Identification and determination of the major constituents in Traditional Chinese Medicinal formula Danggui-Shaoyao-San by HPLC-DAD-ESI-MS/MS

Abstract

Danggui-Shaoyao-San (DSS), a famous traditional Chinese medicine formula consisting of six herbal medicines (Paeonia lactiflora, Angelica sinensis, Ligusticum chuanxiong, Poria cocos, Atractylodis macrocephalae and Rhizoma Alismatis), has been used as a classical gynecological remedy in China for centuries. However, its active substances have remained unknown. In this paper, an HPLC/DAD/ESI-MS/MS method was developed for the qualitative and quantitative analysis of the major constituents in DSS. The ESI-MS/MS fragmentation behavior of the reference compounds was proposed for aiding the structural identification of components in DSS extract. Forty-one compounds including monoterpene glycosides, phenolic acids, phathalides, sesquiterpenoids and triterpenes were identified or tentatively characterized by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data, and 14 of them (gallic acid, albiflorin, paeoniflorin, ferulic acid, benzoic acid, senkyunolide I, coniferyl ferulate, senkyunolide A, 3-butylphthalide, Z-ligustilide, Z-butylidenephthalide, atractylcnolide II, atractylcnolide I and levistolide A) were determined by HPLC-DAD using a C18 column and gradient elution of acetonitrile/water-formic acid (100:0.1, v/v). The linearity, precision, accuracy, LOD and LOQ were validated for the quantification method, which proved sensitive, accurate and reproducible. The study might provide a basis for the quality control of DSS extracts and preparations.

Full text

Journal of Pharmaceutical and Biomedical Analysis 50 (2009) 127–137
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Journal of Pharmaceutical and Biomedical Analysis
journal homepage: www.elsevier.com/locate/jpba
Identification and determination of the major constituents in Traditional Chinese
Medicinal formula Danggui-Shaoyao-San by HPLC–DAD–ESI-MS/MS
Linlin Chen, Jin Qi, Yan-xu Chang, Danni Zhu, Boyang Yu∗
Department of Complex Prescription of TCM, China Pharmaceutical University, No. 1 Shennong Road, Nanjing 210038, PR China
article info
Article history:
Received 27 November 2008
Received in revised form 26 March 2009
Accepted 30 March 2009
Available online 8 April 2009
Keywords:
Traditional Chinese Medicinal formula
Danggui-Shaoyao-San
Major constituents
HPLC–DAD–ESI-MS/MS
abstract
Danggui-Shaoyao-San (DSS), a famous traditional Chinese medicine formula consisting of six herbal
medicines (Paeonia lactiflora,Angelica sinensis,Ligusticum chuanxiong,Poria cocos,Atractylodis macro-
cephalae and Rhizoma Alismatis), has been used as a classical gynecological remedy in China for centuries.
However, its active substances have remained unknown. In this paper, an HPLC/DAD/ESI-MS/MS method
was developed for the qualitative and quantitative analysis of the major constituents in DSS. The ESI-
MS/MS fragmentation behavior of the reference compounds was proposed for aiding the structural
identification of components in DSS extract. Forty-one compounds including monoterpene glycosides,
phenolic acids, phathalides, sesquiterpenoids and triterpenes were identified or tentatively characterized
by comparing their retention times, UV and MS spectra with those of authentic compounds or litera-
ture data, and 14 of them (gallic acid, albiflorin, paeoniflorin, ferulic acid, benzoic acid, senkyunolide I,
coniferyl ferulate, senkyunolide A, 3-butylphthalide, Z-ligustilide, Z-butylidenephthalide, atractylcnolide
II, atractylcnolide I and levistolide A) were determined by HPLC–DAD using a C18 column and gradient
elution of acetonitrile/water–formic acid (100:0.1, v/v). The linearity, precision, accuracy, LOD and LOQ
were validated for the quantification method, which proved sensitive, accurate and reproducible. The
study might provide a basis for the quality control of DSS extracts and preparations.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Traditional Chinese medicine (TCM) has been widely used in
China due to its special efficacy in some instances in which the
conventional Western therapies failed or proved to be insuffi-
cient to provide a palliative cure [1]. Commercially available TCM
preparations are usually composed of several herbs with numerous
constituents. Thus the analysis of such a complex mixture brings
a great challenge to pharmaceutical analysts. Liquid chromatogra-
phy coupled with DAD and electrospray ionization tandem mass
spectrometry (LC/DAD/ESI/MS/MS) is a powerful analytical tool for
the analysis of the known compounds and elucidation of unknown
compounds in complex matrix, showing suitability for the analysis
of TCM products [2,3].
Danggui-Shaoyao-San (DSS), comprising Radix Paeoniae Alba,
Radix Angelica sinensis, Rhizoma Chuanxiong,Poria cocos, Rhizoma
Atractylodis macrocephalae and Rhizoma Alismatis, is a widely used
formula of TCM derived from “Jingui Yaolue”, a medical classic writ-
ten by Zhongjing Zhang in the Eastern Han Dynasty. This medicine
has been used in China as a blood-activating and stasis-eliminating
drug to treat gynecological disorders such as dysmenorrhea, amen-
∗Corresponding author. Tel.: +86 25 85391042; fax: +86 25 85391042.
E-mail address: boyangyu59@163.com (B. Yu).
orrhea and infertility for thousands of years. It has also been
widely prescribed for the clinical practice in China and Japan
[4,5]. Recent studies show that it also possesses the capability
of treating neural dysfunctions such as senile dementia, memory
loss, and other cognitive disorders, thus the formula is used as a
remedy for Alzheimer’s disease in Japan [6,7]. Although so many
beneficial effects have been shown, the actual bioactive compo-
nents of DSS are still unclear. Recently, some active ingredients
related to pharmacological functions are gradually being revealed
[8]. Among them, monoterpene glycosides, phenolic compounds
and phthalides are the most representative components of DSS
as far as both the contents and their biological activities are con-
cerned. Monoterpene glycosides are responsible for the efficacy of
R. Paeoniae Alba. A case in point is albiflorin and paeoniflorin, which
exhibits analgesia, spasmolysis, anti-inflammation and anticoagu-
lation activities [9–12]. Phenolic acids and phthalides in R. Angelica
sinensis and R. Chuanxiong also have vasodilatative, antithrom-
botic, antioxidative, anti-inflammatory and muscle relaxant effects
[13–18]. In addition, atractylenolides from R. Atractylodis showed
gastrointestinal inhibitory, anti-inflammatory and antioxidative
activity [23]. Meanwhile, cytotoxic, anti-inflammatory and antiox-
idant activity of triterpenes in R. Alismatis and Poria cocos have also
been documented [24–26]. Current experimental evidences sug-
gest a close relationship between these components and bioactive
mechanism of DSS [19–22]. Although a number of studies on the
0731-7085/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jpba.2009.03.039

128 L. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 50 (2009) 127–137
quality evaluation of individual herb in DSS have been carried out
using TLC [27],CE[28], HPLC–DAD [13,23,29,30], HPLC–ELSD [25],
GC–MS [31] and LC–MS [30–33], the available methods of quality
control for DSS products were still mainly based on the contents
of one or two indicative compounds [34], while the simultane-
ous determination of multiple constituents in DSS has not been
reported so far. For a complex formula, the comprehensive qual-
ity evaluation method should be based on the identification of its
chemical components in a full spectrum. However, current stud-
ies on the constituents of the formula are inadequate to objectively
assess the bioactive components of DSS. Therefore, it is necessary to
develop a rapid and sensitive method to identify and quantify the
compounds in DSS, which is beneficial to searching the bioactive
substances and controlling the quality of the medicine. In our study,
an LC/DAD/ESI/MS/MS method was developedto identify and quan-
tify the major constituents of this formula for the first time. A totalof
41 compounds in the formula were identified or tentatively charac-
terized. In addition, quantification of 14 bioactive components was
performed with HPLC–DAD and 9 commercial samples were ana-
lyzed, which is expected to provide comprehensive information for
quality control of DSS preparations.
2. Experiment
2.1. Reagents and materials
HPLC grade acetonitrile was from Tedia (Fairfield, OH, USA);
water for HPLC analysis was purified by a Milli-Q academic water
purification system (Milford, MA, USA). Methanol and formic acid
were of analytical grade (Jiangsu Hanbon Sci.&Tech. Co. Ltd., Jiangsu,
China).
Crude drugs were purchased from Fengyuan Tongling crude
drug company (Anhui, China) and were identified by Professor
Boyang Yu. DSS products were purchased from six different brand
manufacturers in China and Japan.
Gallic acid, protocatechuic acid, catechin, phthalic acid, vanilic
acid, paeoniflorin, ferulic acid, benzoic acid and atractylenolide I,
II, III were purchased from the National Institute for the Control of
Pharmaceutical and Biological Products (Beijing, China). Ligustilide
was obtained from Tianjing Zhongxin Pharmaceutical Co. Ltd. (Tian-
jing, China). Albiflorin, lactiflorin and benzoylpaeoniflorin were
isolated from Paeonia lactiflora. Senkyunolide I, coniferyl ferulate,
senkyunolide A, 3-butylphthalide, Z-butylidenephthalide and lev-
istolide A were isolated from essential oil of Angelica sinensis. Alisol
C 23-acetate, alisol F, alisol B and alisol B 23-acetate were isolated
from Rhizoma Alismatis, and pachymic acid was isolated from Poria
cocos in the authors’ laboratory. Their structures were elucidated
by their spectral data (MS, 1H NMR and 13C NMR). The purity of
each compound was determined to be higher than 98% by HPLC.
Each reference compound was accurately weighed and dissolved
in methanol as stock solutions.
2.2. Preparation of standard solutions
Stock solutions of the 14 standard substances for the determi-
nation were prepared in methanol at the concentration (mg/ml)
of: gallic acid (0.40), albiflorin (1.60), paeoniflorin (2.30), fer-
ulic acid (0.216), benzoic acid (0.104), senkyunolide I (0.32),
coniferyl ferulate (1.12), senkyunolide A (1.85), butylphthalide
(0.30), ligustilide (2.16), butylidenephthalide (0.15), atractylenolide
II (0.021), atractylenolide I (0.036) and levistolide A (0.218). A series
of working standard solutions with gradient concentration were
obtained by diluting the mixed standard stock solution. All the solu-
tions were stored in the refrigerator at 4 ◦C and brought to room
temperature before use.
2.3. Preparation of sample solutions
One gram powder of the crude drugs compounded accord-
ing to the formula in “Jin Gui Yao Lue” was extracted in 20ml
methanol–water (75:25, v/v) for 30 min in an ultrasonic water bath.
The extraction was repeated twice. The extracted solutions were
combined and concentrated nearly to dryness at 50 ◦C in vacuo. The
evaporated residue was dissolved with methanol–water (75:25,
v/v) into a 25 ml volumetric flask. The commercial preparations of
DSS were uncoated (for capsule and tablet), powdered (for tablet),
and extracted as described above. Since the contents of each ana-
lyte could vary considerably among different products, the extract
solutions were diluted to appropriate concentrations to fit the val-
idated calibration range for HPLC analysis. The extract was filtered
through a 0.45 ␮m membrane and then 10␮l of the filtrate was
analyzed by LC.
2.4. HPLC–DAD–ESI-MS system
The HPLC system consisted of an Agilent 1100 series HPLC
with a Diode Array Detector. The column was an Alltima C18
(250 mm ×4.6 mm i.d., 5 ␮m, Alltech, USA) maintained at 30 ◦C. The
eluents were acetonitrile (A) and water–formic acid (100:0.1, v/v)
(B) at a flow rate of 1 ml/min. The following multi-step linear gradi-
ent was applied: 0–40 min, 5–30% A; 40–65min linear increased to
55% A; 65–85 min linear increased to 100% A, and then maintained
at that level for another 5min. Total time of analysis was 90min.
The DAD spectra were recorded between 190 and 400nm and the
chromatographic profiles were recorded at 254 nm for qualitative
analysis, while 231 and 275 nm for quantitative analysis.
The above HPLC system was interfaced with an Agilent 1100
LC/MSD Trap XCT ESI (Agilent Technologies, MA, USA). The same
conditions were used during the HPLC–MS analysis. The ESI-MS
spectra were acquired in both negative and positive ionization
modes recorded on a mass range of m/z100–800. Capillary voltage
was 3500 V. Drying gas temperature was set at 350◦Cwithaflow
rate of 9.0 l/min and nebulising pressure was of 40psi. Data were
processed by LC/MSD Trap Software 4.2 and Data Analysis 2.2.
2.5. Qualitative analysis of peaks
Identification of constituents in DSS extract was carried out by
HPLC/DAD and LC/ESI/MS analysis. In orderto obtain MS fragmenta-
tion patterns of constituents, standards and samples were analyzed
by LC/ESI/MS/MS in both negative and positive ion modes. In the
full scan mass spectra, most of the authentic compounds exhib-
ited quasi-molecular ions [M+H]+in positive mode or [M−H]−in
negative mode and more detailed structural information could be
obtained via collision-induced dissociation (CID). The fragmenta-
tion patterns were proposed and theywere helpful for the structural
identification of constituents.
2.6. Validation of quantitative analysis
The prepared mixed standard stock solution containing 14 ana-
lytes was diluted to a series of appropriate concentrations for the
construction of calibration curves. Six different concentrations of
the mixed standard solution were injected in triplicate. The LODs
and LOQs were determined at signal-to-noise ratios (S/N) of 3 and
10, respectively.
The precision of the method was determined for intra-and inter-
day variations. The intra-day variability was performed in triplicate
on the same sample extracted during a single day, while the inter-
day precision was carried out in triplicate in another independent
sample extracted on three different days. The ratios of observed
concentration and nominal concentration of the mixed standard

L. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 50 (2009) 127–137 129
Table 1
HPLC–DAD–ESI-MS identification of the constituents in DSS extract.
Peak no. TR(min) Positive ions (m/z) Negative ions (m/z)max (nm) Identification Crude
drug
1*7.6 – 169[M−H]−, 125[M-H-CO2]−, 97[M-H-CO2-CO]−214, 270 Gallic acid 1
2 9.9 – – 283 Unidentified 1
3*12.6 – 153[M−H]−, 109[M-H-CO2]−310 Protocatechuic acid 3
4 13.4 725, 591, 563, 545, 383, 325, 261, 197 705, 659, 583, 543, 421, 381, 259 231, 274 Unidentified 1
5 14.2 545[M+H]+, 401[M+H+H2O-Glc]+, 543[M−H]−, 497[M-H-HCOOH]−, 421[M-H-BA]−, 375[M-H-HCOOH-BA]−
259[M-H-BA-Glc]−, 215[M-H-CO2-BA-Glc]−, 177[aglycone-H-H2O]−
232, 274 Paeoniflorin sulfonate 1
383[M+H-Glc]+, 285[BA+Glc+H]+,
261[M+H-BA-Glc]+, 197[M+H-BA-Glc-SO2]+
6 16.1 – 495[M−H]−, 451[M-H-CO2]−, 258 Oxypaeoniflorin 1
357[M-H-pOHBA]−, 333[M-H-Glc]−,
281[Glc+pOHBA-H-H2O]−, 195[aglycone-H]−, 167[aglycone-H-CO]−,
137[ pOHBA-H]−
7*18.4 – 289[M−H]−, 271[M-H-H2O]−, 278 (+)-Catechin 1
245[M-H-C2H4O]−, 227[M-H-C2H4O-H2O]−
8*19.3 – – 277 Phthalic acid 2
9*20.1 – 167[M−H]−, 137[M-H-HCHO]−, 123[M-H-CO2]−260, 292 Vanillic acid 2, 3
10 22.4 661[M+H+H2O]+, 643[M+H]+, 625[M+H-H2O]+,
539[M+H+H2O-BA]+, 485[M+H-2H2O-BA]+,
463[M+H-H2O-Glc]+, 341[M+H-H2O-Glc-BA]+,
325[2Glc+H]+, 301[M+H-H2O-2Glc]+
687[M+HCOO]−, 611[M-H-HCHO]−, 623[M-H-H2O]−, 593[M-H-H2O-HCHO]−, 232, 274 Isomaltopaeoniflorin 1
519[M-H-BA]−, 489[M-H-HCHO-BA]−, 445[M-H-aglycone]−, 323[2Glc-H]−,
283[BA+Glc-H]−, 269[M-H-H2O-HCHO-2Glc]−
11*22.8 4 81[M+H]+, 359[M+H-BA]+, 319[M+H-Glc]+,
197[M+H-BA-Glc]+, 161[M+H-BA-Glc-2H2O]+,
133[M+H-BA-Glc-2H2O-CO]+,
525[M+HCOO]−, 479[M−H]−, 435[M-H-CO2]−, 357[M-H-BA]−,
327[M-H-HCHO-BA]−,
231, 273 Albiflorin 1
283[BA+Glc-H]−, 195[aglycone-H]−
12 23.6 – 695, 649, 573, 525, 463, 395, 313 222, 274 Unidentified 1
13*24.7 503[M+Na]+, 463[M+H-H2O]+, 525[M+HCOO]−, 479[M−H]−, 449[M-H-HCOH]−, 327[M-H-HCOH-BA]−,
121[BA-H]−
232, 274 Paeoniflorin 1
319[M+H-Glc]+, 301[M+H-Glc-H2O]+,
179[M+H-Glc-BA-H2O]+,
161[M+H-Glc-BA-2H2O]+
14 27.9 – 787[M−H]−, 635[M-H+H2O-GA]−, 220, 270 Tetragalloyglucose 1
617[M-H-GA]−, 573[M-H-GA-CO2]−, 465[M-H+H2O-2GA]−, 447[M-H-2GA]−,
313[M-H+2H2O-3GA]−, 295[M-H+H2O-3GA]−,
169[GA-H]−, 125[GA-H-CO2]−
15*30.5 195[M+H]+, 177[M+H-H2O]+, 145[M+H-H2O-CH3OH]+,– 295, 322 Ferulic acid 2, 3
117[M+H-H2O-CO-CH3OH]+
16 32.3 – 939[M−H]−, 769[M-H-GA]−, 617[M-H+H2O-2GA]−, 447[M-H+H2O-3GA]−,
169[GA]−, 125[GA-CO2]−
220, 280 Pentagalloylglucose 1
17 33.9 633[M+H]+, 471[M+H-Glc]+, 631[M−H]−, 613[M-H-H2O]−, 509[M-H-BA]−, 491[M-H-BA-H2O]−, 479[M-H-gallic
carbonyl]−
220, 279 Galloylpaeoniflorin 1
349[M+H-Glc-BA]+, 301[M+H-Glc-GA]+,
197[aglycone+H]+, 153[GA+H-H2O]+
313[GA+Glc-H-H2O]−, 169[GA-H]−, 125[GA-H-CO2]−
18*34.4 – – 238, 272 Benzoic acid 1
19 35.8 481[M+H]+, 435[M+H-HCOOH]+, 359[M+H-BA]+,
319[M+H-Glc]+, 197[aglycone+H]+,
179[aglycone+H-H2O]+,
525[M+HCOO]−, 479[M−H]−, 357[M-H-BA]−, 283[BA+Glc-H]−, 195[aglycone-H]−228 Mudanpioside I 1
161[aglycone+H-2H2O]+,
133[aglycone+H-2H2O-CO]+
20*37.5 247[M+Na]+, 225[M+H]+, 207[M+H-H2O]+,
189[M+H-2H2O]+, 179[M+H-H2O-CO]+,
165[M+H-H2O-C3H6]+
– 270 Senkyunolide I 2, 3
21*37.9 485[M+Na]+, 481[M+H+H2O]+, 463[M+H]+,
341[M+H-BA]+, 301[M+H-Glc]+, 273[M+H-Glc-CO]+,
179[M+H-BA-Glc]+,
507[M+HCOO]−, 461[M−H]−, 443[M-H-H2O]−, 234 Lactiflorin 1
151[M+H-BA-Glc-CO]+
431[M-H-HCOH]−, 371[M-H-HCOH-HOAc]−,
339[M-H-BA]−, 308[M-H-BA-CH3O]−, 299[M-H-Glc]−, 283[M-H-C6H10O6]−,
195[C6H9O6+H2O]−, 177[C6H9O6]−, 159[C6H9O6-H2O]−
22 39.3 – – 275 Unidentified 2, 3
23*49.6 585[M+H]+, 463[M+H-BA]+, 319[M+H-BG]+, 583[M−H]−, 553[M-H-HCHO]−, 461[M-H-BA]−, 387[M-H-aglycone]−238 Benzoylpaeoniflorin 1
267[M+H-aglycone-BA]+, 197[M+H-BA-BG]+,
165[aglycone+H-CH3OH]+, 105[benzoyl]+

130 L. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 50 (2009) 127–137
Table 1 (Continued)
Peak no. TR(min) Positive ions (m/z) Negative ions (m/z)max (nm) Identification Crude
drug
24 54.5 505[M+H]+, 487[M+H-H2O]+, 469[M+H-2H2O]+,
415[M+H-H2O-C4H8O]+, 397[M+H-2H2O-C4H8O]+
– 244 16-Oxoalisol A 6
25 56.8 – – 246 Unidentified 2, 3
26*63.1 – 193[M-H-C10H10 O2]−, 178[M-H-C10H10 O2-CH3]−, 268, 317 Coniferyl ferulate 2, 3
149[M-H-C10H10 O2-CO2]−,
134[M-H-C10H10 O2-CH3-CO2]−
27 64.4 487[M+H]+, 469[M+H-H2O]+, 451[M+H-2H2O]+,– – Alisol C 6
397[M+H-H2O-C4H8O]+, 353[M+H-H2O-C4H8O-CO2]+
28*64.5 489[M+H]+, 471[M+H-H2O]+, 453[M+H-2H2O]+,– – Alisol F 6
399[M+H-H2O-C4H8O]+
29*65.2 249[M+H]+, 231[M+H–H2O]+,– 235 Atractylenolide III 4
213[M+H-2H2O]+, 203[M+H-H2O-CO]+,
175[M+H-H2O-2CO]+, 163[M+H-H2O-C5H8]+,
135[M+H-H2O-C5H8-CO]+,
117[M+H-H2O-C5H8-HCOOH]+,
107[M+H-H2O-C5H8-2CO]+,
79[M+H-H2O-C5H8-2CO-C2H4]+
30*66.1 193[M+H]+, 175[M+H-H2O]+, 147[M+H-H2O-CO]+– 280 Senkyunolide A 2, 3
31*67.6 191[M+H]+, 173[M+H-H2O]+, 145[M+H-H2O-CO]+– 230, 276 3-Butylphthalide 2, 3
32 69.8 471[M+H]+, 453[M+H-H2O]+, 443[M+H-CO]+,
435[M+H-2H2O]+, 395[M+H-H2O-CO-HCHO]+,
381[M+H-H2O-C4H8O]+,
– – 11-Deoxyalisol C 6
33 70.2 191[M+H]+, 173[M+H-H2O]+, 155[M+H-2H2O]+– 280, 326 E-Ligustilide 3
34*70.8 529[M+H]+, 511[M+H–H2O]+, 469[M+H-HOAc]+,
451[M+H-H2O-HOAc]+, 433[M+H-2H2O-HOAc]+
–247 Alisol C 23-acetate 6
35 71.3 195[M+H]+, 177[M+H–H2O]+, 149[M+H-H2O-CO]+,
125[M+H-C5H10]+, 121[M+H-H2O-CO-C2H4]+
107[M+H-H2O-CO-C3H6]+, 93[M+H-H2O-CO-C4H8]+
– – Cnidilide 3
36*72.1 191[M+H]+, 173[M+H-H2O]+,– 281, 328 Z-Ligustilide 2, 3
163[M+H-CO]+, 155[M+H-2H2O]+,
149[M+H-C3H6]+, 145[M+H-H2O-CO]+,
117[M+H-H2O-CO-C2H4]+,
105[M+H-H2O-CO-C3H4]+, 91[M+H-H2O-CO-C4H6]+
37*72.2 473[M+H]+, 455[M+H-H2O]+,– – Alisol B 6
383[M+H-H2O-C4H8O]+, 365[M+H-2H2O-C4H8O]+
38*72.7 189[M+H]+, 171[M+H–H2O]+,– 262, 313 Z-Butylidenephthalide 2, 3
153[M+H-2H2O]+, 128[M+H-H2O-CO-CH3]+,
115[M+H-H2O-CO-C2H4]+
39*73.8 233[M+H]+, 215[M+H-H2O]+, 205[M+H-CO]+,
187[M+H-H2O-CO]+, 177[M+H-C4H8]+,
151[M+H-C4H8-C2H2]+
– 220 Atractylenolide II 4
40*78.0 231[M+H]+, 203[M+H–CO]+, 185[M+H-H2O-CO]+,
175[M+H-2CO]+, 143[M+H-H2O-CO-C3H6]+
– 276 Atractylenolide I 4
41 80.0 381[M+H]+, 191[C12 H15 O2]+, 173[C12H15 O2-H2O]+– 280 Riligustilide 2, 3
42 80.3 381[M+H]+, 191[C12H15 O2]+, 173[C12H15 O2-H2O]+– 281 Tokinolide B 2, 3
43*80.7 381[M+H]+, 191[C12H15 O2]+,– 276 Levistolide A 2, 3
173[C12H15 O2-H2O]+, 163[C12H15 O2-CO]+,
155[C12H15 O2-2H2O]+, 145[C12H15 O2-CO-H2O]+,
135[C12H15 O2-CO-C2H4]+
44 81.1. 383[M+H]+, 365[M+H-H2O]+– 226, 282 Senkyunolide P 2, 3
355[M+H-CO]+, 191[C12H15 O2]+
45*84.0 515[M+H]+, 497[M+H-H2O]+, 479[M+H-2H2O]+,
455[M+H-HOAc]+, 437[M+H-H2O-HOAc]+,
– Alisol B 23-acetate 6
419[M+H-2H2O-HOAc]+,
381[M+H-H2O-HOAc-C4H8]+,
365[M+H-H2O-HOAc-C4H8O]+,
339[M+H-H2O-HOAc-C4H8O-C2H2]+

L. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 50 (2009) 127–137 131
46*84.6 – 563[M+Cl]−, 527[M−H]−, 481[M-H-HCOOH]−, 242 Pachymic acid 5
467[M-H-HOAc]−, 465[M-H-H2O-CO2]−,
449[M-H-HOAc-H2O]−,
397[M-H-HOAc-C5H10]−,
355[M-H-H2O-C9H14 O2]−,
313[[M-H-HOAc-C9H14O2]−,
287[M-H-HOAc-C9H14O2-C2H2]−
1. Radix Paeoniae Alba, 2. Radix Angelica sinensis, 3. Rhizoma Chuanxiong, 4. Rhizoma Atractylodis macrocephalae,5.Poria cocos, 6. Rhizoma Alismatis. BA: Benzoic acid; GA: gallic acid; Glc: glucosyl group; BG: glucosyl group with
benzoate group on C-6.
*Compared with authentic compounds.
solutions were calculated as accuracy. The accuracy of this method
was further evaluated by recovery test. The analogs were added at
three concentration levels (approximately equivalent to 0.8, 1.0 and
1.2 times of the amount of the matrix) with two parallels at each
level and then extracted and analyzed as described in Section 2.3.
3. Results and discussion
3.1. Optimization of HPLC–DAD–ESI-MS conditions
The great structural diversity of compounds in the formula
makes it difficult to give good responses to all chemical compo-
nents in MS analysis. Monoterpene glucosides could be analyzed
in both positive and negative ionization. However, most organic
acids were detected only in negative ion mode, and the signals
of phthalides, lactones and triterpenes were obvious in the posi-
tive ion mode. Therefore, both positive and negative ion modes had
to be employed to identify the corresponding signals. In addition,
trace amount of formic acid was also added in the mobile phase to
improve the ionization responses for some compounds.
The quantification of constituents in DSS was achieved at 231
and 275 nm, where the UV spectra of the 14 analytes exhibitedmax-
imum absorbance, in which better response and less interference
could be accomplished. High-gradient slope and aqueous formic
acid in the mobile phase were applied to acquire good resolution
within reasonable time as well. Compounds 1, 11, 13 and 15 showed
better peak forms when 0.5% formic acid was included, while 0.1%
formic acid was utilized to ameliorate the baseline drift at 231 nm.
50%, 75% and 100% ethanol and methanol were tested as extrac-
tion solvents, and the extraction time was also investigated. One
gram DSS powder was extracted three times with 20 ml of each
solvent system under ultrasonic for 15, 30 and 60 min, respectively.
The results showed that all the 14 components were almost com-
pletely extracted by ultrasonication with 75% methanol three times
for 30 min each.
3.2. Identification of constituents in DSS by HPLC–DAD–ESI-MS
The authentic compounds could be classified into five groups
according to their chemical structures and their dominant fragmen-
tation pathways were studied. Most of the authentic compounds
exhibit [M−H]−and/or [M+H]+ions of sufficient abundance that
could be subjected to MSnanalysis. MS/MS and MSndata were
obtained by collision-induced dissociation (CID), and utilized for
the structural identification of compounds with similar fragmenta-
tion patterns. Twenty-six peaks in the HPLC–DAD and HPLC–MS
(TIC) chromatograms were unequivocally identified by compar-
isons of their retention times, MS data and UV spectra with those
of authentic compounds. The other 15 peaks were identified ten-
tatively by comparing their UV spectrum, molecular weight and
structural information from MSnspectra with reference data from
literature. Table 1 listed the retention time (RT), UV max, MS data
and the most characteristic fragments of the reference compounds
and identified peaks. Their chemical structures are shown in Fig. 1.
The UV chromatograms at 254nm and MS TIC chromatograms of
DSS extract were presented in Fig. 2a–c, respectively.
For most of the constituents in the MS, ions of [M+H]+,[M+Na]
+,
[M−H]−, [M+Cl]−, and [M+HCOO]−were observed. Peaks 5, 6, 10, 11,
13, 17, 19, 21 and 23 were identified as monoterpene glucosides in
Paeoniae alba, which showed similar fragmentationpatterns such as
losses of a benzoic acid (122 Da), a glucosyl group (162 Da) and their
combined loss (284 Da). The aglycone ions at m/z195 or 197 could
be observed occasionally and their fragmentations of losing H2O
and CO were also detected in some cases such as compounds 5, 6,
13,19 and 23 [35–38]. Additionally,fragment ions of the compounds

132 L. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 50 (2009) 127–137
with substitutive groups and their corresponding losses were often
observed in MS2or MS3spectra, which provided very useful infor-
mation for structural identification. Peak 5 yielded [M+H]+and
[M−H]−at m/z545 and 543, respectively. Besides the character-
istic UV spectrum and MS fragmentations of paeoniflorin derivates,
loss of SO2was also observed in positive ion mode. Compared with
literature data, it was identified as paeoniflorin sulfonate, an artifact
generated in the processing of white peony root by sulfur dioxide
[32]. Peak 4 exhibited a molecular mass of 706, the similar fragmen-
tations with the ions of 162Da larger than that of compound 5, and
the fragments of losing 2 glucosyl (706 →503 →381), indicating a
product of a glucosyl linked to paeoniflorin sulfonate, which was
not found previously. Another analog of peak 10 gave the [M+H]+
at m/z643 and fragment pattern resembled paeoniflorin with the
Fig. 1. Chemical structures of compounds identified in DSS extract.

L. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 50 (2009) 127–137 133
Fig. 1. (Continued ).
appearance of [2Glc+H]+at m/z325 and [M+H-H2O-2Glc]+at m/z
301, showing a glucose linked to paeoniflorin, which is in agree-
ment with the structure of isomaltopaeoniflorin [39]. Peaks 17 and
23 showed typical fragmentations of substituted paeoniflorin, as
well as the diagnostic ions produced by substituent groups [2].For
instance, peak 17 afforded the fragment ions at m/z479 by losing
gallic carbonyl from the [M−H]−ion, together with the ions at m/z
313, 169 and 125, which could be easily identified as galloylpaeoni-
florin [2,36]. Peaks 14 and 16 displayed intense UV absorptions in
270nm and successive losses of 170Da, with the presence of the

134 L. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 50 (2009) 127–137
Fig. 2. HPLC–DAD–ESI-MS chromatograms of DSS extract. (a) HPLC–DAD chromatogram at 254nm. (b) TIC chromatogram in positive ion mode. (c) TIC chromatogram in
negative ion mode.
ions at m/z125 [GA-CO2]−and 161 [Glc-H]−, suggesting that they
might be glucoses attached by several GA units in their structures.
Actually, they were regarded respectively as tetragalloyglucose and
pentagalloylglucose, two components in Paeoniae alba previously
reported in literature [36].
The characteristic ions of organic acids including gallic acid, pro-
tocatechuic acid, phthalic acid, vanillic acid, ferulic acid and benzoic
acid were mainly formed by the losses of CO2, CO, HCHO, CH3OH
and their combinations. Peak 26 was an ester between ferulic acid
and ciniferol with a dominant fragment ion of m/z193, which could
be attributed to the loss of coniferyl [40]. It should be mentioned
that organic acids and their esters showed low responses under
this MS condition, especially phthalic acid and benzoic acid, which
could only be identified according to the retention time and UV
characteristics in comparison with those of their standards. On
the contrary, intense quasi-molecule ions [M+H]+were found for
phthalides, whose fragments were usually generated by the losses
of H2O (18Da), CO (28 Da) and side chains such as CH4(16Da),
C2H4(28 Da) and C3H6(42 Da), etc. [41,42]. Moreover, protonated
dimeric ions [2M+H]+were also observed for phthalide monomers.
Peaks 33 and 36 had the same UV and MS behaviors, indicating
that they should be typical phthalide isomers. After the structure
of peak 36 was identified as Z-ligustilide compared with authen-
tic standard, peak 33 was assigned as E-ligustilide, the cis-isomer
of Z-ligustilide [41,42]. Peaks 41–43 exhibited similar mass spectra
with the protonated molecular ions [M+H]+at m/z381 as well as
obvious fragment ions at m/z191, indicating that they were dimeric
phthalide with the same molecular mass of 380 Da. With authen-
tic standard, peak 43 was identified as levistolide A, and the other
two were tentatively assigned to be riligustilide and tokinolide B
by the comparison of their UV max with reported data [43,44].
Three lactones in R. Atractylodis macrocephalae were elucidated as

L. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 50 (2009) 127–137 135
atractylenolide I (peak 40), II (peak 39) and III (peak 29), respec-
tively, based on comparisons with standard compounds. Similar
to phthalides, the basic fragment ions of these compounds were
[M+H-H2O]+, [M+H-CO]+and [M+H-H2O-CO]+, with other charac-
teristic ions produced by the cleavage of lipid rings.
Compounds 24, 27, 28, 32, 34, 37, and 45 were all protostane-
type triterpenoids from R. Alismatis. Their common fragmentation
pattern was the loss of H2O followed by a cleavage of the side chain
C4H8O (72 Da). Meanwhile, loss of CH3COOH (60Da) might occur
in acetylated alisols. Their structures were determined by reference
substances while compounds 24, 27 and 32 were deduced by liter-
ature data [33]. A triterpenoid in Poria cocos named pachymic acid
(peak 46) was detected with [M+Cl]−and [M−H]−ions at m/z563
and 527, respectively. The MS spectrum with negative mode for this
compound also afforded diagnostic ions at m/z481, 467, 465, 355
and 313, in which the product ions at m/z355 and 313 were orig-
inated by losing the side chains, which was compatible with the
typical fragmentation of lanostane triterpene [45]. However, some
peaks, such as peaks 2, 22 and 25, showed good UV responses but
no signals in MS spectrum.
3.3. Quantitative determination of constituents in DSS by
HPLC–DAD
Fourteen peaks in chromatogram of DSS with reasonableheights
and good resolution were chosen as mark substances, includ-
ing gallic acid (GA), albiflorin (AF), paeoniflorin (PF), ferulic acid
(FA), benzoic acid (BA), senkyunolide I (SI), coniferyl ferulate (CF),
senkyunolide A (SA), 3-butylphthalide (BP), Z-ligustilide (LL), Z-
butylidenephthalide (BE), atractylcnolide II (AII), atractylcnolide
I (AI) and levistolide A (LA). They were generally considered as
active components except benzoic acid, which was regarded as
a toxic component. Additionally, some characteristic compounds
in the herbs, such as alisol B 23-acetate, atractylcnolide III and
pachymic acid were not determined in the preparations for their
poor ultraviolet absorption or extremely low content. Fig. 3 dis-
played representativeHPL C profiles of DSS and standard substances
detected at 231 and 275 nm. Owing to the great polarity difference
of these components, gradient elution and the long HPLC run time
were applied for the complete separation of the marker compo-
nents.
As shown in Table 2, the HPLC–DAD method provided a good
reproducibility for the quantification of the analytes,with intra- and
inter-day precision of less than 1.64% and 2.27%, respectively. The
intra- and inter-day accuracy was in the range of 95.32–104.30%.
The LODs and the LOQs for the analytes were less than 215 and
684 ng/ml, respectively. The overall recoveries ranged from 96.8%
to 103.3%, with the R.S.D. ranging from 1.63% to 4.78%. These results
demonstrated that the quantitative method was precise, accurate,
and sensitive for the determination of the 14 components in DSS
samples.
3.4. Application
The established method of quantification has been applied to
the analysis of 9 kinds of commercial DSS products including pow-
der, granula,tablets and capsules from 6 different manufacturers. As
listed in Table 3, the 14 constituents were comprised in most prepa-
rations and their total amounts varied from 8.28 to 32.43mg/g.
Among these compounds, PF was found to be an abundant and
essential component, which showed the highest amount in most
samples with the relative contents ranged from 16.11–56.54%. GA,
AF, SA and LL were also quite common in the drug products. In addi-
tion, products from the same manufacturer have a definite ratio of
components for using the same intermediate. For instance, the con-
tents of 14 analytes in sample 3 (intermediate) were about twice as
much as in sample 4, while they were similar in samples 6 and 7.
According to Table 3, the contents of the 14 constituents var-
ied remarkably from different dosage forms and manufacturers. For
example, samples 1 and 2 were DSS powder produced in China and
Taiwan, respectively, while their contents of components differed
remarkably because both the sources of crude drugs and formula
were different in the two regions. Even in the products of same
dosage form from one region such as samples 5 and 6, their con-
tents could be various for the discrepancy in raw materials and
processing procedures. The extraction process was another key fac-
tor affecting the contents of ingredients. Sample 9 showed high
amount of most ingredients, indicating that the procedure of cap-
Fig. 3. HPLC–DAD chromatograms of 14 analytes in DSS extract at 231 and 275 nm. (a) Chromatogram of the mixed standard. (b) Chromatogram of DSS extract.

136 L. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 50 (2009) 127–137
Table 2
Detection wavelength, linear regression data, LOD, LOQ, precision and accuracy of 14 constituents in DSS extract (n= 6).
Analyte (nm) Regression equationaR2Linear range (␮g/ml) LOD (ng/ml) LOQ (ng/ml) Precision R.S.D. (%) Accuracy (%)
Intra-day Inter-day Intra-day Inter-day
Gallic acid 275 y=27264x+1373.3 1.0000 2.00–80.00 20 74 0.73 0.74 99.49 100.12
Albiflorin 231 y=10348x+ 18289 0.9997 8.00–320.0 215 684 0.22 1.51 100.57 104.30
Paeoniflorin 231 y=12092x+ 23453 0.9997 11.60–464.0 176 459 0.42 1.31 96.34 99.76
Ferulic acid 275 y= 24045x−13.491 1.0000 1.08–43.20 44 203 0.25 0.80 100.18 99.61
Benzoic acid 231 y = 36830x+ 8162.3 0.9990 0.52–20.80 52 169 1.64 1.78 100.98 98.90
Senkyunolide I 275 y= 19981x−2023.7 1.0000 1.60–64.00 105 338 0.19 0.60 100.99 99.70
Coniferyl ferulate 275 y= 2352.4x−1331.9 0.9996 11.2–224.0 190 589 0.52 0.96 100.61 98.93
Senkyunolide A 275 y= 2912.8x+ 3656.1 0.9999 9.24–369.6 117 332 0.58 1.61 101.64 103.01
3-Butylphthalide 231 y= 6058x−351.54 0.9993 1.50–60.0 145 425 1.62 2.27 98.86 98.60
Z-Ligustilide 275 y=11662x+ 7176.5 0.9999 10.8–432.0 100 353 0.47 1.22 100.07 99.85
Z-Butylidenephthalide 275 y= 13982x+1772.9 0.9986 0.75–30.0 85 273 0.76 1.89 99.66 95.32
Atractylenolide II 231 y=26174x+156.67 0.9991 0.26–4.14 61 208 1.38 2.04 100.22 101.19
Atractylenolide I 275 y= 49975x+ 2004.7 0.9997 0.18–7.20 22 72 0.15 0.85 103.52 99.65
Levistolide A 275 y= 8540.4x+ 189.47 1.0000 1.09–43.52 178 633 0.30 0.86 100.05 99.73
aIn the regression equation y=ax+b,xrefers to the concentration (␮g/ml), yindicates the peak area, and R2is the correlation coefficient of the equation.
Table 3
The contents (mg/g) of 14 constituents in DSS preparations (n= 3).
Products GA AF PF FA BA SI CF SA BP LL BE AII AI LA
Danggui-Shaoyao-Sana0.48 1.86 2.69 0.25 0.12 0.90 0.64 2.14 0.35 2.54 0.16 0.01 0.04 0.26
Tang Kuei Shao Yao Sanb0.22 0.82 1.97 0.18 0.14 0.38 2.38 0.60 0.08 1.07 0.15 0.03 0.04 0.21
Toki Shakuyaku San Exc0.85 4.07 11.32 0.36 1.01 0.20 0.18 1.01 0.74 0.52 0.04 – 0.06 0.53
Toki Shakuyaku San Granulad0.51 2.40 6.58 0.21 0.52 0.12 – 0.48 0.27 0.26 0.02 0.01 0.02 0.25
DSS Conc. Granulae1.24 1.63 1.65 0.14 1.25 0.92 0.63 0.81 0.52 0.99 0.10 0.07 0.07 0.25
DSS Conc. Granulaf1.51 3.49 15.21 0.49 0.32 1.45 2.73 2.40 0.75 1.87 0.23 0.01 0.01 0.64
DSS Conc. Tabletg1.47 4.30 16.77 0.53 0.33 1.35 2.02 2.09 0.42 1.74 0.21 0.01 0.01 0.62
Guishao Tiaojing Tableth4.43 9.69 15.05 0.14 1.60 0.38 – 0.02 0.86 0.01 – – – 0.24
Danggui Shaoyao Capsulei1.43 4.81 13.75 0.57 0.32 1.85 0.83 4.67 0.68 3.26 0.31 0.05 0.05 0.80
DSS Description Granulaj3.11 6.39 15.30 0.93 0.43 0.43 0.24 0.44 – 0.18 0.01 – – 0.04
“–”: below the LOD.
aProvided by author’s laboratory.
bProvided by Cheng Yung Pharmaceutical Co., Taoyuan, Taiwan. Batch No. AI006014.
cProvided by Tsumura Pharmaceutical Co., Tokyo, Japan. Batch No. 2070023010.
dProvided by Tsumura Pharmaceutical Co., Tokyo, Japan. Batch No. B10371.
eProvided by Tong Yang Pharmaceutical Co., Taichung, Taiwan. Batch No. 550397.
fProvided by Sun Ten Pharmaceutical Co., Taichung, Taiwan. Batch No.131642.
gProvided by Sun Ten Pharmaceutical Co., Taichung, Taiwan. Batch No. 271841.
hProvided by Sanjiu Nankai Pharmaceutical Co., Hunan, China. Batch No. 20070802.
iProvided by Huquan Pharmaceutical Co., Hubei, China. Batch No. 071101.
jProvided by Jianyin Tianjiang Pharmaceutical Co., Jiansu, China. Batch No. 0808177.
sule could comparably yield high extraction of most of its bioactive
components. As to samples 8 and 10, the content of PF fulfilled the
national standard of China, but the levels of other components such
as SA, LL, BE, AII and AI were obviously lower than those of other
products, which might result from the low extraction efficiency of
lipid-soluble constituents in extraction process. In addition, the rel-
ative contents of BA were less than 5% in all products except 5, in
which it reached to 12.15%. Although its amount in product 5 was in
the safe range for clinical applications, monitoring this component
and choosing appropriate herbs are still recommended in order to
increase the safety of the drug product. These results suggested
that the occurrence and contents of the compounds in commercial
preparations depend on the raw materials and are also influenced
by dosage form, extraction processand formula. To ensure its stabil-
ity, safety and efficacy for clinical use, guidelines and quality control
for commercial products of DSS should be standardized.
4. Conclusion
A reliable and simple analytical method was developed for
the qualitation and quantitation of components in DSS by using
LC–DAD–MS/MS. Forty-one components including monoterpene
glycosides, phenolic compounds, phthalides, sesquiterpenoids and
triterpenes in the formula were successfully identified based
on retention time, UV and MS spectra compared with those of
authentic compounds or literature data. Fourteen components
were simultaneously determined by LC–DAD at two different
wavelengths. The developed method was validated to have good
precision, accuracy, and repeatability, thus could be used to evalu-
ate the quality of the drug products. The results demonstrated that
the present method could readily provide full-scale qualitative and
quantitative information for the quality evaluation of DSS inter-
mediates and final preparations. Furthermore, the method can be
modified to analyze the ingredients of individual herbal medicine in
DSS and some other complex prescriptions containing these herbs.
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (Grant No. 30772792) and the grant of “111 Project”
from the Ministry of Education of China and the State Administra-
tion of Foreign Expert Affairs of China (No. 111-2-07).
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