全 文 ：Vol． 5 No． 3，Sept． 2014 Journal of Measurement Science and Instrumentation Sum No． 19
Ｒapid analysis and identification for the alkaloids from
Ｒanunculus japonicus by UPLC /Q-TOF -MS
＊
ZHONG Yan-mei1，FENG Yi-fan1，TAN Yu-zhi2
(1． Central Laboratory，Guangdong Pharmaceutical University，Guangzhou 510006，China;
2． Department of Pharmacology，Guangdong Pharmaceutical University，Guangzhou 510006，China)
Abstract:To establish a rapid and effective method for analysis and identification of the alkaloids from Ｒanunculus japonicus
Thunb by ultra-performance liquid chromatogaraphy with quadruple-time-of-flight mass spectrometry (UPLC /Q-TOF-MS)and dis-
cuss their fragmentation regularity，the UPLC /Q-TOF-MS was used to identify the alkaloids from Ｒanunculus japonicus Thunb by
their MS data，tandem characteristic fragment ions and standards． In the end，12 alkaloids were identified from Ｒanunculus japoni-
cus for the first time，and their fragmentation regularity was discussed． Thus，a rapid and effective analysis and identification meth-
od for the alkaloids from Ｒanunculus japonicus by UPLC /Q-TOF-MS is established．
Key words:alkaloids;ultra-performance liquid chromatogaraphy with quadruple-time-of-flight mass spectrometry (UPLC /Q-TOF-
MS);Ｒanunculus japonicas
CLD number:Ｒ284． 1 Document code:A
Article ID:1674-8042(2014)03-0087-07 doi:10． 3969 / j． issn． 1674-8042． 2014． 03． 017
0 Introduction
In recent years，with the proposing of material foun-
dation of modern Chinese medicine，finer expression
and analysis of total chemical characteristics of material
foundation of Traditional Chinese Herbal Medicines are
required［1-2］． An effective and efficient analytical tech-
nology for the extraction and isolation of active compo-
nents in herbal plants or extracts is an important area of
research in the current push towards the modernization
of traditional Chinese medicine (TCM)［3-4］． Compared
with the recent trend of development，such as silica gel
column chromatography method，gel-filtration chroma-
tography method and high performance liquid chroma-
tography method(HPLC)，the traditional analysis and
separation method used by chemical research on TCM
seems to be time-wasting，purposeless，steps-tedious，
reagent and sample wasting． It cannot find out some
compounds which cannot be found by traditional separa-
tion method，thus they may be left out;Or，even some
compounds can be obtained by aimless and complicated
extracting and isolating，but they are meaningless to re-
search［5-6］．
Compared with the traditional separation method，the
combination technique of ultra-performance liquid chro-
matography with quadruple-time-of-flight mass spec-
trometry (UPLC /Q-TOF-MS) has the advantages of
sensitivity in separation，accuracy in mass measurement
and tandem mass spectrometry． It is a rapider and more
effective method to analyze and identify the composi-
tions from TCM［7］．
Ｒanunculus japonicas Thunb (Ｒanunculaceae) is a
widely spread perennial herb in China． It is used as Chi-
nese fork medicine in the treatment of jaundice，tubercu-
losis and hepatitis［8］． It has pharmacological activities ，
such as antichloristic，anti-inflammatory，antibacterial
and antitumor［9］． However，up to now，what the corre-
sponding constituents are has not been clear． There are
only a few literatures on its chemical constituents［5，8］，
which indicated that flavoneglycosides，lactone glyco-
sides and flavonoids were the main constituents in it．
There are few studies on the alkaloids constituents from
it． Alkaloids are the effective constituents of most TCM
for pharmacological activities，such as antimicrobial ac-
tivity and anti-inflammatory［1-2，10］． Therefore， it is
meaningful to study in this field．
In this study，we used the combination technique of
UPLC /Q-TOF-MS to analyze the alkaloids from Ｒanun-
culus japonicus in order to discover some active alka-
loids in Ｒanunculus japonicas for further research，espe-
cially for searching some new compositions． As a re-
sult，12 alkaloids were identified in Ｒanunculus japoni-
cus，which is a good attempt to analyze and identify the
composition from TCM．
*Ｒeceived date:2014-05-28
Corresponding author:FENG Yi-fan (tfengyf@ 163． com)
1 Experiment
1 ． 1 Materials and chemicals
Acetonitrile (HPLC grade)was purchased from Mer-
ck (Germany)． Formic acid was purchased from DIMA
(USA)． Water was purified by an Arium 611UV sys-
tem (Satorius，Germany)． Other solvents and chemi-
cals were bought from Guangzhou Chemical Agent Fac-
tory (Guangzhou，China)． The standards of berberine，
coptisine，palmatine and jatrorrhizine were purchased
from National Institutes for Food and Drug Control
(China)．
1 ． 2 Plant materials and alkaloids extrac-
tion from Ｒanunculus japonicas
Ｒanunculus japonicus was collected from Jiujiang
(Jiangxi province，China)and preserved in the depart-
ment of pharmacology of Guang Dong Pharmaceutical
University (991231)． The herb was identified by LIU
Jin-zhou of Guangdong Pharmaceutical University． The
air-dried and cut bodies of Ｒanunculus japonicus were
extracted with 85% ethanol under reflux thrice． The so-
lutions were mixed，filtered，and then were evaporated
under vacuum to obtain an EtOH extract． 25 g EtOH ex-
tract of was dissolved with 20 mL water，and then 36%
HCl was added till the solution reached pH2． The solu-
tion was kept over night at 4 ℃ to obtain the acidic solu-
tion． It was extracted with equivalent volume petroleum
ether three times，and the petroleum ether layer was dis-
carded． The acidic solution was filtered，extracted it
four times with 50 mL of chloroform after alkalinisation
with ammonia (pH10)． The chloroform layers were
washed with distilled water，filtered and evaporated un-
der reduced pressure to get the alkaloids extract．
1 ． 3 Sample and standard preparation
The alkaloids extract was dissolved in methanol and
filtered through 0． 22 μm membranes for analysis．
Standard solutions of berberine，coptisine，palmatine
and jatrorrhizine were respectively prepared as follows:
dissolved approximate 10 mg of each accurately weigh-
ted standard compound in 10 mL methanol，precisely
absorbed 1 mL of each standard solution 1 mL，mixed
and filtered them through 0． 22 μm membranes for anal-
ysis．
1 ． 4 Instruments and method
Waters Acquity Ultra Performance LC system (Wa-
ters Co．，USA)was equipped with binary solvent man-
ager， autosampler manager， tunable UV detector
(TUV)． The chromatographic conditions were as fol-
lows:Waters UPLC BEH C18 column (1． 7 μm，50
mm × 2． 1 mm) ;mobile phase:a gradient elution sys-
tem of 0． 1% formic acid aqueous solution (A)and
acetonitrile (B) ，0 － 2 min，98% －75%A，2 － 9 min，
75%A，9 － 10 min，75% －55%A，10 － 12 min，55%
－30%A，12 － 17 min，50% － 0% A;flow rate:0． 2
mL /min;column temperature:30 ℃; injection vol-
ume:5 μL，detecting wavelength was 270 nm． Waters
micro mass Q-TOF micro system (Waters Co．，UK)
was equipped with the Lock Spray and electrospray ioni-
zation (ESI) interface operating in both positive ion
mode and negative ion mode． The source parameters
were electrospray capillary voltage 3． 0 kV both for posi-
tive and negative ion modes;source temperature is set at
100℃ and desolation temperature is set at 350 ℃ ． The
cone voltage was set at 15 V for positive ion mode and
20 V for negative ion mode． Nitrogen and argon were
used for cone and collision gases． The cone and desol-
vation gas flows were 60 L /h and 600 L /h，respective-
ly． The TOF mass spectrometer was calibrated routinely
in the positive electrospray ionization (ESI +)mode u-
sing the solution of sodium formate． Data were collected
from m /z 10 to 1 000 in continuum mode，using inde-
pendent reference lock-mass ions via the Lock Spray in-
terface． The solution of chloramphenicol (Sigma Chem-
ical Co．)was used as lock-mass，with an［M + Na］+
ion of m /z 345． 002 1 and［M + H］－ ion of m /z 321．
004 5． The MS /MS experiments were performed using
a variable collision energy (15 － 60 eV) ，which was
optimized for each individual compound． The Lock
Spray frequency was set at 10 s． The data were pro-
cessed using MassLynx 4． 1 software．
2 Ｒesults and discussion
2． 1 TOF-MS analysis
The base peak ion (BPI)chromatography in positive
ion mode (Fig． 1(a) )and the UV chromatography
(Fig． 1(b) )of the compounds from Ｒanunculus japoni-
cus indicated that each compound of the sample was sep-
arated in 15 min in the chromatogram by UPLC /Q-TOF-
MS．
The compounds of the sample have better sensitivity
in positive ion mode than in negative mode，therefore，
positive ion mode was chosen to analyze the sample． To
get better resolution and ionization，0． 1% formic acid
was added in the mobile phase．
The compounds of the sample appeared mainly in two
groups，compounds 1 － 8 in the front of the chromatog-
raphy，and compounds 9 － 14 in the back of the chroma-
tography (Fig． 2)． The peaks 1 － 8 were very close to
each other，suggesting that these compounds may have
similar structures．
88 Journal of Measurement Science and Instrumentation Vol． 5 No． 3，Sept． 2014
Fig． 1 Chromatogram of Ｒanunculus japonicus sample by UPLC/Q-TOF-MS
Fig． 2 Structure of compounds identified by UPLC-Q-TOF-MS
Each peak of the sample in chromatography was se-
lected to process in TOF-MS analysis． The data were
processed using MassLynx 4． 1 software． The measured
accurate mass of each compound molecular ion was giv-
en． The software in TOF-MS is capable of calculating
all possible molecular formula satisfying the observed
data． In TOF-MS，mass error tolerance can be set at 5
× 10 －6 besides mass measurement． The isotopic mass
distributions also provide information about the composi-
tion of elements． The empirical formula obtained from
the measured accurate mass was used to identify formu-
las of the compounds，as shown in Table 1．
For example，the measured accurate mass of com-
pound 8 was 336． 122 7 (see Fig． 3) ，and the empirical
formula obtained from the measured accurate mass was
C20H18NO4，with the － 2． 7 ppm of the calculated mass
336． 123 6，which agreed with Ｒefs．［3］and［10-13］．
By searching in the database of scifinder and chemspi-
der，the substance corresponding to the formula of［C20
H18 NO4］
+ was berberine． Consequently compound 4
98ZHONG Yan-mei，et al． / Ｒapid analysis and identification for the alkaloids from Ｒanunculus japonicus． ． ．
was preliminarily identified as berberine． The further i-
dentification research will be carried out in the next
MS /MS experiment and the standards control experi-
ment．
By indexing the empirical formula in scifinder data-
base，compounds 1，3 － 8 were initially identified as
thalifendine，jatrorrhizine，epiberberine，coptisine，o-
methylcorypalline，palmatine and berberine，which were
proberberine quaternary ammonium alkaloids［7，10-11，13］．
It was found that accurate mass of compounds 12，
14，9 and 15 were approximately 15 Da more than those
of compounds 3，5，7 and 8． According to the elemen-
tal composition operated by the MassLynx 4． 1 software，
we deduced that compounds 9，11，12，14，and 15
were 8-oxopalmatine，8-oxoEpiberberine，8-oxojatrorrh-
izine，8-oxocoptisine and 8-oxoberberine，which were
8-oxoproberberine quaternary ammonium alkaloids［13-14］．
Table 1 (+)ESI MS data and identification of the alkaloids from Ｒanunculus japonicus
Peak
NO．
TＲ
(min)
Identification Selected ion Empiricalformula
Calculated
mass (m /z) (+)ESI MS /MS
1 3． 48 thalifendine
［M］〗+ +
［M + H］+
［C19H16NO4］+
［C19H17NO4］+
322． 107 9
323． 115 8
322［M］+，307［M － CH3］+，292［M － 2CH3］+，279［M
－ CH3 － CO］+，278［M － CH3 － CO － H］+
2 3． 73 uncertain ［M］+ ［C11H18NO2］+ 196． 133 8 197［M］+ 146［M －51］+，133［M －146］+
3 3． 90 jatrorrhizine ［M］
+
［M + H］+
［C20H20NO4］+
［C20H21NO4］+
338． 139 2
339． 147 1
338［M］+，322［M － CH4］+，308［M － 2CH3］+，294［M
－ CH4 － CO］+，280［M － 2CH3 － CO］+，265［M － CH3
－ CO － CH2O］+，250［M －2CH3 － CO － CH2O］+
4 3． 97 epiberberine ［M］+ ［C20H18NO4］+ 336． 123 0
336［M］+，320［M － CH4］+，292［M － CH4 － CO］+，
263［M － CH3 － CH2O － CO］+
5 4． 17 coptisine ［M］+ ［C19H14NO4］+ 320． 092 3
320［M］+，292［M － CO］+，262［M － CO － CH2O］+，
234［M －2CO － CH2O］+，204［M －2CO － 2CH2O］+
6 4． 44 O-methylcorypalline ［M］+ ［C12H17NO2］+ 207． 125 9
207［M］+，208［M + H］+，192［M － CH3］+，191［M －
CH4］+，163［M － CH4 － CO］+，164［M + H － CH4 －
CO］+
7 4． 91 palmatine ［M］+ ［C21H22NO4］+ 352． 154 9
352［M］+，337［M － CH3］+，336［M － CH4］+，322［M
－2CH3］+，308［M － CH4 － CO］+，294［M － 2CH3 －
CO］+，279［M － CH3 － CO － CH2O］+
8 5． 22 berberine ［M］+ ［C20H18NO4］+ 336． 122 7
336［M］+ 321［M-CH3］+，320［M － CH4］+，306［M －
2CH3］+，292［M － CH4 － CO］+，278［M － 2CH3 －
CO］+，262［M － CH4 － CO － CH2O］+，234［M － CH4 －
2CO － CH2O］+
9 11． 54 8-oxopalmatine ［M］+ ［C21H21NO5］+ 367． 142 0
368［M］+，353［M － CH3］+，338［M － 2CH3］+，324［M
－ CH4 － CO］+
10 12． 43 uncertain ［M］+ ［C18H38NO3］+ 316． 285 2
316［M］+，298［M －18］+，280［M － H2O］+，250［M －
66］+
11 12． 62 8-oxoepiberberine ［M］
+
［M + H］+
［C20H17NO5］+
［C20H18NO5］+
351． 110 7
352． 118 5
352［M + H］+ 336［M + CH4〗+，322［M － 2H － CO］+，
309［M + H － CH3 － CO］+，256［M-96］+
12 12． 73 8-oxojatrorrhizine ［M］
+
［M + H］+
［C20H19NO5］+
［C20H20NO5］+
353． 126 3
354． 134 1
353［M］+，354［M + H］+，339［M － CH3］+，324［M －
2CH3］+，310［M － CO － CH3］+
13 13． 28 8-oxocoptisine ［M］+ ［C19H13NO5］+ 335． 195 2
335［M］+，336［M + H］+，308［M + H － CO］+，293［M
+ H － CO － CH3］+
14 14． 01 8-oxoberberine ［M］
+
［M + H］+
［C20H17NO5］+
［C20H18NO5］+
351． 110 7
352． 118 5
352［M + H］+，337［M + H － CH3］+，322［M + H －
2CH3］+，309［M + H － CH3 － CO］+
15 14． 98 uncertain ［M］+ ［C31H46NO4］+ 496． 342 7
497［M + H］+，478［M － H2O］+，419［M － 77］+，327
［M －69］+
09 Journal of Measurement Science and Instrumentation Vol． 5 No． 3，Sept． 2014
Fig． 3 MS spectrum and the empirical formula analysis of compound 4
2 ． 2 MS /MS analysis
The structures of the detected compounds were de-
duced by their MS /MS fragment ions，which is the veri-
fication for the results of TOF-MS．
In the tandem mass，it was found that the collision
energy and the cone voltage had a strong effect on the
tandem mass spectrum． The collision energy (CE)is
the most effective parameter for MS /MS of the alkaloids
from Ｒanunculus japonicus． The value of CE was evalu-
ated from 15 eV to 40 eV to obtain adequate characteris-
tic fragmentations for the most compounds of the alka-
loids from Ｒanunculus japonicus． The cone voltage was
evaluated at 15，20，25 and 30 V． As a result，the cone
voltage at 15 V in the positive ion mode was applied for
minimal fragment and higher sensitivity．
The considerable fragmentation information obtained
by MS /MS is very useful for the structure elucidation．
According to the MS /MS data，it was found that the i-
ons ［M-15］+ ( ［M-CH3］
+ ) ， ［M-30］+ ( ［M-
2CH3］
+) ，［M-44］+( ［M-CH4-CO］
+) ，［M-58］+
( ［M-2CH3-CO］
+)were the characteristic ions of the
compounds［12，16-19］(see Table 1)． Combined with the
result of TOF-MS analysis，these ions may be produced
by the elimination of the substituent groups of the com-
pounds that were identified as protoberberine alkaloids
and 8-oxoprotoberberine alkaloids． In the experiment，
there were very low abundance product-ions obtained
from ＲDA reaction which corresponds to the skeleton of
six-membered rings of quaternary alkaloids． The results
were very agreeable with Ｒefs．［12］ and［16-19］． As
an example，clearage pathways of compounds 3，7，and
8 were shown in Fig． 4．
Taking compound 7 (m /z 352)as an example，the
predominant MS /MS ions of compound 7 includes ions
at m /z 337，m /z 336，m /z 338，m /z 322，m /z 308
and m /z 294(see Fig． 3)． The fragment ion at m /z 337
was obtained from the loss of -CH3 ． There are three
methyl groups in compound 7，but this de-methyl reac-
tion clearagemay occurred in the predominate position 1
(see Fig． 5)． Because the three methyl groups were in
different chemical environment． This α-cleavage bonds
with oxygen was affected by the electronic effects of N
in ring A． Accordingly，position 1 is the advantage po-
sition． Then the ion at m /z 322 was observed for the
loss of two methyl radicals． The ions at m /z 336 and
m /z 322 had unstable structure such as two carbonyl
groups in a six member ring and -O-C-O- group in five
member rings;with the effect of collision energy，the
loss of -CO reached the steady state． The ion at m /z
308 was formed by the loss of -CO fragment from the
ion at m /z 336;and the ion at m /z 294 was generated
as the loss of -CO fragment from the ion at m /z 322．
All of them are in agreement with the literatures［3，11］．
Meanwhile，the compounds 9，12，and 14 have similar
cleavage pathways to compounds 3，7 and 8［15］．
Fig． 4 MS /MS spectrum of compound 3
19ZHONG Yan-mei，et al． / Ｒapid analysis and identification for the alkaloids from Ｒanunculus japonicus． ． ．
Fig． 5 Cleavage pathways of compounds 3，7 and 8 in positive ion mode
The characteristic ions of the MS /MS spectrum and
their potential clearage pathways proved that most of
these compounds were identified as protoberberine alka-
loids and 8-oxoprotoberberine alkaloids (see Fig． 1 and
Table 1)．
2 ． 3 Authentication by reference sub-
stance compounds
We adopted some standard reference substance com-
pounds to authenticate the result above． The standard
reference substance compounds were presented as fol-
lows． The peaks in the Ｒanunculus japonicus sample
chromatography at 3． 90 min，4． 17 min，5． 22 min，4．
91 min (compounds 3，5，7，and 8)were confirmed as
jatrorrhizine，coptisine，palmatine， and berberine by
standard compounds comparison respectively． It is evi-
dence that confirmed the accuracy of analysis result by
TOF-MS and MS /MS．
3 Conclusion
In this study，a rapid and sensitive UPLC-Q-TOF-MS
analysis method has been established to identify alkloids
from Ｒanunculus Japonicus． Twelve alkaloids including
seven proberberine quaternary ammonium alkaloids and
five 8-oxoproberberine quaternary ammonium alkaloids
were first identified from Ｒanunculus japonicus by UP-
LC-Q-TOF-MS，all of which have not been isolated pre-
viously from Ｒanunculus japonicus．
In this research，the fragmentation regularity of alka-
loids from Ｒanunculus japonicus was discussed． It indi-
cated that the characteristic ions in their MS /MS data
were mainly generated by the loss of small groups such
as methyl radicals，CO． The ions obtained from ＲDA
reaction were not obvious． These results showed the
fragmentation behaviour conformed to the quaternary
ammonium alkaloids and 8-oxoproberberine quaternary
ammonium alkaloids．
In this study，traditional steps-tedious isolation and
separation was not employed． It will be a good attempt
to discover new alkaloids using modern combination
technique (UPLC /Q-TOF-MS)from TCM． This tech-
nique may expand our analysis research of TCM for
more significant in the future．
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基于 UPLC /Q-TOF-MS 技术的毛茛生物碱类
成分快速分析鉴定
钟艳梅1，冯毅凡1，谭毓治2
(1． 广东药学院 中心实验室，广东 广州 510006;2． 广东药学院 药理学教研室，广东 广州 510006)
摘 要: 应用液质联用技术 UPLC /Q-TOF-MS 建立一种有效快速分析鉴定毛茛生物碱类成分的方法并研
究其质谱裂解规律。采用液质联用技术 UPLC /Q-TOF-MS，根据毛茛生物碱类成分的一级质谱数据，二级特
征裂解碎片及标准品比对鉴定毛茛生物碱类成分。共鉴定了 12 种生物碱类成分，并探讨了其裂解规律。首
次从毛茛中分离鉴定了 12 种生物碱类物质，建立了一种有效的基于 UPLC /Q-TOF-MS 鉴定毛茛生物碱类成
分的方法。
关键词: 生物碱;超高效液相色谱-四级杆-飞行时间串联质谱技术 (UPLC /Q-TOF-MS);毛茛
引用格式: ZHONG Yan-mei，FENG Yi-fan，TAN Yu-zhi． Ｒapid analysis and identification for the alkaloids
from Ｒanunculus japonicus by UPLC /Q-TOF-MS． Journal of Measurement Science and Instrumenta-
tion，2014，5(3):87-93． ［doi:10． 3969 / j． issn． 1674-8042． 2014． 03． 017］
39ZHONG Yan-mei，et al． / Ｒapid analysis and identification for the alkaloids from Ｒanunculus japonicus ． ． ．