全 文 ： 2013年 1月 第 11卷 第 1期 Chin J Nat Med Jan. 2013 Vol. 11 No. 1 63

Chinese Journal of Natural Medicines 2013, 11(1): 00630070
doi: 10.3724/SP.J.1009.2013.00063
Chinese
Journal of
Natural
Medicines







Characterization of thirty-nine polymethoxylated
flavonoids (PMFs) in the branches of Murraya
paniculata by HPLC-DAD-ESI-MS/MS
ZHANG Jia-Yu1, 2, LU Jian-Qiu2, GAO Xiao-Yan2, ZHANG Qian1, LI Ning3,
TU Peng-Fei3, 4*, QIAO Yan-Jiang1*
1School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China;
2Center of Scientific Experiment, Beijing University of Chinese Medicine, Beijing 100029, China;
3State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center,
Beijing 100191, China;
4Modern Research Center of Traditional Chinese Medicines, Beijing University of Chinese Medicine, Beijing 100029, China
Available online 20 Jan. 2013
[ABSTRACT] AIM: To investigate the polymethoxylated flavonoids (PMFs) in the branches of Murraya paniculata (L.) Jack.
METHODS: A sensitive HPLC-DAD-ESI-MS/MS method was established to screen PMFs in the branches of M. paniculata based on
the analysis of six PMF standards in the positive mode by CID-MS/MS. RESULTS: The diagnostic fragmentation pathways for poly-
methoxylated flavones, polymethoxylated flavanones, polymethoxylated chalcones and PMF glycosides were summarized, respec-
tively. According to the MS fragmentation pathways, 39 PMFs, including 24 flavones, 10 flavanones or chalcones and 5 PMFs gly-
cosides were screened. CONCLUSION: The results indicated that the developed analytical method could be employed as a rapid,
effective technique for the chemical screening of PMFs in TCMs extracts.
[KEY WORDS] HPLC-DAD-ESI-MS/MS; characterization; polymethoxylated flavonoids (PMFs); Murraya paniculata
[CLC Number] R917 [Document code] A [Article ID] 1672-3651(2013)01-0063-08

1 Introduction
Murraya paniculata (L.) Jack, named Qianlixiang in
Chinese, is a traditional Chinese medicine (TCM) officially
listed in the Chinese Pharmacopoeia[1], whose dried leaves or
tender branches have been used worldwide as a folk medicine
with many strong medical effects, such as febrifuge, astrin-
gent, antidysenteric, toothache remedy, antidiarrheal and
stimulant, among others[2-3].
Previous studies indicated that polymethoxylated fla-
vonoids (PMFs) were considered to be the representative

[Received on] 11-June-2012
[Research funding] This project was supported by the Research
Platform for Quality Standard of TCMs and Information System
Building (No. 2009ZX09308-004) and National S & T Major Pro-
ject-Created Major New Drugs Projects (No. 2010ZX09502-002).
[*Corresponding author] TU Peng-Fei: Prof., Tel: 86-010-82802750.
E-mail address: pengfeitu@vip.163.com; QIAO Yan-Jiang: Prof.,
86-010-84738621. E-mail: yanjiangqiao@sina.com.
These authors have no conflict of interest to declare.
constituents of the plant[4], which possess a number of bio-
logical properties, such as anti-allergic, anti-oxidant,
anti-bacterial, anti-proliferative and anti-inflammatory activi-
ties[5-10]. A sensitive method to screen and identify 70 PMFs
in the leaves of M. paniculata was recently established in our
laboratory[11]. However, the constituents in its branches are
apparently still unknown. It was therefore important to screen
the PMFs of the branches of M. paniculata, which may pro-
vide a wider outlook on the applications of this Chinese herb.
It is well-known that some individual constituents could
not be detected owing to low abundance, co-elution and high
background of HPLC. Therefore, high-resolution chroma-
tographic methods coupled to highly sensitive and selective
detectors are needed. Mass spectrometry, especially coupled
to soft ionization-source, such as electrospray ionization (ESI),
has turned the possibility of coupling with HPLC instrument
into reality and provides rich information, including molecular
weight and structural information, on-line. Recently,
HPLC-ESI-MS and HPLC-ESI-MS/MS have become very
powerful approaches for the rapid identification of constitu-
ents in botanical extracts and the crude plant materials of
ZHANG Jia-Yu, et al. /Chinese Journal of Natural Medicines 2013, 11(1): 6370
64 Chin J Nat Med Jan. 2013 Vol. 11 No. 1 2013年 1月 第 11卷 第 1期

TCMs[12-16].
There has been no study dealing with the systematic re-
port of PMFs in the branches of M. paniculata until now.
Therefore, an HPLC-DAD-ESI-MS/MS method is described
in this paper to evaluate the constituents in the branches of M.
paniculata, which could provide evidence for its medicinal
application.
2 Experimental
2.1 Chemicals and materials
Six PMFs reference compounds, 5, 6, 7, 3,
4-pentamethoxyflavanone (10), 6-hydroxy-3, 4, 5, 2, 4,
5-hexamethoxychalcone (13), 5, 7, 8, 3, 4,
5-hexamethoxyflavone (16), 5, 7, 3, 4,
5-pentamethoxyflavone (22), 5-hydroxy-6, 7, 3, 4,
5-pentamethoxyflavone (32), 5-hydroxy-6, 7, 8, 3,
4-pentamethoxyflavone (34), were previously extracted, iso-
lated and identified from M. paniculata in our laboratory. Their
structures (shown in Fig. 1) were fully elucidated by compari-
son of their spectra data (ESI-MS and 1H, 13C NMR) with
those published literature values[17-19]. The purities of the six
PMFs standards were determined to be no less than 95% by
HPLC-UV.


Fig. 1 Structures of six PMFs reference standards isolated from Murraya paniculata

HPLC-grade acetonitrile and methanol were purchased
from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid
was purchased from Sigma Aldrich (St. Louis, MO, USA).
Ultrapure water (Wahaha, Hangzhou, China) was used
throughout the experiments. The 0.22 µm membranes used
were purchased from Xinjinghua Co. (Shanghai, China).
The branches of M. paniculata were collected from
Longzhou in Guangxi Province, China. The sample was au-
thenticated by Prof. TU Peng-fei , and a voucher specimen
deposited at the Department of Natural Medicines, Peking
University, China.
2.2 Sample preparation for analysis
Powdered dried branches of M. paniculata were weighed
accurately (0.5 g) and placed into a 50 mL flask containing 5 mL
of methanol/water (70 : 30, V/V), then the mixture was ex-
tracted in an ultrasonic bath (Eima Ultrasonics Corp., Ger-
many) at room temperature for 0.5 h. The methanol solution
was filtered through a 0.22 µm membrane, and then an ali-
quot of 10 µL of the filtrate was injected into the HPLC-MS
system for analysis.
2.3 HPLC-DAD-ESI-MS/MS analysis
The HPLC-DAD analysis was carried out on an Agilent
1100 series liquid chromatograph system (Agilent Technolo-
gies, USA), equipped with a binary pump, an auto-sampler, a
photodiode array detector and a column temperature control-
ler. The analytical column was an Agilent Zorbax Extend C18
(250 mm × 4.6 mm, i.d., 5 m) with the oven temperature
maintained at 25 ºC. 0.1% formic acid aqueous solution (V/V,
solvent A) and acetonitrile (solvent B) were used as mobile
phase for the LC separation. The elution conditions were
applied with a linear gradient as follows: 0−5 min, 20%−28%
B; 5−70 min, 28%−42% B; 70−90 min, 42%−64% B; 90−95
min, 64%−100% B. The flow rate was at 1.0 mL·min−1 and
peaks were detected at 330 nm.
For ESI-MS/MS analysis, a 6320 ion trap mass spec-
trometer (Santa Clara, CA, USA) was connected to the same
Agilent 1100 HPLC instrument via an electrospray ionization
(ESI) interface. The HPLC effluent was introduced into the
ESI source in a post-column splitting ratio of 1:3. The
ESI-MS was performed in a positive ionization mode with
source settings as follows: nebulizer gas pressure of 30.0 psi;
dry gas flow rate of 12.0 L·min−1; electrospray voltage of the
ion source of 3 000 V; capillary temperature of 350 ºC; capil-
lary exit of 121.0 V; skimmer of 40.0 V; compound stability of
50%; trap drive level of 100%; target mass of m/z 400; scan
range of m/z 100−700; AutoMS(n) operation mode; collision
energy of 1 V; SmartFrag Start Ampl of 30%, SmartFrag End
Ampl of 200%. A data-dependent program was used in the
HPLC-ESI-MSn analysis so that the protonated ions could be
selected for further MSn analysis. Nitrogen (> 99.99%) and He
(> 99.99%) were used as sheath and damping gas, respectively.
The Agilent 6300 Series Trap Control workstation (Version 6.1)
was used for the data processing.
3 Results and Discussion
3.1 Optimization of HPLC conditions
In order to obtain satisfactory extraction efficiency for
all the constituents, extraction conditions, including extrac-
tion methods, extraction solvents and extraction time were
assessed based on single factor experiments. The best extrac-
tion efficiency was obtained by ultrasonication extraction
with 70% ethanol for 30 min. Meanwhile, it was found that
the choice of detection at 330 nm could provide an optimum
S/N for most of PMFs compounds. Because the ingredients in
the sample could not be separated with isocratic elution, gra-
ZHANG Jia-Yu, et al. /Chinese Journal of Natural Medicines 2013, 11(1): 6370
2013年 1月 第 11卷 第 1期 Chin J Nat Med Jan. 2013 Vol. 11 No. 1 65

dient elution was carried out. The different HPLC parameters,
including mobile phases (methanol/water and acetoni-
trile/water), the concentration of formic acid in water (0.05%,
0.1% and 0.3%, V/V), category of RP-ODS columns (Agilent
Zorbax Eclipse SB C18 column, 250 mm × 4.6 mm i.d., 5 m;
Agilent Zorbax Extend C18, 250 mm × 4.6 mm i.d., 5 m and
Waters Symmetry Shield C18 column, 250 mm × 4.6 mm i.d.,
5 m), column temperature (20, 25 and 30 ºC) and flow rate
(0.8, 1.0 and 1.2 mL·min−1), were examined. The addition of
formic acid was advantageous to obtain the best resolution of
adjacent peaks during chromatographic separation.
3.2 Optimization of ESI-MS/MS conditions
In order to achieve optimum conditions, all factors re-
lated to MS performance, including ionization mode, nebu-
lizer gas pressure, electrospray voltage of the ion source and
collision energy, were assessed. The results showed that ESI
in positive ion mode was more sensitive for PMFs than in the
negative ion mode. The major constituents were well de-
tected, and most of the investigated compounds exhibited
quasi-molecular ions [M + H]+ and product-ions with rich
structural information in the positive mode of Colli-
sion-Induced Dissociation (CID)-MS/MS.

Table 1 Characterization of PMFs in the branches of Murraya paniculata by HPLC-DAD-ESI-MS/MS
MS2/(m/z) MS3/(m/z) MS4/(m/z)
No. tRa/min
[M + H]+
(m/z) P-ion (%, loss)b P-ion (%, loss)b P-ion (%, loss)b
1 6.21 509 347* (100, 162) 332*(100, 15) 289(100, 43), 317(28.4, 15)
2 7.04 461 299*(100, 162) 268*(100, 33), 238(78.0, 61), 269(29.7, 30) -
3 7.31 491 329*(100, 162) 314*(100, 15), 299(15.6, 30) -
4 9.26 493 331*(100, 162) 316*(100, 15) 301(100, 15), 168(73.0, RDA),298(7.9, 18)
5 17.10 521 359*(100, 162) 344(100, 15), 313(16.0, 46), 329(9.9, 30) -
6 17.39 329 314*(100, 15) 286(100, 28), 285(54.4, 29) -
7 18.42 389
328* (100, 61), 345 (29.0,
44),
374 (22.5, 15), 373(21.0, 16),
359(20.1, 30), 356(12.5, 33)
312(100, 16), 313(61.5, 15) -
8 19.93 331
181* (100, RDA), 177(43.9,
RDA),
313(7.3, 18)
121(100, 60) -
9 20.33 359 344*(100, 15), 343(55.9, 16),298(11.1, 61), 326(3.1, 33)
298(100, 46), 328(54.0, 16),
299(49.9, 45),
326(41.0, 18), 315(32.5, 29)
-
10△ 21.35 375
211*(100, RDA),191(37.9,
RDA),
357(16.1, 18)
196* (100, 15), 178(23.0, 33),
183(15.5, 28) 150(100, 46), 178(88.5, 18)
11 21.81 389 374*(100, 15), 359(87.5, 30),341(25.2, 48), 356(10.7, 33)
359(100, 15), 341(44.7, 33),
356(17.0, 18) -
12 22.90 373 343*(100, 30), 358(69.0, 15) 315(100, 28), 325(13.5, 18) -
13△ 24.14 405 221*(100, RDA), 387(31.5, 18), 211(28.2, RDA)
193*(100, 28), 190(51.7, 31),
191 (42.2, 30),
206 (31.7, 15)
163(100, 30)
14 25.85 389 374*(100, 15), 359(98.6, 30),328(20.2, 61) 359*(100,15) 288(100, 61), 341(91.0, 18)
15 27.62 343 328*(100, 15), 327(59.5, 16),299(15.1, 44)
299*(100, 29), 312(33.3,
16),298(5.8, 30),
300(5.4, 28)
284(100, 15)
16△ 28.72 403 373*(100, 30), 343(33.9, 60),388(28.4, 15)
345*(100, 28), 340(34.5, 33),
312(20.1, 61),
343(10.2, 30), 358(12.0, 15)
317(100, 28)
17 29.05 403 373*(100, 30), 342(43.3, 61),388(27.6, 15) 345(100, 28), 327(76.2, 46) -
18 30.56 373
312*(100, 61), 358(71.6, 15),
329(28.1, 44), 343(22.1, 30),
340(20.3, 33)
297*(100, 15), 269(35.1, 43),
281(23.1, 31) 175(100, RDA)
19 31.38 315 297*(100, 18), 285(70.2, 30),257(52.3,58), 255(12.9, 60)
255(100, 42), 241(65.4, 56),
187(58.4),
177(54.8), 227(48.9),
145(35.2), 203(31.6),
201(26.9), 133(26.5)
-

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66 Chin J Nat Med Jan. 2013 Vol. 11 No. 1 2013年 1月 第 11卷 第 1期

Continued
MS2/(m/z) MS3/(m/z) MS4/(m/z)
No. tRa/min
[M + H]+
(m/z) P-ion (%, loss)b P-ion (%, loss)b P-ion (%, loss)b
20 31.77 345
181*(100, RDA), 191(77.5,
RDA),
327(14.3, 18)
125(100, 56), 166(67.9, 15),
107(45.1, 74) -
21 32.29 343 313*(100, 30), 328(92.4, 15) 285(100, 28), 183(47.5, RDA) -
22△ 33.54 373 313*(100, 60), 358(23.2, 15),343(15.9, 30), 357(11.7, 16)
284*(100, 29), 283(62.4, 30),
297(3.9, 16) 268(100, 16)
23 35.07 375 221*(100, RDA), 181(18.8, RDA)
193*(100, 28), 190(72.3, 31),
206(37.0, 15),
191(11.8, 30)
163(100, 30)
24 35.68 375 211*(100, RDA) 168(100, 33), 196(72.9, 15) -
25 37.13 403
342(100, 61), 373(28.2, 30),
359(24.8, 44), 388(18.6, 15),
370*(16.9, 33), 387(16.1, 16)
327*(100, 15), 151(51.4,
RDA), 281(48.0, 61),
312*(20.3, 30), 309(17.9,
33),298(14.0, 44)
151(100, RDA), 284(38.4, 43),
281(31.1, 46), 299(29.9, 28),
312(25.5, 15), 296(21.7, 31)
26 41.01 403 373*(100, 30), 388(69.4, 15),342(11.0, 31), 355 (7.2, 18)
327*(100, 46), 358(52.8, 15),
355(36.5, 18),
345(26.8, 28),330(26.0, 43)
312(100, 15), 297(29.2, 30),
299(22.3, 28), 284(18.3, 43),
298(16.9, 29)
27 42.27 343
282*(100, 61), 328(65.2, 15),
310(18.2,33), 299*(17.9, 44),
313(12.8, 30)
254 (100, 28) -
28 48.09 433 403*(100, 30), 418(55.7, 15),417(19.8, 16), 385(12.9, 48)
388*(100, 15), 373(58.0,
30),375(41.7, 28),
387(39.7, 16),
385(23.7, 18), 360(21.2,
43),371 (20.6, 32)
360(100, 28), 345(83.6, 43),
357(72.7, 31), 206(65.3, RDA),
327(62.5, 61)
29 49.27 433 403*(100, 30), 418(62.1, 15),372(14.0, 61), 385(11.4, 48)
388*(100, 15), 357(98.8, 46),
360(70.6, 43),
342(62.6, 61), 375(57.4,
28),373(32.6, 30), 385(31.9,
18),
370(27.3, 33)
-
30 54.11 373 358*(100, 15), 343(61.9, 30),312(10.9, 61), 325(6.5, 46)
343*(100,15), 312(13.6, 46),
325(5.5, 33)
297(100, 46), 328(52.8, 15),
325(44.4, 18), 300(30.8, 43),
315(21.1, 28)
31 54.55 373 358*(100, 15), 343(57.8, 30),312(13.5, 61)
343*(100, 15), 325(11.8, 33),
312(11.7, 46),
329 (10.8, 29)
297(100, 61)
32△ 61.07 389 356*(100, 33), 328(68.4, 61),374(35.8, 15), 359(10.5, 30)
328*(100, 28), 295(8.5, 61),
341(8.0, 15) -
33 64.40 389
359*(100, 30), 341(45.2, 48),
374(42.6, 15), 356(31.4, 33),
328(16.1, 61)
344*(100, 15), 341(79.8, 18),
343(56.7, 16),
331(49.5, 28), 316(44.5, 43)
-
34△ 64.53 389
359*(100, 30), 341(43.3, 48),
374(39.5, 15), 356(23.9, 33),
328(18.6, 61)
341*(100,18), 328(62.6, 31),
329(26.5, 30),
344(20.6, 15)
326(100, 15)
35 65.91 389
359*(100, 30), 341(48.9, 48),
374(39.9, 15), 356(33.3, 33),
328(22.2, 61)
197 (100, RDA), 344 (72.5,
15) -
36 68.33 375
211*(100, RDA), 191(41.2,
RDA),
357(17.1, 18)
196(100, 15) -
37 74.95 405 221*(100, RDA), 211(29.2, RDA)
193* (100, 28), 190 (55.0,
31), 206 (37.0, 15),
191(28.9, 30), 178(12.7, 43)
107(100, 86)
38 84.86 345 181*(100), 191(70.3), 327 (10.1), 199(4.9) 122(100, 59), 124.8(39.2, 56) -
39 87.19 375 221*(100), 181(24.6), 357(9.3), 193(6.9)
193*(100, 28), 190 (58.6, 31),
206 (32.6, 15),
191 (31.4, 30)
135(100), 163(66.1),
119(55.1), 107(49.6)
atR, retention time;
bP-ion (%, loss), the product ions, the relative intensity and the loss (Da);
*Precursor-ion for next stage MS;
△Compounds identified by comparison with reference standards.

3.3 HPLC-DAD-MS/MS analysis of authentic compounds
In order to identify the structures of the constituents in M.
paniculata, six reference compounds were first analyzed by
HPLC-DAD-ESI-MS/MS techniques. According to their
ZHANG Jia-Yu, et al. /Chinese Journal of Natural Medicines 2013, 11(1): 6370
2013年 1月 第 11卷 第 1期 Chin J Nat Med Jan. 2013 Vol. 11 No. 1 67

chemical structures, UV absorption maxima and dominant
fragmentation pathways, the authentic compounds could be
classified into three groups, including polymethoxylated
flavones, flavanones and chalcones. In the full scan mass
spectra, most of PMF standards exhibited [M + H]+ ions of
sufficient abundance that could be subsequently isolated
automatically and subjected to CID-MS/MS analysis (shown
in Table 1). The proposed fragmentation patterns were help-
ful to clarify the structural identification of the constituents in
M. paniculata. The nomenclature commonly used for the
mass fragments of flavonoids was adopted in this work[20].
In the CID-MS/MS experiment, four polymethoxylated
flavone standards were analyzed first. Comparing the prod-
uct-ion spectra of the standards, some characteristic dissocia-
tion pathways could be summarized for further characteriza-
tion of the other polymethoxylated flavones. First, most of
the [M + H]+ ions of the standards, except compound 32,
could lose one to four methyl radicals (CH3·) in their MS/MS
spectra, and formed the base peaks of [M + H – n × 15]+.
However, compound 32 also eliminated both one methyl
radical and one H2O to yield the [M + H − 33]+ ion as the
base peak of MS spectrum. This fragmentation pathway
could be taken as the major diagnostic characteristic for po-
lymethoxylated flavones. Meanwhile, the other dissociation
pathways by loss of 16 (CH4), 18 (H2O), 28 (CO), 29 (HCO·),
31 (CH4 + CH3·), 33 (H2O + CH3·), 43 (CO + CH3·), 44
(CO2), 46 (H2O + CO), and 61 (CO + H2O + CH3·) were also
frequently detected as diagnostic fragments in their MS/MS
and MS/MS/MS spectra. These main product-ions mentioned
above could form the characteristic ESI-MSn “fingerprint” of
PMFs, which could be used to separate out the polymethoxy-
lated flavones from the complex extract of TCMs rapidly.
The “fingerprint” set up in the study was highly similar to the
one that was achieved by APCI-MS/MS[21]. Some diagnostic
fragment losses, such as 18, 28 and 44 amu, detected in the
product-ion spectra were also frequently reported in the
characterization of ordinary flavonoids[22].
As for polymethoxylated flavanone derivatives, com-
pound 10 gave the [M + H]+ ion at m/z 375 in the
CID-MS/MS experiment, which further generated the
prominent ion at m/z 211 as base peak in its MS/MS spec-
trum. It could be deduced that the dominating fragmentation
pathway was retro-Diels-Alder (RDA) cleavage from the
1,3-position of the C-ring. Meanwhile, the minor ion at m/z
191 was also detected, owing to the RDA fragmentation from
the 1, 4-position of the C-ring. The loss of 15 (CH3·), 18
(H2O), 28 (CO), 33 (H2O + CH3·) and 61 (CO + H2O + CH3·)
amu from the base peak at m/z 211, could be also detected as
minor fragmentation ions in the CID-MS/MS spectrum. This
kind of fragmentation pathway, namely that the [M + H]+ ion
underwent RDA reaction prior to the neutral losses of CH3·,
H2O, CO, etc., was different noticeably from ordinary fla-
vanones. Therefore, it could be adopted as a shortcut to dis-
tinguish polymethoxylated flavanones from ordinary flavones
rapidly.
Compound 13 was taken as an example to summarize
the fragmentation pathways of polymethoxylated chalcones
by the CID-MS/MS method. The RDA cleavage at bond X to
yield the base peak ion XB+ at m/z 221 and at bond Y to yield
the minor ion YA+ at m/z 211 could also be simultaneously
detected in the MS/MS spectrum first. The fragmentation
pathway was highly similar with what happened to fla-
vanones. This is reasonable because cyclization of
6-hydroxychalcones to flavanones has been reported in a
number of studies demonstrating the presence of an in-
tramolecular equilibrium between a flavanone-type and a
chalcone-type molecular ion[23-24]. At the same time, the loss
of 15 (CH3·), 16 (CH4), 18 (H2O), 28 (CO), 30 (2 CH3·) and
31 (CH4 + CH3·) amu from the base peak at m/z 221 could
also be detected. Thus, according to their fragmentation
pathways, it was easy to tell the difference between poly-
methoxylated chalcones and flavones, but difficult to distin-
guish them from polymethoxylated flavanones. However, the
differences of the UV spectra between polymethoxylated
chalcones and polymethoxylated flavanones provided a
method to classify them, because the maximum UV absorp-
tion of chalcones usually ranged from 330 to 370 nm,
whereas the flavanones maintained their UV maximum at
about 320 nm.
3.4 HPLC-DAD-MS/MS analysis of the PMFs in M. pani-
culata
The purpose of this study was to separate and evaluate
the PMFs in the branches of M. paniculata. PMFs have regu-
larity in elemental composition as they have the basic agly-
cone structure with a maximum of seven substituents, such as
methoxyl group (OCH3) and/or hydroxyl group (OH) on their
A, B and C rings. The MWs of the basic structures of agly-
cone are 222, 224 and 224 Da for flavones, flavanones and
chalcones, respectively, which are increased by 30 or 16 Da,
respectively when a methoxyl or hydroxyl was attached.
Based on the numbers and the types of the substituent groups,
the chemical formula and mass of every possible PMFs iso-
mer can be designated in advance.
Because of the complexity and the similarity of the in-
gredients in M. paniculata, EIC-MS (extracted ion chroma-
togram) method was employed to analyze the PMFs in the
plant (shown in Fig. 2 and Table 1).
In the study, the abundances of most of the unknown
peaks especially the chalcones and flavanones were too low
to afford the online UV absorption spectra, so it was difficult
to distinguish them from flavanones and chalcones. Therefore,
they were evaluated together.
Meanwhile, from the results of the full-scan of LC-MS,
some compounds with the MWs (molecular weights) be-
tween 450 and 550 had the distinct possibility to be PMF
glycosides, owing to the fragmentation pathways of their
[aglycone + H]+ ions which were similar to the diagnostic
characteristics of PMFs. In their MS/MS spectra, all of the
ZHANG Jia-Yu, et al. /Chinese Journal of Natural Medicines 2013, 11(1): 6370
68 Chin J Nat Med Jan. 2013 Vol. 11 No. 1 2013年 1月 第 11卷 第 1期

[M + H]+ ions readily eliminated the sugar moiety to produce
the corresponding [aglycone + H]+ ions as the base peak.
Neutral loss scan of 162 Da revealed the presence of hexose
moieties, such as glucose, in their molecules. Then the
[aglycone + H]+ ions were selected to trace the structural
information of the PMF aglycones. The neutral losses de-
tected in their MS/MS spectra were in accord with the “fin-
gerprint” of the corresponding polymethoxylated flavones,
demonstrating they were probable PMF glycosides present in
M. paniculata. The study provided significant information for
the phytochemical research on the branches of M. paniculata
and the plants of the genus Murraya in general.
After screening the molecular masses with the EIC-MS
method, 39 PMFs, including 24 flavones (4 known), 10 fla-
vanones or chalcones (2 known) and 5 PMF glycosides were
tentatively identified (as shown in Table 2). Some EIC-MS
peaks were too weak to be seen clearly in the total ion chro-
matogram (TIC) spectra. Meanwhile, the retention times of
some EIC-MS peaks were so similar that they could not be
identified simultaneously in the TIC spectra. Thus, the
EIC-MS method adopted in this study was confirmed to be a
powerful method to evaluate the ingredients preliminarily in
highly complex extracts of TCMs, and other medicinal
plants.



Fig. 2 The EIC-MS peaks of all possible PMFs in the branches of Murraya paniculata. (A) m/z 521, 509, 491, 433, 389, 329, 315;
(B) m/z 493, 461, 375, 359, 345, 331; (C) m/z 405, 403, 373, 343.
ZHANG Jia-Yu, et al. /Chinese Journal of Natural Medicines 2013, 11(1): 6370
2013年 1月 第 11卷 第 1期 Chin J Nat Med Jan. 2013 Vol. 11 No. 1 69


Table 2 The MWs and structural identification of all possible PMFs detected in the branches of Murraya paniculata
Peaks Number PMFs No. of -OCH3 No. of -OH MW
1 1 Tetrahydroxy-dihydroxyflavone glycoside 2 4 508
2 1 Monohydroxy-dimethoxyflavone glycoside 2 1 460
3 1 Monohydroxy-trimethoxyflavone glycoside 3 1 490
4 1 Trihydroxy-dimethoxyflavone glycoside 2 3 492
5 1 Monohydroxy-tetramethoxyflavone glycoside 4 1 520
6 1 Monohydroxy-trimethoxyflavone 3 1 328
7, 11, 14, 32-35 7 Monohydroxy-pentamethoxyflavone 5 1 388
8 1 Monohydroxy-trimethoxyflavanone or Monohy-droxy-trimethoxychalcone 3 1 330
9 1 Monohydroxy-trimethoxyflavone 3 1 358
10, 23, 24, 36, 39 5 Pentamethoxyflavanone or Pentamethoxychalcone 5 0 374
12, 18, 22, 30, 31 5 Pentamethoxyflavone 5 0 372
13, 37 2 Hexamethoxyflavanone or Hexamethoxychalcone 6 0 404
15, 21, 27 3 Tetramethoxyflavone 4 0 342
16, 17, 25, 26 4 Hexamethoxyflavone 6 0 402
19 1 Dihydroxy-dimethoxyflavone 2 2 314
20, 38 2 Tetramethoxyflavanone or Tetramethoxychalcone 4 0 344
28, 29 2 Heptamethoxyflavone 7 0 432

4 Conclusions
A sensitive HPLC-DAD-ESI-MS/MS method was estab-
lished to screen the PMFs present in the branches of M. pa-
niculata. Six PMF standards, including four flavones, one
flavanone and one chalcone, were analyzed by CID-MS/MS
first to obtain the respective characterizations of the fragment
pathways, which could be adopted as the basis for further
analysis the PMFs in the extract. Meanwhile, owing to regu-
larities of PMFs in elemental composition, the EIC-MS
method by MWs was employed to screen the homoeomor-
phic PMFs from the extract. In the end, 34 PMFs and five
PMF glycosides were screened preliminarily. Among them,
six PMFs could be unambiguously identified by comparison
with reference substances. The results indicated that the de-
veloped analytical method could be employed as a rapid,
effective technique for the structural characterization of
PMFs in complex mixtures.
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千里香枝中 39个多甲氧基黄酮的 HPLC-DAD-ESI-MS/MS分析
张加余 1, 2, 卢建秋 2, 高晓燕 2, 张 倩 1, 李 宁 3, 屠鹏飞 3, 4*, 乔延江 1*
1北京中医药大学中药学院, 北京 100102;
2北京中医药大学科研实验中心, 北京 100029;
3北京大学天然药物及仿生药物国家重点实验室, 北京 100191;
4北京中医药大学中药现代研究中心, 北京 100029
【摘 要】 目的：定性分析千里香枝中的多甲氧基黄酮类成分。方法：建立一种灵敏的 HPLC-DAD-ESI-MS/MS 方法，筛
选千里香枝中的多甲氧基黄酮。结果：分别研究总结了多甲氧黄酮、二氢黄酮、查耳酮以及多甲氧基黄酮苷的诊断性断裂途径。
结合这些特征和 EIC-MS/MS实验，筛选了 39个多甲氧基黄酮类成分，包括 24个多甲氧基黄酮、10个多甲氧基二氢黄酮或查
耳酮、5个多甲氧基黄酮苷。结论：本研究为从复杂物质体系中筛选多甲氧基黄酮提供了一种快速有效的方法。
【关键词】 HPLC-DAD-ESI-MS/MS; 定性鉴定; 多甲氧基黄酮; 千里香枝

【基金项目】 中药材质量标准研究平台(No. 2009ZX09308-004); 国家“重大新药创制”科技重大专项(No. 2010ZX09502-002)资
助项目