Journal of Pharmaceutical and Biomedical Analysis 199 (2021) 114039

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Journal of Pharmaceutical and Biomedical Analysis

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

Short communication

Ultra-high performance supercritical fluid chromatography method

for separation and quantitation of saikosaponins in herbal medicine
Tingting Sun a,b , Jiaoyang Luo b , Yuanyuan Xu a , Xinqi Sun b , Shihai Yang a,∗ ,
Meihua Yang b,∗
a
College of Traditional Chinese Medicine, Jilin Agricultural University, Changchun, 130118, China
b
Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant
Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193, China

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

Article history: Saikosaponins are the main active ingredients of Bupleuri Radix and have been shown to have hep-
Received 30 March 2020 atoprotective, immunomodulatory and anti-viral activities. Among the saikosaponins, saikosaponin a
Received in revised form 22 March 2021 (SSa), saikosaponin b1 (SSb1 ) and saikosaponin b2 (SSb2 ) are a group of isomers, which are difficult to
Accepted 23 March 2021
separate by HPLC. In this study, a new method for separation and quantitation of saikosaponins was
Available online 26 March 2021
established by using ultra-high performance supercritical fluid chromatography (UHPSFC). A Torus Diol
column (100 mm × 3 mm, 1.7 ␮m) was applied in this study. The mobile phase CO2 (A) was the main
Keywords:
solvent with MeOH (B) as co-solvent. The results showed that the five saikosaponins were success-
Ultra-high performance supercritical fluid
chromatography (UHPSFC)
fully separated within 22 min through optimization of chromatographic conditions. Besides, the UHPSFC
Saikosaponin method was applied for the quantitation of saikosaponins in a patent medicine Chaihu Dropping Pills,
Isomer and demonstrated a good correlation coefficient (R2 ) ≥ 0.9990 in the range of 0.025 – 0.25 mg/mL. The
Separation recoveries of the five saikosaponins at three different concentrations were in the range of 90.23–99.84%.
This study indicates that the proposed method has high separation efficiency in analyzing saikosaponins,
which provides a new way for the separation and quantitation of saikosaponins in herbal medicines.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction
Bupleuri Radix is the dried root of Bupleurum chinense DC., which
is one of the most commonly used traditional Chinese medicines
and has a long history of application in China. The main compo-
nents of Bupleuri Radix are saikosaponins, which are pentacyclic
triterpenoid oleanane derivatives. The most important saponin
structures have been identified as saikosaponin a, b, c and d (SSa,
SSb, SSc and SSd). These saponins have been extensively stud-
ied in the fields of pharmacology and immunology. For example,
they have anti-tumor [1,2], immune regulation [3], liver protection
[4], anti-depression [5,6] and neurological (function protection)
activities [7]. Besides, SSa and SSd can be converted to secondary
saponins by aqueous solvent or mild acid treatment because they
contain unstable allyloxide linkage. Saikosaponin b1 (SSb1 ) can
be derived from SSa, and saikosaponin b2 (SSb2 ) is derived from
∗ Corresponding authors.
E-mail addresses: jlyangs@163.com (S. Yang), yangmeihua15@hotmail.com
(M. Yang).
SSd [8,9]. That is the main reason why sometimes SSd cannot be
detected in patent medicines.
As far as we know, the analytical methods for saikosaponins are
mainly liquid chromatography and mass spectrometry. For exam-
ple, HPLC-UV method was used to determine SSa, SSb1 , SSc and
SSd in Chaihuang granules [10]; LC-DAD-ESI/MS method was used
to determine SSa, SSc and SSd in Bupleuri Radix [11]; high perfor-
mance liquid chromatography-mass spectrometry (HPLC-MS) was
used to determine SSa and SSd in a Chinese traditional compound
medicine [12]. However, the isomers such as SSa and SSb2 are dif-
ficult to separate by HPLC method, and it always needs longer time
to reach a satisfactory resolution in saponin analysis. Therefore, it is
very meaningful to find a simpler, higher separation ability method
for saikosaponin analysis.
Supercritical fluid chromatography (SFC) is a promising sepa-
ration technology widely used in analytical chemistry due to its
high separation efficiency, environmental friendliness and low cost
[13–15]. For example, Huang et al. used SFC-MS to rapidly sepa-
rate triterpenoid saponins such as saponin and ginsenosides in Ilex
latiflia [16]; Zhao et al. used SFC to analyze 25 (r/s)- spirostanol
saponin diastereomers in Trigonella foenum graecum L. [17]. As the
latest chromatography technology for instruments, UHPSFC inte-

https://doi.org/10.1016/j.jpba.2021.114039
0731-7085/© 2021 Elsevier B.V. All rights reserved.
T. Sun, J. Luo, Y. Xu et al.
grates SFC and UPLC technologies. Its mobile phase is composed of
supercritical CO2 , which has the advantages of low viscosity, fast
molecular diffusion, high separation efficiency and environmental
protection. This technology opens up a new field of separation that
is becoming a complementary technology for gas chromatography
and liquid chromatography, providing a wide range of adaptabil-
ity and selectivity for chromatographic analysis [18,19]. It has been
applied to vitamins [20], sterols [21], gestagens [22], betaine (index
compound) [23], etc. In this study, UHPSFC was applied to the anal-
ysis of the five saikosaponins including SSa, SSb1 , SSb2 , SSc and
saikosaponin f (SSf), among which, SSa, SSb1 and SSb2 are a group
of isomers. This study was designed to develop a high separation
efficiency method for the separation and determination of saikos-
aponins in complex matrix.
2. Materials and methods
2.1. Chemicals and reagents
Liquid carbon dioxide (99.99 % purity) for SFC separations was
supplied from Beijing Yongsheng Gas Technology Co., Ltd. HPLC
grade methanol (MeOH) and Formic acid was purchased from
Thermo Fisher (Shanghai, China). The distilled water was filtered
through 0.22 ␮m membrane before use. Standards (purity≥98 %) of
SSa, SSb1 , SSb2 , SSc and SSf were obtained from Chengdu Chroma-
Biotechnology Co., Ltd., China. Chaihu Dropping Pills were collected
in pharmacy. All other chemicals and solvents used in the study
were of analytical grade.
2.2. Preparation of standard solutions
The mixed stock solution of SSa, SSb1 , SSb2 , SSc, and SSf
(0.25 mg/mL for each compound) was prepared by dissolving with
methanol in amber glass vials, and was stored at −20 ◦ C. The mixed
stock solution was diluted in methanol to generate a series of work-
ing solutions at the following concentrations: 0.25, 0.2, 0.15, 0.1,
0.05, and 0.025 mg/mL. The structure of the five saikosaponins are
shown in Fig. 1.
2.3. Sample preparation
About 0.5 g sample powder of Chaihu Dropping Pills were accu-
rately weighed into a 50 mL centrifuge tube, 25 mL of 5% ammonia
solution -methanol (1:4, v/v) mixed solution was accurately added
and weighed. The sample was extracted by ultrasound (200 W,
40 kHz) for one hour, the weight loss was made up, and then fil-
tered. The filtrate was transferred to a 100 mL round bottom flask,
and evaporated to dryness using a rotary evaporator. The residue
Fig. 1. The structure diagram of five kinds of saikosaponins.
2
Journal of Pharmaceutical and Biomedical Analysis 199 (2021) 114039
was dissolved in 5 mL of methanol and filtered through a 0.22 ␮m
microporous filter.
2.4. UHPSFC and HPLC conditions
UHPSFC analysis was performed on an ACQUITY UHPSFC sys-
tem (Waters, MA, USA), which was equipped with a column
thermostat, a binary solvent delivery pump, a fixed-loop autosam-
pler, and an automated backpressure regulator (ABPR). Waters
Empower TM Pro 3 software was used for data acquisition, process-
ing and instrument control of UHPSFC system. Torus Diol column
(100 mm × 3 mm, 1.7 ␮m) was obtained from Waters (MA, USA). A
linear gradient mobile phase containing scCO2 (A) and MeOH (B)
was applied as follows: 20 % B to 30 % B for 16 min, 30 %B to 40
%B for 6 min. The flow rate was 1.0 mL/min, the injection volume
was 5 ␮L, the column temperature was 45 ◦ C, and the backpressure
was 2000 psi. A diode array detector was used with the detection
wavelength of 210 nm.
HPLC analysis was performed on an LC-20AT HPLC system (SRLS,
JPN), equipped with SPD-M20A detector, SIL-20A automatic sam-
pling device, DGU-20A5 online degassing, CT0-10AS VP column
temperature box. Agilent Extend-C18 (4.6 × 250 mm, 5 ␮m) was
obtained from Agilent (USA). A linear gradient mobile phase con-
taining acetonitrile (A) and H2 O (B) was applied as follows: 34 % A
to 34 % A for 10 min, 34 %A to 50 %A for 42 min. The flow rate was
1.0 mL/min, the injection volume was 5 ␮L, the column tempera-
ture was 35 ◦ C. A diode array detector was used with the detection
wavelength of 210 nm.
2.5. Quantitative method validation
2.5.1. Linearity and LOQ
A calibration curve was generated in order to determine the
linearity of the saikosaponins detection range. Standard samples
were analyzed at concentrations of 0.25, 0.2, 0.15, 0.1, 0.05, and
0.025 mg/mL. The linear equation and the correlation coefficient
were determined using the least squares linear regression. The LOD
and LOQ were defined by ICH Guidelines according to the S/N of 3
and 10.
2.5.2. Precision, repeatability, and accuracy
Intra-day precision was calculated from the successive six injec-
tions of mixed standard solution at three different concentrations
on the same day, while the repeated analysis was performed six
times per day over three consecutive days for inter-day precision.
Repeatability was obtained via parallel preparation of six sample
extracts from the same batch.
The recoveries of the five saikosaponins were evaluated in trip-
licate at three different concentration, which were calculated using
T. Sun, J. Luo, Y. Xu et al.
Table 1
Parameters of the UHPSFC method for determination of saikosaponins.
Analyte Calibration curve
SSa y = 1,000,000x-2071.5
SSb1 y = 1,000,000x-7177.2
SSb2 y = 1,000,000x+9274.4
SSc y = 1,000,000x+9642.5
SSf y = 776,364x+1131
Calibration curve: y = ax + b, where y is the integrated peak area and x is the concentration in mg/mL.
Fig. 2. The UHPSFC chromatograms under different column temperatures. Exper-
imental conditions: mobile phase components: (A) scCO2 ; (B) MeOH. Gradient:
0 min, 80 % A; 16 min, 70 % A; 22 min, 60 %A; backpressure: 2000 psi; flow rate:1.0 mL
min−1 .
calibration curves performed in a representative sample spiked
with mixed standard solutions.
3. Results and discussions
3.1. Optimization of chromatographic conditions
3.1.1. Optimization of co-solvents
The mobile phase of UHPSFC was mainly a supercritical fluid
composed of CO2 . In order to adjust the solubility to the target, a
relatively small proportion of polar solvent, commonly referred to
as modifiers or cosolvents, is usually added to the mobile phase
to facilitate the adaptation to different solubility of different tar-
get compounds. Methanol, ethanol and isopropanol have H-bond
acceptor and donor properties and their use should be privileged as
they can provide advantages such as minimizing interactions [13].
In this test, methanol and acetonitrile were investigated as liquid
phase solvents, and there was no significant difference on retention
time and peak area of the chromatograms. As is known, methanol
is often adopted as a modifier for the analysis of saponins by SFC
[24,25], and it is cheaper than acetonitrile. Therefore, methanol was
finally chosen as the cosolvent for further research.
3.1.2. Optimization of gradient
The gradient was essential for the separation of isomers. In this
study, a total of five mobile phase gradients containing scCO2 (A)
and MeOH (B) were investigated: (1) 20 % B to 30 % B for 10 min, 30
%B to 50 %B for 10 min; (2) 25 % B to 30 % B for 8 min, 30 % B to 35 %
B for 6 min, 35 %B to 40 %B for 2 min; (3) 20 % B to 25 % B for 6 min,
25 % B to 30 % B for 2 min, 30 % B to 35 % B for 6 min, 35 % B to 40 %
B for 2 min; (4) 20 % B to 25 % B for 6 min, 25 % B to 30 % B for 4 min,
30 % B to 40 % B for 5 min; (5) 20 % B to 30 % B for 16 min, 30 % B
to 40 % B for 6 min. The backpressure of 2000 psi and the column
temperature of 35 ◦ C were adopted in this test. In order to achieve
satisfied peak shape and resolution of ssa and ssb1 , the gradient of
(5) was optimized.
Journal of Pharmaceutical and Biomedical Analysis 199 (2021) 114039
R2 Ranger (mg/mL) LOQ (mg/mL)
0.999 2 0.025−0.25 0.025
0.999 0 0.05 - 0.25 0.05
0.999 2 0.025−0.25 0.025
0.999 3 0.025−0.25 0.025
0.999 0 0.05 - 0.25 0.05
Fig. 3. The UHPSFC chromatograms under different formic acid concentration.
Experimental conditions: mobile phase components: (A) scCO2 ; (B) MeOH 0-0.2 %
(v/v) FA. Gradient: 20 % B to 30 % B for 16 min, 30 % B to 40 % B for 6 min; temperature:
45 ◦ C; backpressure: 2000 psi; other experimental conditions as in Fig.3.
3.1.3. Optimization of column temperature
As is known, when the temperature increases, the density of the
supercritical fluid decreases, and the solvation ability decreases, so
that the retention time of the target is prolonged. In order to obtain
better separation of the five saikosaponins, the effect of column
temperature on the separation of the target was investigated in
the range of 30–45 ◦ C while maintaining other chromatographic
conditions. The results are shown in Fig. 2, which illustrated that
when the temperature gradually increased, the retention time of
the target components prolonged, and the resolution improved.
The resolution was 1.07 when the temperature was 30 ◦ C; the res-
olution increased to 1.51 when the temperature reached 45 ◦ C.
Therefore, the column temperature of 45 ◦ C was adopted for further
study.
3.1.4. Optimization of backpressure
In UHPSFC analysis, the main function of backpressure is to con-
trol the supercritical state of CO2 during the whole process. When
the back pressure increases, the density of supercritical fluid, sol-
vation ability and the column pressure will increase subsequently.
In this experiment, the backpressure of 1700–2300 psi was inves-
tigated. As is shown in Fig. S1, the larger the back pressure, the
higher the column pressure, and shorter the retention time of the
target peaks. In addition, the background noise reduced along with
the increase of backpressure, however, the resolution of the analyte
did not change significantly. Finally, the backpressure of 2000 psi
was chosen for further study.
3.1.5. Optimization of formic acid concentration
In order to improve the resolution and peak shape for saponins,
low concentrations of additives, such as formic acid, TFA, ammo-
nium acetate and water, were added during UHPSFC analysis. Since
saikosaponin is weakly acidic, the peak shape can be appropriately
improved under acidic conditions, so that a small amount of formic
acid was added as a modifier in the mobile phase. The formic acid
concentrations of 0.05 %, 0.1 %, 0.15 % and 0.2 % were investigated
3
T. Sun, J. Luo, Y. Xu et al. Journal of Pharmaceutical and Biomedical Analysis 199 (2021) 114039

Fig. 4. Chromatograms of the five saikosaponins by using HPLC and UHPSFC techniques. HPLC conditions: Agilent Extend-C18 (4.6 × 250 mm, 5 ␮m); mobile phase: (A)
acetonitrile; (B) H2 O. Gradient: 0 min, 34 % A; 10 min, 34 % A; 42 min, 50 %A. Flow rate:1.0 mL min−1 ; injection volume: 5 ␮L; column temperature: 35 ◦ C; injection volume:
5 ␮L. UHPSFC conditions: Torus Diol column (100 mm × 3 mm, 1.7 ␮m); mobile phase: (A) ScCO2 ; (B) H2 O. Gradient: 0 min, 80 % A; 16 min, 70 % A; 22 min, 60 %A. Flow rate:
1.0 mL min−1 ; column temperature: 45 ◦ C; injection volume: 5 ␮L; backpressure: 2000 psi.

Table 2
Intra-day and inter-day precision and accuracy.

Analyte Concentration Intra-day accuracy Inter-day accuracy Intra-day precision Inter-day

(mg/mL) (recovery, %) (recovery, %) RSD (%) precision RSD (%)

0.025 97.12 95.87 2.59 2.62

SSa 0.15 99.84 98.03 1.74 2.04
0.25 97.92 96.54 2.45 2.29
0.025 92.45 90.23 2.65 2.74
SSb1 0.15 93.22 90.75 1.43 2.32
0.25 92.91 90.26 2.52 2.95
0.025 96.53 95.02 2.37 2.56
SSb2 0.15 98.34 95.49 1.66 1.13
0.25 98.03 95.64 2.02 1.01
0.025 94.87 92.72 2.92 2.98
SSc 0.15 96.13 93.46 2.05 2.12
0.25 95.89 92.54 2.91 1.49
0.025 96.01 93.68 1.81 2.05
SSf 0.15 96.35 94.77 1.19 1.73
0.25 96.43 94.16 1.61 1.99

in this study. As is shown in Fig. 3, when the formic acid concen-
tration increased, the peak shape of saikosaponin was improved,
i.e., the chromatographic peaks became sharper with no splitting
peaks. Since higher concentration and long-term use of acid will
damage the column and reduce column efficiency, therefore, no
higher concentration was investigated, and 0.2 % formic acid was
adopted.
3.2. Method validation
3.2.1. Linear range and LOQ of the assay
The regression equations and correlation coefficients (R2 ), as
well as the linear range are shown in Table 1. The results showed
that the linear range of saikosaponins were 0.025 - 0.25 mg/mL, and
all the R2 values were >0.999. The LOQs ranged from 0.025 mg/mL
to 0.05 mg/mL.
3.2.2. Precision, repeatability and accuracy
The intra-day precisions calculated for SSa, SSb1 , SSb2 , SSc and
SSf at three different concentrations ranged from 1.19 to 2.92; while
the inter-day precisions ranged from 1.01 to 2.98 (Table 2). Six por-
tions of the same parallel samples were pretreated and analyzed to
estimate repeatability. The RSDs of repeatability were in the range
of 1.68–2.05 %, indicating a satisfactory repeatability. To test the
intra- and inter-day accuracy, the standard solutions with three
different concentrations were added to Chaihu Dropping Pill pow-
der from the first step of extraction. The mean recoveries for SSa,
SSb1 , SSb2 , SSc and SSf were in the range of 90.23–99.84% (Table 2).
3.3. Comparative analysis of UHPSFC and HPLC
For HPLC analysis, 5 ␮L of each single standard and mixed
standard (2.5 mg/mL) were injected into Shimadzu LC-20AT HPLC
system. Initially, a mobile phase gradient containing acetonitrile
(A) and H2 O (B) was used as follows: 25 % A to 90 % A for 50 min,
and 90 % A to 90 % A for 10 min. Under this condition, the chromato-
graphic peaks of SSa and SSb2 were completely coincident. In order
to improve the resolution and shorten the analysis time, the HPLC
method conditions were optimized and shown in Section 2.4, and
the chromatogram is shown in Fig. 4. For UHPSFC analysis, the chro-
matogram obtained using the optimized UHPSFC method is shown
in Fig. 4. Chromatograms of HPLC and UHPSFC of saikosaponin stan-
dards were obtained. Because the used separation technique was
different for both methods and there was a significant difference
in method parameters, the peak order of the five saikosaponins in
UHPSFC was different from that of HPLC. By using HPLC, the col-
umn length 250 mm (which has better separation ability than that
of 150 mm column when the stationary phase is the same) were
applied, but the isomer of SSa and SSb2 failed to separate within
45 min. In contrast, the five saikosaponins reached baseline separa-
tion within 22 min by using a 100 mm Torus Diol column, indicating
satisfactory resolution. In addition, the Torus Diol column has high-

4
T. Sun, J. Luo, Y. Xu et al.
Fig. 5. Chromatogram of a representative sample of Chaihu Dropping Pills.
density diol group bonding, which is more suitable for saponins in
UHPSFC analysis.
Previous studies have indicated that UHPSFC has an obvious
advantage in separating isomers. Yamada et al. described for the
first time an SFC-FT-MS method for lipid analysis [26]. Zhu et al.
compared UHPSFC with UHPLC in isolating spirostanol saponins
[27], which indicated that UHPSFC could be performed automati-
cally and quickly, however, the resolution of spirostanol saponins
with different aglycones and the same sugar moiety by UHPSFC was
not ideal.
3.4. Real sample analysis
The validated method was applied to determine the content of
saikosaponins in Chaihu Dropping Pills. Since SSb1 , SSc and SSf
were barely detected in real samples, so that only SSa, and SSb2
were determined in this study. The results showed that the con-
tents were 2.60–3.32 mg/g and 1.80–1.89 mg/g for SSa, and SSb2 in
two different batches of pills, respectively (Fig. 5). According to the
literatures, it is not difficult to understand that the majority of SS
are not found. As Li et al. indicated, SSa and SSd are converted into
secondary saponins after mild acid treatment because they contain
very unstable epoxy ether bonds [28], and this is the main reason
why SSd or SSa are barely detected in Chinese patent medicines.
4. Conclusion
In this study, UHPSFC method was established for separation
and quantitation of saikosaponins, and it and HPLC techniques were
compared, which indicates that UHPSFC has significant advan-
tages in separating saikosaponins. Besides, the mobile phase,
column temperature, backpressure and formic acid concentration
were systematically investigated and optimized. The five saikos-
aponins were successfully separated within 22 min. In addition, the
quantitation method was established and validated, indicating sat-
isfactory repeatability and recovery. This study indicates that the
proposed method has high separation efficiency and provides a new
way for the separation and quantitation of saikosaponins in herbal
medicines.
CRediT authorship contribution statement
Tingting Sun: Investigation, Writing - original draft. Jiaoyang
Luo: Methodology, Writing - review & editing. Yuanyuan Xu: Data
curation. Xinqi Sun: Data curation. Shihai Yang: Supervision. Mei-
hua Yang: Supervision, Writing - review & editing.
5
Journal of Pharmaceutical and Biomedical Analysis 199 (2021) 114039
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by grants from the State key R & D
Program (2018YFC1707904, 2018YFF0214202), High-End Foreign
Experts Project (G20200001511), and CAMS Innovation Fund for
Medical Sciences (2016-I2M-3-010).
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at doi:https://doi.org/10.1016/j.jpba.2021.
114039.
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