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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma
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
48
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).
49
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
50
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.
51
52
Table 1
Compounds identied in G-A1 by HPLCMS.
No. Compounds
tR (min) Formula
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
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
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
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
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
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].
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
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
14.86
17.97
19.60
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
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.
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
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
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.
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
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.
4. Conclusions
References
Our study proposed an analysis strategy to identify and characterize chemical constitutes from G. jasminoides by combining
AB-8 resin column chromatography and LC-IMS-MS/MS. The prefractionation by using AB-8 resin column chromatography could
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