全 文 ：
91 Journal of Chinese Pharmaceutical Sciences http://www.jcps.ac.cn
Rapid characterization of 96 chemical constituents in Citri Reticulatae
Folium (leaves of ‘Fuju’) using HPLC-DAD-ESI-MSn
Guihua Cao, Qingrong Fu, Cangman Zhang, Hong Wang*, Shizhong Chen*
Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
Abstract: In order to systematically investigate the chemical constituents of Citri Reticulatae Folium (leaves of „Fuju‟), an analytical
method that included high-performance liquid chromatography, diode array detection, electrospray ionization, and ion -trap
time-of-flight mass spectrometry (HPLC-DAD-ESI-MSn) was used to separate and identify the individual chemical components of
Citri Reticulatae Folium. As a result, 96 compounds were tentatively identified in this study: including 31 phenolic acids, 4 flavonoid
aglycones, 6 flavonoid mono-O-glycosides, 10 flavonoid-O-diglycosides, 5 flavonoid mono-C-glycosides, 5 flavonoid di-C-glycosides,
6 flavonoid O,C-glycosides, 5 (3-hydroxy-3-methylglutaryl) glycosyl flavonoids, 1 flavan-3-ol, and 2 alkaloids. In addition,
21 polymethoxy flavonoids (PMFs) were identified in this paper. Among these compounds, 52 compounds, which were previously
found in other Citrus plants, have been identified for the first time in Citri Reticulatae Folium. 15 compounds have not been
previously found in the Citrus genus were identified. Moreover, 9 potentially new compounds have also been detected in this
paper. This is the first report of the full characterization of chemical components of Citri Reticulatae Folium (leaves of „Fuju‟) by
HPLC-DAD-ESI-MSn.
Keywords: Citri Reticulatae Folium (leaves of „Fuju‟), HPLC-DAD-ESI-MSn, Flavonoid, O,C-Glycosides, PMFs
CLC number: R284 Document code: A Article ID: 1003–1057(2016)2–91–20
Received: 2015-09-21, Revised: 2015-10-24, Accepted: 2015-11-15.
Foundation item: Beijing Natural Science Foundation (Grant No.
7142088).
*Corresponding author. Tel./Fax: 86-10-82802723,
E-mail: chenbjmu@163.com, hw9505@bjmu.edu.cn
http://dx.doi.org/10.5246/jcps.2016.02.010
1. Introduction
The health benefits of Citrus plants have been used for
centuries in foods, drinks, flavorants, and deodorant[1].
Over the past decades, numerous studies have been
carried out to isolate and identify the chemical compo-
nents present in different Citrus plants. The chemical
components and nutritional properties of Citrus plants
have been frequently reported, and it includes flavonols,
flavones, flavanones, catechins, anthocyanidins, and
isoflavones with antioxidant, anti-inflammatory, anti-
ageing, anti-cancer activities, and so on[2–5]. Additionally,
the phenolic compounds that are present in Citrus
peels, such as ferulic acid, sinapic acid, carboxylic acid,
p-cinnamic acid, etc., have also been analyzed[6–8].
Polymethoxy flavonoids (PMFs) are the most common
constituent and have been widely described in the Citrus
genus, which includes the tangerine[9], tangelo[10], Citrus





reticulate Blanco[11], and so on, and it has been reported
that PMFs possess strong health-promoting biological
activities[12]. In addition, other types of compounds, such
as alkaloids, terpenoids, coumarins, etc., have been
detected in Citrus plants as well[4,13–16].
For over 400 years, Citri Reticulatae Folium (the
leaves of Citrus plants) has been used in Chinese medicine
to treat hypochondriac pain and mastitis. However, to
the best of our knowledge, its chemical constituents have
yet to be systematically investigated, and only several
flavonoids and some PMFs have been reported[17–19].
There are also few studies on the nutritional properties
and biological activities of Citri Reticulatae Folium.
There are a variety of bioactive compounds in the other
Citrus plants, and a systematic study of the chemical
components of Citri Reticulatae Folium will provide
scientific data for further developing and utilizing this
particular resource.
The aim of this paper is to characterize the variation
of the small molecules found in Citri Reticulatae Folium
(leaves of „Fuju‟) by using HPLC-DAD-ESI-MSn, which
has been proven to be a rapid and reliable method
for analyzing and identifying chemical compounds. In

92 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
addition, the authors carry out a comprehensive chemical
constituent analysis that will help establish a better
basis for understanding the biological activities and
nutritional properties of Citri Reticulatae Folium.
2. Materials and methods
2.1. Materials, reagents, and apparatus
Citri Reticulatae Folium (leaves of „Fuju‟) was collected
from Fujian City, China. The sample was authenticated
by Prof. Shizhong Chen, Department of Natural
Medicines, School of Pharmaceutical Sciences, Peking
University. Apigenin, Diosmetin, Hesperitin, 5-CQA,
Diosmetin-7-O-glucoside, Isorhamnetin-3-O-glucoside,
Hesperitin-7-O-glucoside, Eriocitrin, Naringenin-7-O-
rutinoside, Quercetin-3-O-rutinoside, Nicotiflorin, Luteolin-
6-C-glucoside, Luteolin-8-C-glucoside, Apigenin-6-
C-glucoside, Isosinensetin, Tangeretin, 5-Desmethylnobiletin,
and Nobiletin were obtained from the National Institutes
for Food and Drug Control (Beijing, China).
LC-MS grade methanol and acetonitrile (Merck,
Darmstadt, Germany) and analytical grade formic acid
(FA) (Suzhou Yacoo Chemical Reagent Corporation,
Jiangsu, China) were used in the preparation of the mobile
phase. Analytical grade methanol (Beijing Chemical
Works, Beijing, China) was used for sample preparation.
Deionised water (18 MΩ) was purified by a Milli-Q
system (Millipore, MA, USA).
The Citri Reticulatae Folium extract was analyzed
using a Shimadzu analytical HPLC system (Kyoto, Japan)
that consisted of two LC-20AD pumps, a CTO-20A
column oven, a DGU-20A3 degasser, an SPD-M20AD,
and an SIL-20AC auto injector. The HPLC system was
connected to a hybrid IT-TOF mass spectrometer
(Shimadzu MS-IT-TOF, Kyoto, Japan) via an ESI
interface. The ultrasonic extraction of Citri Reticulatae
Folium was performed with a KQ250DE ultrasonic
cleaner (Kunshan, China).
2.2. Sample preparation
Powdered Citri Reticulatae Folium (1.0 g, 60 mesh)
was accurately weighed and extracted with 40 mL of
70% MeOH in an ultrasonic bath (40 kHz, 250 W) for
40 min. The extract mixture was filtered through a 0.22 μm
membrane prior to use, and a 10 μL aliquot was directly
injected into the HPLC.
2.3. HPLC-DAD-ESI-MSn analysis
Separation was performed on a ThermoTM C18 (4.6 mm×
250 mm, 5 μm) column (MA, USA) at 40 °C. The linear-
gradient elution was performed using a mobile phase
composed of mixture of solutions A (water containing
0.1% FA), B (ACN containing 0.1% FA), and C (MeOH
containing 0.1% FA) under the following conditions:
0–15 min, 95% A, and 5% B; 15–30 min, 95%–91% A,
and 5%–9% B; 30–40 min, 91%–88% A, and 9%–12% B;
40–50 min, 88% A, and 12% B; 50–60 min, 88%–83% A,
and 12% B; 60–70 min, 83%–77% A, and 12%–13% B;
70–90 min, 77%–76% A, and 13%–14% B; 90–100 min,
76%–74% A, and 14%–16% B; 100–110 min, 74%–
70% A, and 16%–20% B; 110–120 min, 70%–65% A,
and 20%–30% B; 120–130 min, 65%–50% A, and
30%–50% B; 130–140 min, 50%–30% A, and 50%–
70% B; 140–150 min, 30%–20% A, and 70%–80% B;
150–160 min, 20%–0% A, and 80%–100% B. The flow
rate was 1 mL/min. The UV spectra were recorded between
200 and 400 nm, and the diode array detection (DAD)
was set to 285 nm.
The optimized MS operating conditions were as
follows: negative and positive ionization mode, scan
spectra from m/z 100 to 800, and a nebulizing flow rate
of nitrogen at 1.5 L/min. The curved desolvation line
(CDL) temperature and block-heater temperature were
maintained at 200 °C. The capillary voltage, CDL voltage,
and detector voltage were fixed at 4.5 kV, 10 V, and
1.75 kV, respectively. Data were acquired and processed
using the LCMS Solutions software (version 3, Shimadzu,
Kyoto, Japan), which included a chemical formula
predictor. The data-dependent acquisition was set
such that the most abundant ions in the full-scan MS
would trigger multiple-stage mass spectrometry (MSn,
n = 2–4). The collision energy for MSn was adjusted to
50% of the HPLC-MS analysis, and the isolation width
of precursor ions was 3.0 U. All of the produced ions
were introduced into the TOF-MS instrument for accurate
mass determination.

93 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
3. Results and discussion
Methanolic extracts of Citri Reticulatae Folium were
analyzed by reverse phase HPLC and ESI-IT-TOF
mass spectrometry. This process allowed for the iso-
lation and identification of the chemical constituents.
The HPLC trace with the peaks assigned is shown
in Figure 1, and the chemical identities discussed
below are summarized in Figure 2 and Tables 1–2.
The main fragments observed for the flavonoid
glycosides in tandem mass spectrometry are shown
in Figure 3.
Figure 1. Chromatograms and peak assignments of extract of Citri Reticulatae Folium from HPLC-DAD-ESI-MSn experiments.
Figure 2. Chemical structures of the major compounds identified in Citri Reticulatae Folium.
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 min
2.25

2.00

1.75

1.50

1.25

1.00

0.75

0.50

0.25

0.00
mAU (×100)
285 nm, 4 nm (1.00)
120.0 122.5 125.0 127.5 130.0 132.5 135.0 137.5 140.0 142.5 min
2.50
2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
mAU (×10)
285 nm, 4 nm (1.00)
74
71
75+76
72+73
78
77
79
80+81
82
83
84+85
87+88
89
91
86
90 92
93
94
95 96
COOH
H OR1
R2O H
H OR3
H OR4
COOH
COOH
H OR1
R2O H
R3O H
H OR4
COOH
or
OR =
OH
OH
OR =
OH
OR =
OH
OCH3
OR =
Caffeoyl p-Coumaroyl Feruloyl Benzoyl
R1 or R2 or R3 or R4 = Caffeoyl,
p-Coumaroyl, Feruloyl, Benzoyl
O
OH
HOH2C
OHHO
Gentisoyl glucoside
O OH
OH
O
O
OH
HOH2C
OHHO
p-Coumaroyl glucoside
O
O
OH
O
OR2
R1O
OH
O
1,3A
1,3B
0,2A
0,2B
OH
1,2A
Kaempferol methylether
R1 = H, R2 = CH3 or
R1 = CH3, R2 = H
O
OH
HO
OH
O
1,3A
1,3B
0,2A
0,2B
R2
R1
OCH3
Chysoeriol-6,8-di-C-glucoside
R1 = R2 = Glc
O
OH
HO
O
O
OHO
OH
(3-Hydroxy-3-methyl) glutaryl-
3-O-5,7-dihydroxy-flavan-3-ol
O
OH
R1O
OH
O
1,3A
1,3B
0,2A
0,2B
R3
R2
OH
Luteolin-7-O-rutinoside
Luteolin-6-C-glucoside
Luteolin-8-C-glucoside
Luteolin-6,8-di-C-glucoside
OH
H3CO OCH3
O
O
O OH
HO
OH
OH
Sinapoyl-6-O-glucoside
O
OH
RO
OCH3
O
1,3A
1,3B
0,2A
0,2B
1,2A
3,5,7-Trihydroxy-4-
methoxyflavanonol-7-O-rutinoside
R = Rutinosyl
OH
1,4A
OH
O
OCH3
H3CO
H3CO
OCH3
OCH3
O
OCH3
5,6,7,8,3,4-Hexamethoxyflavanone R1 = Rutinosyl, R2 = R3 = H
R1 = H, R2 = Glc, R3 = H
R1 = R2 = H, R3 = Glc
R1 = H, R2 = R3 = Glc

94 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110

Figure 2. Continued.
O
OH
R1O
OCH3
O
1,3A
1,3B
0, 2A
0,2B
R3
R2
OH
Diosmetin
Diosmetin-7-O-glucoside
Diosmin
Diosmetin-6-C-glucoside
Diosmetin-8-C-glucoside
Diosmetin-6,8-di-C-glucoside
Diosmetin-6-C-glucosyl-7-O-rutinoside
R1 = Rutinosyl, R2 = Glc, R3 = H
O
OH
R1O
R5
O
1,3A
1,3B
0,2A
0,2B
1,2A
Hesperitin
Hesperitin-7-O-glucoside
Hesperidin
Eriocitrin
Naringenin-7-O-rutinoside
Naringenin-6,8-di-C-glucoside
R2
R3
R4
1,4A
1,4B-2H
O
OH
R1O
OH
O
1,3A
1,3B
0,2A
0,2B
R3
R2
Isorhoifolin
R1 = Rutinosyl, R2 = R3 = H
Apigenin-6-C-glucoside
R1 = H, R2 = Glc, R3 = H
Apigenin-6,8-di-C-glucoside
R1 = H, R2 = R3 = Glc
Apigenin-6-C-glucosyl-2-O-glucoside
R1 = H, R2 = (2-O-Glucosyl)glc, R3 = H
Apigenin-8-C-glucosyl-2-O-glucoside
R1 = R2 = H, R3 = (2-O-Glucosyl)glc
Apigenin-6-C-glucosyl-2-O-xyloside
R1 = H, R2 = (2-O-Xylosyl)glc, R3 = H
Apigenin-8-C-glucosyl-2-O-xyloside
R1 = R2 = H, R3 = (2-O-Xylosyl)glc
Apigenin-8-C-glucosyl-2-O-rhamnoside
R1 = R2 = H, R3 = (2-O-Rhamosyl)glc
O
OH
R1O
R6
O
1,3A
1,3B
0,2A
0,2B
R3
R2
R5
Nicotiflorin
R1 = R2 = R3 = R5 = H, R4 = Rutinosyl, R6 = OH
Quercetin-3-O-rutinoside
R1 = R2 = R3 = R5 = R6 = H, R4 = Rutinosyl
8-Methoxyquercetin-3-O-glucoside
R1 = R2 = H, R3 = OCH3, R4 = Glucosyl, R5 = R6 = OH
8-Methoxyquercetin-3-O-[6-(3-hydroxy-3-methylglutaroyl)]-glucoside
R1 = R2 = H, R3 = OCH3, R4 = [6-(3-Hydroxy-3-methylglutaroyl)]glucosyl, R5 = R6 = OH
Limocitrin-3-O-glucoside
R1 = R2 = H, R3 = OCH3, R4 = Glucosyl, R5 = OCH3, R6 = OH
Isorhamnetin-3-O-glucoside
R1 = R2 = R3 = H, R4 = Glucosyl, R5 = OCH3, R6 = OH
Isorhamnetin-7-O-rutinoside
R1 = Rutinosyl, R2 = R3 = R4 = H, R5 = OCH3, R6 = OH
Isorhamnetin-3-O-[6-(3-hydroxy-3-methylglutaryl)]-glucoside
R1 = R2 = R3 = H, R4 = [6-(3-Hydroxy-3-methylglutaroyl)]glucosyl, R5 = OCH3, R6 = OH
5,7,4-Trihydroxy-8,3-dimethoxy flavonol-3-O-[2/6-(3-hydroxy-3methylglutaryl)]-glucoside
R1 = R2 = H, R3 = OCH3, R4 = [2/6-(3-Hydroxy-3-methylglutaroyl)]glucosyl, R5 = OCH3, R6 = OH
Limocitrol or Isolimocitrol-3-O-[6-(3-hydroxy-3-methylglutaryl)]-glucoside
R1 = H, R2 = R3 = OCH3, R4 = [6-(3-Hydroxy-3-methylglutaroyl)]glucosyl, R5 = OH, R6 = OCH3 or R5 = OCH3, R6 = OH
OR4
1,2A
O
R1
R2
R3
R4
R6
R5
O
Tetra-O-methylscuteuarein/isoscuteuarein
R1 = R2 = R3 = R6 = OCH3, R4 = R5 = R7 = H or
R1 = R3 = R4 = R6 = OCH3, R2 = R5 = R7 = H
5-Hydroxy-7,8,3,4-tetramethoxyflavone
R1 = OH, R2 = R7 = H, R3 = R4 = R5 = R6 = OCH3
Gardenin B
R1 = OH, R2 = R3 = R4 = R6 = OCH3, R5 = R7 = H
6/7-O-Desmethyltangeritin
R1 = R4 = R6 = OCH3, R5 = R7 = H, R2 = OH,
R3 = OCH3 or R2 = OCH3, R3 = OH
Isosinensetin
R1 = R3 = R4 = R5 = R6 = OCH3, R2 = R7 = H
5,7,3,4,5-Pentamethoxyflavone
R1 = R3 = R5 = R6 = R7 = OCH3, R2 = R4 = H
Tangeretin
R1 = R2 = R3 = R4 = R6 = OCH3, R5 = R7 = H
5-Desmethylnobiletin
R1=OH,R2=R3=R4=R5=R6=OCH3,R7=H
3/4-Desmethylnobiletin
R1 = R2 = R3 = R4 = OCH3, R7 = H, R5 = OH,
R6 = OCH3 or R5 = OCH3, R6 = OH
Nobiletin
R1 = R2 = R3 = R4 = R5 = R6 = OCH3, R7 = H
R7
O
R1
R2
R3
R4
R6
O
R7
5,4-Dihydroxyl-3,7,8,3-tetramethoxyflavonol
R1 = R6 = OH, R2 = H, R3 = R4 = R5 = R7 = OCH3
3-Hydroxy-5,6,7,3,4-pentamethoxyflavonol
R1 = R2 = R3 = R6 = R7 = OCH3, R4 = H, R5 = OH
3,5-Dihydroxy-6,7,8,3,4-pentamethoxyflavonol
R1 = R5 = OH, R2 = R3 = R4 = R6 = R7 = OCH3
Hexa-O-methylgossypetin
R2 = H, R1 = R3 = R4 = R5 = R6 = R7 = OCH3
Natsudaidain
R1 = R2 = R3 = R4 = R6 = R7 = OCH3, R5 = OH
3,5,6,7,8,3,4-Heptamethoxyflavonol
R1 = R2 = R3 = R4 = R5 = R6 = R7 = OCH3
3,5-Dihydroxy-6,7,8,3,4-pentamethoxyflavonol-3-O-[6-(3-hydroxy-3-methylglutaryl)]-glucoside
R1 = OH, R2 = R3 = R4 = R6 = R7 = OCH3, R5 = [6-(3-Hydroxy-3-methylglutaroyl)]glucosyl
Natsudaidain-3-O-[6/4-(3-hydroxy-3-methylglutaryl)]-glucoside
R1 = R2 = R3 = R4 = R6 = R7 = OCH3, R5 = [6/4-(3-Hydroxy-3-methylglutaroyl)]glucosyl
R5
R1 = R2 = R3 = H, R4 = OH, R5 = OCH3
R1 = Glucosyl, R2 = R3 = H, R4 = OH, R5 = OCH3
R1 = Rutinosyl, R2 = R3 = H, R4 = OH, R5 = OCH3
R1 = Rutinosyl, R2 = R3 = H, R4 = R5 = OH
R1 = Rutinosyl, R2 = R3 = H, R4 = H, R5 = OH
R1 = H, R2 = R3 = Glc, R4 = H, R5 = OH
R1 = R2 = R3 = H
R1 = Glucosyl, R2 = R3 = H
R1 = Rutinosyl, R2 = R3 = H
R1 = H, R2 = Glc, R3 = H
R1 = R2 = H, R3 = Glc
R1 = H, R2 = R3 = Glc

95 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
O
OH O
OH
O
O
O
HO
HO
O
O
HO
HO
H3C
OH
H
Y0

[Y0 -H]
.
Y1

Isorhoifolin
O
OOH
HO
OH
OH
O
O O
OH
OH
O
O
OH
OH
OH
H3C
H
[Y0 -H]
.
Y0

Y1

Quercetin-3-O-rutinoside
O
OOH
HO
OHO
O
CH2OH
OH
HO
HO
OHHO
OH
HOH2C
120 Da
90 Da
60 Da
60 Da
90 Da
120 Da
Apigenin-6,8-di-C-glucoside
O
OH
O
O
OCH3
OH
O
OH
HOH2C
HO OH
O
O
OH
HO
O
O
OH
OH
OH
CH3
H
120 Da
90 Da
60 Da
(162+146) Da
146 Da
(163+146) Da
Diosmetin-6-C-glucosyl-7-O-rutinoside
O
OH
HO
OH
O
O
OH
HOH2C
OHO
O
OHHOH2C
OH
OH
120 Da
90 Da
60 Da
162 Da
180 Da
Apigenin-6-C-glucosyl-2-O-glucoside
O
OH
HO
OH
O
O
OH
HOH2C
HO OH
120 Da
(Ag+41)
90 Da
(Ag+71)
60 Da
Apigenin-6-C-glucoside
Figure 3. Main fragmentation observed for flavonoid glycosides in tandem mass spectrometry.
Table 1. Summary of chemical compostion detected in Citri Reticulatae Folium in negative ion scan modea
Peak No.
Rt
(min)
UV λmax
(nm)
[M-H]–
(m/z)
Formula
(–) ESI-MSn
P-ions (m/z) (RA%)
Proposed compounds
Phenolic acids
1b 8.887 285/320 371.0594 C15H16O11
MS2: 353 (6), 209 (100), 191 (39)
MS3: 191 (100)
Caffeoyl glucarate or galactarate
(isomers)
3b 12.980 296/325 371.0594 C15H16O11
MS2: 353 (2), 209 (100), 191 (32), 173 (2)
MS3: 191 (100)
5b 14.690 296/– 371.0587 C15H16O11
MS2: 209 (100), 191 (41)
MS3: 191 (100)
6b 16.207 290 (sh)/324 371.0611 C15H16O11
MS2: 353 (4), 209 (100), 191 (30)
MS3: 191 (100)
4b 14.393 –/302 355.0679 C15H16O10
MS2: 337 (5), 209 (65), 191 (100), 163 (2), 147 (3)
MS3: 85 (100) p-coumaroyl glucarate or galactarate
(isomers)
8b 18.973 210//313 355.0679 C15H16O10
MS2: 337 (6), 209 (76), 191 (100), 163 (1), 147 (5)
MS3: 85 (100), 147 (14), 173 (21)
9b 21.947
242/299 (sh)/
324
385.0741 C16H18O11
MS2: 367 (4), 209 (7), 192 (12), 191 (100), 193 (3),
173 (3), 147 (1)
MS3: 85 (100)
Feruloyl glucarate or galactarate
(isomers)
12b 26.123
233/298 (sh)/
327
385.0724 C16H18O11
MS2: 367 (3), 209 (16), 192 (12), 191 (100), 193 (5),
173 (2), 147 (2)
MS3: 85 (100)
14b 29.157
237/299 (sh)/
326
385.0765 C16H18O11
MS2: 367 (4), 209 (9), 192 (10), 191 (100), 193 (3),
173 (3), 147 (1)
MS3: 147 (6), 129 (5), 85 (100)
15b 29.523 299 (sh)/325 385.2480 C16H18O11
MS2: 367 (4), 209 (8), 192 (12), 191 (100), 193 (4),
173 (3), 147 (2)
MS3: 129 (7), 85 (100)
16b 31.840
238/299 (sh)/
327
385.0745 C16H18O11
MS2: 367 (4), 209 (8), 192 (11), 191 (100), 193 (3),
173 (3), 147 (1)
MS3: 129 (8), 85 (100)
22b 37.423
235/299 (sh)/
327
385.0763 C16H18O11
MS2: 367 (4), 209 (8), 192 (10), 191 (100), 193 (4),
173 (3), 147 (1)
MS3: 129 (6), 85 (100)

96 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110

Table 1. Continued
Peak No.
Rt
(min)
UV λmax
(nm)
[M-H]–
(m/z)
Formula
(–) ESI-MSn
P-ions (m/z) (RA%)
Proposed compounds
7d 17.098 230/282/312 313.0395 C13H14O9
MS2: 295 (12), 277 (2), 251 (2), 191 (100), 173 (12),
147 (10), 129 (16), 121 (<1)
MS3: 129 (34), 85 (100)
Benzoyl glucarate or galactarate
(isomers)
10d 22.658 228/285/326 313.0374 C13H14O9
MS2: 295 (9), 191 (100), 173 (2), 147 (15), 129 (8),
121 (1)
MS3: 85 (100)
11d 24.447 228/275/– 313.0381 C13H14O9
MS2: 295 (14), 191 (100), 173 (12), 147 (10), 129 (15),
121 (1)
MS3: 85 (100)
13d 27.180 227/285/326 313.0385 C13H14O9
MS2: 295 (11), 191 (100), 173 (8), 147 (14), 129 (14),
121 (3)
MS3: 85 (100)
17d 32.773 226/285/324 313.0384 C13H14O9
MS2: 295 (8), 191 (100), 173 (9), 147 (10), 129 (16),
121 (<1)
MS3: 85 (100)
21d 35.900 230/282/325 313.0399 C13H14O9
MS2: 295 (13), 191 (100), 173 (13), 147 (10), 129 (14),
121 (<1)
MS3: 85 (100)
2c 11.133 298 315.0700 C13H16O9
MS2: 225 (2), 207 (3), 179 (2), 153 (100), 152 (83),
109 (4), 108 (9)
MS3: 109 (72), 108 (100)
Gentisoyl glucoside
19c 33.377 285 325.0916 C15H18O8
MS2: 163 (100), 119 (16)
MS3: 119 (100)
p-Coumaroyl glucoside
24c 40.120
230/290 (sh)/
324
399.0901 C17H20O11
MS2: 223 (41), 205 (100), 193 (2)
MS3: 111 (100)
Sinapoyl glucuronide or its isomers
28c 42.730 –/290 (sh)/324 399.0905 C17H20O11
MS2: 223 (44), 205 (100), 193 (2), 187 (3)
MS3: 111 (100)
35c 48.473 285/327 385.1115 C17H22O10
MS2: 325 (47), 295 (100), 265 (86), 223 (18)
MS3: 223 (100)
MS4: 208 (100), 164 (50)
Sinapoyl-6-O-glucoside
18e 32.937
245/299 (sh)/
325
353.0860 C16H18O9
MS2: 191 (100), 179 (3), 173 (1), 161, 135 (<1)
MS2: 173 (10), 127 (66), 93 (91), 85 (100) 5-CQA
27b 42.310 –285 (sh)/311 337.0905 C16H18O8
MS2: 191 (100), 163 (3)
MS3: 173 (23), 127 (34), 111 (27), 93 (100), 85 (84)
5-pCoQA
20b 35.277 285 (sh)/323 367.0988 C17H20O9
MS2: 193 (100), 134 (14)
MS3: 134 (100)
1-FQA
26b 41.953 285 (sh)/324 367.1046 C17H20O9 MS2: 193 (24), 173 (100), 129 (41) 4-FQA
30b 44.943 285 (sh)/324 367.0636 C17H20O9
MS2: 323 (100), 193 (36), 191 (8), 173 (11), 147 (26),
129 (7)
MS3: 147 (100) 3-FQA (isomers)
31b 45.207 285 (sh)/330 367.0652 C17H20O9 MS2: 323 (68), 193 (100), 191 (80), 129 (23)
34b 47.913 298 (sh)/324 367.1035 C17H20O9
MS2: 191 (100), 173 (4)
MS3: 173 (37), 127 (80), 93 (73), 85 (100)
5-FQA (isomers)
40b 57.790 285 (sh)/330 367.0628 C17H20O9 MS2: 323 (38), 193 (23), 191 (100), 129 (15)
Flavonoid aglycones
73e 125.633 282/324 269.0423 C15H10O5
MS2: 225 (80), 201 (50), 197 (36), 183 (53), 181 (24),
159 (42), 151 (54), 149 (100), 121 (11), 117 (12),
107 (8)
Apigenin
74e 126.317 287/330 (sh) 301.0726 C16H14O6
MS2: 286 (28), 283 (35), 268 (20), 258 (35), 242 (100),
240 (17), 227 (31), 199 (85), 164 (32), 155 (<1),
125 (39)
Hesperitin
75e 127.123
270/285 (sh)/
324
299.0563 C16H12O6
MS2: 284 (100)
MS3: 256 (100), 227 (7), 151 (4), 133 (1)
MS4: 227 (100), 213 (17), 185 (10), 149 (15)
Diosmetin
29c 44.803 290 (sh)/327 299.1470 C16H12O6
MS2: 255 (11), 237 (3), 195 (100), 177 (1), 163 (2)
MS3: 179 (80), 177 (100), 165 (87), 151 (77), 149 (73),
137 (80), 123 (67)
Kaempferol methylether

97 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110

Table 1. Continued
Peak No.
Rt
(min)
UV λmax
(nm)
[M-H]–
(m/z)
Formula
(–) ESI-MSn
P-ions (m/z) (RA%)
Proposed compounds
Flavonoid mono-O-glycosides
63b,e 97.490 271/285/334 461.1024 C22H22O11
MS2: 446 (46), 299 (100), 298 (23), 285 (16), 284 (60),
283 (16), 269 (1), 255 (5)
MS3: 285 (18), 284 (100), 283 (1), 269 (6)
Diosmetin-7-O-glucoside
64b,e 98.467
252/266/345
(low)
477.1058 C22H22O12
MS2: 315 (29), 314 (68), 300 (33), 299 (100), 271 (17),
163, 151, 149 (<1)
MS3: 271 (100), 255 (8), 243 (7), 227 (7), 199 (5)
MS4: 243 (7), 227 (24), 199 (100)
Isorhamnetin-3-O-glucoside
50b 72.413 269/334 (low) 493.0929 C22H22O13
MS2: 331 (100), 330 (28)
MS3: 316 (100), 315 (44), 209 (10), 181 (10)
MS4: 287 (93), 271 (73), 259 (11), 243 (23), 227 (17),
194 (16), 179 (11), 166 (100)
8-Methoxyquercetin-3-O-glucoside
59b 88.200 265/333 (low) 507.1087 C23H24O13
MS2: 492 (21), 345 (16), 344 (31), 330 (32), 329 (100),
301 (11), 181 (<1)
MS3: 314 (16), 301 (59), 286 (100), 258 (14)
Limocitrin-3-O-glucoside
62b 96.053 285/341 (low) 537.1185 C24H26O14
MS2: 522 (32), 375 (12), 374 (17), 360 (30), 359 (100),
344 (10), 211 (1)
MS3: 345 (16), 344 (97), 316 (100)
MS4: 302 (31), 300 (91), 232 (16), 188 (100), 160 (34),
145 (21)
Limocitrol or Isolimocitrol-3-O-
glucoside
67b,e 104.637 285/330 (sh) 463.1245 C22H24O11
MS2: 301 (100)
MS3: 286 (21), 283 (10), 268 (12), 258 (37), 242 (84),
227 (12), 199 (100), 164 (26), 125 (20)
Hesperitin-7-O-glucoside
Flavonoid-O-diglycosides
33b 47.593 286/320 (sh) 625.1680 C28H34O16
MS2: 597 (96), 317 (100), 313 (51), 299 (28), 289 (77),
284 (9)
MS3: 299 (54), 289 (100), 284 (4), 274 (19), 258 (24),
180 (4), 152 (6), 151 (4), 125 (36)
MS4: 274 (100), 152 (71), 151 (50), 125 (100)
3′,5,7-Trihydroxy-4′-methoxyflavanonol-
7-O-rutinoside
43e 65.667 281/337 (sh) 595.1577 C27H32O15
MS2: 287 (100)
MS3: 269 (2), 151 (100), 135 (12), 125 (4)
MS4: 107 (100)
Eriocitrin
54e 80.017
271/285 (sh)/
334
579.1620 C27H32O14
MS2: 271 (100)
MS3: 151 (100), 125 (2), 119 (13), 107 (6)
Naringenin-7-O-rutinoside
61e 92.950 284/330 (sh) 609.1751 C28H34O15
MS2: 301 (100), 242 (4)
MS3: 286 (31), 283 (25), 268 (18), 258 (21), 242 (76),
240 (17), 227 (36), 199 (100), 164 (35), 155 (1),
151 (14), 125 (28)
Hesperidin
48b, e 71.250
255/267/352
(low)
609.1373 C27H30O16
MS2: 463 (<1), 343 (8), 301 (100), 300 (40)
MS3: 301 (100), 271 (17), 255 (12), 179 (68), 151 (68),
107 (3)
Quercetin-3-O-rutinoside
57b, e 86.527 264/333 (low) 593.1423 C27H30O15
MS2: 285 (100), 284 (8)
MS3: 267 (52), 257 (100), 255 (13), 241 (23), 239 (29),
229 (82), 163 (40), 151 (19)
MS4: 227 (100), 183 (19)
Nicotiflorin
60b 92.100 255/267/351 623.1547 C28H32O16
MS2: 315 (100), 314 (2), 300 (38)
MS3: 300 (100), 271 (30), 163, 151 (1)
MS4: 271 (100), 255 (57), 243 (5)
Isorhamnetin-7-O-rutinoside
51b 73.420 254/265sh/347 593.1429 C27H30O15
MS2: 447 (<1), 327 (<1), 285 (100)
MS3: 243 (25), 217 (34), 199 (73), 175 (100), 151 (28),
107 (1)
MS4: 131 (100)
Luteolin-7-O-rutinoside
58 88.060 267/334 577.1479 C27H30O14
MS2: 311 (<1), 269 (100)
MS3: 225 (45), 201 (20), 197 (58), 183 (53), 181 (36),
159 (31), 155 (9), 151 (25), 149 (39), 121 (7),
117 (100), 107 (12)
Isorhoifolin
65 98.787
252/267 (sh)/
347
607.1644 C28H32O15
MS2: 299 (100), 284 (33)
MS3: 284 (100)
MS4: 256 (100), 227 (36), 183 (16), 151 (9), 107 (2)
Diosmin

98 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110

Table 1. Continued
Peak No.
Rt
(min)
UV λmax
(nm)
[M-H]–
(m/z)
Formula
(–) ESI-MSn
P-ions (m/z) (RA%)
Proposed compounds
Flavonoid mono-C-glycosides
38b, e 55.873 269/341 447.0897 C21H20O11
MS2: 357 (53), 327 (100)
MS3: 299 (100), 284 (17), 255 (3)
MS4: 175 (100), 165 (29), 133 (20)
Luteolin-6-C-glucoside
39b, e 57.067 269/333 447.0891 C21H20O11
MS2: 357 (37), 327 (100)
MS3: 299 (100), 284 (42), 255 (8)
MS4: 175 (100), 165 (61), 133 (51)
Luteolin-8-C-glucoside
47e 71.137 255/265/348 431.0924 C21H20O10
MS2: 341 (25), 311 (100)
MS3: 283 (100)
MS4: 183 (100), 165 (29), 163 (42), 117 (80)
Apigenin-6-C-glucoside
53b 77.387 267/333 461.1025 C22H22O11
MS2: 371 (9), 341 (100)
MS3: 326 (4), 313 (2), 299 (14), 298 (100)
MS4: 282 (30), 269 (100), 255 (37), 163 (22), 135 (4),
133 (7)
Diosmetin-6-C-glucoside
55b 80.863 267/341 461.1025 C22H22O11
MS2: 383 (2), 371 (23), 353 (1), 341 (100), 326 (1),
299 (2), 298 (28)
MS3: 299 (12), 298 (100)
MS4: 282 (33), 269 (100), 255 (38), 163 (26), 135 (5),
132 (4)
Diosmetin-8-C-glucoside
Flavonoid di-C-glycosides
25b 41.410
257
(sh)/270/348 609.1394 C27H30O16
MS2: 519 (21), 489 (100), 471 (8), 429 (9), 399 (31),
369 (31)
MS3: 399 (36), 369 (100)
MS4: 341 (52), 313 (100), 269 (3), 179 (<1), 177 (3),
133 (6)
Luteolin-6, 8-di-C-glucoside
32 45.643 271/330 593.1428 C27H30O15
MS2: 503 (11), 473 (45), 455 (3), 383 (53), 353 (100)
MS3: 325 (34), 297 (100)
MS4: 177 (100), 149 (70), 135 (70)
Apigenin-6, 8-di-C-glucoside
36b 49.540 255/271/345 623.1524 C28H32O16
MS2: 533 (7), 503 (70), 413 (47), 383 (100), 312 (17)
MS3: 368 (3), 355 (7), 340 (1), 327 (3), 312 (100)
Chysoeriol-6, 8-di-C-glucoside
37b 52.883 271/341 623.1548 C28H32O16
MS2: 533 (5), 503 (63), 413 (39), 383 (100), 312 (27)
MS3: 368 (1), 355 (1), 340 (2), 312 (100)
MS4: 283 (100), 177 (14), 149 (5), 133 (14)
Diosmetin-6, 8-di-C-glucoside
23c 39.643 285/330 (sh) 595.1592 C27H32O15
MS2: 505 (4), 475 (16), 457 (7), 415 (20), 385 (71),
355 (100), 313 (6)
MS3: 235 (100), 249 (33), 221 (20), 209 (43), 145 (12)
Naringenin-6, 8-di-C-glucoside
Flavonoid O, C-glycosides
41b 65.247 272/341 769.2111 C34H42O20
MS2: 609 (3), 461 (100)
MS3: 371 (23), 341 (100)
MS4: 298 (100)
Diosmetin-6-C-glucosyl-7-O-rutinoside
42c 65.350 273/341 593.1461 C27H30O15
MS2: 503 (1), 473 (8), 413 (97), 341 (5), 311 (5),
293 (100)
MS3: 265 (11), 251 (12), 249 (14), 221 (17), 193 (11),
175 (83), 173 (100), 145 (18), 131 (61)
Apigenin-6-C-glucosyl-2′′-O-glucoside
44c 66.770 269/330 593.1441 C27H30O15
MS2: 473 (2), 413 (100), 341 (<1), 311 (6), 293 (99)
MS3: 293 (100)
MS4: 265 (14), 249 (25), 221 (23), 193 (29), 175 (74),
173 (100), 145 (17), 131 (35), 117 (30)
Apigenin-8-C-glucosyl-2′′-O-glucoside
45 68.017 270/337 563.1323 C26H28O14
MS2: 443 (5), 431 (1), 413 (100), 341 (4), 311 (4),
293 (62)
MS3: 293 (100)
MS4: 275 (10), 265 (30), 249 (39), 221 (36), 193 (24),
175 (100), 173 (99), 165 (4), 145 (18), 131 (33),
117 (23)
Apigenin-6-C-glucosyl-2′′-O-xyloside
46 69.697 268/336 563.1331 C26H28O14
MS2: 443 (3), 431 (<1), 413 (100), 341 (2), 311 (7),
293 (50)
MS3: 293 (100)
MS4: 275 (13), 265 (36), 249 (41), 221 (35), 193 (22),
175 (100), 173 (93), 165 (1), 145 (18), 131 (32),
117 (33)
Apigenin-8-C-glucosyl-2′′-O-xyloside

99 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
Table 1. Continued
Peak No.
Rt
(min)
UV λmax
(nm)
[M-H]–
(m/z)
Formula
(–) ESI-MSn
P-ions (m/z) (RA%)
Proposed compounds
49b 72.227 270/334 577.1481 C27H30O14
MS2: 413 (67), 353 (2), 341 (7), 323 (10), 311 (6),
293 (100)
MS3: 275 (2), 265 (18), 249 (16), 221 (10), 193 (11),
163 (1), 175 (100), 173 (50), 145 (7), 131 (45),
117 (13)
MS4: 173 (66), 145 (7), 131 (100)
Apigenin-8-C-glucosyl-2′′-O-rhamnoside
(3-Hydroxy-3-methylglutaryl)glycosyl flavonoids
56b 83.013
256/272/347
(low)
637.1321 C28H30O17
MS2: 575 (10), 535 (33), 493 (100)
MS3: 331 (100), 330 (31)
MS4: 316 (100), 315 (21), 287 (2), 271 (1), 209 (11),
194 (1), 181 (9), 166 (6)
8-Methoxyquercetin-3-O-[6′′-(3-hydroxy-
3-methylglutaroyl)]-glucoside
66b 101.213 275/333 (low) 651.1467 C29H32O17
MS2: 589 (17), 549 (31), 507 (85), 345 (100)
MS3: 345 (100), 330 (18)
MS4: 330 (100), 315 (1), 302 (12)
5,7,4′-Trihydroxy-8,3′-dimethoxy
flavonol-3-O-[2′′/6′′-(3-hydroxy-3-
methylglutaryl)]-glucoside
68b 106.887 271/341 (low) 651.1483 C29H32O17
MS2: 589 (9), 549 (35), 507 (71), 345 (100)
MS3: 330 (100), 315 (14)
MS4: 287 (97), 259 (71), 243 (100), 231 (41), 215 (50),
187 (44), 178 (65), 175 (47), 165 (53)
69c 107.810
254/267 (sh)/
352 (low)
621.1453 C28H30O16
MS2: 559 (31), 519 (50), 477 (100)
MS3: 315 (100), 314 (6)
MS4: 300 (100), 299 (2), 287 (5), 271 (22), 255 (16),
243 (2), 163, 151, 149, 135, 123 (<1)
Isorhamnetin-3-O-[6′′-(3-hydroxy-3-
methylglutaryl)]-glucoside
70d 108.093
255/270 (sh)/
348 (low)
681.1575 C30H34O18
MS2: 579 (36), 537 (100), 375 (39), 360 (4)
MS3: 375 (100), 360 (44), 345 (4), 317 (4), 302 (<1)
MS4: 360 (100), 345 (8), 317 (9)
Limocitrol or Isolimocitrol-3-O-
[6′′-(3-hydroxy-3-methylglutaryl)]-
glucoside
Flavan-3-ol
52d 75.800 285/330 (sh) 401.1819 C21H22O8
MS2: 339 (13), 299 (29), 257 (100), 213 (85)
MS3: 213 (100), 195 (15), 185 (5), 165 (2), 155 (94),
137 (3), 109 (1)
MS4: 155 (100)
(3-Hydroxy-3-methyl)glutaryl-3-O-
5,7-dihydroxy-flavan-3-ol
Alkaloids
72b 125.503 271/285/324
728.2680/
726.3737
MS2: 729 (100), 728 (87), 711 (35), 710 (73),
700 (47), 615 (30), 587 (5), 474 (5),
405 (1)
MS3: 615 (100), 587 (24), 474 (10)
MS2: 698 (9), 696 (100)
MS3: 591 (52), 590 (82), 476 (100),
381 (22), 333 (23), 293 (46),
275 (38)
Citrusin III
77b 129.447 271/282/324
704.2676/
702.3743
MS2: 686 (100), 668 (94), 514 (17)
MS3: 669 (64), 668 (84), 573 (27), 555 (21)
MS2: 659 (21), 658 (53), 614 (100)
MS3: 447 (53), 427 (87), 410 (100)
Citrusin I
a Rt, retention time. P-ions (m/z) (RA%), the product ions and relative intensity. Bold numbers represent the precursor ions for next stage MS. Sh, shoulder; Low, low
intensity; –, not detected. b Reported in Citrus plants, but not in Citri Reticulatae Folium. c Firstly identified in Citrus genus. d Potential novel compounds. e Retention
time, UV and MSn data equal to those of reference standards.
Table 2. Summary of polymethoxy flavonoids (PMFs) identified in Citri Reticulatae Folium in positive ion scan modea
Peak
No.
Rt
(min)
UV λmax
(nm)
[M+H]+
(m/z)
Formula
(+) ESI/MSn (m/z)
P-ions (RA%, loss)
Class Tentative assignment Ref
84b 132.900
260 (sh)/268/
280 (sh)/334
343.1162 C19H18O6
MS2: 328 (13, 15), 313 (100, 30), 299 (4, 44),
285 (5, 58), 181 (<1, [1,3A]+)
MS3: 270 (43, 15), 242 (100, 43), 153 (27,
[1,3A-CO]+), 135 (19, [1,3A-CO-H2O]
+),
133 (17, [1,3B+])
MS4: 153 (100, [1,3A-CO]+)
Tetramethoxyflavone
Tetra-O-methylscuteuarein/
Tetra-O-methylisoscuteuarein
16
86c 135.480 278/336 375.1037 C19H18O8
MS2: 360 (27, 15), 345 (100, 30), 342 (22, 33),
327 (53, 48), 314 (18, 61)
MS3: 330 (100, 15), 317 (31, 28), 302 (47, 43),
197 (32, [1,3A]+), 179 (2, [1,3B]+),
169 (14, [1,3A-CO]+), 151 (7, [0,2B]+),
149 (13, [1,3B-2CH3]
+)
MS4: 169 (100, [1,3A-CO]+)
Dihydroxy-tetramethoxyflavonol
5,4′-Dihydroxyl-3,7,8,3′-
tetramethoxyflavonol
61

100 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
Table 2. Continued
Peak
No.
Rt
(min)
UV λmax
(nm)
[M+H]+
(m/z)
Formula
(+) ESI/MSn (m/z)
P-ions (RA%, loss)
Class Tentative assignment Ref
95b 140.923 285/356 359.1114 C19H18O7
MS2: 344 (100, 15), 329 (6, 30)
MS3: 329 (80, 15), 315 (14, 29), 301 (100, 43),
298 (13, 46), 283 (12, 61)
MS4: 286 (59, 15), 258 (55, 43), 245 (39, 56),
167 (100, [1,3A]+), 163 (6, [1,3B]+),
111 (6, [1,3A-2CO]+)
Monohydroxy-
tetramethoxyflavone
5-Hydroxy-7,8,3′,4′-
tetramethoxyflavone
62
96b 142.447 285/324 359.1090 C19H18O7
MS2: 344 (31, 15), 329 (100, 30), 326 (29, 33),
311 (54, 48), 298 (20, 61)
MS3: 314 (58, 15), 311 (61, 18), 301 (58, 28),
286 (100, 43), 283 (23, 46), 271 (12, 58),
197 (75, [1,3A]+), 169 (65, [1,3A-CO]+),
151 (4, [1,3A-CO-H2O]
+), 133 (40, [1,3B]+)
Monohydroxy-
tetramethoxyflavone
Gardenin B 63
90b 137.530 282/341 359.1139 C19H18O7
MS2: 344 (100, 15), 329 (90, 30), 326 (17, 33),
311 (32, 48)
MS3: 329 (100, 15), 311 (36, 33)
MS4: 314 (42, 15), 311 (65, 18), 301 (16, 28),
286 (53, 43), 283 (59, 46), 197 (100,
[1,3A]+), 169 (36, [1,3A-CO]+), 151 (40,
[1,3A-CO-H2O]
+), 135 (58, [0,2B]+),
133 (87, [1,3B]+)
Monohydroxy-
tetramethoxyflavone
6-O-Desmethyltangeritin/
7-O-Desmethyltangeritin
64
65
92c 138.810 280/334/353 359.1102 C19H18O7
MS2: 344 (100, 15), 343 (72, 16), 315 (48, 44),
165 (2, [0,2B]+), 164 (3, [1,3B-CH3]
+),
151 (3, [1,3A]+)
MS3: 327 (100, 16), 315 (39, 28), 299 (52, 44),
282 (20, 61), 181 (2, [1,2A-CO]+)
MS4: 311 (17, 16), 299 (39, 28), 297 (100, 30),
281 (26, 46), 269 (25, 58),
149 (2, [1,3B-2CH3]
+), 123 (1, [1,3A-CO]+)
Monohydroxy-
tetramethoxyflavonol
Uncertain

82c 132.460
267/280 (sh)/
324
359.1085 C19H18O7
MS2: 344 (20, 15), 343 (13, 16), 329 (26, 30),
326 (13, 33), 315 (30, 44), 298 (100, 61)
MS3: 283 (100, 15), 282 (13, 16), 255 (60, 43),
151 (5, [1,3B-CO]+/[1,3A]+), 193 [0,2A]+,
165 [0,2A-CO]+, 149 [1,3B-2CH3]
+,
123 [1,3A-CO]+ (<1)
Monohydroxy-
tetramethoxyflavonol

78e 130.187 270/341 373.1263 C20H20O7
MS2: 358 (20, 15), 343 (100, 30)
MS3: 315 (100, 28), 299 (14, 44), 181 (12,
[1,3A]+), 163 (20, [1,3B]+), 153 (27,
[1,3A-CO]+), 148 (5, [1,3B-CH3]
+)
Pentamethoxyflavone Isosinensetin
16
66
83b 132.693
258/268/
280 (sh)/330
373.1274 C20H20O7
MS2: 358 (12, 15), 357 (17, 16), 343 (26, 30),
340 (13, 33), 329 (30, 44), 312 (100, 61)
MS3: 297 (71, 15), 296 (100, 16), 281 (14, 31),
269 (56, 43), 268 (71, 44), 251 (31, 61),
151 (96, [1,3A]+)
MS4: 266 (100, 30)
Pentamethoxyflavone
5,7,3′,4′,5′-Pentamet
hoxyflavone
67
85c 133.060
255/267 (sh)/
367
389.1255 C20H20O8
MS2: 374 (19, 15), 359 (100, 30)
MS3: 344 (20, 15), 331 (72, 28), 329 (24, 30),
315 (28, 44), 303 (20, 56), 181 (5, [1,3A]+),
165 (100, [0,2B]+), 153 (9, [1,3A-CO]+),
137 (18, [0,2B-CO]+)
MS4: 137 (100, 28), 122 (23, 15), 109 (44, 56),
107 (23, 58), 79 (50, 86)
Monohydroxy-
pentamethoxyflavonol
3-Hydroxy-5,6,7,3′,4′-
pentamethoxyflavo
nol
68
88e 135.273 253/270/333 373.1264 C20H20O7
MS2: 358 (38, 15), 343 (100, 30), 325 (10, 48),
312 (12, 61)
MS3: 328 (58, 15), 300 (100, 43), 211 (13, [1,3A]+),
183 (18, [1,3A-CO]+), 133 (4, [1,3B]+)
Pentamethoxyflavone Tangeretin 17
93c 139.407
258/280 (sh)/
353/379
405.1209 C20H20O9
MS2: 390 (23, 15), 375 (100, 30), 357 (46, 48),
347 (18, 58)
MS3: 347 (100, 28), 332 (14, 43)
MS4: 332 (100, 15), 317 (22, 30), 165 (1, [0,2B]+),
137 (2, [0,2B-CO]+), 135 (2, [0,2B-2CH3]
+)
Dihydroxy-
pentamethoxyflavonol
3,5-Dihydroxy-6,7,8,3′,4′-
pentamethoxyflavonol
69

101 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
Table 2. Continued
Peak
No.
Rt
(min)
UV λmax
(nm)
[M+H]+
(m/z)
Formula
(+) ESI/MSn (m/z)
P-ions (RA%, loss)
Class Tentative assignment Ref
94e 139.867 282/341 389.1221 C20H20O8
MS2: 374 (26, 15), 359 (100, 30), 356 (16, 33),
341 (32, 48), 328 (15, 61)
MS3: 344 (69, 15), 343 (100, 16), 331 (77, 28),
329 (20, 30), 316 (88, 43), 314 (14, 45),
197 (65, [1,3A]+), 169 (13, [1,3A-CO]+),
163 (50, [1,3B]+), 148 (10, [1,3B-CH3]
+)
Monohydroxy-
pentamethoxyflavone
5-Desmethylnobiletin 17
79b 130.777 268/334 389.1204 C20H20O8
MS2: 374 (42, 15), 359 (100, 30), 341 (11, 48),
328 (14, 61)
MS3: 344 (91, 15), 331 (33, 28), 316 (100, 43),
301 (16, 58), 211 (13, [1,3A]+), 183 (18,
[1,3A-CO]+), 151 (6, [0,2B]+)
Monohydroxy-
pentamethoxyflavone
4′-Desmethylnobiletin/3′-
desmethylnobiletin
70
81 131.963 282/333 (sh) 405.1161 C21H24O8
MS2: 390 (82, 15), 375 (100, 30), 357 (17, 48)
MS3: 347 (100, 28), 332 (9, 43), 211 [1,3A]+,
191 [1,4B]+, 176 [1,4B-CH3]
+, 163 [1,4B-CO]+,
161 [1,4B-2CH3]
+ (<1)
MS4: 332 (100, 15), 329 (6, 18), 317 (21, 30),
301 (8, 46), 286 (6, 61)
Hexamethoxyflavanone
5,6,7,8,3′,4′-
Hexamethoxyflavanone
19
80b 131.870 270/350 403.1368 C21H22O8
MS2: 388 (21, 15), 387 (11, 16), 374 (11, 29),
373 (100, 30), 370 (25, 33), 342 (30, 61)
MS3: 358 (77, 15), 357 (22, 16), 355 (12, 18),
344 (12, 29), 343 (54, 30), 330 (87, 43),
329 (12, 44), 328 (18, 45), 327 (100, 46),
313 (19, 60), 181 (6, [1,3A]+), 165 (58, [0,2B]+)
MS4: 299 (100, 28), 297 (47, 29), 284 (28, 43),
271 (59, 56)
Hexamethoxyflavonol Hexa-O-methylgossypetin 71
87e 135.217 270/333 403.1397 C21H22O8
MS2: 388 (29, 15), 373 (100, 30), 355 (11, 48),
342 (13, 61)
MS3: 358 (53, 15), 345 (29, 28), 329 (32, 44),
327 (100, 46), 301 (75, 72), 211 (15, [1,3A]+),
183 (15, [1,3A-CO]+)
MS4: 163 (100, [1,3B]+), 148 (23, [1,3B-CH3]
+)
Hexamethoxyflavone Nobiletin 17
91 137.810
257/270
(sh)/337/370
419.1385 C21H22O9
MS2: 404 (47, 15), 389 (100, 30), 371 (9, 48),
361 (5, 58)
MS3: 374 (17, 15), 361 (100, 28), 346 (67, 43),
328 (68, 61), 165 (5, [0,2B]+), 163 (1, [1,3A]+)
MS4: 346 (100, 15), 328 (99, 33), 315 (5, 46),
300 (13, 61)
Monohydroxy-
hexamethoxyflavonol
Natsudaidain 17
89 136.820
255/270
(sh)/340
433.1496 C22H24O9
MS2: 418 (25, 15), 403 (100, 30), 385 (17, 48)
MS3: 388 (67, 15), 373 (100, 30), 360 (62, 43),
345 (72, 58), 211 (6, [1,3A]+), 183 (2,
[1,3A-CO]+), 165 (12, [0,2B]+)
MS4: 358 (34, 15), 345 (100, 28), 327 (20, 46)
Heptamethoxyflavonol
3,5,6,7,8,3′,4′-
Heptamethoxyflavonol
17
71d 123.837
270/324
(low)
711.0893/
709.1842
C32H38O18
MS2: 405 (100, 306)
MS3: 390 (49, 15), 375 (100, 30),
MS4: 347 (100, 28), 332 (61, 43), 329 (18, 46),
314 (69, 33)
MS2: 647 (4, 62), 607 (15, 102),
565 (52, 144), 403 (100, 306)
MS3: 388 (100, 15), 373 (40, 30),
345 (44, 58), 330 (4, 73)
MS4: 373 (100, 15), 345 (1943),
330 (10, 58), 315 (2, 73)
3,5-Dihydroxy-6,7,8,3′,4′-
pentamethoxyflavonol-
3-O-[6′′-(3-hydroxy-3-
methylglutaryl)]-glucoside
69
76b 127.020 267/340
725.1004/
723.2032
C33H40O18
MS2: 419 (100, 306) MS3: 389 (100, 30),
361 (6, 58), 343 (3, 76)
MS4: 361 (100, 28), 346 (68, 43), 343 (16, 46),
330 (16, 59), 328 (85, 61), 213 (1,
[1,3A-CO]+), 165 (5, [0,2B]+)
MS2: 649 (1), 607 (4), 417 (100,
306), 403 (8), 402 (10),
345 (1)
MS3: 403 (24), 402 (100, 15),
387 (53, 30), 359 (58, 58),
344 (6)
MS4: 387 (100, 15), 359 (17, 43),
344 (7, 58)
Natsudaidain-3-O-[6′′/4′′-(3-
hydroxy-3-methylglutaryl)]-
glucoside
16
47
a Rt, retention time. P-ions (RA%, loss), the product ions, relative intensity and loss Da. Bold numbers represent the precursor ions for next stage MS. yA+, xB+ stand
for the fragment ions from the RDA cleavage in the C-ring of PMFs. Sh, shoulder; Low, low intensity; Ref., references. b Reported in Citrus plants, but not in Citri
Reticulatae Folium. c First identified in the Citrus genus. d Potential new compounds. e Retention time, UV and MSn data equal to those of reference standards.

102 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
3.1. Characterization of phenolic acids
Peaks 1, 3, 5, 6, 9, 12, 14, 15, 16 and 22 showed two
absorption maxima: one at 320–327 nm (band I) and one
at 233–242 nm (band II) with a characteristic shoulder at
285–299 nm. Peaks 4 and 8 were monitored at UVmax
302–313 nm. Comparison with literature data[20–23]
revealed that the former peaks may be caffeic or ferulic
acid derivatives, and the latter compounds were consistent
with p-coumaric acid derivatives.
In the negative ion mode, peaks 1, 3, 4, 5, 6, 8, 9, 12,
14, 15, 16 and 22 exhibited fragments at m/z 209
([aldaric acid-H]–) and m/z 191 ([aldaric acid-H-H2O]
–
in the MS2 mode, which suggests the presence of
glucaric acid or galactaric acid. Peaks 1, 3, 5, and 6
showed a deprotonated molecular ion ([M-H]–) at m/z
371.06, and cleavage of a caffeoyl unit from the
molecular ion yielded m/z 209 [M-H-162]– in the MS2
experiment. Peaks 4 and 8 showed a [M-H]– at m/z
355.07. The fragment with m/z 163 ([p-coumaric acid-H]–)
indicated the presence of a p-coumaroyl moiety in the
MS2. Peaks 9, 12, 14, 15, 16 and 22 had a molecular
ion ([M-H]–) at m/z 385.07, and the fragment ion at m/z
193 ([ferulic acid-H]–) in the MS2 spectra is indicative
of feruloyl-depside. In agreement with the preceding
data, peaks 1, 3, 5, and 6 were assigned as caffeoyl
glucarate or galactarate (isomers), peaks 4 and 8
were identified as p-coumaroyl glucarate or galactarate
(isomers), and peaks 9, 12, 14, 15, 16 and 22 were
assigned as feruloyl glucarate or galactarate (isomers).
Similar compounds have been previously found in
Citrus plants[22,24] but not in Citri Reticulatae Folium.
Peaks 7, 10, 11, 13, 17 and 21 had a negatively charged
molecular ion ([M-H]–) at m/z 313.04. The MS2 spectra
gave a base peak ion at m/z 191 ([M-H-122]–) and a
fragment ion at m/z 121 ([benzoic acid-H]–). Additionally,
further fragmentation ions of m/z 191 (Table 1) were also
found in MS2 and MS3, indicating that m/z 191 is the
aldaroyl fragment, which forms upon glucaric acid or
galactaric acid dehydratation. According to literature[16],
the compounds were tentatively identified as benzoyl
glucarate or galactarate (isomers), however, it is possible
they may potentially be novel compounds.
Peak 2 had a [M-H]– at m/z 315.0700, MS2 ions at m/z
153 ([M-H-162]–), and m/z 109 ([M-H-162-CO2]
–), which
indicates that it could be gentisoyl glucoside[25]. Peak
19 gave a [M-H]– at m/z 325.0916, MS2 ions at m/z 163
([M-H-162]–), and m/z 119 ([M-H-162-CO2]
–). This com-
pound was proposed as to be p-coumaroyl glucoside[26–28].
These two compounds have not been previously identi-
fied in other plants in the Citrus genu.
Peaks 24 and 28 had a m/z 399.09 ([M-H]–), which
produced the MS2 base peak at m/z 205 ([M-H-176-H2O]
–)
upon loss of the glucuronic acid moiety (194 Da). They
also produced fragment ions at m/z 223 ([M-H-176]–)
and m/z 193 ([M-H-176-2CH3]
–), which were consistent
with the presence of sinapic acid. Thus, peaks 24 and
28 were assigned as sinapoyl glucuronide (isomers)[26,27,29].
Peak 35 was detected at m/z 385.1115 ([M-H]–), which
produced the MS2 base peak at m/z 295([M-H-90]–) as
well as secondary peaks at m/z 265 ([M-H-120]–), m/z
325 ([M-H-60]–), and m/z 223 ([sinapic acid-H]–) upon
loss of a glucosyl residue moiety (162 Da). The fragment
ions at m/z 325, 295, and 265 suggested that the sinapic
acid was connected with the glucosyl residue by an
ester linkage (C-6‟)[26]. Based upon these arguments,
compound 35 was identified as sinapoyl-6-O-glucoside
[27,30]. This is the first instance in which these three
compounds have been found in Citrus.
Peak 18 gave a molecular ion ([M-H]–) at m/z 353.0860,
MS2 ions at m/z 191 ([quinic acid-H]–) upon loss of a
caffeoyl residue (162 Da), and m/z 179 ([caffeic acid-H]–)
with a relative abundance of 3%. By comparing to a
reference compound, this peak was identified as 5-O-
caffeoyl quinic acid (5-CQA). Peak 27 gave a [M-H]–
at m/z 337.0905, which produced the MS2 base peak at
m/z 191 ([quinic acid-H]–) upon loss of the p-coumaroyl
moiety (146 Da), and the base peak ion m/z 191, which
revealed that compound 27 should be 5-O-p-coumaroyl
quinic acid (5-pCoQA)[31], which is a known constituent
in lemon juice[32].
Peaks 20, 26, 30, 31, 34 and 40 had an absorption
maximum at ~325 nm and a shoulder at ~285 nm,
which suggested these peaks could potentially be
caffeoyl quinic acid or feruloyl quinic acid [21]. All of
these peaks had the same molecular ion ([M-H]–) at

103 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
m/z 367.10. Peak 26 showed the MS2 base peak at m/z
173 ([quinic acid-H-H2O]
–), which was caused by the
loss of a ferulic acid moiety (194 Da), in addition to
a secondary peak at m/z 193 ([ferulic acid-H]–). Further-
more, due to the peak intensity at m/z 173 (100%),
compound 26 was assigned as 4-O-feruloyl quinic
acid (4-FQA). Peaks 30 and 31 produced ions at m/z
193 ([ferulic acid-H]–) and m/z 191 ([quinic acid-H]–)
in the MS2. According to the peak intensity at m/z 193,
they were tentatively identified as 3-O-feruloyl quinic
acid (3-FQA) (isomers). Peaks 34 and 40 produced the
MS2 base peak at m/z 191 ([quinic acid-H]–) and were
thus identified as 5-O-feruloyl quinic acid (5-FQA)
(isomers). Peak 20 showed a base peak ion at m/z
193 ([ferulic acid-H]–) in the MS2 and a m/z 134
([M-H-CO2-CH3]
–) in the MS3 was observed, and
this compound was identified as 1-O-feruloyl quinic
acid (1-FQA). All of these justifications were based
on data found in the literature[33,34], and furthermore,
these compounds have been previously identified in
lemon juice[32].
3.2. Characterization of flavonoid aglycones
Peak 73 gave a [M-H]– ion at m/z 269.0423, peak 74 at
m/z 301.0726, and peak 75 at m/z 299.0563. Because these
peaks possessed the same spectral and chromatographic
properties as the reference compounds, peak 73 was
assigned as apigenin (MW = 270), 74 as hesperitin
(MW = 302), and 75 as diosmetin (MW = 300). These
compounds have been previously reported to appear in
Citri Reticulatae Folium[17,18].
Peak 29 had a [M-H]– ion at m/z 299.1470, which
produced the MS2 base peak at m/z 195 ([M-H-CH2O2-
CO-CH2O]
–), and the MS3 spectrum revealed a base peak
at m/z 177[0,2A]– and other ions at m/z 149[0,2A-CO]–, m/z
163[0,2A-CH2]
–, and m/z 165[1,3A]–. This suggested that
compound 29 could be a flavonol with a methoxy group
substitution in the A-ring. Furthermore, the MS and
UV spectral data were highly consisted with a literature
compound[35]. Peak 29 was identified as kaempferol
methylether (5 or 7-methoxykaempferol), which has not
been found in other Citrus plants.
3.3. Characterization of flavonoid mono-O-glycosides
In the negative ion mode, peaks 50, 59, 62, 63 and
64 showed a heterolytic ion Y0
– ([M-H-162]–) and a
hemolytic ion [Y0-H]
–• ([M-H-163]–) from the frag-
mentation of the deprotonated molecular ions, and
these signals were diagnostic for the identification of a
flavonoid with a 3- or 7-O-glycosyl substitution with a
relative abundance of Y0
– to [Y0-H]
–•[36–40]. In addition,
in the UV spectra, the flavonoid-3-O-glycosides band I
(330–345 nm) had a lower intensity than band II (250–
280 nm), whereas for the flavonoid-7-O-glycosides,
band I and band II have similar absorption intensities[41].
According to the previously mentioned data, peak 63
was proposed to be 7-O-glucoside (Y0
–, 100%; [Y0-H]
–•,
23%), and peaks 50, 64, 59 and 62 were identified as
3-O-glucoside (Y0
–<[Y0-H]
–•, low band I). Peak 63 had
a [M-H]– ion at m/z 461.1024, and compared to a standard
compound, this peak was assigned as diosmetin-7-O-
glucoside, which is a compound that has been identified
in bergamot peels[42]. Peak 64 gave a [M-H]– ion at m/z
477.1058, which produced the MS2 peaks at m/z 315 (Y0
–),
m/z 314 ([Y0-H]
–•), m/z 151 [1,3A]–, m/z 163 [0,2A]–, and
m/z 149 [0,2B]–. The peak was identified as isorhamnetin-
3-O-glucoside after being compared with a reference
compound. This molecule has been, which was previously
found in Citrus fruits[43]. Peak 59 showed a [M-H]– ion
at m/z 507.1087, which produced the peaks at m/z 345
(Y0
–), m/z 344 ([Y0-H]
–•), m/z 329 ([Y0-H-CH3]
–), and
m/z 314 ([Y0-H-2CH3]
–) in the MS2 and MS3 modes.
This indicates that this compound contains two methoxy
groups, and furthermore, the additional ion at m/z 181
[1,3A]– from a retro-Diels-Alder fragmentation in the
C-ring of the aglycone revealed that the A-ring and
B-ring had a methoxy group substitution, respectively.
Therefore, peak 59 was assigned as limocitrin-3-O-
glucoside in reference to the literature compound in
Citrus juices[44]. Peak 62 exhibited a [M-H]– ion at m/z
537.1185, which produced the ions at m/z 375 (Y0
–), m/z
374 ([Y0-H]
–•), m/z 359 ([Y0-H-CH3]
–), m/z 316 ([Y0-H-
2CH3-CO]
–), and m/z 302 ([Y0-H-2CH3-CO-CH2]
–•).
This suggests that this compound contains 3 methoxy
groups, and additionally, the fragment m/z 211 [1,3A]–

104 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
indicates two methoxy groups on the A-ring and one
on the B-ring. By comparing these results to data
from literature[44], this peak was identified as limocitrol
or isolimocitrol-3-O-glucoside. Peak 50 produced a
[M-H]– ion at m/z 493.0929 as well as product ions
at m/z 331 (Y0
–), m/z 330 ([Y0-H]
–•), m/z 209 [1,2A]–,
m/z 181 [1,3A]–, and m/z 166 [1,3A-CH3]
–. Similar to
the literature data[45], this compound was identified as
8-methoxyquercetin-3-O-glucoside. Compounds 50, 59
and 62 have been previously reported to appear in
Citrus but were first found in Citri Reticulatae Folium.
Peak 67 had a [M-H]– ion at m/z 463.1245, which
produced the MS2 base peak at m/z 301 (Y0
–) upon loss
of a glucosyl residue (162 Da), and the further frag-
mentation of the aglycone ion (Y0
–) was consistent with
peak 64 (hesperitin) in the MS3 mode. Thus, this peak
was identified as hesperitin-7-O-glucoside by referencing
with a standard compound, and it has been previously
found in grapefruit peels[46].
3.4. Characterization of flavonoid-O-diglycosides
Peaks 33, 43, 54 and 61 showed the characteristic
UV absorption of flavanones: the presence of band I
at 320–337 nm (sh) and band II at 281–286 nm
(maximum)[41]. In the MS2 mode, every peak showed
the aglycone ion Y0
– ([M-H-146-162]–), which likely
indicated the O-diglycoside unit, rhamnosyl-glucose.
However, the diagnostic ions [M-H-120]– and [M-H-164]–
that result from the cleavage of the rhamnosyl-glucose
moiety were not observed. This data can help distinguish
the difference between a 1→6 linkage and a 1→2 linkage
in the glycan moiety[36–40], and the glycan sequence
of the 4 compounds should be identified to a 1→6
linkage (rutinosyl residue). Peak 33 had a [M-H]– ion
at m/z 625.1680, which produced the fragments at
m/z 317 (Y0
–), m/z 125 [1,4A]–, m/z 151 [1,3A]–, and
m/z 180 [1,2A]– and, thus, it could be a flavanonol with
a methoxy group in the B-ring. In agreement with this
data, peak 33 (also detected in tangerine orange juice[47])
was tentatively identified as 3′,5,7-trihydroxy-4′-methoxy-
flavanonol-7-O-rutinoside. Peak 43 gave a [M-H]– ion
at m/z 595.1577, which produced the fragments at m/z
287 (Y0
–), m/z 151 [1,3A]–, m/z 135 [1,3B]–, and m/z 125
[1,4A]– and, in reference to a standard, was identified
as eriocitrin. Peak 54 showed a [M-H]– ion at m/z
579.1620, which produced the ions at m/z 271 (Y0
–),
m/z 151 [1,3A]–, m/z 125 [1,4A]–, and m/z 119 [1,3B]– in
the MS2 and MS3 spectras. Using a reference compound,
these results readily led to the identification of this
peak as naringenin-7-O-rutinoside (narirutin). Peak 61
revealed a [M-H]– ion at m/z 609.9259 which produced
the MS2 base peak at m/z 301 (Y0
–), and additionally,
further fragmentation of the aglycone ion (Y0
–) showed
the same fragment patterns as peak 74 (hesperitin) in
the MS3 experiment. Furthermore, by comparison to a
standard, this compound was confirmed as hesperidin.
Compounds 43, 54 and 61 have previously been
reported in literature as present in the leaves of
Citrus plants[17].
Peaks 48, 51, 57, 58, 60 and 65 showed typical frag-
ments of a rutinosyl residue (1→6 linkage) in the MS2
mode. Based on the UV spectra profile and the abun-
dance ratios of Y0
– to [Y0-H]
–•, peaks 48 and 57 were
assigned as 3-O-rutinosides, whereas peaks 51, 58, 60
and 65 were assigned as 7-O-rutinosides[36–41].
Peak 48 showed a [M-H]– ion at m/z 609.1373, which
produced fragments at m/z 301 (Y0
–), m/z 300 ([Y0-H]
–•),
m/z 151 [1,3A]–, and m/z 179 [1,2A]– and was confirmed
as quercetin-3-O-rutinoside with a standard. Peak 57
had a [M-H]– ion at m/z 593.1423, which produced peaks
at m/z 285 (Y0
–), m/z 284 ([Y0-H]
–•), m/z 163 [0,2A]–, and
m/z 151 [1,3A]– and was unambiguously identified as
kaempferol-3-O-rutinoside (nicotiflorin) upon comparison
with a reference compound. Peak 60 gave a [M-H]– ion
at m/z 623.1547, which produced the ions at m/z 315 (Y0
–),
m/z 300 ([Y0
–-CH3]
–), m/z 151 [1,3A]–, and m/z 163 [0,2A]–.
In contrast to peak 64 (isorhamnetin-3-O-glucoside),
this peak was assigned as isorhamnetin-7-O-rutinoside.
These three compounds have previously been found in
Citrus fruit juices[48].
Peak 51 exhibited a [M-H]– ion at m/z 593.1429, which
produced the peaks at m/z 285 (Y0
–) and m/z 151 [1,3A]–
and was tentatively assigned to be luteolin-7-O-rutinoside,
which has previously been detected in tropical Citrus
fruits[1]. Peak 58 showed a [M-H]– ion at m/z 577.1479,

105 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
which produced the MS2 base peak at m/z 269 (Y0
–),
and the same fragment ions as peak 73 (confirmed
as apigenin) were observed in the MS3 mode. Thus,
compound 58 was deduced as apigenin-7-O-rutinoside
(isorhoifolin). Similarly, peak 65 was identified as
diosmetin-7-O-rutinoside (diosmin) in reference to peak
75 (diosmetin). Compounds 58 and 65 have been reported
to appear in the leaves of Citrus plants[17].
3.5. Characterization of flavonoid mono-C-glycosides
Peaks 38, 39, 47, 53, and 55 showed characteristic
fragment patterns of the flavonoid mono-C-glucoside
that results from the cleavage of the glucosyl residue:
for instance, [M-H-60]–, [M-H-90]–, [M-H-120]–, and
[M-H-120-CO]–, and so on[49–51]. Peaks 38 and 39 gave
the same molecular ion ([M-H]–) at m/z 447.089 and
produced similar peaks in the MS2 and MS3 spectrums.
According to m/z 165 [1,3A]– from the RDA fragmentation
of the precursor ion [Ag+41-CO]– and to m/z 133 [1,3B]–,
they were proposed to be luteolin-6/8-C-glucoside. Upon
comparison to standards, peaks 38 and 39 were established
as luteolin-6-C-glucoside(isoorientin) and luteolin-8-C-
glucoside (orientin), respectively[10,48,52]. Peak 47 showed
a [M-H]– ion at m/z 431.0924, which produced the
fragments at m/z 165 [1,3A]– , m/z 163 [0,2A]– , and m/z
117 [1,3B]– from the aglycone residue ([Ag+41-CO]–)
in the MS4. Using a standard, we confirmed this peak
to be apigenin-6-C-glucoside (isovitexin), which has
also been described in Citrus aurantifolia leaves[53].
Peaks 53 and 55 provided the molecular ion ([M-H]–)
at m/z 461.1025 and fragments at m/z 163 [1,3A]– , m/z
135 [1,3A-CO]– , and m/z 133[1,3B-CH2]
– from [Ag+11]–;
thus, this could be diosmetin-6/8-C-glucoside. Because
of the differing HPLC retention times (compared to
peaks 38 and 39), they were assigned as diosmetin-6-C-
glucoside (peak 53) and diosmetin-8-C-glucoside (peak
55), respectively[43]. Compounds 38, 39, 53, and 55
have previously been identified in Citrus fruit juices.
3.6. Characterization of flavonoid di-C-glycosides
The characteristic fragment patterns of the flavonoid
di-C-glucoside that results from the cleavage of two
glucosyl units, [M-H-90]–, [M-H-120]–, [M-H-90-120]–,
[M-H-2×120]–, [M-H-120-CO]–, [M-H-2×120-2CO]–,
and so on, were observed for peaks 23, 25, 32, 36, and
37 in the MS spectras[49–51]. Peak 25 showed a [M-H]–
ion at m/z 609.1394 which produced peaks at m/z
177 [0,2A]– from the fragmentation of [Ag+2×41-2CO]–
and m/z 133 [1,3B]–. Based on the above ions, this peak
was identified as luteolin-6,8-di-C-glucoside[48]. Peak
32 gave a [M-H]– ion at m/z 593.1428, which produced
the ions at m/z 177 [0,2A]– and m/z 135 [1,3A-CO2]
–,
therefore, compound 32 was identified as apigenin-6,8-
di-C-glucoside, which was previously detected in Citrus
leaves[18]. Peaks 36 and 37 had a molecular ion ([M-H]–)
at m/z 623.15, which produced fragments at m/z 177
[0,2A]– and m/z 133 [1,3B-CH2]
–. Consistent with the
elution order that is given in literature [48], peak 36
was assigned as chysoeriol-6,8-di-C-glucoside and peak
37 as diosmetin-6,8-di-C-glucoside. According to the
UV spectra absorption, which showed a maximum at
285 nm and shoulder at 330 nm, peak 23 was deduced
as a flavanone. In the MSn spectrums, it showed a
[M-H]– ion at m/z 595.1592, which produced the
fragments at m/z 235 [1,3A]–, m/z 249 [1,2A]–, and m/z
209 [1,4A]– that is attributed to the fragmentation of
[Ag+2×41]–. Thus, compound 23, present in the Vicia
faba L. (Fabaceae) seed[54], was characterized as naringenin-
6,8-di-C-glucoside.
3.7. Characterization of flavonoid O,C-glycosides
Peaks 41, 42, 44, 45, 46, and 49 demonstrated the
diagnostic fragment patterns of flavonoid O,C-glycosides.
Peak 41 gave a [M-H]– ion at m/z 769.2111, which pro-
duced the MS2 base peak at m/z 461 ([M-H-146-162]–),
whereas it was absent at [M-H-120]– and [M-H-164]–,
which suggested the existance of an O-rutinosyl residue
(1→6 linkage). Moreover, the base peak at m/z 341
([M-H-146-162-120]–) and secondary peak at m/z 371
([M-H-146-162-90]–) in the MS3 led to the identification
of the peak as diosmetin-6-C-glucosyl-7-O-rutinoside,
which has been described in sweet orange peels[55].
Peaks 42 and 44 gave a molecular ion ([M-H]–) at m/z
593.14, the MS2 peaks at m/z 293 ([M-H-120-180]–),

106 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
m/z 413 ([M-H-180]–) with higher intensity (~100%),
which led to the identification of C-glucosyl-2″-O-
glucoside, m/z 473 ([M-H-120]–), and m/z 311 ([M-H-
120-162]–). In addition, there are MS3 ions at m/z
175 [1,3A]– and m/z 173 [0,2A]– resulting from the
fragmentation of the aglycone residue ([Ag+41-H2O]
–),
and both the peaks were proposed as apigenin-6/8-C-
glucosyl-2″-O-glucoside. Further, the HPLC elution order
distinguished peak 42, which was assigned to be
apigenin-6-C-glucosyl-2″-O-glucoside, from peak 44,
which was assigned to be apigenin-8-C-glucosyl-2″-O-
glucoside (peak 44). These peaks were similar to peaks
38 and 39, and these compounds corresponded to
literature compounds in the Trollius species[56]. Peaks
45 and 46 showed the same [M-H]– ion at m/z 563.13,
which generated the peaks at m/z 413 ([M-H-150]–),
m/z 443 ([M-H-120]–), m/z 431 ([M-H-132]–), and m/z
293 ([M-H-120-150]–), and is characteristiczation of
a C-glucosyl-2″-O-xyloside. Additionally, MS4 ions
at m/z 175 [1,3A]–, m/z 117 [1,3B]–, and m/z 173 [0,2A]–
derived from the fragmentation of [Ag+41-H2O] further
established the proposed assignment of compound 45
as apigenin-6-C-glucosyl-2″-O-xyloside and compound
46 as apigenin-8-C-glucosyl-2″-O-xyloside. This was
also confirmed by comparing the HPLC retention
times[18]. In the same pathway, peak 49 was identified
as apigenin-8-C-glucosyl-2″-O-rhamnoside based on
the observation of ions at m/z 577.1481 ([M-H]–), m/z
293 ([M-H-146-120-H2O]
–), m/z 413 ([M-H-164]–),
and m/z 311 ([M-H-146-120]–). This compound has
been identified in tropical Citrus fruits[1].
3.8. Characterization of (3-hydroxy-3-methylglutaryl)
glycosyl flavonoids
In this work, there were a total of seven (3-hydroxy-
3-methylglutaryl) glycosyl flavonoids that were deteceted
in Citri Reticulatae Folium: peaks 56, 66, 68, 69, 70,
71, and 76. Among them, compounds 56, 66, 68, and 76
have been previously reported to occur in Citrus plants,
Citrus fruits and peels, and Citrus molasses. Compound
69 has been found in Sphaerophysa salsula, whereas
compounds 70 and 71 are potentially previously
unidentified compounds.
The characteristic fragment ions of (3-hydroxy-3-
methylglutaryl) glycosyl flavonoids were the ions at
m/z [M-H-62]–, [M-H-102]–, and [M-H-144]– in the MS2
spectrum, which were crucial for determining whether
the flavonoid glycosides contained a 3-hydroxy-3-
methylglutaryl group[16]. Furthermore, peaks 56, 66,
68, 69, 70, 71, and 76 were deduced as 3-O-(3-hydroxy-
3-methylglutaryl) glucosyl flavonoids due to the UV
spectra profile, in addition to the abundance ratios of
Y0
– ([M-H-144-162]–) to [Y0-H]
–• ([M-H-144-163]–)[36–41],
peak 56 exhibited similar fragment ions that were
produced from the 3-O-glycosyl flavonoid unit as
peak 50 in the MSn spectras and, thus, could be
identified as 8-methoxyquercetin-3-O-[6″-(3-hydroxy-
3-methylglutaroyl)]-glucoside[45]. Peaks 66 and 68 had
the same [M-H]– ion at m/z 651.14, which generated
fragments at m/z 330 ([Y0
–-CH3]
–), m/z 315 ([Y0
–-2CH3]
–),
m/z 178 [0,2A-CH3]
–, and m/z 165 [0,2A-CO]–. In reference
to literature compounds, both were tentatively assigned
to be 5,7,4′-trihydroxy-8,3′-dimethoxyflavonol-3-O-
[2″/6″-(3-hydroxy-3-methylglutaryl)]-glucoside[57,58].
Similar to peak 64, peak 69 was assigned as isorhamnetin-
3-O-[6″-(3-hydroxy-3-methylglutaryl)]-glucoside[59].
Analogously, peak 70 was proposed as either limocitrol or
isolimocitrol-3-O-[6″-(3-hydroxy-3-methylglutaryl)]-
glucoside by comparing it with peak 62. Peaks 71 and 76
showed the characteristic fragmentation of polymethoxy
flavonoids (Table 2) and, therefore, were tentatively
identified as 3,5-dihydroxy-6,7,8,3′,4′-pentamethoxy-
flavonol-3-O-[6″-(3-hydroxy-3-methylglutaryl)]-glucoside
and natsudaidain-3-O-[6″or 4″-(3-hydroxy-3-methyl-
glutaryl)]-glucoside, respectively.
3.9． Characterization of a flavan-3-ol
Peak 52 had a quasi-molecular ion at m/z 401.1819
in the negative ion mode. The base peak ion at m/z
257 (Y0
–, [M-H-144]–), m/z 339 ([M-H-62]–), and m/z
299 ([M-H-102]–) in the MS2, and the MS3 spectrum
exhibited fragments at m/z 213 ([Y0
–-CO2]
–), m/z 155
([Y0
–-CO2-C3H6O]
–), m/z 165 [1,2A]–, m/z 137 [1,3A]–,
and so on. This allowed us to make the unambiguous

107 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
identification of a flavan-3-ol. In conjuction with the
UV spectra profile (maximum at 285 nm and shoulder
at 330 nm), this compound was tentatively assigned as
(3-hydroxy-3-methyl) glutaryl-3-O-5,7-dihydroxy-flavan-
3-ol[60] (tentative name) (Fig. 2 and 4).
3.10. Characterization of polymethoxy flavonoids
The characterized dissociation pathways of polymethoxy
flavonoids in the (+)ESI-MSn experiment have been
summarized in the literatures[48–51]. First, the majority
of the PMFs‟ precursor ions lose between one to four
methyl radicals (CH3
•), which leads to a change in the
molecular weight of 15 (CH3
•), 30 (2CH3
•), 45 (3CH3
•),
or 60 (4CH3
•), and the formation of base peak ions in
their multi-stage MS spectra. Second, the fragmentation
ions that derived from the C-ring breakage of the
PMFs skeleton, at m/z [1,3A]+, [1,3B]+, [0,2B]+, [1,4A]+, or
[0,2A]+, were frequently observed as diagnostic fragments
and were used to classify the PMFs skeleton type. Lastly,
the other dissociation pathways resulted in a molecular
weight reduction of 16 (CH4), 18 (H2O), 28 (CO), 29
(HCO•), 31 (CH4+CH3
•)/(CH3O), 33 (H2O+CH3
•), 43
(CO+CH3
•), 44 (CO2), 46 (H2O+CO), 48 (H2O+2CH3
•),
56 (2CO), 58 (CO+2CH3
•), 60 (4CH3
•), 61 (H2O+CO+CH3
•),
and 86 (2CO+2CH3
•) were always seen in the MSn
spectras. These aforementioned main product ions can
be used as the major diagnostic characteristic for PMFs.
Isosinensetin was used as an example to explain some
of the PMFs‟ fragmentation behavior (Fig. 5).
Figure 4. The fragmentation pathway of compound 52.
O
OH
HO
O
O
O OH
OH
O
OH
HO
O
O
O
OH
HO
O
O
O
OH
HO
OH
OH
HO O
O
O
OH
HO
O
OH
O
OO
OH
HO
C17H15O5
 m/z 299.1498
C21H21O8
 m/z 401.1819
C20H19O5
 m/z 339.1800
C7H5O3
 m/z 137.1037C15H13O4
 m/z 257.1387C8H5O4
 m/z 165.1239
C11H7O
 m/z 155.1081 C14H13O2
 m/z 213.1497 C6H5O2
 m/z 109.0647
C14H11O
 m/z 195.1397C13H13O
 m/z 185.1548
102 Da 62 Da
144 Da
1,2A 1,3A
H2O
H
COCO2
C3H6O
CO
H H
H
H
H
H
H
H
HH

108 Cao, G.H. et al. / J. Chin. Pharm. Sci. 2016, 25 (2), 91–110
According to the fragmentation rules presented above,
21 total polymethoxy flavonoids were tentatively
identified in Citri Reticulatae Folium in this work and
are shown in detail in Table 2.
3.11. Characterization of alkaloids.
Peaks 72 and 77 have been previously identified in
Citri Reticulatae Pericarpium[4,14]. Furthermore, based
on similar compound fragmentation patterns that were
found in the literature, peaks 72 and 77 were assigned
as citrusin III (cal. C36H53N7O9) and citrusin I (cal.
C34H53N7O9), respectively.
4. Conclusions
This work‟s most important contribution is the com-
prehensive characterization of chemical constituents
and the successful identification and characterization
of 96 compounds in Citri Reticulatae Folium (leaves of
„Fuju‟) by HPLC-DAD-ESI-MSn. Among those identi-
fied in this study, 52 compounds that were previously iden-
tified in other Citrus plants have been identified for the
first time in Citri Reticulatae Folium. 15 compounds
were characterized in the Citrus genus for the first time.
Moreover, 9 potentially new compounds were also
identified in this study. Thus, we hope that this work will
establish the scientific basis for a further investigation
of Citri Reticulatae Folium.
Acknowledgements
This work was supported by a grant from Beijing
Natural Science Foundation (Grant No. 7142088).
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HPLC-DAD-ESI-MSn技术对橘叶(福橘叶)中96个化学成分的
快速分离和鉴定
曹规划, 付庆荣, 张藏曼, 王弘*, 陈世忠*
北京大学医学部 药学院 天然药物化学系, 北京 100191
摘要: 为了对橘叶(福橘叶)中的化学成分进行较为系统地研究, 我们首次采用液质联用技术((HPLC-DAD-ESI-MSn)
系统地分离和鉴定了橘叶中的化学成分。最终, 我们在橘叶中解析了96个化学成分, 包括: 31个酚酸类化合物, 4个黄酮苷元,
6个黄酮单氧苷, 10个黄酮双氧苷, 5个黄酮单碳苷和5个黄酮双碳苷化合物, 以及6个黄酮碳氧苷, 5个3-羟基-3-甲基戊二酰
基取代的黄酮苷, 1个黄烷-3-醇和2个生物碱类化合物, 并且初步鉴定出了21个多甲基黄酮类化合物(PMFs)。在上述解
析的96个化合物中, 52个化合物为首次在橘叶中发现, 15个化合物为首次在柑橘属植物中发现。同时, 我们在橘叶中也发
现了9个潜在的新化合物。
关键词: 橘叶(福橘叶); HPLC-DAD-ESI-MSn; 黄酮碳氧苷; 多甲基黄酮类化合物