Journal of Pharmaceutical and Biomedical Analysis 128 (2016) 322–332

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Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Method development and application of offline two-dimensional

liquid chromatography/quadrupole time-of-flight mass
spectrometry-fast data directed analysis for comprehensive
characterization of the saponins from Xueshuantong Injection
Wenzhi Yang a,1 , Jingxian Zhang a,1 , Changliang Yao a , Shi Qiu a , Ming Chen b , Huiqin Pan a ,
Xiaojian Shi a , Wanying Wu a,∗ , Dean Guo a
a
Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, People’s Republic of China
b
Guangxi Wuzhou Pharmaceutical (Group) Co., Ltd., Wuzhou 543000, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Xueshuantong Injection (XSTI), derived from Notoginseng total saponins, is a popular traditional Chi-
Received 12 March 2016 nese medicine injection for the treatment of thrombus-resultant diseases. Current knowledge on its
Received in revised form 19 May 2016 therapeutic basis is limited to five major saponins, whereas those minor ones are rarely investigated.
Accepted 20 May 2016
We herein develop an offline two-dimensional liquid chromatography/quadrupole time-of-flight mass
Available online 3 June 2016
spectrometry-fast data directed analysis (offline 2D LC/QTOF-Fast DDA) approach to systematically char-
acterize the saponins contained in XSTI. Key parameters affecting chromatographic separation in 2D
Keywords:
LC (including stationary phase, mobile phase, column temperature, and gradient elution program) and
Xueshuantong Injection
Minor saponins
the detection by QTOF MS (involving spray voltage, cone voltage, and ramp collision energy) were
Offline two-dimensional liquid optimized in sequence. The configured offline 2D LC system showed an orthogonality of 0.84 and a
chromatography theoretical peak capacity of 8976. Total saponins in XSTI were fractionated into eleven samples by the
Quadrupole time-of-flight mass first-dimensional hydrophilic interaction chromatography, which were further analyzed by reversed-
spectrometry phase UHPLC/QTOF-Fast DDA in negative ion mode. The fragmentation features evidenced from 36
Fast data directed analysis saponin reference standards, high-accuracy MS and Fast-DDA-MS2 data, elemental composition (C < 80,
Structural elucidation H < 120, O < 50), double-bond equivalent (DBE 5–15), and searching an in-house library of Panax notogin-
seng, were simultaneously utilized for structural elucidation. Ultimately, 148 saponins were separated
and characterized, and 80 have not been isolated from P. notoginseng. An in-depth depiction of the chem-
ical composition of XSTI was achieved. The results obtained would benefit better understanding of the
therapeutic basis and significant promotion on the quality standard of XSTI as well as other homologous
products.
© 2016 Published by Elsevier B.V.

1. Introduction components difficult to be detected, analyzed, and isolated. Aside

Clarification of the chemical composition of traditional Chinese
medicine (TCM) is a premise for elucidation of the mechanism of
action and also a key segment for TCM modernization and inter-
nationalization. However, this work is often hindered because of
the ubiquitous interference from the chemical matrix. Additionally,
the significantly altered content difference renders those minor
∗ Corresponding author.
E-mail address: wanyingwu@simm.ac.cn (W. Wu).
1
These authors contributed equally to this work.
from the conventional phytochemical approaches, liquid chro-
matography coupled to mass spectrometry (LC–MS) has been
proven as powerful in rapid characterization of herbal compo-
nents, in particular for discovery of minor and potentially new ones
[1]. Unfortunately, due to the limited peak capacity and the sin-
gle separation mechanism used, only tens of compounds (typically
<50) can be characterized by once chromatographic analysis [2–4].
Despite the ongoing development of ultra-high performance liq-
uid chromatography (UHPLC) coupled with high-resolution mass
spectrometry (such as TOF, FT-ICR, Orbitrap, QTOF, IT-TOF, and LTQ-
Orbitrap, etc) can greatly improve the efficiency in separation and
the reliability in structural elucidation of herbal components, the

http://dx.doi.org/10.1016/j.jpba.2016.05.035
0731-7085/© 2016 Published by Elsevier B.V.
 W. Yang et al. / Journal of Pharmaceutical and Biomedical Analysis 128 (2016) 322–332
characterized compounds by one-dimensional chromatographic
separation are, in general, still less than 100 [5–8], in which
many minor, structurally novel, possibly pharmacologically active
molecules fail to be exposed and identified.
This situation has been greatly improved in recent years due
to the development of enhanced chromatographic separation
techniques and hybrid mass spectrometric scan strategies: (1) two-
dimensional liquid chromatography coupled with multistage mass
spectrometry (2D LC–MSn ) [9–11]; (2) fractionation and enrich-
ment effects by column chromatography or knockout of major
components prior to LC–MS analysis [12–15]; and (3) enhanced,
integral targeted or nontargeted scan methods performed on a
triple quadrupole (QqQ) or a hybrid triple quadrupole/linear ion-
trap (QTrap) mass spectrometer [16–18]. Amongst them, 2D LC,
with significantly extended peak capacity and versatile separation
mechanisms hybridation, has arisen much interest of the chemists
in resolution of a complex mixture, such as an herbal extract. 2D LC
can operate in either online or offline mode. Complicated pump-
ing and switching valves are always necessary for configuration of
an online 2D LC system to achieve heart-cutting or comprehensive
analysis [9]. Alternatively, flexible integration of different separa-
tion mechanisms is easily accomplished in the offline mode for 2D
LC without instrumental limitation [19]. A multiple heart-cutting
2D LC approach was recently developed in our lab that enabled the
simultaneous quality evaluation of Panax notoginseng in eight dif-
ferent Chinese patent medicines [20]. Offline comprehensive 2D LC
coupled with LTQ-Orbitrap MS was developed to comprehensively
characterize ginsenosides from the stems/leaves of Panax ginseng
and quinochalcone C-glycosides from Carthamus tinctorius [10,21].
Offline comprehensive 2D LC is deemed as a desirable solution to
the exposure and characterization of minor herbal components.
Panax notoginseng (Burk.) F.H. Chen is used as a traditional herbal
medicine or as functional food worldwide [22,23], which has been
officially recorded in USP Herbal Medicines Compendium and China
Pharmacopeia [24,25]. Diverse categories of primary and secondary
metabolites, such as saponins, nonprotein amino acids, polysaccha-
rides, polyacetylenes, phytosterols, and flavonoids, etc, have been
reported or isolated from P. notoginseng. Saponins are considered
closely related to the pharmacology and clinical effects of P. notogin-
seng on the cardiovascular and digestive systems [26], which refer
to the oligo-glycosides of dammarane type protopanaxadiol (PPD),
protopanaxatriol (PPT), and versatile derivatives [27]. Given the
significant bioactivities of Notoginseng saponins on blood circula-
tion system, diverse therapeutic agents derived from Notoginseng
total saponins are clinically available, and Xueshuantong Injection
(XSTI; “Zhusheyongxueshuantong” or “Xueshuantong Lyophilized
Powder”) is ranked among the most famous.
XSTI is a very popular TCM injection agent in China. Accord-
ing to the statistic information provided by the vendor, the
average annual sales between 2012 and 2014 reached 360 mil-
lion US dollars. XSTI could improve cerebral blood perfusion
and enhance hematoma adsorption and neurological function in
patients [28,29]. However, systematic chemical analysis of XSTI has
been rarely reported. Our previous research characterized thirty
saponins from XSTI by use of a well-developed HPLC/IT-MS method
[30]. Very recently, Wang et al. used UHPLC/QTOF-MS combined
with peaks-knockout to characterize 94 saponins [15]. We believe
that, by integration of different separation mechanisms in 2D LC,
more minor, unknown saponin molecules can be discovered and
identified from XSTI.
In this study, we develop an offline 2D LC/QTOF MS method
to achieve the comprehensive characterization of minor saponins
from XSTI following a protocol we recently established [10]. Dif-
ferent from the former studies [10,21], the MS detection was
performed on a Waters Xevo® G2-S QTOF mass spectrometer in
the current study. It facilitates multiple scan methods, among
323
which MSE and Fast DDA (fast data directed analysis) are two rou-
tine ones [31]. Comparatively, MSE is data-independent allowing
MS/MS fragmentation of all precursor ions without ion selec-
tion or separation [32], whilst Fast DDA as a data-dependent
mode enables automatic intensity-ranking of precursors-triggered
MS/MS fragmentation [6,31], and can greatly ease data process-
ing for structural elucidation. First, the parameters essential for
establishment of an offline 2D LC–MS system were optimized.
Orthogonality evaluation was performed using asterisk equa-
tions proposed by Camenzuli and Schoenmakers [33]. Second,
the total saponins contained in XSTI were fractionated by the
first-dimensional (1 D) separation. Third, each fraction sample was
analyzed by the second-dimensional (2 D) separation coupled with
QTOF MS detection by data-dependent Fast DDA in negative mode.
Fourth, the fragmentation pathways evidenced from 36 reference
standards, high-accuracy MS and MS/MS data, elemental composi-
tion (EC), and double bond equivalent (DBE), were simultaneously
employed to characterize the substructures (including sapogenin,
sugar residue, and acyl substituent). Fifth, an in-house library of P.
notoginseng was constructed and used to discern potentially new
saponin structures. Ultimately, 148 saponins were identified or
tentatively characterized, including 80 not isolated from P. notogin-
seng, which demonstrates the remarkable superiority of the offline
2D LC/QTOF-Fast DDA approach over the conventional methods
applied for the qualitative analysis of XSTI. This study focuses on
the method development, by systematic optimization of the key
parameters in both offline 2D LC and QTOF MS.
2. Experimental
2.1. Chemicals and reagents
A total of 36 saponins (purity >95%) isolated from the roots
of P. notoginseng were used as the reference standards, involving
notoginsenoside-R1 , −R2 , −R3 , −R4 , −G, −K, −M, −T, −T5 , 20(R)-
noto-R2 , ginsenoside-Ra3 , −Rb1 , −Rb2 , −Rc, −Rd, −Re, −Re3 , −Rf,
−Rg1 , −Rg2 , −Ro, −F2 , −Rk3 , 20(S)-Rg3 , 20(R)-Rg3 , 20-O-glc-Rf,
20(S)-Rh1 , 20(R)-Rh1 , 20(S)-Rh2 , 20(R)-Rh2 , 20(S)-sanchirhinoside-
A3 , −A4 , −A5 , vinaginsenoside-R4 , 5,6-didehydroginsenoside-Rb1 ,
and compound K. Detailed information with regard to the isolation
and structural elucidation is offered as Electronic Supplemen-
tary Data. Fig. 1 displays structures of these reference standards.
Notably, Ro, Re3 , F2 , 20(S)-Rh2 , 20(R)-Rh2 , and compound K, are
first isolated from the roots of P. notoginseng, while 20(R)-noto-R2
is first obtained from plants. The XSTI sample (Batch No. 14010709,
150 mg/bottle) was kindly provided by Guangxi Wuzhou Pharma-
ceutical (Group) Co., Ltd., with the voucher specimen deposited
at the author’s laboratory (ID: XSTI-14010709). HPLC-grade ace-
tonitrile, methanol (Merck, Darmstadt, Germany), formic acid (ROE
Scientific Inc., USA; FA), and ammonium acetate (Sigma-Aldrich,
MO, Switzerland; AA), and ultra-pure water (18.2 M cm at 25 ◦ C)
prepared by a Millipore Alpha-Q water purification system (Milli-
pore, Bedford, USA), were applied in this study. Leucine-enkephalin
was purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Sample preparation
A stock solution was prepared by dissolving a quantity of the
lyophilized power in 50% aqueous methanol (v/v) to reach a con-
centration of 10 mg/mL. The solution was then filtered through a
0.2-␮m PTFE filter (Agilent Technologies, USA) and stored at 4 ◦ C
prior to use.
324 W. Yang et al. / Journal of Pharmaceutical and Biomedical Analysis 128 (2016) 322–332

Fig. 1. Structure of 36 saponin reference standards used in this study. Glc: glucose; GlurA: glucuronic acid; Rha: rhamnose; Ara(p)/Ara(f): pyran-/furan-arabinose; Xyl: xylose.

2.3. Offline 2D LC separation of the saponins in XSTI
Combination of HILIC × RPLC was used to separate the saponin
components contained in XSTI. In detail, the total saponins were
initially separated in HILIC mode on an Agilent 1100 HPLC system
(Agilent Waldbronn, Germany) using a Waters Xbridge Amide col-
umn (4.6 × 150 mm, 3.5 ␮m) maintained at 25 ◦ C. A binary mobile
phase consisting of acetonitrile (A) and water (B) at a flow rate
of 1.0 mL/min was used following a gradient elution program:
0–12 min: 90%–83% (A); 12–25 min: 83%–70% (A); 25–28 min:
70%–50% (A); 28–30 min: 50% (A); 30–31 min: 50%–90% (A); and
31–41 min: 90% (A). The VWD detector was set at 203 nm for detec-
tion of saponins. Eluate collection was based on time-dependent
fractionation, giving eleven fractions: 0–5 min as Fr. 1, 5–10 min
as Fr. 2, 10–11 min as Fr. 3, 11–13 min as Fr. 4, 13–16 min as Fr. 5,
16–18.5 min as Fr. 6, 18.5–20.5 min as Fr. 7, 20.5–21.5 min as Fr. 8,
21.5–24 min as Fr. 9, 24–27 min as Fr. 10, and 27–30 min as Fr. 11.
Each 40 ␮L stock solution of XSTI (10 mg/mL) was injected onto the
column, with five replications. Pooled eluate for each fraction was
dried under a steady flow of nitrogen (N2 ) at ambient temperature.
The dried residues of eleven fractions were separately reconstituted
in 400 ␮L 50% aqueous methanol and filtered through a 0.2-␮m
PTFE filter ahead of RP-UHPLC/QTOF-Fast DDA analysis.
Afterwards, eleven fractionated samples were subjected to RPLC
separation on a Waters ACQUITY I-Class UPLC® system (Waters,
Milford, MA, USA) equipped with a binary solvent manager, a sam-
ple manager, a column manager, and a PDA detector. An ACQUITY
UPLC HSS T3 column (2.1 × 100 mm, 1.8 ␮m) at 25 ◦ C was used for
chromatographic separation with a binary mobile phase consisting
of acetonitrile (A) and water containing 0.1% formic acid (B) at a flow
rate of 0.3 mL/min. An optimized gradient elution program was as
follows: 0–3 min: 20%–23% (A); 3–6 min: 23%–24% (A); 6–6.5 min:
24%–30% (A); 6.5–11.5 min: 30%–32% (A); 11.5–16 min: 32%–65%
(A); 16–17.5 min: 65%–95% (A); 17.5–21 min: 95% (A); 21–21.5 min:
 W. Yang et al. / Journal of Pharmaceutical and Biomedical Analysis 128 (2016) 322–332
95%–20% (A); and 21.5–25 min: 20% (A). The PDA detector was set
between 190 and 400 nm and at 203 nm for detection of saponins.
An injection volume of 5 ␮L was set for each sample.
2.4. QTOF-Fast DDA
High-accuracy MS data of saponins were acquired on a Waters
Xevo® G2-S QTOF mass spectrometer (Waters, Manchester, UK)
connected to UPLC system via a ZprayTM ESI source. In order to
obtain more reliable characterization results, data-dependent Fast
DDA in negative, Resolution mode was utilized. The ESI source
parameters were set as follows: spray voltage, 2.5 kV; cone volt-
age, 140 V; source offset voltage, 80 V; source temperature, 100 ◦ C;
desolvation temperature, 400 ◦ C; cone gas flow rate, 40 L/h; and
desolvation gas flow rate, 800 L/h. For Fast DDA settings, the mass
analyzer scanned over a mass range of 400–1500 Da in full scan with
a scan time of 0.15 s under the collision energy of 6 V, and over m/z
100–1400 for MS/MS by the same scan time. Dual-dynamic colli-
sion energies, 20–40 V for LM CE (low-mass collision energy) and
60–80 V for HM CE (high-mass collision energy), were employed
to acquire more balanced fragmentation independent of molecular
mass difference. Automatic switching to MS/MS mode was enabled
when TIC intensity rose above 5000 intensity/s, and switching off
when 0.45 s had elapsed. Tolerance window of ±3.0 Da and peak
extract window of 7.0 Da were set in deisotope peak detection
mode. Data calibration was performed using an external reference
(LockSprayTM ) by constant infusion of the leucine-enkephalin solu-
tion (1 ng/␮L) at a flow rate of 5 ␮L/min. MS data were referenced
to m/z 554.2615. MassLynx V4.1 software (Waters, Milford, USA)
was used in data acquisition and processing. For prediction of EC of
an observed precursor ion, the ranges of EC and DBE were strictly
predefined: (1) C < 80, H < 120, O < 50; and (2) DBE 5–15. An in-
house library that records the structure information of 95 saponins
ever isolated from P. notoginseng was established and employed in
structural elucidation.
2.5. Method validation for offline 2D LC/QTOF-Fast DDA
The established offline 2D LC/QTOF-Fast DDA method was val-
idated in respect of intra-day/inter-day precision (both 1 D and 2 D
separation), repeatability, and limit of detection (LOD). Precision
was evaluated by six consecutive injections on the first day and
another three injections separately on the second and third days.
Repeatability was assessed by analysis of six reduplicative samples
of Fr. 5 (containing noto-R1 , Re, and Rd). LOD, defined as the low-
est amount at which a saponin compound can be characterized by
the Fast DDA method, was determined using five major saponins
(noto-R1 , Rg1 , Re, Rb1 , and Rd).
2.6. Orthogonality evaluation
Orthogonality of the offline 2D LC system was assessed by cal-
culating the distribution of 50 index components using an in-house
executable program we have reported [21]. Peak distribution was
depicted according to a series of asterisk equations reported in lit-
erature [33]. In brief, normalized retention time (tR ,norm(i) ) of each
index component in each dimension was calculated according to
Eq. (1) (tD : dead time; : tG total gradient elution time). Spreading
of all the index components (Sz- , Sz+ , Sz1 , and Sz2 ) around four
lines were calculated based on Eqs. (2)–(5) (␴: standard deviation
of the values of the bracketed equations for all 50 index compo-
nents), which were then used to determine four Z parameters by
325
Eqs. (6)–(9) for indication of peaks spreading degrees. Ultimately,
A0 was employed to assess the orthogonality according to Eq. (10).
tI − tD
tR,norm(i) = (1)
tG − tD
Sz- = {1 t R,norm(i) − 2 t R,norm(i) } (2)
Sz+ = {2 t R,norm(i) − (1 − 1 t R,norm(i) )} (3)
1
Sz1 = { t R,norm(i) − 0.5} (4)
Sz2 = {2 t R,norm(i) − 0.5} (5)
Z- = |1 − 2.5|Sz - − 0.4|| (6)
Z+ = |1 − 2.5|Sz + − 0.4|| (7)
Z1 = 1 − |2.5 × Sz 1 × 2 − 1| (8)
Z2 = 1 − |2.5 × Sz 2 × − 1| (9)

A0 = Z− × Z+ × Z1 × Z2 (10)
3. Results and discussion
3.1. Parameters optimization for the offline 2D LC system
3.1.1. Selection of chromatographic columns
Given that integration of different separation mechanisms is
able to enhance the selectivity and thus benefits the exposure of
minor components in a larger number, different stationary phases
representing versatile separation mechanisms were examined in
the first step. Meanwhile, taking into account the compatibil-
ity with mass spectrometer and the retention information as
important evidence for unknown saponins characterization and
isomers discrimination by having recourse to literature, we initially
employed RPLC for 2 D separation due to its wide application in
Ginseng analysis [2,3,7,12–15,18,30,34]. Six columns suitable for
UHPLC separation from three different vendors (Waters, Agilent,
and Phenomenex) were tested in respect of selectivity and reso-
lution (Supplementary Fig. A.1). Apparently, HSS T3 and CSH C18
columns exhibited stronger retention capacity to saponins than the
other four, in particular, the most polar ginsenosides (eluted before
noto-R1 , tR 5.20 min) were resolved on the HSS T3 column. Addi-
tionally, the largest number of medium-polarity saponins (eluted
between Rg1 and Rb1 ) were separated by HSS T3. It suggested HSS
T3 had the potential to resolve the most minor saponins in 2 D
separation.
Subsequently, another six columns having different separation
mechanisms or bonded with different functional groups were com-
pared in terms of their separation difference to HSS T3, determined
by linearity regression correlation coefficient (R2 ) using fourteen
saponins as the index components. As shown in Supplementary
Fig. A.2, BEH Phenyl, CSH Phenyl-Hexyl, HSS Cyano, and HSS PFP
columns, all showed R2 > 0.9, indicating their similar selectivity to
HSS T3 on separation of saponins. In contrast, two HILIC columns
exhibited high orthogonality to HSS T3, with R2 < 0.1. We finally
selected the Xbridge Amide column in 1 D separation because of
its greater separation difference from HSS T3, consistent with our
previous report in comprehensive characterization of ginsenosides
from the stems and leaves of P. ginseng [10]. The results demon-
strated the combined use of HILIC and RPLC was potent in resolution
of polar or medium-polarity herbal components [10,19,21,35].
3.1.2. The 1 HILIC separation
One of the advantages of offline mode of 2D LC over the online
mode is embodied in the convenient, individual optimization of
chromatographic conditions in each dimension, without consid-
ering solvent effect and limitations in sampling frequency and
326 W. Yang et al. / Journal of Pharmaceutical and Biomedical Analysis 128 (2016) 322–332

injection volume [10,21]. The 1 D HILIC condition was optimized in

terms of different compositions of mobile phase, column tempera-
ture, and gradient elution program, on an Xbridge Amide column. It
was found that the resolution of XSTI saponins was seldom affected
by use of different mobile phases (CH3 CN-H2 O, CH3 CN-0.1%FA,
and CH3 CN-2 mM AA) (Supplementary Fig. A.3). Acyl ginsenosides
(such as malonyl-ginsenosides) in the roots of P. notoginseng have
been removed in XSTI, leaving neutral saponins [15,30]. Retention
of neutral saponins was rarely affected by different pH values of
the mobile phase due to the addition of different additives. On the
other hand, when the column temperature increased from 25 ◦ C to
40 ◦ C, the retention times of major saponins only slightly decreased
(Supplementary Fig. A.4). Low column temperature favored the
separation of a small peak eluted between Re and noto-R1 . Con-
sequently, the 1 D HILIC separation was performed on an Xbridge
Amide column maintained at 25 ◦ C with a binary mobile phase
composed of CH3 CN-H2 O at a flow rate of 1.0 mL/min.

3.1.3. The 2 D chromatographic separation

The 2 D chromatographic separation condition, including the
mobile phase, column temperature, and gradient elution program,
was optimized. As shown in Supplementary Fig. A.5, different
compositions of mobile phase (CH3 CN-H2 O, CH3 CN-0.1% FA, and
CH3 CN-2 mM AA) remarkably influenced both the chromato-
graphic separation and ion response of saponins by using QTOF MS
as the detector. The largest number of peaks were resolved by use
of CH3 CN-0.1% FA as the mobile phase, despite the most abundant Fig. 2. Orthogonality evaluation of the offline 2D LC system by a series of asterisk
equations.
peak Rg1 was weaker than that obtained using CH3 CN-2 mM AA as
the mobile phase. In addition, much less complicated ion species
corresponding to the same compound (such as Ra3 , Supplementary 3.2. Parameters optimization of QTOF-Fast DDA
Fig. A.6) was generated by addition of 0.1% FA in the mobile phase,
which is crucial for intensity-ranked automatic selection of precur- Even if a few of reports are available that employ Fast DDA
sors in Fast DDA. On the other hand, little influence was observed for characterization of herbal components, a systematic parame-
when different column temperature was set (Supplementary Fig. ters optimization study has not been reported hitherto. Herein, the
A.7). Taking into account the resolution of minor peaks around Rb1 , ESI source parameters and ramp collision energy were optimized,
we performed the 2 D separation with the HSS T3 UPLC® column aiming to achieve sensitive detection and characterization of XSTI
maintained at 25 ◦ C and a binary mobile phase of CH3 CN-0.1% FA saponins based on abundant precursor-product ions information
at a flow rate of 0.3 mL/min. recorded by Fast DDA.

3.2.1. ESI source parameters

3.1.4. Peak capacity and orthogonality evaluation
The separation potency of the established offline 2D LC system
was assessed by peak capacity and orthogonality. Theoretical peak
capacity of a comprehensive 2D LC system (n2D ) is the product of the
peak capacity in 1 D and 2 D separation (n1 and n2 ) [35]. The aver-
age base-line peak width of three peaks eluted at the beginning,
the middle, and the end of the chromatograms in 1 D and 2 D sep-
aration were 0.29 min and 0.24 min, respectively, based on which
n1 and n2 were calculated to be 102 and 88. Thereby, n2D should
be 8976. Clearly, compared to the conventional one-dimensional
chromatography, even in the UHPLC mode, the peak capacity of
the 2D LC system was enhanced by approximate 100-fold. Improve-
ment on peak capacity is beneficial to the exposure of more minor
components, laying a solid foundation for the completion of a com-
prehensive elucidation of the therapeutic basis of XSTI.
Orthogonality evaluation was based on a series of asterisk
equations proposed by Camenzuli and Schoenmakers [33] using
normalized retention time of 50 saponins (Table A.1). As a conse-
quence, the spreading degrees (SZ ) of 50 saponins around lines Z+ ,
Z- , Z1 , and Z2 , were 0.97, 0.81, 0.91, and 0.98, respectively (Fig. 2).
The orthogonality was calculated to be 0.84, ultimately, indicating
a powerful 2D LC system for exposure of minor saponins contained
in XSTI.
The ESI source parameters, including spray voltage (SV) and
cone voltage (CV), affect ions generation in terms of relative inten-
sity of the molecular ions, adduct ions, and product ions, due to
in-source fragmentation. The objective in this step was to obtain
the best ion response and, meanwhile, to suppress the adduct
ions and avoid the occurrence of in-source fragmentation. Tak-
ing into account the molecular mass coverage (600–1400 Da) of
saponins ever isolated from P. notoginseng [27,36], four representa-
tive saponins, comprising noto-R2 (C41 H70 O13 , 770; a biglycoside),
noto-R1 (C47 H80 O18 , 932; a triglycoside), Rb1 (C54 H92 O23 , 1108;
a tetraglycoside), and Ra3 (C59 H100 O27 , 1240; a pentaglycoside),
were taken as index components.
Influence of different SV settings ranging from 1.0 kV to 3.5 kV
was assessed by comparing the ion response of four index com-
ponents. In general, the peak areas positively correlated with the
increasing of SV from 1.0 kV to 2.5 kV, however, slightly decreased
to a low level when 3.0 kV and 3.5 kV were set (Supplementary Fig.
A.8). No remarkable in-source fragmentation occurred under these
SV settings (1.0–3.5 kV). As a result, SV was set at 2.5 kV.
Subsequently, different CV settings (from 130 V to 180 V) were
compared by observing the transition tendency of adduct ions,
molecular ions, and in-source fragmentation product ions. As evi-
denced in Supplementary Fig. A.9, with the increasement of CV
from 130 V to 160 V, FA-adduct ions of noto-R1 , Rb1 , and Ra3 ,
 W. Yang et al. / Journal of Pharmaceutical and Biomedical Analysis 128 (2016) 322–332
exhibited decreasing tendency. Significant in-source fragmenta-
tion (m/z 637.43, due to the neutral elimination of an external
Xyl residue) was observed for noto-R2 when CV was set higher
than 140 V, which was not detected for the tri- (noto-R1 ), tetra-
(Rb1 ), and penta- (Ra3 ) glycosides. Therefore, CV was set at 140 V,
under which no in-source decay occurred and the adduct ions were
suppressed in lower abundance than the genuine molecular ions.
3.2.2. Ramp collision energies
Ramp (or dynamic) collision energy (RCE) setting enables
mass-dependent collision-induced dissociation (CID) of molecu-
lar ions, conducing to the generation of more balanced MS/MS
spectra independent of molecular mass difference. Constant col-
lision energy (CCE) was initially set from 20 V to 70 V to acquire
the fragmentation behaviors of noto-R2 , noto-R1 , Rb1 , and Ra3 .
From Supplementary Fig. A.10, rather different CID features were
observed. Considering the diversity of product ions and generation
of sapogenin ions, we could infer the appropriate collision energies
were 30–50 V for noto-R2 , 40–50 V for noto-R1 , and 60–70 V for Rb1
and Ra3 . Based on these findings and considering the mass range for
full scan (400–1500 Da), as a consequence, RCE was set at 20–80 V
in this study, by which rich CID information concerning the pre-
cursor ions, product ions, sapogenin ions, and even the secondary
product ions of sapogenins, were obtained sufficient for structural
elucidation of the saponin structures.
3.3. Validation of the offline 2D LC/QTOF-Fast DDA method
The established offline 2D LC/QTOF-Fast DDA method was sim-
ply validated in precision, repeatability, and LOD. The intra-day
and inter-day precision evaluated by five major saponins for 1 D
HILIC-UV (RSD, in%) varied from 0.19%–1.48% and 0.62%–3.13%,
respectively, while 9.39%–11.14% of intra-day precision and
12.94%–16.84% of inter-day precision were determined for the 2 D
UPLC/QTOF-Fast DDA. RSD of repeatability was less than 7%. LOD
was determined at 0.1 ng for noto-R1 and Rd, and 0.5 ng for Rg1 , Re,
and Rb1 . These data indicated an acceptable qualitative analysis
approach by means of offline 2D LC/QTOF-Fast DDA.
3.4. CID features of 36 saponin reference standards
Despite the CID features of ginsenosides have been extensively
studied [3.4,7,10,12–15,17,18,30], different fragmentations may
occur on different mass spectrometers. Herein, the negative mode
CID of 36 saponin reference standards (Fig. 1), representing five
different subclasses, that is, protopanaxatriol (PPT, 17 in total),
protopanaxadiol (PPD, 14), oleanolic acid (OA, 1), C17-side chain
varied (C17-SCV, 2), and miscellaneous (2), were comparatively
investigated to outline the characteristic fragmentation pathways
and potential diagnostic product ions (DPIs) for characterization of
the sapogenins and sugar residues. Fig. 3 displays the base peak
chromatograms (BPCs) of mixed reference standards and an XSTI
sample. Full-scan MS and Fast DDA-MS2 spectra of four represen-
tative saponins (comprising 20-O-glc-Rf, Rd, Ro, and noto-T5 ) are
interpreted as shown in Fig. 4. In general, the CID behaviors of
saponin reference standards obtained on the QTOF mass spectrom-
eter were similar to those on an LTQ-Orbitrap mass spectrometer
as we previously reported [10].
3.4.1. Characterization of sapogenins
Sequential elimination of the attached sugar residues could gen-
erate DPIs associated with the sapogenins, involving m/z 475.38
for PPT, m/z 459.38 for PPD, m/z 455.35 for OA, and m/z 457.36 for
dehydrated PPT (Fig. 4). Furthermore, relatively weak secondary
product ions of deprotonated PPD and PPT, due to the neutral elimi-
nation of C6 H12 (84.09 Da), were observed at m/z 391.29 and 375.29,
327
respectively. In addition, miscellaneous sapogenins, 5-ene-PPD in
5,6-didehydro-Rb1 and 7-OH-5-ene-PPD in noto-G exhibited rich
sapogenin ions at m/z 457.36 and m/z 473.37, respectively.
3.4.2. Characterization of sugar residues
The negative mode CID of ginsenosides offers indirect evidence
(masses of neutral elimination) for the sugar moieties, and, mean-
while, the low-mass region (m/z 100–400) in the Fast DDA-MS2
spectra informs direct information (DPIs) for the nature, composi-
tion, and even attachment patterns of the oligosaccharide chains.
The neutrally lost masses of 162.05 Da, 146.05 Da, 132.04 Da, and
176.03 Da, were separately consistent with the sugars of glucose
(Glc), rhamnose (Rha), arabinose (furan- or pyran-, Ara)/xylose
(Xyl), and glucuronic acid (GlurA) [10,15]. By comparing the MS2
spectra of 20(S)-Rh1 , Rf, 20-O-glc-Rf, and Rb1 that contain 1–4 Glc
residue(s), the DPIs at m/z 161.05, 113.02, and 101.03, were indica-
tive of a single Glc residue, while m/z 221.07 (not detected for
Re3 that contains Glc(4,1)Glc), was informative of Glc(2,1)Glc,
which might result from the 0,2 X0 fragmentation (Supplementary
Fig. A.11). Likewise, the product ions at m/z 163.06 and 205.07,
observed for 20(S)-Rg2 and Re, were correlated to the Rha residue
and the oligosaccharide chain of Glc(2,1)Rha, respectively, and m/z
337.08 in Ro and 353.11 in noto-T were ascribed to GlurA(2,1)Glc
and Glc(2,1)Glc(2,1)Xyl, respectively. Differently, the DPIs associ-
ated with Glc(2,1)Ara in 20(S)-sanchi-A5 , Glc(2,1)Xyl in noto-R2 ,
Glc(6,1)Ara(p) in Rb2 , and Glc(3,1)Xyl in noto-T5 , were all detected
at m/z 191.06, nonspecific for a disaccharide chain of a Glc and a
pentose. These DPIs and the eliminated masses are complementary
for characterization of the sugar moieties.
3.5. Systematic characterization of the saponins contained in XSTI
The total saponins in XSTI were initially separated by 1 D HILIC
separation yielding eleven fractions, which were then individually
analyzed by an optimized UHPLC/QTOF-Fast DDA method (Fig. 5).
Because of the orthogonal separation facilitated by the offline 2D LC,
numerous minor saponins that were easily covered up by the dom-
inant ones were well exposed, and thus got characterized. Under
the current 2 D chromatographic condition, the saponins were ion-
ized into predominant FA-adduct ions ([M+HCOO]− ), concomitant
with [M+H2 PO4 ]− ions (except 50, 53, 90, 113, 130, 134, 138, 142,
and 143 that have the richest [M−H]− ). Thereby, the structural
characterization work was carried out based on a workflow consist-
ing of four steps: (1) simultaneous detection of dominant adduct
ions of [M+CH3 COO]− and [M+H2 PO4 ]− informs a saponin com-
pound; (2) EC of the precursor ion is deduced by strict atoms setting
(C < 80, H < 120, and O < 50), DBE screening (5–15), and isotope dis-
tribution matching (i-FIT); 3) the fragmentation pathways and DPIs
detected from the Fast DDA-MS2 spectra are interpreted to charac-
terize the sapogenins and sugar moieties; 4) possible structures
are retrieved in the in-house library of P. notoginseng (record-
ing 95 saponins, Table A.2), to discern potentially new saponins.
Glc, Rha, GlurA, and Xyl (used to represent all possible pentose
residues), are employed to interpret the observed neutral loss of
162.05 Da, 146.06 Da, 176.03 Da, and 132.04 Da, in characterization
of unknown saponins from XSTI [10].
The characteristic fragmentations, m/z 475.38 → 391.29 and
459.38 → 375.29, were employed as major diagnostic informa-
tion to identify PPT and PPD type saponins, respectively, whilst
the sapogenin ion at m/z 455.35 and neutral loss of 176.03 Da
were informative of the OA-type characterization. Compound 76
(tR 7.84 min in Fr. 4) is a typical PPT-type saponin, giving rich
[M+HCOO]− and [M+H2 PO4 ]− ions at m/z 1169.59 and 1221.56,
respectively (Fig. 6). The molecular formula was thus determined as
C54 H92 O24 . High intensity of [M−H]− at m/z 1123.59, [M−H−2Glc]−
at m/z 799.49, [M−H−3Glc]− at m/z 637.43, the sapogenin ion at m/z
328 W. Yang et al. / Journal of Pharmaceutical and Biomedical Analysis 128 (2016) 322–332

Fig. 3. Negative mode base peak chromatograms (BPC) of 36 reference standards and the XSTI sample. 30: noto-R3 ; 33: Re3 ; 34: noto-M; 39: 20-O-glc-Rf; 49: noto-R1 ; 54:
20(S)-sanchi-A5 ; 60: Rg1 ; 62: Re; 79: 20(S)-sanchi-A4 ; 80: noto-G; 89: 20(S)-sanchi-A3 ; 97: vina-R4 ; 104: noto-T; 110: Rf; 113 noto-R4 ; 118: 20(S)-noto-R2 ; 123: Ra3 ; 125:
5,6-didehydro-Rb1 ; 127: 20(R)-noto-R2 ; 1*: 20(S)-Rg2 ; 128: 20(S)-Rh1 ; 130: Rb1 ; 131: 20(R)-Rh1 ; 132: Rc; 134: Ro; 135: Rb2 ; 141: Rd; 144: noto-K; 2*: noto-T5 ; 3*: F2 ; 145:
Rk3 ; 147: 20(S)-Rg3 ; 148: 20(R)-Rg3 ; 4*: compound K; 5*: 20(S)-Rh2 ; 6*: 20(R)-Rh2 . Peaks 1*–6* represent six saponins isolated from the roots of P. notoginseng, but are rare
or not detected from XSTI in this study.

475.39, together with the oligosaccharide chain fragments at m/z the PPT framework. Thereby compound 76 was characterized as
323.10 and 221.07, were readily obtained by CID-MS/MS. All these PPT-Glc-Glc-(Glc-Glc). Compound 142 (tR 14.29 min in Fr. 6) is a
fragments informed the presence of four Glc residues attached to typical OA-type saponin since the characteristic sapogenin ion at

Fig. 4. The negative ion mode full-scan MS and Fast DDA-MS2 spectra of four saponin reference standards, representative of the PPT-type, PPD-type, OA-type, and C17-side
chain varied type. The dashed box region shows diagnostic product ions of sugar moieties.
 W. Yang et al. / Journal of Pharmaceutical and Biomedical Analysis 128 (2016) 322–332 329

Fig. 5. Fractionation of XSTI saponins by the first-dimensional HILIC. A-the UV spectrum (at 203 nm) of XSTI; B-the base peak chromatograms (BPC) of mixed reference
standards and eleven fraction samples determined by UHPLC/QTOF MS.

m/z 455.36 and neutral loss of 176 Da were acquired by CID of the
deprotonated molecules at m/z 793.43 (Fig. 6), as those occurring to
the reference standard Ro. We thus tentatively characterized 142 as
chikusetsusaponin IVa (OA-GlurA-Glc), an OA ginsenoside reported
from P. japonicum [37]. Consequently, 67 PPT-type, 24 PPD-type,
and 2 OA-type saponins, were characterized. Notably, this is the
first report of OA saponins (134 and 142) from XSTI, which are
generally considered absent in P. notoginseng [36,38].
Thirty-seven saponins encompassing varied C17-side chains
were characterized from XSTI, accounting for 31.1% of the total
amount. However, owing to the unexpected structural variation
and lack of relevant reference standards, their characterization
is putative in this study. Characterization of compound 63 (tR
6.87 min in Fr. 7) was taken as an example (Fig. 6). The adduct ions
at m/z 1025.65 and 1077.52 could inform the molecular formula
C48 H84 O20 . The MS/MS product ions at m/z 979.54, 799.48, 637.42,
496.39, and 475.39, were ascribed to [M−H]− , [M−H−Glc−H2 O]− ,
[M−H−2Glc−H2 O]− , [sapogenin−H]− , and [sapogenin−H−H2 O]− ,
respectively, based on which it could be inferred that H2 O-addition
PPT and three Glc residues were involved in compound 63. By
330 W. Yang et al. / Journal of Pharmaceutical and Biomedical Analysis 128 (2016) 322–332
Fig. 6. Interpretation of the MS and Fast-DDA-MS/MS spectra of compounds 76, 142, 63, and 42, representative of the PPT, OA, C17-SCV, and 7-OH-5-ene-PPD type, respectively.
comparative analysis of the fragmentation behaviors, the modifi-
cations on C17-side chains were primarily characterized to involve
dehydrogenation of PPD (−H2 ; 71), hydrogenation of PPT (+H2 ;
68), H2 O-addition of PPT (+H2 O; 16, 17, 35, 50, 63, and 70) or PPD
(100), hydroxylation of PPT (+O; 12–15, 19, 20, 22, 38, and 44),
and combination of above reactions or unknown variations (such
as hydroxylation and H2 O-addition of PPT in 1, 3, 5, 6, 8, and 10).
Saponins with variations on Ring B, including four contain-
ing 5-ene-PPD (90, 99, 125, and 138) and fourteen involving
7-OH-5-ene-PPT (42, 57, 58, 64, 66, 73, 77, 80, 87, 91, 94, 96,
112, and 115), were characterized from XSTI as well. Compound
42 (tR 4.67 min in Fr. 10) gave abundant adduct ions at m/z
1299.62 and 1351.59, and accordingly its molecular formula was
Fig. 7. A 2D scatter plot of all the characterized saponins from XSTI. The blue ellipse
region shows certain linearity between the molecular weight and the retention time
of PPD-type saponins on the HSS T3 UPLC® column. (For interpretation of the ref-
erences to colour in this figure legend, the reader is referred to the web version of
this article.)
determined as C59 H98 O28 . CID of the adduct precursor ion at m/z
1299.62 generated MS/MS product ions at m/z 797.47, 635.42,
473.35, and 353.11, which were assigned as [M−H−2Glc−Xyl]− ,
[M−H−3Glc−Xyl]− , [sapogenin−H]− ([M−H−4Glc−Xyl]− ), and
[GlcGlcXyl + H2 O−H−120 Da]− , respectively. Given its weaker
retention than noto-G (Fig. 1) on the 2 D RPLC column, we speculat
they both have the 7-OH-5-ene-PPD sapogenin. The sugar fragment
at m/z 353.11, as that obtained by CID of noto-T (Supplementary Fig.
A.11), inferred a possible trisaccharide chain of Glc-Glc-Xyl. Based
on these evidences, compound 42 was tentatively characterized as
7-OH-5-ene-PPD-Glc-Glc-(Glc-Glc-Xyl).
As a result, a total of 148 saponins were identified or tentatively
characterized (Table A.3), comprising 30 unambiguously identi-
fied by comparing with the reference standards (including tR , MS,
and MS/MS fragments) (Fig. 3). By further searching the in-house
library, 80 of them have not been isolated from P. notoginseng. The
versatile variations on C17-side chains, different compositions of
the sugar chains or linkage patterns may result in the novelty of
these potentially new saponins. Moreover, a chemical depiction by
plotting the tR -m/z information for all characterized saponins is
implemented as shown in Fig. 7. The structural features of saponins
in XSTI can be outlined as follows: (1) PPT and C17-SCV are two
most common subclasses; (2) polarization on the C17-side chains
weakens the retention of some saponins; (3) both PPT and PPD
types exhibit a general relationship between the molecular weight
and the retention behavior, particularly the PPD-type saponins
(scattered in the blue ellipse of Fig. 7), which is useful for their
auxiliary characterization.
4. Conclusion
Comprehensive characterization of the components contained
in XSTI by conventional LC–MS approaches is confronted with
 W. Yang et al. / Journal of Pharmaceutical and Biomedical Analysis 128 (2016) 322–332
increasing challenges arising from the cover-up of five major
saponins, and the limited peak capacity and selectivity, which are
also a common issue that impedes elucidation of the therapeutic
basis for most herbal medicines as well as their products. Here,
we developed an offline 2D LC/QTOF-Fast DDA approach with the
view of systematic exposure and characterization of the minor
saponins contained in XSTI. Hybridation of HILIC and RPLC in offline
2D LC showed high orthogonality for separating XSTI saponins and
approximate 100-fold improvement in peak capacity, in contrast to
conventional one-dimensional RPLC separation. After parameters
optimization, the negative mode data-dependent Fast DDA facili-
tated a more reliable structural characterization of minor, unknown
saponins from XSTI, particularly by searching an in-house library
of P. notoginseng. As many as 143 minor saponins were separated
and characterized, comprising 80 not ever isolated from P. noto-
ginseng. Remarkable superiority, due to orthogonal separation and
high structural characterization capacity facilitated by QTOF MS,
was embodied over the reported LC–MS methods and knock-out
strategies in separation and characterization of minor components
from XSTI. Integration of offline 2D LC and high-resolution mass
spectrometry is proven as a promising vehicle for elucidation of the
therapeutic basis for herbal medicines together with their prod-
ucts and composite preparations. As the continuous studies, on
one hand, the molecular descriptors mainly responsible for the
observed relationship between structures and retention features
will be disclosed, and, on the other hand, the chemical difference
between XSTI and its homogenous products, such as Xuesaitong,
will be systematically compared.
Acknowledgements
We are grateful to acknowledge the financial support from
the 56th China Post-doctoral Science Foundation (2014M560364),
National Natural Science Foundation of China (81473344 and
81503240), and National Science and Technology Major Project for
Major Drug Development (2014ZX09304-307-001-007).
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.jpba.2016.05.035.
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