Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 130–141

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Journal of Pharmaceutical and Biomedical Analysis

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

HPLC/qTOF-MS-oriented characteristic components data set and

chemometric analysis for the holistic quality control of complex TCM
preparations: Niuhuang Shangqing pill as an example
Dan-dan Wang a,1 , Jian Liang b,1 , Wen-zhi Yang a , Jin-jun Hou a , Min Yang a , Juan Da a ,
Ying Wang c , Bao-hong Jiang a , Xuan Liu a , Wan-ying Wu a,∗ , De-an 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, Haike Road 501, Shanghai 201203, China
b
Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China
c
Agilent Technologies Inc., Shanghai 200131, China

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

Article history: The quality control of Da-Fu-Fang (DFF), referring to the traditional Chinese medicine (TCM) preparations
Received 29 July 2013 comprising more than 10 TCMs, is challenging due to their extreme chemical complexity. In this study, a
Accepted 27 October 2013 strategy is proposed for the holistic quality control of DFFs based on HPLC/qTOF-MS-oriented characteris-
Available online 11 November 2013
tic components data set (CCDS) and chemometric analysis. Niuhuang Shangqing pill (NHSQP), composed
of 19 TCMs, is used to illustrate this strategy. The fingerprint profiling of NHSQP by HPLC/qTOF-MS
Keywords:
resulted in the characterization of 190 compounds, comprising 47 unambiguously identified by reference
Complex traditional Chinese medicine
standard comparison. A CCDS containing 60 characteristic components was constructed by analyzing the
preparation
Characteristic components data set
MS spectral differentiation of the crude drugs, a laboratory-made NHSQP powder, and negative control
High performance liquid preparations. With the established CCDS, it was possible to simultaneously monitor 16 out of the 19 drugs
chromatography/quadrupole time-of-flight involved in NHSQP. Subsequently, 26 NHSQP samples from different vendors were evaluated by the qual-
mass spectrometry itative and semi-quantitative analyses of their LC/MS fingerprint data. The 60 characteristic components
Chemometrics were detected in all of the NHSQP samples, which demonstrated their authenticity. When compared
Niuhuang Shangqing pill with the standard sample No. 3, however, 15 of the NHSQP samples exhibited inferior quality. Samples
No. 21 and No. 13 differed significantly based on a PCA score plot, and the components responsible for
the differentiation were confirmed to originate from different TCMs. This strategy is a powerful and easy
method to implement and provides a potential approach to establishing the holistic quality control of
complex TCM preparations.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction preparations, rather than single or several compounds, which act on

The currently available literature regarding the quality control
of traditional Chinese medicines (TCM) is mostly focused on single
herbal medicines or simple TCM preparations (comprising between
two to six TCMs) [1–4]. For those rather complex preparations
which contain more than 10 TCM ingredients, known as Da-Fu-
Fang (DFF), their quality evaluation is confronted with significant
challenges based on the approaches applicable to the analysis of a
single plant TCM or simple TCM preparations. In this laboratory, it
is believed that it is the multiple components contained in the TCM
∗ Corresponding authors. Tel.: +86 21 20231000x2221; fax: +86 21 50272789.
E-mail addresses: wanyingwu@mail.shcnc.ac.cn, wanyingwu@simm.ac.cn
(W.-y. Wu), daguo@simm.ac.cn (D.-a. Guo).
1
These authors contributed equally to this work.
multiple-targets or interact with different biochemical pathways
to synergistically perform the overall therapeutic functions [5,6].
Therefore, it is critically important to establish an adequate analyt-
ical method which enables the holistic quality control of DFFs so as
to ensure their safety, efficacy, and batch-to-batch consistency.
The compendial approaches adopted by the latest Chinese Phar-
macopeia also exhibit remarkable deficiencies in the approaches
to the quality evaluation of TCM preparations. In terms of the
qualitative aspects, the microscopic examination and thin layer
chromatography (TLC) comparison cover the most extensive appli-
cation. However, these two methods are both targeted at simple
TCMs or some major components only to verify their presence
or absence. In addition, the fingerprint assays based on high-
performance liquid chromatography coupled with UV detector
(HPLC-UV) and gas chromatography coupled with hydrogen flame
ionization detector (GC-FID) are applied to identify some major

0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jpba.2013.10.042
 D.-d. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 130–141
bioactive components in 38 and 29 TCM preparations, respectively,
among the 2855 collected ones [7]. Even for the high resolution
HPLC and GC, satisfactory chromatographic separation is some-
times difficult to achieve in the cases of DFFs. Moreover, the coupled
UV and FID detectors are restricted to the analysis of unsaturated
compounds or volatile constituents, and fail to profile other types of
constituents that might be essential for therapeutic efficacy. With
respect to the quantitative analysis, the conventional approach is
to determine the contents of one to three marker (not necessar-
ily bioactive) compounds in the complex matrix, but the content of
other important bioactive components are not able to be controlled
simultaneously. A multi-component determination assay is there-
fore a good solution, however, it depends on the collection of pure
reference compounds, and the quantitative assay itself is always
time-consuming and relatively laborious.
The well-developed liquid chromatography/mass spectrometry
(LC/MS) system renders it possible to launch a more comprehensive
and thorough chemical profiling by utilizing versatile ionization
techniques and/or different ion modes [8,9]. The recent decade has
witnessed the increasingly important role of LC/MS in the unbi-
ased metabolic profiling and highly sensitive quantitation analyses
of medicinal herbs and biosamples, as detailed in the previous
reviews [10–12]. Substantial progress has been made in using the
LC/MS-based fingerprint technique in conjunction with subsequent
chemometric analysis. In particular, coupled high resolution mass
spectrometry (HR-MS), using the time-of-flight (TOF) [13] or an
orbitrap analyzer [14], provides accurate precursor and/or prod-
uct ions information with a mass error of less than 5 ppm, which
substantially enhances the metabolite characterization reliabil-
ity. Multivariate statistical analysis by classic pattern recognition
enables the chemical groups to be segregated and the rapid identi-
fication of discriminating components achieved. The integrated use
of LC/MS-based fingerprint and chemometrics was demonstrated
to be a powerful tool for the analysis of TCMs with similar chemical
compositions [15–17].
Niuhuang Shangqing pill (NHSQP), which clinically prescribed
to treat headache and dizziness, is a classic DFF composed of 19
TCMs (see Table A.1). Relatively little research has been conducted
with regard to the pharmacological effects, preparation technol-
ogy, or the content determination of more than five components in
NHSQP [18–20]. Qualitative identification methods, based on the
microscopic characteristics and the TLC examination for berberine
hydrochloride and three of the major TCMs (Rhei Radix et Rhizoma,
Coptidis Rhizoma, and Angelicae Sinensis Radix) in NHSQP have
been adopted in the Chinese Pharmacopeia in order to control the
quality of NHSQP [21].
This laboratory aims to develop novel analytical methods
capable of the holistic quality control of DFFs from both the
qualitative and quantitative aspects. Our recent study has estab-
lished a more sensitive dynamic multiple reaction monitoring
method to simultaneously quantify 41 bioactive ingredients in
NHSQP methanol extract [22]. As the continuity but from a com-
pletely different aspect, this study presents a strategy based on
the HPLC/qTOF-MS-oriented characteristic components data set
(CCDS) and chemometric analysis to identify as many as the
compositional TCMs of NHSQP and provide a rapid solution to
semi-quantitative assessing the quality homogeneity. Standard
operational procedures were established for this strategy (Fig. 1),
and are exemplified by the study of NHSQP: (I) to perform the
fingerprint profiling by HPLC/qTOF-MS, (II) to carry out multi-
path chromatographic peak identification, i.e. qTOF-MS/MS data
analysis, reference standard comparison, and in-house library and
related literatures searching, (III) to construct the CCDS by compar-
ing the BPC or EIC of the crude commercial drugs, a laboratory-made
NHSQP sample, and a negative control preparation, (IV) to carry
out a qualitative assessment of unknown NHSQP samples by an
131
examination of all of the characteristic components; (V) to conduct
a semi-quantitative analysis of NHSQP samples based on the rela-
tive peak areas, and to disclose the differentiating components by
PCA. Through the use of this strategy, it was possible to identify 190
compounds from NHSQP. Based on these data, a CCDS comprising
60 characteristic components was established. With this CCDS, the
simultaneous monitoring of 16 out of 19 TCMs involved in NHSQP
was possible, without resorting to a laborious quantitation assay.
2. Experimental
2.1. Materials and reagents
A total of 47 reference compounds were purchased either from
National Institute for the Control of Pharmaceutical and Biologi-
cal Products (Beijing, China) or from Shanghai U-SEA Biotech Co.
Ltd. (Shanghai, China). The structures of these compounds are
presented in Fig. 2. HPLC grade acetonitrile, methanol (Merck,
Darmstadt, Germany), formic acid (Roe Science Inc., USA) and
ultra-pure water, prepared by a Millipore Alpha-Q water system
(Millipore, Bedford, MA, USA), were used in the mobile phase.
Twenty-six batches of NHSQP samples (Table 1) were acquired
from different pharmaceutical manufacturers. The nineteen com-
positional medicines of NHSQP were purchased from the Northeast
districts of China and Shanghai.
2.2. Preparation of the test solutions of NHSQP samples, negative
control preparations, and reference standards
The laboratory-made NHSQP powder and negative control (NC)
preparations were used to create the characteristic components
data set. The 19 crude drug materials were pulverized into fine pow-
ders (particle size <40 mesh). According to the NHSQP prescription
recorded in the Chinese Pharmacopeia, each drug was accurately
weighed and mixed into homogeneity to afford the NHSQP pow-
der. Negative control (NC) preparations, referring to the mixed TCM
powder involving TCMs composing NHSQP except a specific one,
were also prepared. As in the case of Scutellariae Radix NC prepa-
ration (HQNN), Scutellariae Radix was absent in contrast to the
laboratory-made NHSQP powder. All NC preparations of 17 TCMs
(except Gypsum Fibrosum and Borneolum syntheticum in NHSQP)
were made in the laboratory based on the formula without the cor-
responding drug (Table A.2). An aliquot of 0.4 g of each powder was
dispersed in 20 mL 80% aqueous methanol (v/v) and ultrasonicated
in a water bath for 60 min at room temperature to prepare 17 NC
preparation solutions after filtration of the supernatant through a
0.22-␮m filter membrane. The test solutions of the 26 NHSQP sam-
ples were obtained from 1.0 g NHSQP extracted in 40 mL aqueous
methanol and based on the same procedure.
An adequate amount of each reference compound was dissolved
in methanol to prepare the reference standard test solution. Mean-
while, a mixed solution of all of the 47 reference compounds was
made using the same method. All of the test solutions were stored
at 4 ◦ C prior to analysis.
2.3. HPLC/qTOF-MS conditions
The Agilent 1290 Infinity Liquid Chromatography system (Agi-
lent, MA, USA), equipped with a binary pump, an online vacuum
degasser, an autosampler and a thermostated column compart-
ment, was used to perform the fingerprint assay. Desirable
chromatographic separation of NHSQP samples was obtained on
a Poreshell EC-C18 column (150 mm × 2.1 mm, 2.7 ␮m) connected
with an Agilent online filter (filter cartridge, 2.1 mm, 0.5 ␮m) by use
of the mobile phase A (0.1% formic acid–water) and mobile phase
B (0.1% formic acid–acetonitrile) in a gradient elution program:
132 D.-d. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 130–141

Fig. 1. The workflow of the proposed strategy for the holistic quality control of DFFs (illustrated for NHSQP).

0–3.5 min, 5–8% A; 3.5–8 min, 8–16.5% A; 8–16 min, 16.5–18% A;
16–26 min, 18–23% A; 26–40 min, 23–51% A; 40–50 min, 51–70%
A; 51–60 min, 90% A. The column was maintained at 25 ◦ C. The flow
rate was set at 0.2 mL/min, and 2 ␮L of each solution was injected
onto the column for analysis.
The high accuracy mass spectrometric data were recorded on a
6530 quadrupole time-of-flight mass spectrometer (Agilent Tech-
nologies, Germany) equipped with an ESI source in both positive
and negative ion modes. The optimized parameters were obtained
as follows: capillary voltage, 3500 V; nebulizer pressure, 45 psig;
drying gas flow rate, 8 L/min; drying gas temperature, 325 ◦ C; frag-
mentor, 145 V. The analyzer scanned over 250–1500 Da for MS and
100–1200 Da for MS/MS. The acquisition rate was 2 spectra per
second for both MS and MS/MS. Three collision energies at 10 V,
20 V, and 40 V were applied to acquire sufficient product ions. Data
acquisition was controlled by the MassHunter Workstation Soft-
ware (Agilent Technologies, USA).
In addition, the HPLC-IT-MS served as a complementary tool to
detect and analyze those constituents which showed poor response
on the qTOF mass spectrometer, such as the phthalides in Angelicae
Sinensis Radix (see Table A.2 in detail). The optimal HPLC-IT-MS
conditions are presented as Supplementary Material.
2.4. Establishment of the in-house library for NHSQP
To ensure the reliability of peak identification, an in-house
library that covers the known major secondary metabolites
in the 17 TCMs (except Gypsum Fibrosum and Borneolum
syntheticum in NHSQP) was established by researching the
databases of TCM Database @ Taiwan (http://tcm.cmu.edu.tw),
ChemSpider database (http://www.chemspider.com), MassBank
(http://www.massbank.jp/), and the MetFrag (http://msbi.ipb-
halle.de/MetFrag/) data source, also described in the literature
[23]. More than 1100 compounds are included in an Excel
spreadsheet that involves the trivial name, molecular formula,
molecular weight, structure subtype, and natural source. Through
use of the “Find” function of the Microsoft Office Excel, those
compounds which matched the determined molecular formula

Table 1
Information of 26 batches of NHSQP samples.

No. Source Batch No. No. Source Batch No.

S1 Beijing (TRT)a 1015309 S14 Shenyang (HY) 091001

S2 Beijing (TRT) 1015795 S15 Liaoning (JD) 20090102
S3 Beijing (TRT) 1015586 S16 Liaoning (JD) 20090101
S4 Beijing (TRT) 1015511 S17 Inner Mongolia (CFTQ) 20081136
S5 Beijing (TRT) 1015513 S18 Inner Mongolia (CFTQ) 20110601
S6 Beijing (TRT) 1015689 S19 Hebei (CDJFK) 090605
S7 Beijing (TRT) 9015565 S20 Hebei (CDJFK) 090711
S8 Beijing (TRT) 9015511 S21 Hebei (SJZHT) 090901
S9 Beijing (TRT) 9015529 S22 Beijing (YST) 20111001
S10 Beijing (TRT) 9015609 S23 Shanxi (TS) 111208
S11 Shenyang (HY) 090402 S24 Liaoning (DD) 090401
S12 Shenyang (HY) 110901 S25 Wuhan (ZL) 120040
S13 Shenyang (HY) 091202 S26 Hebei (YDZY) 091001
a
Abbreviated for different pharmaceutical companies.
 D.-d. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 130–141 133

Fig. 2. The structures of 47 reference compounds.

were found as potential candidates. Only those metabolite data of the NHSQP sample. Each matrix was loaded from the *.xls file to
which further matched the experimental qTOF-MS/MS data were the SIMCA software.
finally selected for establishing the identity of an unknown con-
stituent.
3. Results and discussion

2.5. Multivariate statistical analysis 3.1. Characterization of the chemical constituents in NHSQP

Unsupervised principal component analysis (PCA) was applied
in the qualitative and semi-quantitative analyses of NHSQP sam-
ples by the commercially available software SIMCA-P+ (Version
13.0, Umetrics, Umea, Sweden). The MS-oriented fingerprint spec-
trum of each sample was exported to a text file using the function
“Export” of the MassHunter Workstation. The text files of all sam-
ples were combined into one spreadsheet to generate a matrix.
Meanwhile, the other matrix was created by the relative peak areas
of the characteristic components in the semi-quantitative analysis
Detailed clarification of the chemical constituents in NHSQP
is essential for holistic quality control. In order to complete the
comprehensive characterization, an in-house library that covers
the major constituents of the 17 herbal medicines comprising
NHSQP was established. Multiple approaches, including database
searching, reference standard comparison, and qTOF-MS and
MS/MS data analysis, were employed for structural characteri-
zation of the NHSQP constituents. The chemical formula of an
unknown structure was deduced based on the high-accuracy
134 D.-d. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 130–141
Fig. 3. The proposed negative ion mode MS/MS fragmentation of the reference compounds geniposidic acid, geniposide, and an unknown compound 15.
[M−H]− /[M+HCOO]− (in the negative ion mode) or
[M+H]+ /[M+Na]+ (positive ion mode) precursor ion. The com-
pound(s) matching the determined chemical formula and
considered to undergo the observed MS/MS fragmentation
was selected as the final identity. As a result, a total of 190
compounds, including 43 flavonoids, 30 terpenoids, 22 alka-
loids, 22 anthraquinones, 19 phenolic acids, 14 phthalides, eight
phenylethanoid glycosides, five coumarins, four lignans, four
steroids, and 19 miscellaneous compounds, were identified or
tentatively characterized from the constituents of NHSQP. Among
these metabolites, 47 compounds were unambiguously identified
by comparison with reference standards in terms of the tR and
MS/MS product fragments. Information regarding the 190 NHSQP
constituents, such as the tR (min), identity, observed m/z values,
mass error (in ppm), molecular formula, and botanical source,
is offered in Table A.3. These compounds originate from the 17
plant-based TCMs, while the typical constituents of Borneolum
Syntheticum (bornel) and Gypsum Fibrosum (CaSO4 ·2H2 O) were
not detected.
3.1.1. Flavonoids
A large number of flavonoids (16 free aglycones and 27 O-/C-
glycosides), were identified from NHSQP. The negative ion MS/MS
fragmentations of flavonoid aglycones are characterized by the
retro-Diels–Alder (RDA) cleavage of ring C (1,3 A− and 1,3 B− ) and
multiple neutral loss pathways, whereas sequential elimination of
sugar residues, combined with aglycone fragments, are diagnostic
for the characterization of glycosidic flavonoids [24,25]. For two of
the flavone reference compounds, the product fragments observed
at m/z 151 ([C7 H3 O4 ]− ) and 117 ([C8 H5 O]− ) of apigenin (Fig. A.1a),
and the fragments at m/z 151 ([C7 H3 O4 ]− ) and 133 ([C8 H5 O2 ]− )
of luteolin (Fig. A.1b) corresponded to their 1,3 A− and 1,3 B− ions,
respectively. In the case of liquiritin (Fig. A.1c), a flavanone O-
glycoside reference compound, the liquiritigenin ion at m/z 255
(aglycone ion) was obtained after eliminating the glucose residue
(−162 Da), which further fragmented into product ions m/z 135 and
119, consistent with the typical 1,3 A− and 1,3 B− fragments, respec-
tively. These MS/MS cleavage features assisted in the identification
of the unknown compound 130 (Table A.3). The [M−H]− precursor
ion at m/z 271 (m/z 271.0824 for [C15 H11 O5 ]− , 0.03 ppm) indi-
cated the possible molecular formula C15 H12 O5 , which matched
naringenin on the basis of library searching. Its MS2 fragmenta-
tion produced two major product ions at m/z 151 ([C7 H3 O4 ]− )
and 119 ([C8 H7 O]− ), in agreement with the classic 1,3 A− and
1,3 B− fragments, indicating the presences two and one hydroxyl
groups substituted on rings A and B of a flavanone, which further
Table 2
The established characteristic components data set (CCDS) for NHSQP.

No. Source Identity tR (min) m/z (+/−) M.F. MS/MS fragments

1(10) DiH Rehmannioside D 3.94 731.2245c C27 H42 O20 MS2 [731]: 685, 665, 649
2(13)a ZZ Geniposidic acid 5.66 373.1389 C16 H22 O10 MS2 [373]: 211, 167, 156, 149, 123, 113, 105, 101, 95, 89
3(14) DiH Leonuride or isomer 5.68 393.141c C15 H24 O9 MS2 [393]: 347, 317, 282, 241, 185, 167, 127, 112, 74
4(15) DiH 8-Epiloganic acid 6.45 375.1547 C16 H24 O10 MS2 [375]: 355, 213, 179, 169, 151, 125, 100, 97, 89
5(25) ZZ Genipin 1-␤-d-gentiobioside 9.21 549.1820 C23 H34 O15 MS2 [549]: 389, 225, 207, 147, 123, 101, 69
6(31)a ZZ Geniposide 10.36 433.1558c C17 H24 O10 MS2 [433]: 387, 207, 147, 123, 113, 101, 89, 69, 59
7(32)a HB Phellodendrine 10.49 342.1721 C20 H24 NO4 MS2 [342]: 192, 177, 149
8(36)a CS Albiflorin 11.27 525.1921c C23 H28 O11 MS2 [525]: 479, 357, 283, 121, 77
9(37) HL, HB Magnoflorine 11.35 342.1712 C20 H24 NO4 MS2 [342]: 297, 282, 237, 191, 58
10(43)a CS Paeoniflorin 12.20 525.1921c C23 H28 O11 MS2 [525]: 479, 449, 327, 165, 121, 77
MS2

D.-d. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 130–141
11(57) HL, HB Methoxy-(Lotusine/(−)-Oblongine) 15.17 344.1856 C20 H26 NO4 [344]: 299, 239, 207, 175, 151, 143, 137, 115, 91, 58
12(63)a GC Liquiritin 15.56 417.1462 C21 H22 O9 MS2 [417]: 255, 135, 119, 91
13(67)a LQ Forsythoside A 16.16 623.1982 C29 H36 O15 MS2 [623]: 461, 443, 179, 161, 135, 112, 68
14(68)a DaH (−)-Epicatechin gallate 16.37 441.0830 C22 H18 O11 MS2 [442]: 289, 245, 203, 175, 169, 149, 137, 125, 109
15(74)a LQ Acteoside 17.46 623.1980 C29 H36 O15 N/A
16(75) HL, HB Groenlandicine 18.58 322.1087 C19 H16 NO4 MS2 [322]: 307, 292, 265, 250, 221, 193
17(86)a JH, JJS Apigenin-7-O-glucoside 22.14 431.0989 C21 H20 O10 MS2 [431]: 269, 268, 151, 119
18(87) BH Diosmin 22.51 607.1686 C28 H32 O15 MS2 [607]: 299, 284
19(93) HL, HB Columbamine 24.04 338.1394 C20 H20 NO4 MS2 [338]: 322, 307, 294, 279, 265, 240, 237
20(97)b BH Rosmarinic acid 24.74 359.0770 C18 H16 O8 N/A
21(99)a HL Coptisine 25.41 320.0922 C19 H14 NO4 MS2 [320]: 307, 292, 277, 262, 249, 219, 204, 191, 177, 161
22(100)a HL, HB Jatrorrhizine 25.45 338.1394 C20 H20 NO4 MS2 [338]: 322, 307, 294, 280, 265, 240, 237, 222
23(103) ZZ 3,5-di-O-caffeoyl-4-O-(3-hydroxy,3- 26.06 659.1619 C31 H32 O16 MS2 [695]: 659, 639, 497, 353, 335, 273, 255, 233, 211, 191, 173, 161, 135, 93, 59
methyl)glutaroylquinic
acid
24(105) DaH 1-O-galloyl-6-O-cinnamoyl-glucose 26.54 461.1099 C22 H22 O11 MS2 [461]: 313, 211, 169, 147, 125, 103
25(106)a HQ Baicalin 28.21 445.0768 C21 H18 O11 MS2 [445]: 891, 621, 543, 445, 345, 269, 175
26(108) HL, HB S/C(Tetradehydroscoulerine/ 28.64 322.1084 C19 H16 NO4 MS2 [322]: 307, 292, 279, 264, 251
tetradehydrocheilanthifolinium)
27(110)a LQ Forsythin 29.90 579.2081c C27 H34 O11 MS2 [579]: 545, 533, 489, 371, 356, 323, 236, 161, 151, 121, 113, 71
28(111)a HL, HB Palmatine 30.44 352.1566 C21 H22 NO4 MS2 [352]: 336, 320, 208, 294, 278, 264, 250, 235
29(113) JH, JJS Eriodictyol 30.75 287.0564 C15 H12 O6 MS2 [287]: 151, 135, 107, 87, 83, 65
30(114)a HL, HB Berberine 30.77 336.1297 C20 H18 NO4 MS2 [336]: 320, 304, 292, 278, 263
31(118)a JH, JJS Luteolin 31.63 285.0626 C15 H12 O6 MS2 [285]: 175, 151, 133, 107, 83, 65, 51
32(120)a HQ Wogonoside 32.10 459.0939 C22 H20 O11 MS2 [459]: 919, 459, 283, 268, 175
33(127)a HQ Oroxylin A-7-O-glu acid 33.12 459.0929 C22 H20 O11 MS2 [459]: 919, 459, 283, 268, 175
34(135d )a JH, JJS Apigenin 34.75 269.0688 C15 H10 O5 MS2 [269]: 225, 151, 117, 107, 83, 65
35(137) GC 22-Acetoxyl-glycyrrhizin 35.03 879.4014 C44 H64 O18 MS2 [879]: 351, 289, 193, 113
36(139) GC 22-Hydroxyl-glycyrrhizin 35.26 837.3909 C42 H64 O17 MS2 [837]: 351, 193
37(144)a HQ Baicalein 36.10 269.0463 C15 H10 O5 MS2 [283]: 268, 266
38(147) GC Licorice-saponin G2 36.66 837.3909 C42 H62 O17 MS2 [837]: 351, 193, 113
39(149)a GC Glycyrrhizin 37.74 821.4377 C42 H62 O16 MS2 [821]: 351, 193, 113, 71
40(151) GC Uralsaponin B 39.15 821.3960 C42 H62 O16 MS2 [821]: 351, 193, 113
41(154)a NH Cholic acid 40.27 407.2815 C24 H40 O5 MS2 [407]: 371, 343, 325, 289, 205, 123, 95
42(155)a HQ Wogonin 40.42 283.0622 C16 H12 O5 MS2 [283]: 268, 266
43(156)a HQ Chrysin 40.77 253.0509 C15 H10 O4 N/A
44(158)a JH, JJS Acacetin 41.05 283.0619 C16 H12 O5 MS2 [283]: 268, 239, 211, 195, 151, 107, 83, 63
45(162) DG, CX Senkyunolide A 42.38 193.1234 C12 H16 O2 MS2 [193]: 175, 147, 105
46(163) DG, CX 3-Butylphthalide 43.13 191.1080 C12 H14 O2 MS2 [191]: 173, 145, 137, 117
47(164) DG, CX E-Ligustilide 44.58 191.1075 C12 H14 O2 MS2 [191]: 173, 149, 108, 95
48(165)a BZ Imperatorin 44.83 271.0982 C16 H14 O4 MS2 [271]: 203, 175, 147, 131, 91, 65
49(166) DG, CX E-Butylidenephthalide 45.12 189.0915 C12 H12 O2 MS2 [189]: 171, 147, 143, 137, 133
50(167) BZ Neocnidilide or Cnidilide 45.18 195.1386 C12 H18 O2 MS2 [195]: 149
51(168)b BZ Xanthotoxol 45.18 203.0347 C11 H6 O4 N/A

135
136 D.-d. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 130–141

The numbering consistent with Table A.3; BH, Menthae Herba; BZ, Angelicae Dahuricae Radix; CS, Paeoniae Radix Rubra; CX, Chuanxiong Rhizoma; DaH, Rhei Radix et Rhizoma; DiH, Rehmanniae Radix; DG, Angelicae Sinensis
Radix; GC, Glycyrrhizae Radix et Rhizoma; HB, Phellodendri Chinensis Cortex; HL, Coptidis Rhizoma; HQ, Scutellariae Radix; JH, Chrysanthemi Flos; JG, Platycodonis Radix; JJS, Schizonepetae Spica; LQ, Forsythiae Fructus; NH,
confirmed the identify of compound 130 as naringenin, a known

[269]: 241, 225, 210, 197, 185, 171, 157, 141, 125, 105, 91, 79
compound in Glycyrrhizae Radix et Rhizoma [26]. With respect to

MS2 [381]: 263, 233, 215, 191, 173, 145, 133, 117, 115, 91, 67
the C-glycosidic flavones, incidental elimination of partial sugars
has been known as their characteristic MS/MS fragmentation fea-
ture [27]. Two unknown isomeric compounds 55 (tR = 15.10 min)

[391]: 373, 345, 327, 312, 295, 259, 203, 164, 69

and 73 (tR = 17.36 min) both exhibited predominant [M−H]− pre-
cursor ions at m/z 547 (Fig. A.2). The [M−H−90]− (m/z 457) and
[M−H−120]− (m/z 427) product ions in their MS2 spectra dis-

[381]: 363, 280, 267, 191, 133, 117, 115

closed the presence of a hexose and pentose residues in these
two compounds, respectively. However, the relative abundances
[191]: 173, 155, 145, 135, 105

of the [M−H−90]− to [M−H−120]− ions were calculated at 300%

and 40% for 55 and 73. This MS/MS fragmentation feature, together
[189]: 164, 143, 115, 97

with their retention difference, permitted the discrimination of

55 and 73 as chrysin 6-C-arabinoside-8-C-glucoside and chrysin
6-C-glucoside-8-C-arabinoside, two major Scutellariae Radix con-
MS/MS fragments

stituents based on an earlier report [28].

[437]: 391

[301]:

3.1.2. Terpenoids
The terpenoids, including 12 iridoids, eight monoterpenes, and
MS2
MS2
MS2
MS2
MS2
MS2

MS2
N/A

ten triterpenes (four sapogenins and six saponins) were character-

ized from NHSQP. Three iridoids, 7 (catalpol), 13 (geniposidic acid),
and 31 (geniposide) were unambiguously identified by comparison
with reference standards. As illustrated in Fig. 3, the deprotonated
iridoid aglycone fragments of 13 and 31 observed at m/z 211 and
C24 H40 O4
C15 H10 O5
C12 H14 O2
C17 H16 O5
C12 H12 O2
C24 H40 O4
C16 H14 O4
C24 H28 O4

C24 H28 O4

225 both underwent 1,4 F cleavage [29]. Additionally, neutral loss

M.F.

of CO2 occurred to 13 due to the presence of a carboxyl func-

tionality. These fragmentation features were applied in order to
characterize the remaining nine unknown iridoids, such as com-
pound 15 (tR = 6.45 min). The [M−H]− precursor ion appearing at
437.2909c

m/z 375.1547 indicated the molecular formula to be C16 H24 O10 ,

m/z (+/−)

391.2851
269.0548
191.1078
301.1110
189.0919

271.0972
381.2069

381.2069

consistent with 8-epiloganic acid in the NHSQP database. Further

evidence was obtained from the negative MS/MS fragmentation of
the aglycone ion m/z 213 ([M−H−Glc]− ), which yielded product
ions at m/z 125 and 89 originating from 1,4 F fragmentation, and
at m/z 169 due to the elimination of CO2 (Fig. 3). In addition, five
monoterpene glycosides were also identified and are typical for
45.38
45.57
45.78

46.13
47.27
46.05
46.10

53.08
52–52.8
tR (min)

Paeoniae Radix Rubra (36, 43, 90, 128, and 131).

The negative MS2 spectra of the reference compounds albiflorin
(36) and paeoniflorin (43) both displayed a benzoic acid product ion
m/z 121 ([C7 H5 O2 ]− ) and the neutral loss of 30 Da (CH2 O) [30]. The
unknown compound 128 underwent similar cleavage pathways as
those of 43, but showed corresponding fragments with an increase
in mass of 104 Da, suggesting one additional benzoyl substituent,
Angelicide/Riligustilide/Z,Z -6,8 7,3 -

and that matched benzoylpaeoniflorin in the NHSQP database. Five

saponins (137, 139, 147, 149, and 151) were characterized as the
constituents of Glycyrrhizae Radix et Rhizoma. They yielded an
diligustilide/tokinolide

abundant product ion at m/z 351 representing a GlurA–GlurA gly-

Z-Butylidenephthalide

Components identified with reference compounds comparison.

Hyodeoxycholic acid

cosyl chain, as in glycyrrhizin (149, Fig. A.4). The transitions from

Deoxycholic acid

[M−H]− to [GlurAGlurA−H]− provide diagnostic information for

Isoimperatorin

Levistolide A
Z-Ligustilide

the sapogenins. Based on the HR-MS information, the differenti-

ated retention behavior (Fig. A.4), and previous literature [31], the
Identity

Cnidilin
Emodin

unknown saponins were characterized.

Bovis Calculus Artifactus; ZZ, Gardeniae Fructus.
B

m/z values of [M+HCOO]− precursor ions.

Components without MS/MS fragments.

3.1.3. Alkaloids
The alkaloids exhibited specific and abundant [M]+ precur-
sor ions, the MS/MS fragmentation of which featured successive
losses of • CH3 , CH4 , and CO, as illustrated by two reference com-
DG, CX

DG, CX

DG, CX

DG, CX
Source

DaH

pounds (114 berberine and 100 jatrorrhizine) shown in Fig. A.5

NH

NH
BZ

BZ

[32]. These fragmentation characteristics, when combined with

database searching, permitted the characterization of 22 alkaloids
from NHSQP. An unknown compound 93 (tR = 24.04 min) displayed
Table 2 (Continued)

the [M]+ precursor ion at m/z 338.1394 (C20 H20 NO4 ), implying
59(178–180)

an isomer of jatrorrhizine, and corresponded to columbamine in

58(175)b
54(171)a
53(170)a
52(169)

55(172)
56(173)
57(174)

60(181)

the NHSQP database. Notably, compound 93 and the reference

compound jatrorrhizine underwent highly analogous MS/MS frag-
No.

mentation pathways, as shown in Fig. A.5. Moreover, its weaker

 D.-d. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 130–141 137

Fig. 4. The base peak chromatograms (BPC) of the laboratory-made NHSQP powder in the negative (a) and positive (b) ion modes showing the deduced 60 characteristic
components for NHSQP.

retention capacity than jatrorrhizine on reversed phase column selected as the characteristic components. Both the negative and
chromatography was consistent with an earlier report [33], and positive ion modes of MS spectra were used to obtain the character-
further confirmed the identification. istic components for the maximum number of TCMs in the NHSQP
Regarding the characterization of the other categories of NHSQP matrix. Following this, a total of 60 characteristic components were
constituents, similar approaches based on reference standard com- distinguished representative of the 16 plant-based TCMs (Fig. 4 (±)
parison, in-house library searching, and MS/MS fragmentation BPC of the laboratory-made NHSQP powder, Table 2). In the imple-
analysis were simultaneously employed. Literature evaluation with mentation of this criterion in order to deduce the CCDS for NHSQP,
respect to the LC/MS analysis of anthraquinones [34], phenolic acids three different situations were encountered.
[35], phthalides [36], phenylethanoid glycosides [37], coumarins
[38], lignans [37], and steroids [39], were also utilized in order to 3.2.1. Unique characteristic components
warrant the identification results. This is the first comprehensive Among the TCMs comprising NHSQP, ten medicines, compris-
chemical profiling of NHSQP, and therefore lays the foundation for ing Scutellariae Radix, Menthae Herba, Paeoniae Radix Rubra,
holistic quality control of this complex preparation. Rhei Radix et Rhizoma, Rehmanniae Radix, Glycyrrhizae Radix et
Rhizoma, Bovis Calculus Artifactus, Forsythiae Fructus, Gardeniae
3.2. The establishment of a characteristic components data set Fructus, and Angelicae Dahuricae Radix, displayed several chro-
matographic peaks that could only be found in one TCM. As a
Due to the HPLC/qTOF-MS fingerprint assay, 190 chemical con- result, those specific constituents were selected as the character-
stituents present in the 17 medicinal plants of NHSQP were clearly istic components. The case of Rhei Radix et Rhizoma is used to
identified. Based on these results, the next step was to construct illustrate the selection process. Fig. 5 exhibits the BPCs of NHSQP,
a characteristic components data set (CCDS) representing all of Rhei Radix et Rhizoma (the crude drug), and DaHNN (the NC prepa-
the compositional TCMs. The characteristic components refer to ration) in the negative ion mode. Through inspection and analysis,
those metabolites which are present in one unique or, at most, NHSQP (Fig. 5a) and the Rhei drug (Fig. 5b) shared multiple peaks,
two of the TCMs in the studied TCM preparation, and mainly con-
tribute to the therapeutic function depicted in previous literature.
To name a few, the iridoid glycosides, such as geniposide, genipin-
gentiobioside and geniposidic acid, have been known as the major
components responsible for the choleretic effect of Gardeniae Fruc-
tus, which are thus locked as potential characteristic components
for Gardeniae Fructus [29].
Through the simultaneous monitoring of these characteristic
components, it was possible to perform the holistic quality control
of DFFs. In the case of NHSQP, the base peak chromatograms (BPC)
or the extracted ion chromatograms (EIC) of the crude drug materi-
als, the laboratory-made NHSQP powder, and the negative control
(NC) preparations (Table A.2) were compared abiding by a proposed
criterion. The initial comparison of the NHSQP powder and a com-
positional TCM indicates some common chromatographic peaks
(showing the same tR and precursor ion). Among them, only those
that are absent in the corresponding NC preparation are poten-
Fig. 5. The base peak chromatograms (BPC) of NHSQP (a), the crude drug, (b), and
tial characteristic candidate components. If multiple components the negative control preparation (c) of Rhei Radix et Rhizoma. Inset shows their
match the conditions, then the most abundant metabolites are extracted ion chromatograms (EIC) based on extracting the ion at m/z 441 (d).
138 D.-d. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 130–141
among which 14 ([M−H]− m/z 441), 24 (m/z 461), and 53 (m/z
269) were the most abundant. Due to their absence in the NC
preparation (Fig. 5c), compounds 24 and 53 could be readily distin-
guished as the characteristic components. However, in the case of
14, since an abundant peak eluting at the same time also appeared
in the NC preparation (Fig. 5c) there may be some confusion. This
problem was solved by comparing their EICs through extracting
the ion at m/z 441 and demonstrating it was absent in the NC
preparation (Fig. 5d). Based on these analyses, compounds 14 [(−)-
epicatechin gallate)], 24 (1-O-galloyl-6-O-cinnamoyl-glucose), and
53 (emodin) were chosen as the characteristic components of Rhei
Radix et Rhizoma.
3.2.2. Characteristic components common for two drugs
Some TCMs derivatized from the contained plant species
possess a somewhat similar secondary metabolite profile. One
example is Chuanxiong Rhizoma and Angelicae Sinensis Radix.
The chormatographoc profile can be diagnostic for their pres-
ence, but is not suitable for their mutual discrimination. In these
cases, those constituents which are specific for the two drugs are
selected as their common characteristic components. Fig. A.6 dis-
plays the EICs of NHSQP, the drugs of Chuanxiong Rhizoma and
Angelicae Sinensis Radix, together with their NC preparations. Eight
phthalide compounds 45 (senkyunolide A), 46 (3-butylphthalide),
47 (E-ligustilide), 49 (E-butylidenephthalide), 54 (Z-ligustilide), 56
(Z-butylidenephthalide), 59 (Angelicide/Riligustilide/Z,Z -6,8 7,3 -
diligustilide/tokinolide B), and 60 (levistolide A) were detected in
NHSQP and these two herbal medicines. However, they were also
detected in the NC preparations because of the presence of Chuanx-
iong Rhizoma in DGNN (the NC preparation of Angelicae Sinensis
Radix) and of Angelicae Sinensis Radix in CXNN (the NC preparation
of Chuanxiong Rhizoma). Through literature analysis it was deter-
mined that these phthalide compounds have not been reported
from the other 17 TCMs present in NHSQP, and were not observed
from their BPCs in our study. Consequently, they were selected
to represent Chuanxiong Rhizoma and Angelicae Sinensis Radix
in NHSQP. The same circumstances occur for Schizonepetae Spica
and Chrysanthemi Flos, which share five characteristic flavonoid
components (17, 29, 31, 34, and 44) that can be used for their identi-
fication in an unknown NHSQP sample (Fig. A.7). On the other hand,
a somewhat different situation was observed between Coptidis Rhi-
zoma and Phellodendri Chinensis Cortex. Aside from the common
characteristic constituents, namely, the eight alkaloids (9, 11, 16,
19, 22, 26, 28, and 30), one another alkaloid, compound 7, and one
other compound 21 were specific for Phellodendri Chinensis Cortex
and Coptidis Rhizoma (see Table 2).
In addition to the above 16 TCMs constituting NHSQP, no con-
stituents typical for Gypsum Fibrosum and Borneolum syntheticum
were detected utilizing the ESI-qTOF-MS technique. In addition, the
saponins present in Platycodonis Radix (platycodins) exhibit a poor
response under the applied MS conditions. However, with respect
to these three TCMs, their identification can be easily realized by
TLC as recorded in the Chinese Pharmacopeia. Regardless of this
minor deficiency, the established CCDS enables the identification of
84.2% (16 out of 19) of the medicinal plants present, thereby estab-
lishing the strategy as a potentially valuable tool which is applicable
to the holistic quality control of NHSQP.
3.3. Qualitative and semi-quantitative analyses of NHSQP
samples
The available approaches for the quality control of complex
TCM preparations are mostly based on the microscopic exami-
nation of diagnostic plant tissues, and the TLC comparison with
reference compounds or reference drug materials. Research efforts
aimed at the determination of several compounds (less than 5 in
general) have been reported. However, these methods are com-
pletely inadequate for the simultaneous metabolite monitoring of
a complex TCM, such as the 17 plant materials in NHSQP. In the
present study, a CCDS, representative of 16 of the plant-based
constituent drugs in NHSQP was developed. These characteristic
components were subsequently employed to evaluate, qualita-
tively and semi-quantiatively, the constituent qualities of 26
NHSQP samples collected from different pharmaceutical manufac-
turers.
3.3.1. Qualitative analysis of NHSQP samples
A total of 26 batches of NHSQP samples from different vendors
(Table 1) were analyzed to evaluate their constituent quali-
ties utilizing the established CCDS, in combination with pattern
recognition chemometric analysis. Firstly, unsupervised principle
components analysis (PCA) was applied to verify the applicability
of the selected CCDS for the qualitative identification of the NHSQP
samples.
Primary analysis revealed that the characteristic components of
the different compositional drugs exhibited significantly differen-
tiated ion response between the negative and positive ion modes.
Therefore, in order to obtain higher peak intensity, the positive ion
mode was selected to analyze Angelicae Dahuricae Radix, Angel-
icae Sinensis Radix, Chuanxiong Rhizoma, Phellodendri Chinensis
Cortex, and Coptidis Rhizoma, while the negative mode was used
to detect the twelve other TCMs in NHSQP. Fig. 6 depicts the PCA
score plot of the 26 NHSQP samples, and the 17 laboratory-made
NC preparations, using different ionization modes. It is apparent
that, despite the different ion modes which were used, the 26
NHSQP samples and the 17 NC preparations were well separated.
This demonstrates that the deduced characteristic components are
powerful and are able to distinguish the presence or absence of
their corresponding parent TCM in an unknown NHSQP sample. For
instance, the negative preparation of Scutellariae Radix (HQNN),
was located far from the NHSQP group and other negative prepara-
tions in the score plot (marked), which made it easy to discriminate
a NHSQP sample for which Scutellariae Radix was absent. These
results also manifest the adaptability of the established CCDS for
performing the qualitative assessment of NHSQP samples.
The HPLC/qTOF-MS spectra of the unknown NHSQP samples
were then analyzed to qualitatively examine the presence of the
16 TCMs by extracting the data on the corresponding ions of their
characteristic components (Table 2). As in the case of identifying
Scutellariae Radix, the ions at m/z 445, 459, 269, 283, and 253,
which are the [M−H]− precursor ions of the proposed six char-
acteristic components baicalin (25), wogonoside (32)/oroxylin A
7-O-glucuronide (33), baicalein (37), wogonin (42), and chrysin
(43), were extracted for each of the test NHSQP samples (Fig.
A.8). It is apparent that they all displayed the chromatographic
peaks of these characteristic components despite their differenti-
ated contents. This verifies the presence of Scutellariae Radix in the
NHSQP samples tested. In the similar manner, the other 15 TCMs
involved in NHSQP were positively identified. Using this approach,
it was therefore possible to conclude that all of the examined
NHSQP samples were of authentic quality.
3.3.2. Semi-quantitative analysis of NHSQP samples
Using a semi-quantitative assay, the quality difference among
the multiple TCM preparation samples could be evaluated based
on the deduced CCDS. With respect to the semi-quantitation of
NHSQP, the precursor ions of the characteristic components in the
26 samples were extracted, and the absolute peak areas of a total
of 55 metabolites (except 20, 46, 47, 49, and 55 in the CCDS due
to their extremely low content) were obtained. Sample No. 3 was
determined to be of good quality based on the previous quantita-
tive study of NHSQP by LC/MS/MS, and was therefore selected as
 D.-d. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 130–141
Fig. 6. Non-supervised PCA score plot of the 26 NHSQP samples and 17 negative control preparations originating from the negative and positive ion mode LC/MS fingerprint
spectra.
the reference sample. The peak areas of each characteristic compo-
nent in the other 25 samples, compared to those in sample No. 3,
were calculated as the relative peak areas (RPAs), and the data are
presented in Table A.4. In order to simplify the resulting data, the
RPA values which were below 0.01 were defined as 0.
A graphical comparison of the 26 NHSQP samples is illustrated
in Fig. A.9. In contrast to the “qualified” sample No. 3, for which the
cumulative RPAs add up to 53, a total of 15 batches of the NHSQP
samples showed inferior quality, among which No. 21 (Fig. A.9, hol-
low bar) is the worst. On the other hand, No. 13 (Fig. A.9, dashed bar)
possesses the superior quality since it contains the most abundant
bioactive constituents originating from the 16 different constituent
TCMs. Through the use of the reference sample, the quality differ-
entiation of the other samples can be readily assessed.
On the basis of the calculated RPAs, the quality difference of
the NHSQP samples from different vendors was further evaluated
by the use of PCA. A preliminary PCA score plot derived from the
LC/MS fingerprint spectra data failed to give remarkable group
segregation (data not shown), while PCA based on the calculated
RPAs of the 55 characteristic components excavated the potential
139
significance of the 26 batches of NHSQP samples. Consistent with
the above direct analysis, in the score plot (Fig. 7a), samples No. 21
and No. 13 were observed as two outliers. Through the analysis of
the loading plot (Fig. 7b), the variables (consistent with the charac-
teristic components) which contribute more to the discrimination
of the NHSQP samples can be further explored.
For the “unqualified” sample No. 21, it was possible to conclude
that the variable compounds marked in Circle I (including 10, 14,
15, 19, 24, 27, 28, 29, 30, 41, 50, 54 and 56) contributed to its role
as an outlier (Fig. 7b). This influenced No. 21 in a negative manner
due to their opposite phase based on the location of the sample
in the score plot (Fig. 7a). Consistently, all of these variables in
sample No. 21, corresponding to the characteristic components
in Table 2, were at a much lower level (peak area ratios less than
0.20 except for 30 and 56). As shown in Table 2, compounds 10,
14, 15, 19, 24, 27, 28, 29, 30, 41, 50, 54 and 56 were characterized
as Paeoniflorin, (−)-Epicatechin gallate, Acteoside, Columbamine,
1-O-galloyl-6-O-cinnamoyl-glucose, Forsythin, Palmatine, Eriod-
ictyol, Berberine, Cholic acid, Neocnidilide/Cnidilide, Z-Ligustilide
and Z-Butylidenephthalide which were the characteristic
140 D.-d. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 130–141
Fig. 7. PCA score plot (a) and loading plot (b) of the 26 batches of NHSQP samples
on the basis of the relative peak areas of 55 characteristic components.
components of the 10 TCMs, Paeoniae Radix Rubra, Chrysan-
themi Flos, Angelicae Sinensis Radix/Chuanxiong Rhizoma,
Angelicae Dahuricae Radix, Rhei Radix et Rhizoma, Forsythiae
Fructus, Bovis Calculus Artifactus, Coptidis Rhizoma/Phellodendri
Chinensis Cortex. In this way, those TCMs which yielded a lower
content of the characteristic components were readily identified.
Analogously, the variable metabolites 1, 5, 8, 13, 17, 31, 34, 35, 38
and 44 in Circle II were responsible for the differentiation of sample
No. 13. These are the characteristic components of Chrysanthemi
Flos/Schizonepetae Spica, Chuanxiong Rhizoma, Paeoniae Radix
Rubra, Rehmanniae Radix, and Gardeniae Fructus, respectively.
Based on these comparisons it can be stated that the integrated
use of CCDS and PCA proved to be powerful and easy to implement
strategy which could be used for the quality assessment of NHSQP
samples in both a qualitative and a semi-quantitative manner.
We have tried two different strategies for the holistic quality
control of complex TCM preparations like NHSQP utilizing LC/MS
followed by chemometric analysis. The previous research docu-
mented a more sensitive dynamic MRM method to simultaneously
determine 41 compounds that enabled the precise evaluation of
15 TCMs in NHSQP [22]. In contrast, the strategy proposed in the
current study, by monitoring the characteristic components, is a
relatively simplified solution to qualitative characterization of TCM
preparations. These two strategies are exemplified by the same
study object but from different aspects, which are thus basically
differentiated. In light of the remarkable merits over currently
available methodologies, we expect to have them popularized in
more research cases and practical use of DFF quality control.
4. Conclusions
As an integral aspect of a long-term project aimed at the explo-
ration of new strategies for applying integrated techniques to the
contemporary quality control of complex TCM preparations, the
present study has outlined a detailed approach based on the char-
acteristic components data set (CCDS) and pattern recognition
chemometrics for the quality evaluation of DFFs. Using the 19-
component, highly complex preparation NHSQP as the paradigm, a
standard operations procedure comprised of five successive steps
was established to qualitatively and semi-quantitatively evalu-
ate the qualities of DFFs. The fingerprint profiling of NHSQP by
HPLC/ESI-qTOF-MS in different ionization modes resulted in the
characterization of 190 compounds covering 10 different structural
subtypes, involving 47 metabolites which were precisely identified
by comparison with reference standards. The CCDS was established,
comprising 60 compounds deemed as representative of 16 of the
19 TCMs, on the basis of a systematic comparison of the MS spec-
tral differences among NHSQP, the constituent crude drugs, and
the laboratory-made negative control preparations. The qualities of
26 NHSQP samples acquired from manufacturers were assessed by
detecting their characteristic components, and by comparison with
a reference sample based on the relative peak areas. The integrated
PCA allowed visualization of the batch-to-batch quality differenti-
ation of the NHSQP samples, and further assisted in determining
which of the compositional TCMs were contributing to the qual-
ity differentiation. It should be noted that 16 out of the 19 TCMs
present in NHSQP could be simultaneously monitored through the
utilization of the proposed strategy. This study indicates that the
integrated use of characteristic components data set and chemo-
metric analysis is a very powerful tool that can be applied for the
holistic quality control of complex TCM preparations in both a qual-
itative and a semi-quantitative manner.
Acknowledgements
This work was supported by National Natural Science Foun-
dation committee of China (90612002) and the Twelfth Five-Year
National Science & Technology Support Program (2012BAI29B06).
We gratefully acknowledge the assistance of Geoffrey A. Cordell,
who is president of Natural Products, in revising the language. We
also thank Agilent Technologies Inc. for the instrument support.
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.2013.10.042.
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