mass Spectrometry Approach
Daimon Syukri*, Ismed, Vioni Derosya, Ririn Fatma Nanda
and Dosmawarni Indah Gultom

Faculty of Agricultural Technology, Andalas University, Limau Manis, Padang, West Sumatera,
25163, Indonesia.
*Corresponding author E-mail: dsyukri@ae.unand.ac.id

http://dx.doi.org/10.13005/ojc/350516

(Received: September 03, 2019; Accepted: October 01, 2019)

Abstract

The aim of this study was to identify the chemical structure of anthocyanins in the fruits of
Ficus aurata. The anthocyanin was detected and characterized using the liquid chromatography
system with UV-Vis detection tandem triple quadrupole mass spectrometer. After UV-Vis detection,
the characterization of anthocyanin was subjected to a triple quadrupole system of mass spectrometer.
The precursor ions of anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, petunidin,
and peonidin) were scanned to identify the distinctive particular anthocyanin. Then, the detected
anthocyanins was further confirmed and their isomers such as glycosides and galactosides forms
were distinguished by the fragmentation pattern on production analysis scan after comparison with
mass spectroscopy online databases. For the first time, it had characterized that in the fruits of
Ficus aurata contains at least seven kinds of anthocyanins with all possible combinations of three
anthocyanidins.

Keywords: Ficus aurata, Mass spectrometry, Structure conformation, Triple quadrupole.

INTRODUCTION Many plants are being discovered as

Anthocyanin is a class of phenolic compounds
that cause certain colors (blue, purple and red) in
natural products1,2. Since the few last decades, there
have been an increasing attention in anthocyanins
research due to its benefits on potential health effect
on human being other than attractive food colorant.
There are many reports indicated the health beneficial
effects of anthocyanins such as antioxidative3,4,5,
anti-inflammatory and anti-obesity6, DNA cleavage7
and cardiovascular protective properties8.
the natural source of anthocyanin where each of
them is showing the attractive color from their peel
such as grape, strawberry, apple, berries and etc9.
Meanwhile, there is a fruit which has no specific
attractive color, namely Ficus aurata, yet might
has a potential as an anthocyanin source since
its seed has a strong specific anthocyanin’s color.
Ficus aurata is a variety of ficus or fig species that
is very commonly seen along the roadside in the
Andalas University, Indonesia, in which it gives great
contribution to the forest ecosystem.

This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC- BY).
Published by Oriental Scientific Publishing Company © 2018
 Syukri et al., Orient. J. Chem., Vol. 35(5), 1579-1583 (2019)
When it is ripe, Ficus aurata can acquire
coloring seed hues to dark-violet that might due to
the presence of high amount of anthocyanin (Fig.1).
We suggest, due to the lack of striking color form
the peel of Ficus aurata, make this fruit has not
recognized as a potential source of anthocyanin.
Fig.1. The fruits of Ficus aurata
The objective of this study was to identify
the anthocyanin in the fruits of the Ficus aurata
because there is no information available for this till
date. Many analytical techniques are applicable for
anthocyanins characterization in natural products.
However, the most popular techniques is liquid
chromatography with multiple UV-Vis detection using
photodiode-array detector (LC UV-Vis) combined
with mass spectrometry (MS) detection10. Although
the utilization of LC UV-Vis especially in the range
of wavelength from 500 to 550 nm has already
known as general condition for identification of
anthocyanin in natural product mixtures, however
this kind of method cannot produce very accurate
results. This detection only indicate the presence
of the red-violet colors as representative of normal
anthocyanin without any consideration of various
forms of anthocyanins11.
Moreover, since the anthocyanins
derivatives have classified as class-targeted
metabolite, the use of MS detection with triple
quadrupole system permits an ideal platform to
produce more accurate analyses of anthocyanin
down to low-level and establishment any kind of
anthocyanin derivatives structure up to oxidation
nor various forms that exhibit no absorbance in the
range from 500 to 550 nm through determination
of specific molecular mass and subsequent its ion
fragmentations12,13.
EXPERIMENTAL
Materials Plant material
The Ficus aurata was identified at
1580
herbarium identification laboratory of Andalas
University (Identification Number: 101/K-ID/ANDA/
III/2019). The fruits of Ficus aurata were sampled
at the botanical garden of Andalas Univesiry and
immediately transported to laboratory for analysis.
Chemicals
Methanol, acetonitrile and water (LC
MS grade) were purchased from J. T. Baker
(Baker Mallinckrodt, Phillipsburg, NJ, USA).
Methanol, acetonitrile, formic acid (LC grade) were
purchased from Smart Lab (SMART-LAB, Tangerang,
Indonesia). Pro analysis grade hidrochloric acid was
also purchased form Smart Lab (SMART-LAB,
Tangerang, Indonesia).
The extraction process of anthocyanins
A 100 g of fruits has cut into small pieces
and put into 1000 mL erlenmeyer flask then acidified
methanol (pH 1.5) was added to the flask. The
extraction of anthocyanins were done by maceration
technique at room temperature for 12 h in a absence
of light. This step was conducted three times.
Furthermore, the extract was concentrated using a
rotary evaporator. About 5 mL of concentrated extract
was then filtered passed through a 0.2 μm millipore
filter for subsequent analysis.
HPLC UV-Vis and LC MS/MS analysis
The anthocyanins in the fruits of Ficus
aurata was separated in reverse phase liquid
chromatography and subsequent identified and
characterized using an photodiode array detector
and a MS/MS detector as describes on Syukri
et al., 201414 and Syukri et al., 201815 with some
modifications, respectively. The utilization of
simultaneous detection modes in triple quadrupole
ion trap mass spectrometer such as precursor ion
scan, multiple and product ion scan were considered
and proposed to get a rapid, sensitive and accurate
data of anthocyanin qualitatively in this study.
A high performance liguid chromatography
(HPLC) series system (Prominence HPLC 20
series Shimadzu, Kyoto, Japan), and a reverse-
phase chromatographic column (a Zorbax SB-
C18, 150 mm×3.0 mm i.d., 5 μm in particle size,
agilent, Canada, USA) coupled to a DAD detector
(Prominence SPD-M20A) were used for HPLC
UV-Vis analysis. The mobile phases were (A) 1%
of formic acid in water and (B) 50% of acetonitrile
 Syukri et al., Orient. J. Chem., Vol. 35(5), 1579-1583 (2019) 1581

in water containing 1% formic acid. The separation and 550 nm, respectively). There were two peaks
of anthocyanin in the sample was eluted based on observed at those wavelengths. The highest peak
the step gradient polarity separation as described intensity of the peak was observed at a wavelength
in Table 1. The detection wavelengths on UV-Vis of 520 nm. Primary anthocyanin was detected at RT
were in the range of 500 to 550 nm. The extract 11.8 min while the second ones was detected at
samples were injected as 5 μL. Moreover, for RT 10.8 minute. Such detection can be used to
LC MS/MS, the anthocyanins were identified confirm anthocyanins in plant material samples16.
and characterization using a prominence HPLC However, referring to previous studies, most plant
20A Shimadzu, Kyoto, Japan that equiped with a material samples should contain more than two
reverse-phase chromatographic column (Unison anthocyanins16,17,18,19. Therefore, another analysis
UK-C8, 150 mm ×2.0 mm i.d., 3 μm in particle
such as mass spectrometry needs to be further
size, Imtakt, Kyoto, Japan). The LC was coupled to
carried out to obtain more comprehensive data on
a triple-quadrupole mass spectrometer (Q-TRAP
anthocyanins in the fruits of Ficus aurata.
4500 AB-Sciex, Framingham, MA, USA). The mobile
phases were 0.1% formic acid in water (solvent A)
and 0.1% formic acid in acetonitrile (solvent B). The
mobile phases were set on anthocyanin separation
similar as the LC UV-Vis detection at flow rate as
0.2 mL/minute. Anthocyanins were ionized using a
Turbo-V™ ion source in positive mode. Precursor
ion scan functions in AB- Sciex, QTRAP®, namely,
PSI scan followed by enhanced product ion (EPI)
scans, were used to study the fragmentation pattern
of anthocyanins in the Ficus aurata extraction solvent
that was separated in retention on a chromatographic
column. For mass spectroscopy scanning, the MS
was operated in positive polarity at a scan rate of
1000 Da/s within the mass range of 200–800 amu.
The MS parameters were set as follows: Collision
Energy Spread (CES) = 15, Declostiring Potential
(DP) = 45, Entrance Potential (EP) = 10, Curtain
gas (CUR) = 10 and Temperature (TEM) = 600. The
Fig. 2. HPLC UV-Vis chromatogram of anthocyanins in the
Analyst software version 1.5 was used to integrate
fruits of Ficus aurata
and analyze all detection component in this study.
Figure 3 indicates the precursor-ion
Table 1: The step gradient polarity program scan (PIS) chromatogram of anthocyanin in
for HPLC-anthocyanin identification
the fruit of Ficus aurata. The PIS analysis was
No Time (min) %B Flow (mL/min) conducted simultaneously for all precursors of six
anthocyanidins (aglycone) i.e. 287 for cyanidin, 303
1 0 6 1.2
for delphinidin, 331 for malvidin, 301 for peonidin,
2 4 10 1.2
3 12 25 1.2 271 for pelargonidin, and 317 for petunidin. It has
4 13 25 1.2 detected three precursor of anthocyanidins such as
5 20 40 1.2
cyanidin (287), pelargonidin (271) and delphinidin
6 35 60 1.2
7 40 100 1.2 (303) that formed in seven anthocyanins derivatives.
8 45 6 1.2 The major and the smallest were identified as
RESULT AND DISCUSSION a cyanidin anthocyanin while pelargonidin and
delphinidin anthocyanin were found in the moderate
Figure 2 shows the UV-Vis chromatogram level. This data can strengthen the previous UV-Vis
of the predicted anthocyanin in the methanol observation that indicate the presence of two kinds
extract from the fruits of Ficus aurata (λ = 500, 520 of anthocyanin in the fruits of Ficus aurata.
 Syukri et al., Orient. J. Chem., Vol. 35(5), 1579-1583 (2019) 1582

as the major anthocyanin. Moreover, delphinidin

3-O-(6”-acetylglycoside), pelargonidin 3-glycoside,
delphinidin 3-O- galactoside, cyanidin 3-O-(6-O-
malonyl-β-D-glycoside), cyanidin 3-O-sophoroside
were identified as minor anthocyanins.

Fig. 3. The chromatogram of product ion scan anthocyanin

analysis in the fruits of Ficus aurata. Precursor of cyanidin,
m/z = 287 (A,B,E and G), Precursor of pelargonidin, m/z =
303 (D) and precursor of delphinidin, m/z = 303 (C and F)

Furthermore, the structural confirmation

of detected anthocyanin was conducted by the
enhanced product ion (EPI) analysis. Fig. 4 indicates
the EPI chromatogram of each detected anthocyanin
in the fruits of Ficus aurata. Each fragmentation
Fig. 4. Fragmentation mass spectra of each detected
was compared to the mass fragmentation of
anthocyanin after enhanced product ion analysis. cyanidin
common anthocyanins from online databases 3-glycoside (A); cyanidin 3-(6''-acetylglycoside) (B);
such as METLIN metabolomics databases and delphinidin 3-O-galactoside (C); pelargonidin 3-glycoside)
LIPIDMAPS. As the results, it can be proposed (D); cyanidin 3-O- (6-O- malonyl -β -D-glycoside) (E);
that the fruits of Ficus aurata contain cyanidin delphinidin 3-O-(6”-acetylglycoside)(F) and cyanidin 3-O-
3-glycoside and cyanidin 3-(6''-acetylglycoside) sophoroside (G)
 Syukri et al., Orient. J. Chem., Vol. 35(5), 1579-1583 (2019)
CONCLUSION
The data described here demonstrating
the combination of UV-Vis detection, precursor-ion
analysis and product-ion analysis tandem mass
spectrometer on liquid chromatography instrument
for viable technique on the screening of anthocyanins
in complex biological matrices rapidly. By employing
these techniques, the anthocyanins in the fruits
of Ficus aurata could be identified and confirmed
only by an injection into the LC-MS/MS system and
without any standard needed. Thus, the efficiency
of time analysis in the identification of anthocyanin
was the main consideration in this study.
In addition, till date, there is no report
indicated the anthocyanin conformation in the fruits of
Ficus aurata, therefore, we describe the anthocyanins
in the fruits of Ficus aurata for the first time. In the
fruits of Ficus aurata, at least, there are seven
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in plants for practical use.
ACKNOWLEDGEMENT
This work was financed by a grant from
the Faculty of Agricultural Technology of Andalas
University, Indonesia (01K/PL/DF-DIPA/FATETA-
2019).
Conflict of Interests
The authors declare that there is no conflict
of interests.
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