identification and determination of the saikosaponins in radix bupleuri by accelerated solvent...

Research Article

Identification and determination of thesaikosaponins in Radix bupleuri byaccelerated solvent extraction combinedwith rapid-resolution LC-MS

A method based on accelerated solvent extraction combined with rapid-resolution LC–MS

for efficient extraction, rapid separation, online identification and accurate determination

of the saikosaponins (SSs) in Radix bupleuri (RB) was developed. The RB samples were

extracted by accelerated solvent extraction using 70% aqueous ethanol v/v as solvent, at a

temperature of 1201C and pressure of 100 bar, with 10 min of static extraction time and

three extraction cycles. Rapid-resolution LC separation was performed by using a C18

column at gradient elution of water (containing 0.5% formic acid) and acetonitrile, and

the major constituents were well separated within 20 min. A TOF-MS and an IT-MS were

used for online identification of the major constituents, and 27 SSs were identified

or tentatively identified. Five major bioactive SSs (SSa, SSc, SSd, 600-O-acetyl-SSa and

600-O-acetyl-SSd) with obvious peak areas and good resolution were chosen as benchmark

substances, and a triple quadrupole MS operating in multiple-reaction monitoring mode

was used for their quantitative analysis. A total of 16 RB samples from different regions of

China were analyzed. The results indicated that the method was rapid, efficient, accurate

and suitable for use in the quality control of RB.

Keywords: Accelerated solvent extraction / MS / Radix bupleuri / Rapid-resolution LC / SaikosaponinDOI 10.1002/jssc.201000100

1 Introduction

Radix bupleuri (RB), the dried roots of Bupleurum chinenseDC. or Bupleurum scorzonerifolium Willd. [1], is a popular

traditional Chinese medicine (TCM) and has been used in

China for thousands of years. Moreover, RB is used as a key

ingredient in many Chinese multi-herbal remedies, such as

Xiaochaihu-tang, a famous multi-herbal remedy renowned

for its possible healing effects on chronic hepatitis [2, 3] and

its beneficial effects in preventing the development of

hepatocellular carcinoma in patients with cirrhosis of the

liver [4]. The major bioactive compounds isolated from RB

are saikosaponins (SSs) [1, 5, 6], which have been proven to

possess significant biological activities, including antihepa-

titis [7], anti-inflammatory [8], antitumor [9] and immuno-

regulatory effects [10]. Among all of these SSs, SSa, SSc,

SSd, 600-O-acetyl SSa, 600-O-acetyl-SSd and other SS deriva-

tives are the main constituents of RB [1, 5, 6].

It has been well recognized that the variation in the

composition of a specific TCM is the foremost cause of

unpredictable clinical effects. Several investigations on RB

have indicated that the contents of SSs show remarkable

differences depending on their genus, origins, growing

conditions, and so on [1, 5]. Therefore, developing a rapid,

efficient and reliable analytical method for the determina-

tion of SSs is crucial for the quality control of RB.

Several methods for the determination of SSs in RB

have been described, including thin-layer chromatography

[11], droplet countercurrent chromatography [12], micellar

electrokinetic capillary chromatography [13] and HPLC

[14–16]. However, these methods were limited due to their

poor sensitivity and lack of specificity [1]. The application of

HPLC and triple quadrupole MS/MS greatly improved the

sensitivity and specificity of the determination of the SSs in

RB [1], but using conventional HPLC was time consuming.

More recently, rapid-resolution LC (RRLC) coupled with

evaporative light-scattering detection had been applied to

analyze SSs [5]. Using high-linear-velocity columns packed

with porous 1.8-mm particles, the separation efficiency of

RRLC was remarkably improved comparing with that of

Yun-Yun Yang1

You-Zhi Tang2

Chun-Lin Fan3

Hui-Tai Luo1

Peng-Ran Guo1

Jian-Xin Chen2

1Guangdong Provincial PublicLaboratory of Analysis andTesting Technology, ChinaNational Analytical CenterGuangzhou, Guangzhou,P. R. China

2Guangdong Provincial KeyLaboratory of VeterinaryPharmaceutics Developmentand Safety Evaluation, Collegeof Veterinary Medicine, SouthChina Agricultural University,Guangzhou, P. R. China

3Institute of TCM & NaturalMedicine, Jinan University,Guangzhou, P. R. China

Received February 13, 2010Revised March 22, 2010Accepted April 6, 2010

Abbreviations: ASE, accelerated solvent extraction; MRM,

multiple-reaction monitoring; m/z, mass-to-charge ratio; RB,

Radix bupleuri; RRLC, rapid-resolution liquid chromatography;

SS, saikosaponin; TCM, traditional Chinese medicine; TIC,

total ion chromatogram

Correspondence: Dr. Jian-Xin Chen, College of VeterinaryMedicine, South China Agricultural University, 483 WushanRoad, Guangzhou 510642, P. R. ChinaE-mail: [email protected]: 1 86-20-8528-3730

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2010, 33, 1933–1945 1933

HPLC. The time required for a full run of the determination

of the SSs in RB by RRLC was about one-fourth of that by

HPLC [5]. However, evaporative light-scattering detection

offered a significantly lower sensitivity and poorer specificity

than MS/MS as detector. Recent success with the applica-

tion of RRLC-MS/MS offered the possibility of developing a

rapid analytical method which could get high sensitivity and

specificity for the determination of SSs in RB and RB-

containing Chinese multi-herbal remedies. Furthermore,

the use of TOF-MS and IT-MS linked to RRLC provided the

possibility of straightforward online identification of the

constituents in RB, due to available exact molecular weight

and abundant MS fragments information.

A fast, efficient and complete extraction method also

played an important role in the quality control of TCM. There

have been several studies focused on the extraction of SSs

from RB by conventional methods, including boiling-water

extraction and ethanol- or aqueous ethanol-soaking extraction

[17–19]. However, these conventional methods were time

consuming, solvent consuming and have low efficiency [20].

Attention has therefore been paid to the development of a

rapid and efficient method to extract the bioactive constitu-

ents from herbal medicines. Accelerated solvent extraction

(ASE) is a new extraction procedure that uses organic

solvents at high pressures and temperatures above their

boiling points [21]. With ASE, a solid or semisolid sample is

enclosed in a sample cartridge that is filled with an extraction

fluid and used to statically extract the sample under elevated

temperature (50–2001C) and pressure (500–3000 psi) condi-

tions for short time periods (5–10 min) [21]. The high

temperature reduces the extraction time and improves the

extraction efficiency because it decreases the viscosity of the

solvent, and thus allowing better penetration of solvent

molecules into the sample matrix [21, 22]. To date, ASE has

rarely found to be used in TCM extraction, although the

technique has been widely applied in environmental, food

and biological samples [23–27].

In this article, we proposed a reliable method for effi-

cient extraction, rapid separation, online identification and

accurate determination of the SSs in RB by ASE combined

with RRLC-MS. The critical parameters, such as the

extraction conditions of ASE and the gradient elution

conditions of RRLC, were optimized. A TOF-MS and an IT-

MS were applied to identify the major constituents online.

Five major bioactive SSs, namely SSa, SSc, SSd, 600-O-acetyl-

SSa and 600-O-acetyl-SSd, were chosen as benchmark

substances and quantitatively analyzed by a triple quadru-

pole tandem MS.

2 Materials and methods

2.1 Materials and chemicals

Acetonitrile and methanol of HPLC grade were purchased

from Burdick & Jackson (Muskegon, MI, USA). Water for

RRLC analysis was purified by a Milli-Q water-purification

system (Milford, MA, USA). Ethanol and formic acid were

analytical grade and purchased from Guangzhou Chemical

Reagent Factory (Guangzhou, China).

SSa, SSc, SSd, SSb1, SSb2, baicalin and wogonoside

were purchased from Sichuan WeiKeQi Bio-Technology

(Sichuan, China). 600-O-Acetyl-SSa and 600-O-acetyl-SSd were

synthesized from SSa and SSd by acetylation using acetic

anhydride. The reaction products were separated and puri-

fied using a preparative HPLC system. The structures of all

compounds (Fig. 1) were confirmed by comparing their UV,

IR, MS, 1H NMR and 13C NMR spectra with those in the

literatures [28–31]. Stock solutions were prepared by

dissolving 5.0 mg (7 0.01 mg) of each reference substance

Figure 1. Chemical structures of seven reference SSs in this study.

J. Sep. Sci. 2010, 33, 1933–19451934 Y.-Y. Yang et al.


in 5.0 mL methanol. A mixed stock solution containing

0.10 mg/mL of SSa, SSc, SSd, 600-O-acetyl-SSa and 600-O-

acetyl-SSd was prepared by diluting 1.0 mL of each stock

solution in methanol. Working solutions were prepared by

diluting the mixed stock solutions with methanol to the

desired concentrations.

The samples of dried roots of Bupleurum chinense DC.

were purchased from different drugstores. They were

collected from the Anhui, Guangdong, Guangxi, Hebei,

Jilin, Shandong, Shanxi and Sichuan provinces of China. All

samples were authenticated by Professor Dan-Yan Zhang,

Guangzhou University of Chinese Medicine, Guangzhou,

China. The samples were dried, crushed and sieved through

a 0.3-mm stainless-steel sieve before extraction. Sample no.

1, which was collected from Guangdong province, was used

in the method-development studies.

2.2 Sample extraction

An SP-100 QSE system (Shanghai Spectrum Instruments,

China) with an 11-mL stainless-steel extraction cell was used

for ASE extraction. In total, 1.0 g of sample was placed in the

extraction cell, and then the cell was placed into the heating

block of the instrument. The sample was extracted under

preset conditions. The extract was transferred to a 50-mL

volumetric flask, brought up to volume with methanol and

filtered through a 0.45-mm nylon filter membrane prior to

injection into the RRLC system.

Reflux extraction was performed in a cooled condenser

and a round-bottomed 100-mL flask. About 1.0 g of sample

and 50 mL of 70% aqueous ethanol v/v were added into the

round-bottomed flask. The suspension was extracted for 1 h

at 801C on a water bath and then filtered. The extraction was

repeated two additional times and the extracts were

combined. Ultrasonic extraction was carried out by mixing

1.0 g of sample and 50 mL of 70% aqueous ethanol v/v in a

flask and sonicating for 30 min followed by filtration. The

extraction was repeated two additional times and the extracts

were combined. The combined extracts of both reflux and

ultrasonic extraction were evaporated at 801C, reduced the

volume to about 20 mL, transferred to a 50-mL volumetric

flask, brought up to volume with methanol and filtered

through a 0.45-mm nylon filter membrane prior to injection

into the RRLC system.

2.3 Instrument analysis

The chromatographic separation was performed on an

Agilent 1200 Series RRLC system (Agilent Technologies,

Waldbronn, Germany), equipped with a binary pump, a

microvacuum degasser, a high-performance autosampler, a

column compartment and a diode-array detector. The

samples were separated on a Zorbax Eclipse XDB-C18

column (50 mm� 2.1 mm id, 1.8-mm) at a temperature of

251C and a flow rate of 0.5 mL/min using water-formic acid

(100:0.5, v/v) (solvent A) and acetonitrile (solvent B) as the

mobile phase. For the identification of the major constitu-

ents in RB, the linear solvent gradient was 0–1 min

(15%, B); 1–13 min (15–50%, B); 13–17 min (50–95%, B)

and 17–20 min (95%, B). For the determination of the

contents of SSa, SSc, SSd, 600-O-acetyl-SSa and 600-O-acetyl-

SSd in RB, the linear gradient was 0–0.5 min (15%, B);

0.5–6.5 min (15–50%, B); 6.5–8.5 min (50–95%, B) and

8.5–10 min (95%, B). The diode-array detector was set to

monitor absorbance at 254 nm, and online UV spectra were

recorded in the range of 190–400 nm.

The above RRLC system was interfaced with an Agilent

6510 TOF-MS (Agilent Technologies, MA, USA) and an

Agilent Trap XCT IT-MS (Agilent Technologies, USA) for

the identification of the major constituents in RB. While for

quantitative analysis of the five SSs in RB, RRLC was

coupled with an Agilent 6410 B triple quadrupole tandem

mass spectrometer (Agilent Technologies, USA). The ESI

source was used for all of the three mass spectrometers. The

capillary voltage was set at 3500 V, drying-gas temperature

was set at 3501C and a flow rate of 10.0 L/min, and nebu-

lizer pressure was 50 psi. RRLC-IT-MS spectra were scan-

ned from mass-to-charge ratio (m/z) 50 to 2000 in the Auto-

MSn mode, with acquisition of both of the positive- and

negative-ion MS1, MS2 and MS3 data. RRLC-TOF-MS

spectra were recorded over a mass range of m/z 50–2000, in

both positive- and negative-ion modes. A reference solution

was used for the elimination of the system bias during the

whole RRLC-TOF-MS analysis procedure. Ions with m/z118.0863 and 922.0098 in positive-ion mode and m/z112.9856 and 1033.9881 in negative-ion mode were selected

for mass calibration, and the mass accuracy of calibration

ions was within 5 ppm. The RRLC-MS/MS operated in

multiple-reaction monitoring (MRM) mode with the ion

pairs of m/z 825/779 and 779/617 for the determination of

SSa and SSd; m/z 971/925 and 925/779 for the determina-

tion of SSc and m/z 867/821 and 821/659 for the determi-

nation of 600-O-acetyl-SSa and 600-O-acetyl-SSd. The cone

voltage was 35 V and the collision energy was 30 eV.

HPLC-TOF-MS analysis was performed on the same

Agilent 1200 system, but the separation was performed on a

Zorbax Eclipse XDB-C18 column (250 mm� 4.6 mm id,

5-mm) at a temperature of 251C and a flow rate of 1.0 mL/

min using water-formic acid (100:0.5, v/v) (solvent A) and

acetonitrile (solvent B) as the mobile phase. The linear

solvent gradient was 0–5 min (15%, B); 5–50 min (15–50%,

B); 50–70 min (50–95%, B) and 70–80 min (95%, B). The

outlet was connected to a split valve in order to divert a flow

rate of 0.4 mL/min into the ESI source. Other TOF-MS

conditions were the same as above.

2.4 Validation of quantitative analysis

The prepared mixed stock solution containing SSa, SSc,

SSd, 600-O-acetyl-SSa and 600-O-acetyl-SSd was diluted to

form a series of concentrations appropriate for the

J. Sep. Sci. 2010, 33, 1933–1945 Liquid Chromatography 1935


construction of calibration curves. Working solutions at six

different concentrations were injected in triplicate. The

LODs and LOQs for each analyte were defined as the

concentrations which generated peaks with S/Ns of 3 and

10, respectively.

Intra- and inter-day variations were evaluated for the

determination of the method’s precision and accuracy. For

the intra-day variability, the mixed working solution at the

concentration of 100 mg/L for each compound was analyzed

six times in 1 day, whereas for inter-day variability it was

examined in duplicate on three consecutive days.

The recoveries were determined using the standard

addition method, in which 1.5 mg SSa, 0.5 mg SSc, 1.0 mg

SSd, 0.5 mg 600-O-acetyl-SSa and 0.5 mg 600-O-acetyl-SSd

were spiked into sample no. 1. The spiked samples were

extracted by ASE and analyzed by RRLC-MS/MS using the

above-described method.

3 Results and discussion

3.1 Optimization of ASE conditions

The parameters affecting extraction efficiency of ASE

include the solvent type, temperature, static extraction time

and number of extraction cycles [21, 22]. These parameters

were optimized by employing a univariate design, and using

the relative extraction ratios of SSa, SSc, SSd, 600-O-acetyl-

SSa and 600-O-acetyl-SSd as performance indicators. The

pressure applied did not have a significant effect on

the extraction efficiency as it was used simply to keep the

solvent in the liquid state during the extraction procedure

[21]. Thus, 10 MPa (100 bar, the system default value) was

set as the extraction pressure. The optimization of the ASE

procedure was performed using RB sample no. 1.

The extraction efficiency was evaluated for ten kinds of

solvents, including pure methanol, pure ethanol and 90, 70,

50 and 30% aqueous ethanol and methanol v/v. The ASE

conditions were as follows: extraction temperature, 1201C;

extraction pressure, 100 bar; static extraction time, 10 min;

and three extraction cycles. Of the ten solvent systems

compared in Fig. 2A, the overall relative extraction ratio of

SSa, SSc, SSd, 600O-acetyl-SSa and 600-O-acetyl-SSd was the

highest while using 70% aqueous ethanol v/v, followed in

decreasing order by 50 and 90% v/v aqueous ethanol.

Therefore, 70% aqueous ethanol v/v was selected as the

extraction solvent.

Temperature affected not only the extraction efficiency

but also the thermal decomposition of SSs. At temperatures

above 1001C, SSa and SSd can be converted into SSb1 and

SSb2, respectively. To find an optimal extraction tempera-

ture, a series of experiments were performed at different

Figure 2. Effects of (A) solvent, (B) temperature, (C) time, and (D) number of cycles on ASE extraction efficiency, and (E) differentextraction methods.



temperatures (80, 90, 100, 110, 120, 130 and 1501C). The

extraction experiments were conducted using 70% aqueous

ethanol v/v as the solvent at a pressure of 100 bar with

10 min of static extraction time and three extraction cycles.

The results of extraction efficiencies are shown in Fig. 2B.

The relative extraction efficiency of SSc increased continu-

ously as the temperature increased from 80 to 1501C. The

extraction efficiencies of SSa, SSd, 600-O-acetyl-SSa and 600-O-

acetyl-SSd increased as the temperature raises from 80 to

1201C, and then decreased at higher temperatures. The

decomposition products SSb1 and SSb2 were readily

observed at temperatures above 1001C, especially at 1501C.

The maximum extraction efficiencies of SSa, SSd, (SSa 1

SSb1), (SSd 1 SSb2), 600O-acetyl-SSa and 600-O-acetyl-SSd

were all at the temperature of 1201C. Thus, 1201C was

chosen as the extraction temperature.

To evaluate the influence of static extraction time on

extraction efficiency, different times (5, 7, 10, 15 and

20 min) were used with the following ASE conditions: 70%

aqueous ethanol v/v as the extraction solvent, an extraction

temperature of 1201C, an extraction pressure of 100 bar and

one extraction cycle. The results are shown in Fig. 2C. The

extraction efficiencies of each compound increased as

extraction time extended from 5 to 10 min, while the

extraction time increased from 10 to 20 min, it did not

influence the extraction efficiencies. Thus, the static

extraction time was set at 10 min for one cycle.

The effect of the number of extraction cycles was

determined by running five consecutive extractions on the

same sample. As shown in Fig. 2D, three extraction cycles

were sufficient to extract the target SSs from RB completely.

Finally, the extraction efficiencies of SSa, SSc, SSd,

600-O-acetyl-SSa and 600-O-acetyl-SSd by ASE were compared

with those obtained by reflux extraction and ultrasonic

extraction. As shown in Fig. 2E, the extraction efficiencies of

the five SSs by ASE were higher than those by both reflux

and ultrasonic extraction. Moreover, ASE has the advantages

of a shorter extraction time and use of less extraction

solvent than either the reflux extraction or the ultrasonic

extraction.

3.2 Optimization of RRLC conditions and

comparison of RRLC with HPLC

In order to identify most constituents in RB, a full

separation by RRLC is necessary. However, SSs usually

occur in plants as a mixture of structurally related forms

with very similar polarities, and the separation is rather a

difficult task [5]. Thus, different elution programs with

different elution solvent systems, including water-methanol,

water-acetonitrile, water (containing 0.5% formic acid)-

methanol and water (containing 0.5% formic acid)-acetoni-

trile, were investigated. The results showed that a linear

gradient elution with water (containing 0.5% formic acid)-

acetonitrile gave the best resolution and most of the

constituents could be efficiently separated within 20 min

(Figs. 3A and B). Acetonitrile remarkably improved the

separation of the isomers of SSs and acetyl-SSs compared

with methanol. The addition of formic acid had a substantial

effect on the formation of [M 1 H] 1 or [M 1 HCOO]� and

strikingly improved the sensitivity of the method.

In order to reach accurate analysis of the five major SSs

(SSa, SSc, SSd, 600-O-acetyl-SSa and 600-O-acetyl-SSd) in RB

by RRLC-MS/MS, an adequate separation for them and

their isomers was needed, because the isomers of SSs

showed the same precursors ions and MS/MS fragmenta-

tion, and the compound resolution cannot be achieved by a

Figure 3. RRLC chromatograms obtained by TIC of (A) positive-and (B) negative-ion ESI-TOF-MS, and (C) HPLC chromatogramobtained by TIC of negative-ion ESI-TOF-MS of RB extract.



mass spectrometer. The five SSs could be efficiently sepa-

rated with their isomers within 10 min when using a linear

gradient elution of water (containing 0.5% formic acid)-

acetonitrile (Fig. 4).

For comparison of the separation efficiency of RRLC

with conventional HPLC, the same RB sample extract was

analyzed by both RRLC-TOF-MS and HPLC-TOF-MS. For

HPLC, a full separation run of the RB extract needed 80 min

(Fig. 3C), whereas for RRLC it needed only 20 min. The

separation time by RRLC was about one-fourth of that by

conventional HPLC. For a TCM which contains complex

compounds, such as RB, a reliable HPLC method for

complete separation was relatively time consuming and

hard to fulfill the requirement of rapid determination.

Moreover, HPLC resulted in inefficiency as they required

the use of a large amount of organic solvent and instrument

time [5]. The RRLC method, which performs the separation

at very high pressure using a shorter analytical column

packed with sorbents of particle size 1.8-mm, has been

introduced to offer high speed, good resolution and sensi-

tivity of analysis for the separation of a complex mixture [5,

32]. With the much shorter analytical time, lower solvent

consumption and satisfactory resolution, the RRLC method

was potent for the comprehensive analysis for large

numbers of RB samples and suitable for the quality control

of RB.

3.3 Identification of constituents in RB extract

The RRLC chromatograms obtained by total ion chromato-

gram (TIC) from positive- and negative-ion ESI-TOF-MS are

shown in Figs. 3A and B, respectively, and most constitu-

ents were efficiently separated. In positive-ion ESI mode

experiments, the sodiated adduct [M 1 Na]1 was detected

for nearly all of the constituents. For most constituents, ion

[M 1 H�H2O]1 was detected as the base peak in the

spectra. In the negative-ion ESI mode experiments, the

deprotonated molecule [M�H]� was detected in all spectra,

and the formate adduct [M 1 HCOO]� was observed as the

Figure 4. RRLC chromato-grams obtained by MRM ofnegative-ion ESI triple tandemquadrupole MS of seven refer-ence SSs.



most intense ion in the spectra of most constituents. These

were similar with the previous studies [1, 6]. The exact

molecular weight of each constituent was easily calculated

according to the exact mass of respective pseudo-molecular

ions, and the molecular formula of those was deduced from

each exact molecular weight obtained by high-resolution

ESI-TOF-MS. Further analysis of the same RB extract using

RRLC-ESI-IT-MS at the same chromatographic conditions

provided more structural information, which was very

useful for the identification of constituents. Especially for

SSs, the link order of glycosidic units could be easily

deduced from IT-MS2 and MS3 data [1, 6]. Table 1 lists the

retention times (tR), molecular formulas, exact molecular

weight, ESI-TOF-MS1 ions, ESI-IT-MS2 and MS3 ions

information of the 37 major peaks in the chromatograms.

Due to the characteristic absorption of heteroannular

diene structure with maximal UV wavelength at 254 nm,

SSs with a heteroannular diene structure, such as SSb and

its derivatives, could be discriminated from SSs which were

lack of that structure, such as SSa, SSc, SSd and their

derivatives, by their typical UV absorption data [6]. Based on

the exact molecular weight, MS2 and MS3 data, the typical

UV absorption and the elution order, a total of 27 SSs from

RB were identified or tentatively identified. Among them, 7

SSs including SSc (10), SSa (16), SSb2 (18), SSb1 (22), 600-O-

acetyl-SSa (24), SSd (26) and 600-O-acetyl-SSd (33) were

unambiguously identified by comparison of their tR, UV

spectra, TOF-MS, IT-MS2 and MS3 data with those of the

reference substances.

The acetylated SSs could be identified by the observa-

tion of the loss of one or two CH2CO (42 Da) groups from

deprotonated molecule in negative-ion ESI-IT-MS2 and

MS3. SSs containing an acetyl group usually exhibited

[M–H–42]� and [M–H–H2O–42]� ions in negative-ion ESI-

MS2, whereas for SSs containing two acetyl groups, the ions

[M–H–84]�, [M–H–H2O–84]�, [M–H–2H2O–84]� together

with [M–H–42]�, [M–H–H2O–42]� were observed in nega-

tive-ion ESI-MS2 and MS3. These behaviors were also

reported in Huang et al.’s study [6]. In this study, 12

compounds (12, 13, 19, 20, 21, 24, 25, 27, 30, 31, 32 and 33)

were classified to monoacetylated SSs, and five compounds

(29, 34, 35, 36 and 37) were classified to diacetylated SSs,

according to the fragmentation rule of acetylated SSs in

negative-ion ESI-MS2 and MS3.

Among SSs from the RB extract, the presence of many

isomers with the same molecular weight and MS frag-

mentation made the identification of SSs more difficult.

Fortunately, the isomeric SSs have much different retention

times on RRLC columns. Figures 3A and B show that SSa

(16) eluted 2.39 min faster than SSd (26) did, and 600-O-

acetyl-SSa (24) eluted 2.42 min faster than 600-O-acetyl-SSd

(33) did. Madl et al. had reported that saponins modifica-

tions within the saccharide moiety had a small effect on

retention time, whereas an exchange of the aglycone shifted

retention time significantly [6, 33]. For isomers of mono-

acetylated SSs with the same saikogenin, Huang et al. had

studied their chromatographic properties in detail and

found that 200-O-acetyl-SSa was eluted first, and then

following the order of 300-, 400-, 600-O-acetyl-SSa on RP-HPLC

columns [6]. Based on the chromatographic properties of

SSs, together with their MS2, MS3 data and UV spectra,

three isomers (19, 20 and 21) with the exact molecular

weight of 822.4766 Da were identified as 200-O-acetyl-SSa

(19), 300-O-acetyl-SSa (20) and 400-O-acetyl-SSa (21), and

another three isomers (30, 31 and 32) with the same exact

molecular weight were identified as 200-O-acetyl-SSd (19),

300-O-acetyl-SSd (20) and 400-O-acetyl-SSd (21). While for the

isomer (25), due to the characteristic absorption with

maximal UV wavelength at 254 nm, it could be identified as

monoacetylated-SSb1 or SSb2. A further comparison of

the tR differences between SSb2 (11.87 min) and SSa

(11.53 min) and compound 25 (13.71 min) to 600-O-acetyl-

SSa (13.46 min) suggested that the most possible structure

of compound 25 was 600-O-acetyl-SSb2. Five isomeric diace-

tylated SSs (29, 34, 35, 36 and 37) with the exact molecular

weight of 864.4871 Da could be discriminated by their

retention times. Compound 29, which eluted 1.95 min

faster than compound 34 did, was identified as diacetyl-SSa.

The other four isomers (34, 35, 36 and 37), which clustered

in retention time window from 16.33 to 16.95 min, were

identified as diacetyl-SSd. However, the exact positions of

the acetyl groups could not be determined due to lack of

authentic diacetyl-SSs references.

The other ten compounds in RB extract were

also identified or tentatively identified as HOSSa (6),

HOSSd (7), buddlejasaponin IV (8), SSf (11), acetyl-SSc

(12), acetyl-SSf (13), SSb3 or SSb4 (14), chinoposaponin

XVIII (15), SSe (23) and acetyl-SSe (27), by comparing

their exact molecular weight, MS2 and MS3 spectra, UV

absorption and retention behaviors with those of the

reported compounds [6, 34].

3.4 Quantitative determination of SSs by

RRLC-MS/MS

To develop a method for the quality control of RB, five

constituents with obvious peak areas and good resolution

were chosen as the marker substances, i.e. SSa, SSc, SSd, 600-

O-acetyl-SSa and 600-O-acetyl-SSd. They were generally

considered to be the major bioactive constituents in RB.

As a triple quadrupole tandem mass spectrometer working

in MRM mode provided better sensitivity, repeatability and

reproducibility than those provided by TOF-MS and IT-MS,

a method for quantitative determination of the five SSs by

RRLC-MS/MS was developed.

Both positive- and negative-ion ESI modes were

compared regarding their sensitivities for the five SSs,

negative-ion mode provided better S/Ns and was thus more

suitable for quantitative determination. The formate adduct

[M 1 HCOO]� and the deprotonated molecule [M�H]�

were the primary and secondary peaks in the spectra of all

the five SSs, and hence they were chosen as the parent ions.

As the isomers showed the same MS/MS fragmentation



Tab

le1.

RR

LC

–ES

I–T

OF–M

San

dR

RLC

–ES

I–IT

–MS

iden

tifi

cati

on

of

the

con

stit

uen

tsin

RB

extr

act

Pea

kt R (m

in)

Mol

ecul

ar

form

ula

Exac

t

mol

ecul

ar

wei

ght

Pos

itive

ESI–

TOF–

MS

1

(m/z

)

Neg

ativ

eES

I–TO

F–M

S1

(m/z

)

Pos

itive

ESI–

IT–M

S2

and

MS

3

(m/z

)

Neg

ativ

eES

I–IT

–MS

2an

dM

S3

(m/z

)

Iden

tifica

tion

13.

48C

27H

30O

1661

0.15

3461

1.16

19[M

1H

]160

9.14

68[M

–H]�

465

[M1

H–1

46]1

,

303

[M1

H–3

08]1

301

[M–H

–308

]�U

nide

ntifi

ed

24.

61C

28H

32O

1662

4.16

9062

5.17

75[M

1H

]162

3.16

21[M

–H]�

479

[M1

H–1

46]1

,

317

[M1

H–3

08]1

315

[M–H

–308

]�U

nide

ntifi

ed

35.

83C

21H

18O

1144

6.08

4944

7.09

21[M

1H

]144

5.07

76[M

–H]�

,

891.

1602

[2M

–H]�

271

[M1

H–g

luco

nic

acid

]126

9[M

–H–g

luco

nic

acid

]�B

aica

lina

)

46.

30C

42H

66O

1581

0.44

0283

3.43

13[M

1N

a]1

809.

4307

[M–H

]�,

855.

4364

[M1

HC

OO

]�77

9[M

–H–3

0]�

617.

2[M

–H–1

92]�

Uni

dent

ified

57.

13C

22H

20O

1146

0.10

0646

1.10

92[M

1H

]145

9.09

28[M

–H]�

285

[M1

H–g

luco

nic

acid

]1,

270

[M1

H–g

luco

nic

acid

–CH

3]1

283

[M–H

–glu

coni

cac

id]�

,

268

[M–H

–glu

coni

c

acid

–CH

3]�

Wog

onos

idea

)

67.

68C

42H

70O

1479

8.47

6676

3.46

42[M

1H

–2H

2O]1

,

821.

4679

[M1

Na]

1

797.

4667

[M–H

]�,

843.

4729

[M1

HC

OO

]�74

5[M

1H

–3H

2O]1

,

601

[M1

H–2

H2O

–Glc

]1,

583

[M1

H–3

H2O

–Glc

]1,

455

[M1

H–2

H2O

–Fuc

Glc

]1,

437

[M1

H–3

H2O

–Fuc

Glc

]1

635

[M–H

–Glc

]�,

489

[M–H

–Fuc

Glc

]�H

OS

Sab

)

77.

92C

42H

70O

1479

8.47

66Th

esa

me

asco

mpo

und

6Th

esa

me

asco

mpo

und

6Th

esa

me

asco

mpo

und

6Th

esa

me

asco

mpo

und

6H

OS

Sdb

)

88.

09C

48H

78O

1894

2.51

8892

5.51

75[M

1H

–H2O

]1,

965.

5097

[M1

Na]

1

941.

5096

[M–H

]�,

987.

5151

[M–H

CO

O]�

907

[M1

H–2

H2O

]177

9[M

–H–G

lc]–

,

617

[M–H

–2G

lc]�

Bud

dlej

asap

onin

IVc

)

99.

03C

18H

34O

533

0.24

0635

3.23

21[M

1N

a]1

329.

2323

[M–H

]�33

5[M

1N

a–H

2O]1

311

[M–H

–H2O

]–,

293

[M–H

–2H

2O

]�U

nide

ntifi

ed

109.

51C

48H

78O

1792

6.52

3990

9.52

23[M

1H

–H2O

]1,

949.

5149

[M1

Na]

1

925.

5146

[M–H

]�,

971.

5195

[M1

HC

OO

]�89

1[M

1H

–2H

2O]1

,

439

[M1

H–H

2O–G

lc(R

ha)G

lc]1

,

421

[M1

H–2

H2O

–Glc

(Rha

)Glc

]1

779

[M–H

–Rha

]�,

763

[M–H

–Glc

]�,

617

[M–H

–Rha

–Glc

]�

SS

ca)

119.

89C

48H

80O

1792

8.53

9691

1.53

77[M

1H

–H2O

]1,

951.

5317

[M1

Na]

1

927.

5292

[M–H

]�,

973.

5353

[M1

HC

OO

]–

893

[M1

H–2

H2O

]1,

441

[M1

H–H

2O–G

lc(R

ha)G

lc]1

,

423

[M1

H–2

H2O

–Glc

(Rha

)Glc

]1

781

[M–H

–Rha

]–,

765

[M–H

–Glc

]�,

619

[M–H

–Rha

–Glc

]�

SS

fb)

1210

.13

C50

H80

O18

968.

5345

951.

5323

[M1

H–H

2O

]1,

991.

5257

[M1

Na]

1

967.

5246

[M–H

]�,

1013

.528

7[M

1H

CO

O]�

933

[M1

H–2

H2O

]1,

439

[M1

H–H

2O–C

2H2O

–Glc

(Rha

)Glc

]1,

421

[M1

H–2

H2O

–C2H

2O–G

lc(R

ha)G

lc]1

925

[M–H

–C2H

2O]�

,

907

[M–H

–C2H

2O–H

2O]�

,

779

[M–H

–C2H

2O–R

ha]�

,

763

[M–H

–C2H

2O–G

lc]�

,

617

[M–H

–C2H

2O–R

ha–G

lc]�

Ace

tyl–

SS

c

1310

.42

C50

H82

O18

970.

5501

953.

5488

[M1

H–H

2O

]1,

993.

5348

[M1

Na]

1

969.

5411

[M–H

]�,

1015

.546

5[M

1H

CO

O]�

935

[M1

H–2

H2O

]1,

441

[M1

H–H

2O–C

2H2O

–Glc

(Rha

)Glc

]1,

423

[M1

H–2

H2O

–C2H

2O–G

lc(R

ha)G

lc]1

927

[M–H

–C2H

2O]�

,

909

[M–H

–C2H

2O–H

2O]�

,

781

[M–H

–C2H

2O–R

ha]�

,

765

[M–H

–C2H

2O–G

lc]�

,

619

[M–H

–C2H

2O–R

ha–G

lc]�

Ace

tyl–

SS

f

1410

.71

C43

H72

O14

812.

4922

795.

4911

[M1

H–H

2O

]1,

835.

4829

[M1

Na]

1

811.

4842

[M–H

]�,

857.

4897

[M1

HC

OO

]�77

7[M

1H

–2H

2O]1

,

633

[M1

H–H

2O–G

lc]1

,

615

[M1

H–2

H2O

–Glc

]1,

649

[M–H

–Glc

]�,

503

[M–H

–Fuc

Glc

]�S

Sb 3

orS

Sb 4

b)



Tab

le1.

Co

nti

nu

ed

.

Pea

kt R (m

in)

Mol

ecul

ar

form

ula

Exac

t

mol

ecul

ar

wei

ght

Pos

itive

ESI–

TOF–

MS

1

(m/z

)

Neg

ativ

eES

I–TO

F–M

S1

(m/z

)

Pos

itive

ESI–

IT–M

S2

and

MS

3

(m/z

)

Neg

ativ

eES

I–IT

–MS

2an

dM

S3

(m/z

)

Iden

tifica

tion

487

[M1

H–H

2O–F

ucG

lc]1

,

469

[M1

H–2

H2O

–Fuc

Glc

]1

1511

.19

C48

H78

O18

942.

5188

925.

5177

[M1

H–H

2O]1

,

965.

5104

[M1

Na]

1

941.

5095

[M–H

]�,

987.

5148

[M1

HC

OO

]�90

7[M

1H

–2H

2O]1

779

[M–H

–Glc

]�,

617

[M–H

–2G

lc]�

Chi

nopo

sapo

nin

XV

IIIc

)

1611

.53

C42

H68

O13

780.

4660

763.

4643

[M1

H–H

2O]1

,

803.

4566

[M1

Na]

1

779.

4572

[M–H

]�,

825.

4627

[M1

HC

OO

]–

745

[M1

H–2

H2O

]1,

601

[M1

H–H

2O–G

lc]1

,

583

[M1

H–2

H2O

–Glc

]1,

455

[M1

H–H

2O–F

ucG

lc]1

,

437

[M1

H–2

H2O

–Fuc

Glc

]1

617

[M–H

–Glc

]�,

471

[M–H

–Fuc

Glc

]�S

Saa

)

1711

.72

C44

H74

O14

826.

5079

849.

4987

[M1

Na]

1,

865.

4731

[M1

K]1

825.

4958

[M–H

]�,

871.

5047

[M1

HC

OO

]�—

663

[M–H

–162

]�,

617

[M–H

–208

]�U

nide

ntifi

ed

1811

.87

C42

H68

O13

780.

4660

763.

4649

[M1

H–H

2O]1

,

803.

4571

[M1

Na]

1

779.

4576

[M–H

]�,

825.

4635

[M1

HC

OO

]�74

5[M

1H

–2H

2O]1

,

601

[M1

H–H

2O–G

lc]1

,

583

[M1

H–2

H2O

–Glc

]1,

455

[M1

H–H

2O–F

ucG

lc]1

,

437

[M1

H–2

H2O

–Fuc

Glc

]1

617

[M–H

–Glc

]�,

471

[M–H

–Fuc

Glc

]�S

Sb 2

a)

1912

.24

C44

H70

O14

822.

4766

805.

4749

[M1

H–H

2O]1

,

845.

4681

[M1

Na]

1

821.

4670

[M–H

]�,

867.

4731

[M1

HC

OO

]�78

7[M

1H

–2H

2O]1

,

601

[M1

H–H

2O–C

2H2O

–Glc

]1,

583

[M1

H–2

H2O

–C2H

2O

–Glc

]1,

455

[M1

H–H

2O–C

2H2O

–Fuc

Glc

]1,

437

[M1

H–2

H2O

–C2H

2O

–Fuc

Glc

]1

779

[M–H

–C2H

2O]–

,

761

[M–H

–C2H

2O–H

2O]–

,

617

[M–H

–C2H

2O–G

lc]–

,

471

[M–H

–C2H

2O–F

ucG

lc]�

200 –

O–a

cety

l–S

Sab

)

2012

.41

C44

H70

O14

822.

4766

The

sam

eas

com

poun

d19

The

sam

eas

com

poun

d19

The

sam

eas

com

poun

d19

The

sam

eas

com

poun

d19

300 –

O–a

cety

l–S

Sab

)

2112

.56

C44

H70

O14

822.

4766

The

sam

eas

com

poun

d19

The

sam

eas

com

poun

d19

The

sam

eas

com

poun

d19

The

sam

eas

com

poun

d19

400 –

O–a

cety

l–S

Sab

)

2212

.75

C42

H68

O13

780.

4660

763.

4655

[M1

H–H

2O]1

,

803.

4579

[M1

Na]

1

779.

4561

[M–H

]�,

825.

4621

[M1

HC

OO

]�74

5[M

1H

–2H

2O]1

,

601

[M1

H–H

2O–G

lc]1

,

583

[M1

H–2

H2O

–Glc

]1,

455

[M1

H–H

2O–F

ucG

lc]1

,

437

[M1

H–2

H2O

–Fuc

Glc

]1

617

[M–H

–Glc

]–,

471

[M–H

–Fuc

Glc

]�S

Sb 1

a)

2313

.04

C42

H68

O12

764.

4711

747.

4689

[M1

H–H

2O]1

,

787.

4621

[M1

Na]

1

763.

4613

[M–H

]�,

809.

4672

[M1

HC

OO

]�72

9[M

1H

–2H

2O]1

,

585

[M1

H–H

2O–G

lc]1

,

567

[M1

H–2

H2O

–Glc

]1,

439

[M1

H–H

2O–F

ucG

lc]1

,

421

[M1

H–2

H2O

–Fuc

Glc

]1

601

[M–H

–Glc

]–,

455

[M–H

–Fuc

Glc

]�S

Seb

)

2413

.46

C44

H70

O14

822.

4766

805.

4739

[M1

H–H

2O]1

,

845.

4671

[M1

Na]

1

821.

4681

[M–H

]�,

867.

4732

[M1

HC

OO

]�78

7[M

1H

–2H

2O]1

,

601

[M1

H–H

2O–C

2H2O

–Glc

]1,

583

[M1

H–2

H2O

–C2H

2O

–Glc

]1,

455

[M1

H–H

2O–C

2H2O

–Fuc

Glc

]1,

437

[M1

H–2

H2O

–C2H

2O

–Fuc

Glc

]1

779

[M–H

–C2H

2O]–

,

761

[M–H

–C2H

2O–H

2O]–

,

617

[M–H

–C2H

2O–G

lc]�

,

471

[M–H

–C2H

2O–F

ucG

lc]�

600 –

O–a

cety

l–S

Saa

)

2513

.71

C44

H70

O14

822.

4766

The

sam

eas

com

poun

d24

The

sam

eas

com

poun

d24

The

sam

eas

com

poun

d24

The

sam

eas

com

poun

d24

600 –

O–a

cety

l–S

Sb 2

b)

2613

.92

C42

H68

O13

780.

4660

763.

4639

[M1

H–H

2O]1

,

803.

4569

[M1

Na]

1

779.

4570

[M–H

]�,

825.

4625

[M1

HC

OO

]�74

5[M

1H

–2H

2O]1

,

601

[M1

H–H

2O–G

lc]1

,

617

[M–H

–Glc

]�,

471

[M–H

–Fuc

Glc

]�S

Sda

)



Tab

le1.

Co

nti

nu

ed

.

Pea

kt R (m

in)

Mol

ecul

ar

form

ula

Exac

t

mol

ecul

ar

wei

ght

Pos

itive

ESI–

TOF–

MS

1

(m/z

)

Neg

ativ

eES

I–TO

F–M

S1

(m/z

)

Pos

itive

ESI–

IT–M

S2

and

MS

3

(m/z

)

Neg

ativ

eES

I–IT

–MS

2an

dM

S3

(m/z

)

Iden

tifica

tion

583

[M1

H–2

H2O

–Glc

]1,

455

[M1

H–H

2O–F

ucG

lc]1

,

437

[M1

H–2

H2O

–Fuc

Glc

]1

2714

.17

C44

H70

O13

806.

4816

789.

4798

[M1

H–H

2O]1

,

829.

4726

[M1

Na]

1

805.

4731

[M–H

]�,

851.

4781

[M1

HC

OO

]�77

1[M

1H

–2H

2O]1

,

585

[M1

H–H

2O–C

2H2O

–Glc

]1,

567

[M1

H–2

H2O

–C2H

2O–G

lc]1

,

439

[M1

H–H

2O–C

2H2O

–Fuc

Glc

]1,

421

[M1

H–2

H2O

–C2H

2O–F

ucG

lc]1

763

[M–H

–C2H

2O]�

,

745

[M–H

–C2H

2O–H

2O]�

,

601

[M–H

–C2H

2O–G

lc]�

,

455

[M–H

–C2H

2O–F

ucG

lc]�

Ace

tyl–

SS

eb)

2814

.26

C42

H66

O13

778.

4503

761.

4489

[M1

H–H

2O]1

,

801.

4417

[M1

Na]

1

777.

4397

[M–H

]�,

823.

4464

[M1

HC

OO

]�74

3[M

1H

–2H

2O]1

,

599

[M1

H–H

2O–G

lc]1

,

581

[M1

H–2

H2O

–Glc

]1,

453

[M1

H–H

2O–F

ucG

lc]1

,

435

[M1

H–2

H2O

–Fuc

Glc

]1

615

[M–H

–Glc

]–,

469

[M–H

–Fuc

Glc

]�U

nide

ntifi

ed

2914

.38

C46

H72

O15

864.

4871

847.

4755

[M1

H–H

2O]1

,

887.

4779

[M1

Na]

1

863.

4772

[M–H

]�,

909.

4836

[M1

HC

OO

]�82

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pattern as their precursors, compound resolution cannot be

achieved spectroscopically. For quantitative analysis of the

investigated compounds, a chromatographic separation was

needed to adequately resolve the isomers, especially SSa and

SSb2 (m/z 5 825/779, 779/617) and the isomers of 600-O-

acetyl-SSa and 600-O-acetyl-SSd (m/z 5 867/821, 821/659). A

linear gradient elution of water (containing 0.5% formic

acid)-acetonitrile gave the best resolution and the analytes

were completely separated within 10 min (Fig. 4).

The linearity and ranges, regressions, LODs, LOQs,

precisions, accuracies and recoveries of the method are

listed in Table 2. The high-correlation coefficient values

(r40.9984) indicated appropriate correlations between the

concentrations of the investigated compound and their peak

areas within the test concentrations. The LODs and LOQs

ranged from 0.4 to 0.7 mg/L and from 1.3 to 2.3 mg/L,

respectively. The intra- and inter-day precisions for each

compound were less than 4.2 and 7.1%, respectively. The

intra- and inter-day accuracies were in the range of

96.6–102.9 and 96.1–102.1%, respectively. The developed

method displayed good accuracy, with overall recoveries

ranging from 93.3 to 103.2%. The results indicated that the

developed method was precise, accurate and sensitive for

quantitative determination of the SSs in RB.

3.5 Application to analysis of the RB samples

The ASE-RRLC-MS/MS method that we developed was

applied to quantify the contents of SSa, SSc, SSd, 600-O-

acetyl-SSa and 600-O-acetyl-SSd in 16 RB samples. All the

contents were calculated by the external standard method,

and the mean values and SDs from three parallel

determinations of each sample are summarized in Table 3.

The five SSs were detected in all of the 16 RB samples. In

some RB samples, acetyl-SSs, including 600-O-acetyl-SSa and

600-O-acetyl-SSd, were shown to be major constituents and

their contents were even higher than SSc. Moreover, the

Table 2. Linear-regression data, LODs, LOQs, precision, accuracy and recovery of five SSs as determined by RRLC-MS/MS

Analytes Regression equation r Linear range

(mg/L)

LOD

(mg/L)

LOQ

(mg/L)

Precision RSD (%) Accuracya) (%) Standard addition

recoveryb) (%)

Intra-day Inter-day Intra-day Inter-day

SSa y 5 4.415 x 1 291.4 0.9984 5 – 5000 0.4 1.3 3.3 5.2 98.1 98.9 98.7

SSc y 5 5.680 x 1 233.1 0.9993 5 – 5000 0.4 1.3 2.1 3.9 102.9 102.2 103.2

SSd y 5 3.798 x 1 250.3 0.9986 5 – 5000 0.6 2.0 3.9 7.1 98.3 97.7 93.3

600-O-acetyl-SSa y 5 4.515 x 1 262.6 0.9988 5 – 5000 0.5 1.7 3.8 6.1 96.6 96.1 97.6

600-O-acetyl-SSd y 5 4.970 x 1 271.1 0.9985 5 – 5000 0.7 2.3 4.2 6.7 98.4 97.8 95.2

a) Accuracy (%) 5 100�mean of measured concentration/nominal concentration.

b) The data are presented as the average of three determinations, where standard addition recovery (%) 5 100� (amount found�original

amount)/amount spiked.

Table 3. Contents of SSs in different RB samples

Sample no. Site of collection Content (mean7 SD, n 5 3, mg/g)

Ssa SSc SSd 600-O-acetyl-SSa 600-O-acetyl-SSd

1 Guangdong province 3.087 0.15 0.747 0.03 2.977 0.13 1.027 0.05 1.537 0.08



4 Guangxi province 1.537 0.05 0.787 0.03 1.477 0.06 0.237 0.01 0.207 0.01

5 Guangxi province 1.377 0.06 0.727 0.04 1.557 0.06 0.867 0.04 0.977 0.05

6 Jilin province 3.617 0.16 0.837 0.05 2.337 0.11 0.797 0.03 1.517 0.04

7 Jilin province 2.127 0.13 0.517 0.02 2.017 0.10 0.337 0.01 0.537 0.02

8 Jilin province 2.897 0.11 0.887 0.05 2.257 0.12 0.597 0.02 0.917 0.05

9 Anhui province 0.937 0.03 0.327 0.01 1.127 0.03 0.177 0.01 0.217 0.01

10 Shandong province 1.737 0.07 0.487 0.02 1.657 0.06 0.367 0.01 0.427 0.02

11 Hebei province 0.717 0.02 0.237 0.01 0.957 0.04 0.287 0.01 0.257 0.01

12 Shanxi province 5.167 0.21 2.067 0.11 3.777 0.16 1.057 0.05 1.837 0.06

13 Shanxi province 2.677 0.11 0.867 0.03 2.337 0.10 0.447 0.02 0.837 0.02

14 Sichuan province 6.077 0.24 1.617 0.05 4.827 0.15 2.337 0.09 3.087 0.12





varied contents of acetylated derivatives might have affected

the contents of their corresponding saponins, due to a

possible transformation of acetylated SSs to corresponding

saponins by hydrolysis [5, 35]. Therefore, the simultaneous

analysis of both SSs and acetylated SSs is much more effective

than the detection of SSa, SSc and SSd alone [5, 36], and it

indicated that the analytical method we developed might be

more capable and comprehensive than previously reported.

In this study, the 16 investigated RB samples were

originated from different eight provinces in China, but the

content of the five monitored SSs in RB samples did not

show varying law based on their origins. In fact, the varia-

tion of the content of SSs in RB was affected by many

factors, especially by its genus, origins, growing and

harvesting conditions. For this reason, a rapid, efficient and

reliable analytical method for the determination of the SSs

is crucial for the quality control of RB samples.

4 Concluding remarks

In this study, a method combining ASE with RRLC–MS was

established for efficient extraction, rapid separation, online

identification and accurate determination of the SSs in RB.

ASE has the advantages of having higher extraction

efficiency, using shorter extraction time and less extraction

solvent than reflux extraction and ultrasonic extraction. The

separation speed and efficiency of RRLC was much higher

than those of conventional HPLC, for a full separation of the

major constituents in RB was completed within 20 min by

RRLC, which is only one-fourth of that by HPLC. RRLC

coupled with TOF-MS and IT-MS can be used for online

identification or tentative identification of the constituents

quickly. The method of ASE combined with RRLC-MS/MS

was simple, fast, showed good linearity, precision and

recovery for quantitative analysis of SSa, SSc, SSd, 600-O-

acetyl-SSa and 600-O-acetyl-SSd in RB. Furthermore, the

established ASE-RRLC-MS/MS method was applied for the

quality evaluation of the RB samples from different regions

of China. The results showed that the proposed method was

suitable for use in the quality control of RB.

This work was financially supported by the NationalNatural Science Foundation of China (No. 30772643) and theScientific and Technological Project of Guangzhou City (No.2008Z1-E341).

The authors declared no conflict of interest.

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identification and determination of the saikosaponins in radix bupleuri by accelerated solvent...

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