Journal of Chromatography A, 1452 (2016) 4757

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

A strategy for identication and structural characterization of

compounds from Gardenia jasminoides by integrating macroporous
resin column chromatography and liquid chromatography-tandem
mass spectrometry combined with ion-mobility spectrometry
Lu Wang a,b , Shu Liu a , Xueju Zhang a,b , Junpeng Xing a , Zhiqiang Liu a , Fengrui Song a,
a
National Center of Mass Spectrometry in Changchun, Jilin Province Key Laboratory of Chinese Medicine Chemistry and Mass Spectrometry, Changchun
Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
b
University of Chinese Academy of Sciences, Beijing 100039, China

a r t i c l e

i n f o

Article history:
Received 16 September 2015
Received in revised form 9 April 2016
Accepted 5 May 2016
Available online 7 May 2016
Keywords:
Gardenia jasminoides Ellis
LCMS/MS
Ion mobility spectrometry
Macroporous resin

a b s t r a c t
In this paper, an analysis strategy integrating macroporous resin (AB-8) column chromatography and
high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLCESI-MS/MS) combined with ion mobility spectrometry (IMS) was proposed and applied for identication
and structural characterization of compounds from the fruits of Gardenia jasminoides. The extracts of G.
jasminoides were separated by AB-8 resin column chromatography combined with reversed phase liquid chromatography (C18 column) and detected by electrospray ionization tandem mass spectrometry.
Additionally, ion mobility spectrometry (IMS) was employed as a supplementary separation technique to
discover previously undetected isomers from the fruits of G. jasminoides. A total of 71 compounds, including iridoids, avonoids, triterpenes, monoterpenoids, carotenoids and phenolic acids were identied by
the characteristic high resolution mass spectrometry and the ESI-MS/MS fragmentations. In conclusion,
the IMS-MS technique achieved the separation of isomers in crocin-3 and crocin-4 according to their
acquired mobility drift times differing from classical analysis by mass spectrometry. The proposed strategy can be used as a highly sensitive and efcient procedure for identication and separation isomeric
components in extracts of herbal medicines.
2016 Elsevier B.V. All rights reserved.

1. Introduction
The Gardenia jasminoides Ellis (G. jasminoides) as traditional Chinese medicines (TCMs) has been used as an antiphlogistic, diuretic,
hemostatic, laxative and choleretic [1]. Geniposide is the main component in G. jasminoides and genipin is its aglycone. Zhang et al.
[2] proved that genipin inhibited uncoupling protein 2 (UCP2)mediated proton leak and consequently improved type 2 diabetes
in mice. Besides geniposide, previous researches of G. jasminoides
revealed the presence of a variety of terpenoids, including iridoids
and their glycosides, monocyclic monoterpenoids and their glycosides, triterpenoids, crocetin and its glycosides, quinic acids and
their derivatives and avonoids [3]. Some of the minor constituents
have been reported to exhibit various activities [3]. Comprehensive identication and structural characterization of the chemical

Corresponding author.
E-mail address: songfr@ciac.ac.cn (F. Song).
http://dx.doi.org/10.1016/j.chroma.2016.05.026
0021-9673/ 2016 Elsevier B.V. All rights reserved.

components from the fruits of G. jasminoides is important for revealing the material basis of its therapeutic effects.
Liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) is a rapid and sensitive alternative approach for
identication of TCMs rather than NMR need sufcient quantity of
an isolated component [4]. The combination of LC and quadrupole
time-of-ight mass spectrometry (Q-TOF) technique, in particular,
can give high resolution mass data thus provide the basis for structural characterization which make it suitable to analyze complex
extracts from Traditional Chinese Medicine. Unfortunately, LC analysis alone is not sufcient to separate all constituents and minor
but signicant constituents are often overlapped [5]. In light of
this situation, a further separation step is warranted. Column fractionation or two-dimensional chromatography followed by LCMS
analysis has been used to characterize minor compounds from
herbal medicines [6,7]. It integrated different separation procedures and improved chromatographic peak capacity effectively [8].
Hence, a strategy integrating AB-8 resin column chromatography
and liquid chromatography-tandem mass spectrometry analysis

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L. Wang et al. / J. Chromatogr. A 1452 (2016) 4757

was proposed to prole chemical constituents of herbal medicines.

G. jasminoides was taken as an example and the strategy could be
applied to other extracts of herbal medicines.
Ion mobility spectrometry (IMS) is an analytical technique that
separates gas-phase ions based on their size and shape, analogous
to electrophoresis in the condensed phase [9]. Mass spectrometry (MS) is also used in the analysis of gas-phase ions on the
basis of their mass-to-charge (m/z) ratios. Compared with MS, the
major benet of IMS is that it can distinguish isomeric species
due to its unique ability to separate ions, permitting determination
of their structural information (for example rotationally averaged
cross-sectional area) [9,10]. Ion mobility coupled to mass spectrometry (IM-MS) was rst described in 1962 [11] and recently
has become an analytical technique to be applied widely, primarily owing to commercialization of the necessary instrumentation.
By combining ion mobility to LCMS, a system composed of LC
separationionizationion mobility separation (IMS)-ion detection and mass analysis can serve as an alternative to traditional
LCMS. As very different time scales (MS in s, IMS in ms and
LC in s), comprehensive data can be obtained and LC-IMS-MS
greatly enhance information and resolution power [12]. In previous studies, IMS-MS has been applied to biomolecules including
peptides [13], proteins (60150 kDa) [14] and large protein complexes (14 MDa) [15]. But the applications for small molecule
in herbal medicines are still rare. To the best of our knowledge,
very limited researches have been done on the undetected natural isomers. Previously researches proved that LC-IMS-MS method
provides a useful approach towards investigating isomers in natural plants, which makes structural elucidation of molecules from
herbal extracts to be more efcient [16]. In this paper, we described
an analysis strategy for separation and detection of various components. Initially, extracts of G. jasminoides was fractionated on
AB-8 resin column, then the obtained fractions were analyzed
by LCMS on Q-TOF mass spectrometer. And the fragmentation
behaviors of different types compounds under MS/MS conditions
were studied and the separations of isomers by LC-IMS-MS were
optimized. To sum up, a strategy for simultaneous separation,
detection, identication, and structural characterization of components was developed which could be used for herbal medicines,
such as extracts of G. jasminoides.

2. Experimental
2.1. Materials and reagents
HPLC grade methanol, acetonitrile and formic acid were purchased from Fisher Scientic (Lough borough, UK); water was
obtained from Milli-Q water purication system (Milford, MA);
Leucine encephalin and sodium formate were purchased from
Waters (Milford, USA). Other chemicals were of analytical grade.
The reference standards, geniposide, genipin, gardenoside,
genipin-1-O-gentiobioside, shanzhiside methyl ester, shanzhiside,
geniposidic acid, chlorogenic acid, hyperoside, isoquercitrin, rutin,
oleanolic acid, ursolic acid, crocin-3 and crocin-4 were obtained
from Shanghai Yuanye Bio-Technology Co. Ltd (Shanghai, China).
The purities of the reference standards were over 98% and their
chemical structures are illustrated in Fig. 1. The dried Gardeniae
Fructus were authenticated as fruits of G. jasminoides by Professor Shumin Wang (Changchun University of Traditional Chinese
Medicine, China).
AB-8 macroporous resin, Shandong lukang Record Pharmaceuticals Co. Ltd, China was used for Gardenia fruits extracts
fractionation. Physical and chemical properties of AB-8 macroporous resin are summarized as follows: weak-polar, a particle
size of 0.31.25 mm, a surface area of 480530 m2 /g, and an

average pore diameter of 13.014.0 nm. Pretreatment of macroporous resins mainly referred to the literatures [17,18].
2.2. Standard solution and sample preparations
An aliquot of 25 g of powdered G. jasminoides (40 mesh) were
extracted under reux in 250 mL of 95% ethanol (v/v) (2 2 h).
Preparative chromatography was equipped with a glass column
(15 mL, 1 cm 20 cm), which was wet packed with selected AB8 resin (10.0 g, dry mass). The mobile phase consisted of ethanol
and water. The crude extracts were concentrated to 0.25 g/mL
and applied to an AB-8 macroporous resin column eluted with
water (45 mL), 30% (75 mL) and 90% (75 mL) ethanol. The eluents
of 30% and 90% ethanol were collected as two fractions (G-A1 and
G-A2), and then concentrated and vacuum dried at 25 C to constant weight for further use. Each fraction (5 mg) was dissolved in
1 mL methanol-water (1:1, v/v) under ultrasonic for 30 min. The
obtained solutions were centrifuged at 10,000 rpm for 10 min and
ltered through a 0.22 m membrane lter before HPLC analysis.
Reference standards were mixed and dissolved in methanol to
achieve a stock solution with concentration of 1.0 mg/mL for each
compound. All sample solutions were stored at 4 C and used at
room temperature.
2.3. HPLC-IMS-MS/MS analysis
LCMS analysis was performed with a Waters ACQUITY UPLC
system (Waters Corp., Milford, MA, USA) connected to a QTOF SYNAPTG2 High Denition mass spectrometer (Waters Corp.,
Manchester, UK). Chromatographic separation was carried out on a
Kromasil C18 column (250 mm 4.6 mm, 5 m particle size) using
gradient elution. The mobile phase was composed of (A) water containing 0.1% (v/v) formic acid and (B) acetonitrile containing 0.1%
(v/v) formic acid. The column temperature was maintained at 25 C,
while the temperature of the auto-sampler was held at 4 C. 5 L
of sample was injected and a ow rate of 600 L/min was applied
during the gradient elution. The elution program was as follows:
05 min, 7% B isocratic; 513 min, 715% B linear; 1320 min, 15% B
isocratic; 2035 min, 1525% B linear; 3542 min, 25% B isocratic;
4250 min, 2590% linear; 5060 min, 90% B isocratic. Data were
acquired in negative ion mode. Source parameters for the MS were
set as follows: capillary voltage, 2.0 kV; sampling cone voltage,
30 V; extraction cone voltage, 4.0 V; source temperature 100 C;
desolvation temperature, 380 C; cone gas ow rate, 50 L/h; desolvation gas ow rate, 700 L/h. Trap and transfer collision energies
were set at 5 V for low energy and 2535 V for high energy. The
lock mass compound used was leucine enkephaline with a reference mass at m/z 554.2615 in negative ion mode. Mass range was
set at 1001200 Da with a scan speed of 0.2 s per scan using the
MassLynx software 4.1 (Waters Corp., Milford, MA, USA).
2.4. T-wave ion mobility separation (IMS)
IMS was optimized by injecting a sample extract spiked with
crocin-3 and crocin-4 directly into the MS in IMS mode using direct
infusion at a ow of 5 L/min. IMS parameters were then optimized to ensure that the isomers could be separated over the entire
drift time range. Bias voltage was set to 40 V. And the traveling
wave (T-wave) ion mobility cell was operated at a wave velocity
of 654 m/s and a wave height of 40 V. In this approach, intact ions
were allowed to pass through the trap cell and separated in the drift
tube. The transfer cell was used to fragment the isomers by applying an energy ramp of 1045 V. IMS data were processed using Drift
Scope software 2.1 (Waters Corp., Milford, MA,USA).

L. Wang et al. / J. Chromatogr. A 1452 (2016) 4757

49

Fig. 1. Chemical structures of the reference standards.

3. Results and discussions

3.1. Application of AB-8 resin column chromatography
With the development of analysis and separation technology,
in particular hyphenations of different types of mass spectrometry with liquid chromatography, more and more compounds
in plant are well separated and characterized. However, chemical diversity of a medical plant is still a big challenge due to
complexity of herbal extracts. In most herbal extracts, chemical
constituents are numerous in number and trace in amount, which
hinders their routine detection. When a few predominant components present in herbal extracts, even two-dimensional liquid
chromatography (2DLC) could hardly improve separation capability remarkably [19]. Before LCMS analysis, AB-8 resin column
chromatography fractionation was tested to remove major components and enrich minor components from G. jasminoides extracts.
Iridoids are the major components in G. jasminoides and geniposide (tR = 19.36 min) was the most predominant component in total
ion current (TIC) chromatography (see Fig. 2(G)). In previous study
[2022], a macroporous resin column was a powerful method to
monitor and separate iridoids as a preliminary separation. In our
study, two types of macroporous resin (AB-8 and D101) were exam-

ined. Also, mobile phase (water, 20% ethanol, 30% ethanol and 50%
ethanol) was optimized. When 20% ethanol was used, AB-8 resin
column could separate irodoids well according to their polarity.
With these optimized conditions, most iridoids and few monoterpenoids can be eluted during 1.0028.00 min (see Fig. 3 (G-A1)).
However, these peaks cannot be observed in G-A2 (see Fig. 3 (GA2)) except for the peak of geniposide, which indicated AB-8 resin
column chromatography was an efcient method to remove predominant components in G. jasminoides. TIC chromatography of
the obtained two fractions (G-A1 and G-A2) and crude extract (G)
are shown in Fig. 3. The Y-axis of G-A1, G-A2 and G are in absolute abundance and the TIC intensity is equal to each other. After
removing the major compounds by AB-8 resin column chromatography, the minor constituents of G. jasminoides could be clearly
observed on TIC chromatography (see Fig. 3). In one-dimensional
HPLC separation, lots of minor constituents could not be well separated due to limited chromatographic resolution. A preliminary
fractionation of crude extracts could improve chromatographic
peak capacity due to the different separation mechanisms [23,24].
AB-8 resin column chromatography could remove iridoids better and enrich minor ingredients such as avonoids, triterpenes,
carotenoids and phenolic acids at the same time. More than 80
peaks were detected in TIC chromatography after enrichment

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L. Wang et al. / J. Chromatogr. A 1452 (2016) 4757

Fig. 2. Total ion current chromatograms displaying the fractionation of G. jasminoides crude extract by AB-8 column chromatography. G, G. jasminoides crude extract; G-A1
and G-A2, two combined fractions after AB-8 macroporous resin column separation.

Fig. 3. Total ion current chromatograms of the G-A1, G-A2 and G with equal intensity.

(see Fig. 3(G-A2)), and thus could help characterize more minor
constituents in G. jasminoides extracts. Consequently, AB-8 resin
column chromatography combined with reversed phase liquid

chromatography (RPLC) effectively facilitated the analytical separation of herbal extracts, which allowed a comprehensive chemical
proling of herbal medicines.

L. Wang et al. / J. Chromatogr. A 1452 (2016) 4757

3.2. HPLCMS/MS analysis of G. jasminoides

A total of 71 components including iridoids, avonoids, triterpenes, monoterpenoids, carotenoids and phenolic acids from the
fruits of G. jasminoides were identied (Tables 17), among which
15 compounds (see Fig. 1) were identied unambiguously by comparing with the retention time and accurate mass data of reference
standards. For the other components, accurate molecular masses
were mainly obtained from negative full scan spectra provided
by Q-TOF-MS. The molecular formula was established by highaccuracy quasi-molecular ion such as [MH] and [M+HCOO]
within mass error of 5 ppm and fractional isotope abundance.
And the most rational molecular formula was searched in chemical databases, such as ChemSpider (www.chemspider.com) and
SciFinder (http://scinder.cas.org). Then fragment ions were used
to further conrm the chemical structures of compounds. Finally,
the structures were identied by comparing with the data or nature
products provided in the literatures.

3.2.1. Iridoids and iridoid glycosides

In this study, 21 iridoids and iridoid glycosides were characterized from the two fractions (G-A1 and G-A2) by their retention
times and MS data respectively shown in Tables 1 and 2. I2 , I4 , I6 ,
I8 , I9 , I11 were determined as shanzhiside, geniposidic acid, gardenoside, genipin-1--gentiobioside, geniposide and genipin by
comparing with the reference compounds, respectively. The typical fragmentation patterns for iridoid glycosides contain the losses
of a glucosyl moiety (162 Da), H2 O and retro Diels-Alder (RDA)
reaction of iridoid skeleton. The hemiacetal group of iridoids is
easily isomerized to two aldehyde groups which lead to the bond
breakage between C-4 and C-5 [25]. To identify other iridoids and
iridoid glycosides from the fruits of G. jasminoides, the characteristic fragmentations of geniposide were investigated and the
possible fragmentation mechanism is shown in Fig. 4. Geniposide
(tR = 19.32 min) gave [M+HCOO] ion at m/z 433.1344 in full scan
mass spectrum in negative ion mode. The [M+HCOO] ion further produced [MH] and [MHGlc] fragments ions at m/z
387 and m/z 225, respectively. The neutral loss like H2 O (18 Da)
was found as well. In addition, the ions at m/z 123 and m/z 101
were observed, originating from a retro-Diels-Alder (RDA) reaction, which occurred simultaneously in three positions including
one double bond and two single bonds.
I1 (tR = 7.01 min) gave [MH] ion at m/z 373.1133. This precursor ion generated aglycone fragment ion at m/z 211 by the loss
of hexose moiety. The characteristic [MHC3 H4 O3 ] ion at m/z
123 was observed as base peak in the MS/MS spectrum. Therefore,
I1 was identied as gardoside. I3 (tR = 5.72 min) gave an [MH]
ion at m/z 389.1076, and was supposed to be a carboxylic acid.
Compared with the MS/MS spectrum, it could be tentatively characterized as deacetylasperulosidic acid. I9 (tR = 13.04 min) and I12
(tR = 30.69 min) produced the same [M+HCOO] and [MH] ions.
Apart from the characteristic fragment ions of iridoids at m/z 139
and 101, [MHGlc] and [MHGlcH2 O] ions were detected.
Therefore, I9 and I12 were tentatively identied as ixoroside and
10-O-acetylgeniposide, respectively.
I16 I18 and I21 showed their [MH] ions at m/z 755.2391,
695.2184, 725.2288 and 725.2299, respectively. They all displayed similar MS fragmentation patterns with genipin-1-gentiobioside in the MS/MS spectra. Moreover, all the above
four compounds exhibited losses of 206 Da (sinapoyl), 146 Da
(courmaroyl), 176 Da (feruloyl) and 130 Da (cinnamoyl), respectively. Therefore, I16 -I18 and I21 were identied as 6 -Otrans-sinapoyl-genipin-gentiobioside, 6 -O-p-coumaroyl-genipingentiobioside, 6 -O-trans-feruloyl-genipin gentiobioside and 6 -Otrans-p-cinnamoyl genipin-gentiobioside by comparison of their

51

retention behavior and MS/MS spectra with the literature data,

respectively [26].
Based on the fragmentation patterns, I14 I15 and I19 I20
were identied as 6 -O-trans-coumaroyl geniposidic acid, 6 O-trans-sinapoyl gardoside, 6 -trans-sinapoyl geniposide and
6 -O-trans-coumaroyl geniposide, respectively.
3.2.2. Flavonoids and their glycosides
A total of 9 avonoids were identied in G-A2 fraction. Most
of these compounds were 5, 7-dihydroxyfavonoid derivatives with
characteristic ion at m/z 151 which was originated from the RDA
reaction of C-ring in negative ion mode [26]. Also, the loss of 162 Da
was the characteristic neutral loss of avonoid glycosides in both
positive and negative ion modes. Hyperoside and isoquercitrin
were two isomers that were identied by comparing with the references. The representative fragmentation pattern of hyperoside is
shown in Fig. 5. Hyperoside showed [MH] ion at m/z 463.0871.
In the MS/MS spectrum of hyperoside, [MHGlc] ion at m/z 301
and [MHGlcCO] ion at m/z 271 were detected. And the RDA
feature fragment ion at m/z 151 in negative ion mode suggested
that F3 was hyperoside.
F4 gave [MH] ion at m/z 593.1522 and produced the
fragment ion at m/z 285.0387 after losing 308 Da (rhamnosyl + glucopyranosyl) in the MS/MS spectrum. Moreover, the
characteristic ion at m/z 151 from the RDA reaction was also
detected. Thus, F4 was identied as nicotiorin.
F7 , F8 and F10 showed [MH] ions at m/z 345.0620, 329.0680
and 375.0727, respectively. All of the three compounds exhibited continuous losses of methyl groups in their MS/MS spectra.
F7 respectively showed [MHCH3 ] and [MH2CH3 ] ions at
m/z 330 and 315. The similar [MHCH3 ] , [MH2CH3 ] and
[MH3CH3 ] fragment ions individually at m/z 360, 345 and
330 were also observed in the MS/MS spectrum of F10 . F10 was
tentatively identied as 4 ,5,6,7-tetrahydroxy-3,3 ,5 -trimethoxy
avone. In the MS/MS analysis of F8 , the fragment ions from the
cleavages of one and two methyl groups from [MH] ion were
detected respectively at m/z 314 and m/z 299 as well. Additionally, in the fragmentations of F7 and F8 , [MH2CH3 CO] ion
was detected at m/z 287 and 271, respectively. All the fragmentation patterns of F7 revealed itself to be 5, 7, 3 , 4 -tetrahydroxy-6,
8-dimethoxy avone. F8 was identied as 2-(3,5-dihyroxy-4methoxyphenyl)-5-hydroxyl-7-methoxy-4H-chromen-4-one.
3.2.3. Triterpenes
In total, 9 triterpene compounds eluted from 40 to 60 min were
identied by the features of their MS and MS/MS fragmentation
patterns in G-A2 fraction. In negative ion mode, the major ion
species were [MH] ions. The characteristic ions were produced
via the losses of H2 O and formaldehyde (HCHO) from the quasimolecule ion [MH] and usually the intensities were also very
high [27,28]. Further fragmentations were produced by the losses
of CH4 and 2CH3 . Ursolic acid and oleanolic acid are two isomers that were identied by comparing with the references. In the
MS/MS analysis of ursolic acid, the [MH] ion at m/z 455.3534
was detected. The fragmentation at m/z 407 was assigned as the
losses of formaldehyde (HCHO) and H2 O. The further losses of CH4
and 2CH3 groups yielded the fragment ions at m/z 391 and 377,
respectively.
T3 and T4 displayed [MH] ions at m/z 485.3262 and 469.3318
in the ESIMS spectra, respectively. In the MS/MS spectrum of T4 ,
[MHH2 O] ion at m/z 471 and [MHCH2 OH2 O] ion at m/z
453 suggested the existence of one carboxyl group and at least one
hydroxyl group. T4 was deduced as 9,19-cyclolanost-24-ene-3,23dione. T3 showed similar fragment ions and the [MHH2 O] ion at
m/z 463 and [MHCO2 H2 O] ion at m/z 441 indicated that it had

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L. Wang et al. / J. Chromatogr. A 1452 (2016) 4757

Table 1
Compounds identied in G-A1 by HPLCMS.
No. Compounds

tR (min) Formula

ESI()measured(m/z) ESI()Calculated(m/z) Delta(ppm) Fragments

I1
I2
I3
I4
I5
I6
I7
I8
I9
I10
I11
I12
M1
M2
M3
M4
M5
M6
P1
P2
P3

7.01
9.07
5.72
10.22
11.35
12.36
13.06
16.20
19.32
13.04
19.34
30.69
14.33
20.49
22.67
23.45
28.45
35.84
17.64
18.78
25.44

373.1133
391.1251
389.1076
373.1144
449.1300
449.1296
449.1294
549.1810
433.1344
405.1397
225.0766
429.2122
345.1555
345.1551
375.1664
375.1659
183.1019
489.1975
353.0869
385.1126
503.1762

gardoside
shanzhiside
deacetylasperulosidic acid
geniposidic acid
scandoside methyl ester
gardenoside
7,8-epoxy-8a-dihydrogeniposide
genipin-1--gentiobioside
geniposide
ixoroside
genipin
10-O-acetylgeniposide
jasminoside D
jasminosideB
jasminoside A/E
jasminoside A/E
jasminodiol
jasminoside R
chlorogenic acid
sinapyglucoside
2-methyl-l-erythritol-4-O-(6-O-transsinapoyl)- -d-glucopyranoside

C16 H22 O10

C16 H24 O11
C16 H22 O11
C16 H22 O10
C17 H24 O11
C17 H24 O11
C17 H24 O11
C23 H34 O15
C16 H24 O11
C16 H24 O9
C11 H14 O5
C12 H34 O9
C16 H26 O8
C16 H26 O8
C17 H27 O9
C17 H27 O9
C10 H16 O3
C22 H34 O12
C16 H18 O9
C17 H22 O10
C22 H32 O13

0.54
1.28
2.01
2.41
1.11
0.22
0.22
1.82
0.46
0.00
1.33
0.61
1.74
0.58
0.27
1.07
1.09
0.61
1.13
2.34
0.60

373.1135
391.1246
389.1084
373.1135
449.1295
449.1295
449.1295
549.1820
433.1346
405.1397
225.0763
429.2125
345.1549
345.1549
375.1655
375.1655
183.1021
489.1972
353.0873
385.1135
503.1765

193,149,123,167,121,101,211
167,149,185,121,229,109,193
209,165,183,147,139,121
149,123,105,211
241,139,223,193,101
241,127,223,193,101
241,139,223,193,101
225,123,101,207
225,123,101,207
359,197,179
101,207,147,123,
191, 209,339,113,249
153,113,101,89,167
165,121,183,101,89
113,89,101,59,85,71,143
113,89,101,59,85,71,143
139,137,109,123,151
165,121,113
191,135
205,223,190,247,265
205,223,190

Table 2
Iridoids identied from G-A2 by HPLCMS.
No.

Compounds

tR (min)

Formula

ESI()measured(m/z)

ESI()calculated(m/z)

Delta(ppm)

Fragments

I13
I14
I15
I16
I17
I18
I19
I20
I21

8-epiapodantheroside
6 -O-trans-coumaroyl geniposidic acid
6 -O-trans-sinapoyl gardoside
6 -O-trans-sinapoyl genipingentiobioside
6 -O-p-coumaroyl-genipin-gentiobioside
6 -O-trans-feruloyl-genipin gentiobioside
6 -trans-Sinapoyl geniposide
6 -O-trans-coumaroyl geniposide
6 -O-trans-p-cinnamoyl genipingentiobioside

7.47
8.18
12.96
16.63
16.83
17.51
21.90
23.60
29.20

C17 H24 O10

C25 H28 O12
C27 H32 O14
C34 H44 O19
C32 H40 O17
C33 H42 O18
C28 H34 O14
C26 H30 O12
C32 H40 O16

387.1291
519.1491
579.1716
755.2391
695.2184
725.2288
593.1866
533.1672
725.2299

387.1283
519.1503
579.1714
755.2399
695.2187
725.2293
593.1870
533.1659
725.2293

2.07
2.31
0.35
1.06
0.43
0.69
0.67
2.44
0.83

123,207,101,332
145,163,119,123,149
205,325,367,385,223,123
123,223,205,101,427,529
469,367,123,225,207
499,123,193,225
367,223,205,123
145,123,307,225
531,225,123,147,101

Table 3
Crocins identied from G-A2 by HPLCMS.
No.

Compounds

tR (min)

Formula

ESI()measured(m/z)

ESI()calculated(m/z)

Delta(ppm)

Fragments

C1
C2
C3
C4
C5
C6
C7
C8

trans-crocin3/cis-crocin3
trans-crocin4/cis-crocin4
trans-crocin4/cis-crocin4
trans-crocin3/cis-crocin3
cis-crocin2/trans-crocin2/cis-crocin2 /trans-crocin2
cis-crocin2/trans-crocin2/cis-crocin2 /trans-crocin2
cis-crocin2/trans-crocin2/cis-crocin2 /trans-crocin2
cis-crocin2/trans-crocin2/cis-crocin2 /trans-crocin2

21.05
16.51
30.71
32.05
31.96
32.08
35.09
35.52

C38 H54 O19

C44 H64 O24
C44 H64 O24
C38 H54 O19
C32 H44 O14
C32 H44 O14
C32 H44 O14
C32 H44 O14

813.3185
975.3705
975.3705
813.3185
651.2651
651.2651
651.2671
651.2665

813.3181
975.3710
975.3710
813.3181
651.2653
651.2653
651.2653
651.2653

0.49
0.51
0.51
0.49
0.31
0.31
2.76
1.84

651,327,283
651,327,283,239
651,327,283,239
651,327,283
327,283,239
327,283,239
327,283,239
327,283,239

Table 4
Monoterpenoids identied from G-A2 by HPLCMS.
No.

Compounds

tR (min)

Formula

ESI()measured(m/z)

ESI()Calculated(m/z)

Delta(ppm)

Fragments

M7
M8
M9
M10
M11
M12
M13
M14

11-(6-O-trans-sinapoylglucopyranosyl)-gardendiol
jasminoside S/H/I
6 -O-trans-sinapoyl jasminoside L
jasminoside T
6 -O-trans-sinapoyl jasminoside A
6 -O-trans-sinapoyl jasminoside C
gadenone
(R)-linalyl-6-O--l-arabinopyranosyl-D-glucopyranoside/bornyl-6-O--Dxylopyranosyl--D-glucopyranoside
crocusatin-C

14.15
17.33
19.73
21.63
29.59
31.01
36.08
28.46

C27 H34 O13

C22 H36 O12
C27 H36 O12
C21 H34 O11
C27 H36 O11
C27 H34 O11
C12 H20 O3
C21 H36 O10

565.1934
491.2124
551.2117
507.2079
535.2177
533.2029
211.1328
447.2226

565.1921
491.2129
551.2129
507.2078
535.2179
533.2023
211.1334
447.2230

2.30
1.02
2.18
0.20
0.37
1.13
2.84
0.89

325,265,223,295
167,323,221,125,263
533,521,265,367
367,173,193
325,265,223,205
205,367,223,165,190
197,167
131,315,191

27.93

C10H16O2

167.1076

167.1072

2.39

M15

an additional hydroxyl group than T4 . Therefore, T3 was identied

as gardenic acid B.

135,121

3.2.4. Carotenoids
Crocins belong to compounds of carotenoid class. It can
form several crocins glucose (Glc = -D-glucopyranosyl) and
gentiobiose (Gnt = -D-glucopyranosyl-D-glucose) esters [29].

L. Wang et al. / J. Chromatogr. A 1452 (2016) 4757

53

Table 5
Flavonoids identied from G-A2 by HPLCMS.
No.

Compounds

tR (min)

Formula

ESI()measured(m/z)

ESI()Calculated(m/z)

Delta(ppm)

Fragments

F1
F2
F3
F4
F5
F6
F7

rutin
isoquercitrin
hyperoside
nicotiorin
genistein
5, 7, 3 ,4 -tetrahydroxy-6, 8-dimethoxy avone
2-(3,5-dihyroxy-4-ethoxyphenyl)-5ydroxy-7-methoxy-4H-chromen-4-one
quercetin
4 ,5,6,7-tetrahydroxy-3,3 ,5 -trimethoxyavone

10.80
12.62
12.33
13.52
45.72
30.11
33.22

C27 H30 O16

C21 H20 O12
C21 H20 O12
C27 H30 O15
C15 H10 O5
C17 H14 O8
C17 H13 O7

609.1448
463.0871
463.0871
593.1522
269.0464
345.0620
329.0680

609.1456
463.0877
463.0877
593.1507
269.0450
345.0610
329.0661

1.31
1.30
1.30
2.53
5.20
2.90
5.77

301,300,151,179
300,301,271,151,255
300,301,271,151,255
285,151,327
241,225
315,330,287
314,299,271,227

30.31
32.60

C15 H10 O7

301.0357
375.0727

301.0348
375.0716

2.90
2.93

F8
F9

151,273,73
360,345,330

Table 6
Triterpenes identied from G-A2 by HPLCMS.
No.

Compounds

tR (min)

Formula

ESI()measured(m/z)

ESI()Calculated(m/z)

Delta(ppm)

Fragments

T1
T2
T3
T4
T5
T6
T7
T8
T9

erubigenin
dikamaliartanes A
gardenic acid B
9,19-cyclolanost-24-ene-3,23-dione
quadrangularic acid E
ursolic acid
oleanolic acid
secaubrytriol
27-O-p-(E)-coumaroyloxyursolic acid

40.58
40.76
45.82
54.69
55.06
59.46
59.00
43.67
47.94

C30 H48 O5
C30 H44 O6
C30 H46 O5
C30 H46 O4
C30 H48 O4
C30 H48 O3
C30 H48 O3
C30 H50 O5
C39 H54 O6

487.3433
499.3071
485.3262
469.3318
471.3487
455.3534
455.3529
489.3576
617.3828

487.3424
499.3060
485.3267
469.3318
471.3474
455.3525
455.3525
489.3580
617.3842

1.85
2.20
1.03
0.00
2.76
1.98
0.87
0.82
2.27

469,437,425,393
455,437,481,393
441,423,467,367
451,407,423
453,423,409
407,391,377
407,391,377
471,441
453,163,119

Table 7
Phenolic acids and other compounds identied from G-A2 by HPLCMS.
No.

Compounds

tR (min)

Formula

ESI()measured (m/z)

ESI()Calculated (m/z)

Delta (ppm)

Fragments

P4
P5
P6

3,5-dicaffeoylquinic acid/3,4-dicaffeoylquinic acid

3,5-dicaffeoylquinic acid/3,4-dicaffeoylquinic acid
3,5-di-O-caffeoyl-4-O-(3-hydroxy-3-methyl)glutaroylquinic acid
5-O-caffeoyl-4-O-sinapoylquinic acid
methyl-5-O-caffeoyl-3-O-sinapoylquinate
(+)-lyoniresinol-3a-O-b-glucopyranoside

14.86
17.97
19.60

C25 H24 O12

C25 H24 O12
C31 H32 O16

515.1202
515.1190
659.1600

515.1190
515.1190
659.1612

2.33
0.00
1.82

353,173,179,191
353,173,179,191
497,335,353,191,161

22.66
29.27
12.00

C27 H28 O13

C28 H30 O13
C28 H38 O13

559.1462
573.1616
581.2233

559.1452
573.1608
581.2234

1.79
1.40
0.17

173,223,397
529,558,369,207
401,389,371,356,265

P7
P8
P9

Fig. 4. Tandem mass spectrum and possible fragmentation pathways of geniposide in negative ion mode.

Thus, the successive losses of glycosides (162 Da or 324 Da) and

CO2 (44 Da) are feature losses in negative ion mode [30,31]. In this
study, crocin-2, 3, 4 were identied in G-A2 fraction and all of them
had at least one isomer. Trans-cis isomerizations are common but
they can hardly be distinguished without reference compounds.
The feature fragment ions of crocin-3 are shown in Fig. 6. Crocin-3
gave [MH] ion at m/z 813.3185. The feature ions ([MHGlc] ,
[MHGnt] and [MHGlcGnt] ) were detected in its MS/MS
spectrum. The product ion at m/z 327 could be detected accompanied by further losses of one and two molecules of CO2 (44 Da and
88 Da).

3.2.5. Monoterpenoids
In this study, 15 monoterpenoids were tentatively identied in
the fruits of G. jasminoides (G-A1 and G-A2 fractions) according to
their retention time, accurate molecular masses and MS/MS spectra. Their fragmentation pathways were quite different from iridoid
glycosides. The losses of glycosides (162 Da), CH3 and H2 O were
the characteristic fragmentations in their MS/MS spectra. The ions
at m/z 179 [glucoseH] and 161 [glucoseHH2 O] are feature
fragment ions in the MS spectra of the monoterpenoid glycosides
[3,32,33].

54

L. Wang et al. / J. Chromatogr. A 1452 (2016) 4757

Fig. 5. Tandem mass spectrum and possible fragmentation pathways of hyperoside in negative ion mode.

Fig. 6. Tandem mass spectrum and possible fragmentation pathways of crocin-3 in negative ion mode.

Fig. 7. Tandem mass spectrum and possible fragmentation pathways of jasminoside B in negative ion mode.

As shown in Fig. 7, the fragmentation of M2 gave the product ions at m/z 183 and m/z 165 which respectively corresponded
to the losses of a glycoside (162 Da) and H2 O. The ion at m/z

183 [MHGlc] further produced a fragment ion at m/z 121

[MHGlcCO2 ] . All the fragmentation information led to the
conclusion that M2 was jasminoside B.

L. Wang et al. / J. Chromatogr. A 1452 (2016) 4757

55

Fig. 8. Tandem mass spectrum and the possible fragmentation pathways of 3, 5-dicaffeoylquinic acid or 3, 4-dicaffeoylquinic acid in negative ion mode.

3.2.6. Phenolic acids and other compounds

The MS data of 9 phenolic acids and other compounds are listed
in Table 7. Most of the phenolic acids are quinic acid derivatives
which produce the diagnostic ions at m/z 191 [quinic acidH] and
m/z 173 [quinicacidHH2 O], respectively [34,35].
P4 (tR = 14.86 min) and P5 (tR = 17.97 min) as isomers displayed
identical MS data in negative ion mode. Apart from the same
[MH] ion at m/z 515.1190, their MS/MS spectra presented
[MHcaffeoyl] ion at m/z 353 and [MH2 caffeoyl] ion at
191, respectively. Another common fragment ion at m/z 173 was
formed by the loss of H2 O. Thus, P3 and P4 were deduced as 3,
5-dicaffeoylquinic acid or 3, 4-dicaffeoylquinic acid. The representative fragmentation pattern of P4 is shown in Fig. 8. P1 in G-A1 was
identied as chlorogenic acid by comparing with the reference. Following the same principle, I6 (m/z 659.1600) with the fragment ions
at m/z 497,335,353,191,161 was named 3,5-di-O-Caffeoyl-4-O-(3hydroxy-3-methyl)-glutaroyl-quinic acid.

3.3. Ion mobility separation of crocin-3 and crocin-4

Crocins constitute a class of structure similar compounds varying in the number of glycosylated sites (one or two) and in the
chain length/type of linkage of the carbohydrate moiety. Crocin

analogues including crocins 1-4 are almost glycosides of transCrocetin, while, cis-crocetin and its glycosides are low-abundant
compared to trans-crocetin. It is reported that all crocin derivatives occur as pairs of cis-trans isomers except crocin-1 [36].
Structures of crocin-3 (gentiobiosyl-glucosyl-crocetin) and crocin4 (gentiobiosyl-crocetin) are shown in Fig. 1. Purity of crocin-3 and
crocin-4 are not guaranteed because both of them are extremely
unstable, especially when existing alone, not in a mixture. As shown
in Fig. 8, the chromatographic peak (A) at a retention time of
21.05 min exhibited three distinct mobility drift times at 4.07 ms,
4.83 ms and 8.28 ms. Extracting their mass spectra, all of them
gave an accurate mass at m/z 813.3185 in negative ion mode (see
Fig. 9(e)). A single chromatographic peak (A) of an analyte at m/z
813 showed the presence of three signals (A1, A2 and A3) in their
mobility drift times, which conrmed the presence of previously
undetected isomeric structures in crocin-3 besides cis-trans isomers. In crocin-4, both terminal carboxylic groups are esteried
with gentiobiose and also can be present in two isomeric forms:
cis- cis- trans-. The HPLC separation of crocin-4 is shown in Fig. 9(b).
Unexpectedly, the chromatographic peak (B) at retention time of
16.51 min showed three ion peaks (B1B3) in ion mobility spectrum
as illustrated in Fig. 9(d), suggesting the presence of unidentied
isomeric structures in crocin-4. However, elucidating in detail the

56

L. Wang et al. / J. Chromatogr. A 1452 (2016) 4757

Fig. 9. HPLC-IMS-MS experimental data for crocin-3 and crocin-4. (a) HPLCMS total ion chromatogram of crocin-3. (b) HPLCMS total ion chromatogram of crocin-4. (c)
Drift time distribution of m/z 813. (d) Drift time distribution of m/z 975. (e) Mass spectrum corresponding to LC peak at 21.05 min. (f) Mass spectrum corresponding to LC
peak at 16.51 min.

structures of the three isomeric crocin-3 and crocin-4 observed in

ion mobility experiment is still a challenge due to the unavailability
of crocin-3 and crocin-4 reference materials.
For obtaining more structural information of crocin isomers, the
primary ions were fragmented in the transfer after ion mobility
separation. Transfer collision energy ramp used in the transfer was
035 V for crocin-3 and 040 V for crocin-4. The results showed
35 V and 40 V collision energy provided an adequate fragmentation
extension. As a result, a fragmentation pattern could be determined
for both isomers at their respective drift times (Fig. S1 and Fig. S2).
The drift time versus m/z contour plots for each collision energy
indicated that isomers (A1A3 and B1B3) were structurally similar
and positional isomerism might exist. The information regarding
precursor ions and product ions obtained by IMS-MS/MS experiments were coherent with those of HPLC-IMS-MS techniques. It is
worth noting that drift time shifts were in the range 0.1525 ms,
and they could be explained by the different conditions when transfer collision fragmentation is activated [12]. Thus, the usage of
IMS coupled with accurate mass spectrometry was a complementary tool to fully understand the chemical diversity of an herbal
medicine.

enrich trace constituents and improve peak capacity. In total, 71

components including iridoids, avonoids, triterpenoids, monoterpenes, carotenoids and phenolic acids were identied or presumed
based on their accurate mass and fragment patterns. Ion mobility
separation provided complementary information for structural elucidation and discovered previously undetected isomeric structures
in G. jasminoides. The strategy proposed in this work is proved reliable and efcient in identication and structural characterization
of herbal medicines extracts. The previously undetected components in this paper signicantly expand our understanding on the
chemical constituents in G. jasminoides.
Acknowledgements
The authors gratefully acknowledge the support by the National
Natural Science Foundation of China (No. 81373952, 81473537).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.chroma.2016.05.
026.

4. Conclusions
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