Separations 09 00163 v2

separations
Review
Recent Advances in Separation and Analysis of Saponins in
Natural Products
Yi Wang 1,† , Yan Ma 1,† , Li Tao 1 , Xiaoyan Zhang 1 , Fusheng Hao 1 , Shipeng Zhao 1 , Lu Han 1,2, *
and Changcai Bai 1,2, *
1 College of Pharmacy, Ningxia Medical University, Yinchuan 750004, China; [email protected] (Y.W.);
[email protected] (Y.M.); [email protected] (L.T.); [email protected] (X.Z.);
[email protected] (F.H.); [email protected] (S.Z.)
2 Key Laboratory of Ningxia Ethnomedicine Modernization, Ministry of Education,
Ningxia Medical University, Yinchuan 750004, China
* Correspondence: [email protected] (L.H.); [email protected] (C.B.)
† These authors contributed equally to this work.
Abstract: To better control the quality of saponins, ensure their biological activity and clinical thera-
peutic effect, and expand the development and application of saponins, this paper systematically
and comprehensively reviews the separation and analytical methods of saponins in the past decade.
Since 2010, the electronic databases of PubMed, Google Scholar, ISI Web of Science, Science Di-
rect, Wiley, Springer, CNKI (National Knowledge Infrastructure, CNKI), Wanfang Med online, and
other databases have been searched systematically. As a result, it is found that ionic liquids and
high-performance countercurrent chromatography are the most popular extraction and separation
techniques for saponins, and the combined chromatography technique is the most widely used
method for the analysis of saponins. Liquid chromatography can be used in combination with differ-
ent detectors to achieve qualitative or quantitative analysis and quality control of saponin compounds
Citation: Wang, Y.; Ma, Y.; Tao, L.; in medicinal materials and their preparations. This paper provides the latest valuable insights and
Zhang, X.; Hao, F.; Zhao, S.; Han, L.; references for the analytical methods and continued development and application of saponins.
Bai, C. Recent Advances in
Separation and Analysis of Saponins Keywords: saponin; ionic liquid; HPCCC; HPLC-MS; QAMS; metabolomics; qualitative and
in Natural Products. Separations 2022, quantitative; quality control
9, 163. https://doi.org/10.3390/
separations9070163
Academic Editors: Yao Shun and

Paraskevas D. Tzanavaras
1. Introduction
Saponin is a kind of natural secondary metabolite, which is composed of sapogenin
Received: 9 May 2022
and a sugar chain [1]. They can be divided into triterpenoid or steroidal types according to
Accepted: 19 June 2022
the sapogenin differences: The triterpenoid saponin’s sapogenin is triterpene, whereas the
Published: 27 June 2022
steroidal saponin’s is spirostane.
Publisher’s Note: MDPI stays neutral The sapogenin of triterpenoid saponins is a triterpenoid derivative composed of 30 car-
with regard to jurisdictional claims in bon atoms and a basic skeleton of six isoprene units. Again, distinguished by sapogenins
published maps and institutional affil- differences, the triterpenoid saponins are divided into tetracyclic and pentacyclic triter-
iations. penoids. The tetracyclic triterpenoid types include lanostane, euphane, dadamane type [2],
cucurbitacin alkanes, cycloxylane-type, meliacanes, and more; the pentacyclic triterpenoid
types include oleanane type [3], ursane, lupane, friedelanes, and others. The sapogenins
of steroidal saponins are steroidal derivatives that generally contain 27 carbon atoms
Copyright: © 2022 by the authors.
and have the basic skeleton of spirostane. Steroidal saponins are divided into spirostanol,
Licensee MDPI, Basel, Switzerland.
This article is an open access article
isospirostanol, furostanol, and deformed spirostanol types. Saponins are widely distributed
distributed under the terms and
in nature and are commonly found in roots, stems, leaves, flowers, and seeds of terrestrial
conditions of the Creative Commons higher plants. Triterpenoid saponins are mainly distributed in the Leguminosae, Araliaceae,
Attribution (CC BY) license (https:// Umbelliferae, Compositae, Polygalaceae, and other plant families. Steroidal saponins are
creativecommons.org/licenses/by/ mostly found in monocotyledons, such as Dioscoreaceae, Liliaceae, and Scrophulariaceae
4.0/). plant families. Saponins represent a main effective component in many kinds of Chinese
Separations 2022, 9, 163. https://doi.org/10.3390/separations9070163 https://www.mdpi.com/journal/separations

However, most saponins have a similar chemical structure and have no UV absorp-
tion or terminal absorption. In addition, the content and proportion of saponins are easily
affected by geographical location, cultivation technology, harvest times, differences in
batches from different manufacturers, and other factors, which all lead to unqualified
Separations 2022, 9, 163 quality, reduced biological activity, and limited clinical applications. Given the deter- 2 of 30
mination challenges, researchers around the globe have proposed different analytical
methods for saponins (Figures 1–3). These methods include spectrophotometry,
thin-layer chromatography
herbal medicines, (TLC), capillary
such as ginseng, astragalus,electrophoresis (CE), infrared
bupleurum, Ophiopogon spectroscopy,
japonicus, notogin-
high-performance liquid chromatography (HPLC), ultra-performance liquid
seng [4], Anemarrhena, Polygala, Platycodon grandiflorus, and licorice. In addition, saponins chroma-
tography
exist in sea(UPLC), quantitative
creatures, such as theanalysis of multicomponents by single marker (QAMS),
sea cucumber.
immunoassay, and metabolomics [17].
Pharmacological studies have shown that saponins have many important biological
In recent
activities years, as significant
and pharmacological pharmacological
actions [5–13], such asaction of saponins
immunity has become
enhancement, well
antitumor,
known and their general
anti-inflammatory, application
antifungal, in variousactions,
and anti-viral fields has increased,
blood glucosetheandquality control of
lipid reduction,
saponins has become a global concern (Table 1). Therefore, based on
antioxidation, cardiovascular function improvement [14,15], hemolysis [16], and more.the qualitative and
In
quantitative perspectives, this article discusses the problems and future
recent years, saponins have been widely used in medicines, health foods, animal feed, development
trends of these
cosmetics, analytical
and other items.methods by reviewing
In addition, saponins arethe used
literature of the
as plant past regulators
growth ten years and
and
provides a valuable reference for choosing appropriate analytical
insect repellents in agriculture. Therefore, saponins have great research value methods to control the
and broad
quality of saponins
developmental (Table(Figures
prospects 2). 1–3).
Separations 2022, 9, x FOR PEER REVIEW 3 of 31

Figure 1. Saponins: distribution, pharmacology, quality control, development and application
Figure 1. Saponins: distribution, pharmacology, quality control, development and application [5–17].
[5–17].
Figure 2.
Figure 2. Chemical
Chemicalstructures
structuresofoften
tenkinds of of
kinds triterpenoid saponins,
triterpenoid (a) lanostane,
saponins, (b) euphane
(a) lanostane, (c)
(b) euphane
dadamane type, (d) cucurbitacin alkanes, (e) cycloxylane-type, (f) meliacanes (g) oleanane type, (h)
(c) dadamane type, (d) cucurbitacin alkanes, (e) cycloxylane-type, (f) meliacanes (g) oleanane type,
ursane, (i) lupine, (j) friedelanes.
(h) ursane, (i) lupine, (j) friedelanes.
Separations 2022, 9, 163 Figure 2. Chemical structures of ten kinds of triterpenoid saponins, (a) lanostane, (b) euphane
3 of (c)
30
dadamane type, (d) cucurbitacin alkanes, (e) cycloxylane-type, (f) meliacanes (g) oleanane type, (h)
ursane, (i) lupine, (j) friedelanes.
Figure 3.
Figure 3. Chemical structures
structures of
offour
fourkinds
kindsofofsteroidal
steroidalsaponins,
saponins,(a)(a)
deformed spirostanol
deformed types,
spirostanol (b)
types,
furostanol,
(b) (c) (c)
furostanol, spirostanol, (d)(d)
spirostanol, isospirostanol.
isospirostanol.
TableHowever,
1. Efficacymost
tablesaponins
of Chinesehave a similar
medicinal chemical
materials structure
mainly and saponins.
containing have no UV absorption
or terminal absorption. In addition, the content and proportion of saponins are easily
Drug Medication
affected
No. by geographical location, Main cultivation technology, harvest
Ingredient times, differences
Efficacy Ref. in
batches from differentSite
Name manufacturers, and other factors, which all lead to unqualified
It can strengthen
quality, reduced biological activity, and limited clinical applications. Given the deter-
mination challenges, researchers around thesaponins, the vitality,
globe have proposed different analytical
Triterpenoid
methods for saponins (Figures 1–3). These methods include strengthen the
spectrophotometry, thin-
ginseng polysaccharides,
layer chromatography (TLC), capillary electrophoresis (CE), body, nourish
infrared blood
spectroscopy, high-
Panax Dry roots ginseng alkynols, amino
performance liquid chromatography (HPLC), ultra-performance and blood,liquid chromatography
nourish
1 ginseng C. and rhi- acid proteins, sugars, [14,18,19]
(UPLC), quantitative analysis of multicomponents by singlethe marker
spleen (QAMS),
and ben- immunoassay,
A. Mey.
and metabolomics [17]. zomes vitamins, organic acids,
efit the lungs, calm
In recent years, as significanttracepharmacological
elements, flavonoidsactiontheofheart,
saponins
calmhasthebecome well
known and their general applicationand peptides
in various fields hasmind
increased, the
and promote quality control
of saponins has become a global concern (Table 1). Therefore, based on the qualitative
wisdom
and quantitative perspectives, this article discusses the problems and future development
Panax Dammar type tetracyclic
trends of these analytical methods by reviewing the literature
Roots and of the
Diffuse past
stasis toten years and
providespseudo- reference fortriterpene
a valuablerhizomes, choosingsaponins,
appropriatefla-analytical methods to control the
stop bleeding, re-
2 ofginseng
quality saponins (Table 2). vonoids, notoginseno- [15,20,21]
leaves, duce swelling and
Wall. Var. sides (amino acids), pro-
flowers relieve pain
Table 1. Efficacy teins, volatile
noto- table of Chinese medicinal oils,
materials acety-containing saponins.
mainly
No. Drug Name Medication Site Main Ingredient Efficacy Ref.

It can strengthen the
Triterpenoid saponins,
vitality, strengthen the
ginseng polysaccharides,
body, nourish blood and
ginseng alkynols, amino
Panax ginseng C. Dry roots blood, nourish the
1 acid proteins, sugars, [14,18,19]
A. Mey. and rhizomes spleen and benefit the
vitamins, organic acids,
lungs, calm the heart,
trace elements, flavonoids
calm the mind and
and peptides
promote wisdom
Separations 2022, 9, 163 4 of 30
Table 1. Cont.

Dammar type tetracyclic
triterpene
saponins, flavonoids,
Panax pseudoginseng notoginsenosides (amino Diffuse stasis to stop
Wall. Var. Roots and rhizomes, acids), proteins, volatile bleeding, reduce
2 [15,20,21]
notoginseng(Burkill) leaves, flowers oils, acetylenes, alcohols, swelling and
Hoo et Tseng polysaccharides, polyols, relieve pain
polyacetylene alcohols,
organic acids, trace
elements, etc.
Oleanane-type pentacyclic
triterpene saponins, Promoting lung,
flavonoids, phenols, relieving asthma,
sterols, polysaccharides, dispelling cold,
Platycodon
polyacetylenes, steroids, benefiting pharynx,
3 grandiflorus (Jacq.) Dry root [5–7,22]
phenolic acids, expectorating phlegm,
A. DC.
polysaccharides, fatty discharging pus,
acids, fatty acids and trace inducing drugs
elements, inorganic to increase
elements, etc.
Fill the air to raise Yang,
fixed surface
Triterpene saponins, antiperspirant, diuretic
4 Astragali Radix Dry root polysaccharides, to poison, discharge pus, [8,23,24]
flavonoids, amino acids collect sore muscle,
benefit water to
reduce swelling
Steroid saponins
Clearing heat and
diphenpyrone, flavonoids,
purging fire, nourishing
lignin, polysaccharides,
Anemarrhena Yin and moistening
5 Dry rhizome alkaloids, amino acids, [9,25–27]
asphodeloides Bunge dryness, quenching
volatile oils, organic acids
thirst and
and trace elements,
eliminating annoyance
inorganic elements, etc.
Antipyretic,
anti-inflammatory,
lowering blood
cholesterol, reconciling
the inside and outside,
Bupleurum saponins,
soothing the liver and
flavonoids, volatile oils,
stagnating depression,
polysaccharides, sterols,
raising Yang, lifting
polyols, coumarins,
depression, protecting
lignans, fatty acids (oleic
Dried roots, liver and boldness,
6 Bupleuri Radix acid, linolenic acid, [10–12,28,29]
whole grasses cooling down, relieving
palmitic acid, stearic
the stasis of the liver qi,
acid, etc.), tryptophan,
relieving qi, relieving
wood sugar alcohol,
pain and reducing
uridine, adenosine and
inflammation,
trace elements, etc.
anticancer, resisting liver
fibrosis, evacuating
fever, soothing liver and
relieving depression,
raising Yang, lifting qi
Table 1. Cont.

Triterpene saponins,
ketones, oligosaccha- Dispelling phlegm,
Polygala tenuifolia rides, alkaloids, reducing swelling,
7 Dry roots [13,30]
Willd phenylpropanoid calming the mind and
flavonoids, lactones, improving intelligence
coumarins, lignin, etc.
Replenishing spleen and
qi, clearing away heat
Triterpenoid saponins
Glycyrrhiza ur alensis Dried roots and detoxifies, expelling
8 flavonoids [31–33]
Fisch. and rhizomes phlegm and cough,
and polysaccharides
relieving pain,
reconciling all drugs
Table 2. Quality standard table of important medicinal materials mainly containing saponins in the
2020 Chinese Pharmacopoeia.
No. Drug Name Content Determination

Total amount of ginsenoside Rg1 (C42 H72 O14 ) and
ginsenoside Re (C48 H82 O18 ) should not be less than
1 Panax ginseng C. A. Mey.
0.27%, and ginsenoside Rb1 (C54 H92 O23 ) should not be
less than 0.18%
Total amount of ginsenoside Rg1 (C42 H72 O14 ),
Panax pseudoginseng Wall. var.
2 ginsenoside Rb1 (C54 H92 O23 ) and notoginseng R1
notoginseng (Burkill) Hoo et Tseng
(C47 H80 O18 ) should not be less than 5.0%
Platycodon grandifloras (Jacq.) Platycodon grandiflorum saponin D (C57 H92 O28 ) shall
3
A. DC. not be less than 0.10%
Astragaloside IV (C41 H68 O14 ) shall not be less than
4 Astragali Radix 0.080%, and calycoflavone glucoside (C22 H22 O10 ) shall
not be less than 0.020%
Mangiferin (C19 H18 O11 ) shall not be less than 0.70%,
5 Anemarrhena asphodeloides Bunge and Anemarrhena saponin BII (C45 H76 O19 ) shall not be
less than 3.0%
Total content of saponin a (C42 H68 O13 ) and saponin d
6 Bupleuri Radix
(C42 H68 O13 ) should not be less than 0.30%
Polygala tenuifolia saponins (C36 H56 O12 ), not less than
2.0%, Polygala ketone III (C25 H28 O15 ) not less than
7 Polygala tenuifolia Willd.
0.15%, containing 3,6’-dierucyl sucrose (C36 H46 O17 )
not less than 0.50%
Glycyrrhizin (C21 H22 O9 ) shall not be less than 0.50%,
8 Glycyrrhiza uralensis Fisch. glycyrrhizic acid (C42 H62 O16 ) shall not be less
than 2.0%
2. Extraction and Separation Methods

Since saponins are complex mixtures with very similar structures and polarities, their
extraction and separation are challenging. Therefore, based on this problem, researchers at
home and abroad have explored green, efficient, solvent-saving and time-saving methods
for the extraction and separation of saponins.
2.1. Extraction of Saponins

2.1.1. ILs
Traditionally, saponins are mainly extracted by decoction, recrystallization and other
methods. However, these methods have many disadvantages, such as time-consuming,
low extraction efficiency, use of toxic and harmful organic solvents, and environmental
pollution. In recent years, with the development of green chemistry, environment-friendly
technologies have attracted more and more attention. Ionic liquids (ILs) are liquid salts
composed of organic cations and inorganic or organic anions with negligible volatility,
low flammability, chemical stability, good environmental friendliness, and good solubility
for organic compounds and extraction ability. By fine-tuning its chemical structure and
properties, selectively distinguishing one compound from other compounds and other
advantages, it has shown great potential to replace traditional organic solvents in many
fields and has been widely used in the extraction and separation of saponins. Some
researchers have explored the use of IL-ATPS to extract ginsenosides (Rg1, Re, Rd and
Rb1) from the crude extract of ginseng root, which has high extraction efficiency and
good selectivity [34]. Other researchers have explored the determination of seven rare
ginsenosides (ginsenosides) in Xuesaitong injection by ILATPE based on imidazolium
ionic liquid (1-butyl-3-methylimidazolium bromide (Bmim)Br) and salt (K2 HPO4 ). Rg6, F4,
20(S)-Rg3, 20(R)-Rg3, Rk3, Rk1, Rg5) potential applications, studies have shown that this
method has a higher extraction rate [35].
2.1.2. UAE
There are currently some auxiliary techniques combined with ILs, such as microwave-
assisted extraction (MAE), ultrasonic-assisted extraction (UAE), which show significant
improvements in the field of extraction and separation. Ionic liquid-based ultrasonic-
assisted extraction (IL-UAE) has been shown to be the most efficient method for the
extraction of saponins from natural plants. Some researchers have established an ionic
liquid ultrasonic extraction of licorice root liquiritigenin (LQ), liquiritigenin apigenin
(LA), isoliquiritigenin (ILQ), isoliquiritigenin apigenin (ILA) and glycyrrhizin (GA), and
compared with the traditional UAE method, the IL-UAE method has higher extraction
efficiency and significantly shortens the extraction time [36]. There are studies using
the method of coupling IL-UAE and ABS to extract eight kinds of ginsenosides from
ginseng flower buds (ginsenoside-Rg1, ginsenoside-Rg2, ginsenoside-Rc, ginsenoside-Rd,
ginsenoside-Re, ginsenoside- Rf, ginsenoside-Rb1 and ginsenoside-Rb2) [37].
2.1.3. MAE
The use of microwave energy enables fast dissolution, drying, acidic digestion, and
extraction of organic compounds from complex matrices. The microwave heats the solvent
or solvent mixture directly, and the direct interaction of microwaves with the free water
molecules present in the glands and vascular systems results in subsequent rupture of the
plant tissue and release of components into the organic solvent. Its main advantages are
reduced solvent volume and time consumption and increased sample throughput. Thus,
MAE provides an alternative method to conventional extraction methods in plants.
2.2. Isolation of Saponins

2.2.1. SFC
SFC is a green chromatographic separation technology. By using supercritical fluid
with low viscosity and high diffusivity, such as supercritical carbon dioxide as the mobile
phase, SFC shows some remarkable features, such as high separation efficiency, high flow
rate, short analysis time, environmental friendliness and low cost, etc. In addition to its
achievements in chiral separations, SFC has also shown great potential in the separation of
saponins from natural products, becoming a complementary technique to gas chromatog-
raphy and liquid chromatography, offering a wide range of adaptability and selectivity
for chromatographic analysis. Some researchers used UHPSFC technology to successfully
separate five saikosaponins, including SSa, SSb1, SSb2, SSc and saikosaponin f (SSf) within
22 minutes, of which SSa, SSb1 and SSb2 are a group of isomers. Furanosterol saponins
have the same aglycone but different sugar chains. SFC is sensitive to the amount and type
of sugars. Therefore, SFC is suitable for separating hydrophilic furosterol saponins and
analyzing traditional Chinese medicines mainly containing steroid saponins [38]. Some
researchers used supercritical fluid chromatography-single quadrupole mass spectrometry
technology to establish a fast and effective method for the separation of matrine saponins
and ginsenosides. Compared with reversed-phase liquid chromatography, the SFC method
shows higher resolution and shorter run time [39]. Other researchers found that UH-
PSFC can effectively separate spirosterol saponins with the same aglycone and different
sugar chains, while ultra-high pressure liquid chromatography (UHPLC) can well separate
spirosterol saponins with the same sugar group and different aglycones. UHPLC and
UHPSFC are complementary in the separation of spirosterol saponins. Considering that
the naturally occurring spirosterol saponins in Chinese herbal medicine are different in
both aglycones and sugar chains, the combination of UHPLC and UHPSFC can achieve
better separation [40].
2.2.2. HSCCC
As an all-liquid partition chromatography technique, it eliminates the irreversible
adsorption loss of samples on solid support matrix columns, has high sample loading
compared with traditional liquid–solid separation methods, and has good reproducible
sample recovery after scale-up, which is a unique advantage over other devices. Therefore,
HSCCC has been widely used to prepare saponin-like active ingredients from natural
products (Figure 4). Nine new triterpenoid saponins (1–9), namely camoreoside A-I, were
extracted and isolated by high-performance countercurrent chromatography and prepar-
ative reversed-phase high-performance liquid chromatography [41]. Some researchers
have used high-speed countercurrent chromatography to successfully separate four minor
saponins from Panax notoginseng leaves, namely Gynostemma saponin XVII, ginsenoside
Rd2, Panax notoginsenoside Fe and Panax notoginsenoside Fd [42]. Some researchers have
used high-speed countercurrent chromatography (HSCCC) and preparative RP-HPLC to
separate and purify 300-O-acetylplatycoside D and polygalactoside D with a purity of more
than 98.9%. Studies have shown that this method can be used for crude extract of Platy-
codon grandiflorum Preparation and rapid separation of medium and trace saponins [43].
Some researchers used high-speed countercurrent chromatography (HSCCC) combined
with evaporative light scattering detection to separate three furosterols and four spirosterol
saponins (parvifloside; methyl protodeltonin; trigofoenoside A-1; zingiberensis saponin I;
deltonin; dioscin; prosapogenin A of dioscin) from Dioscorea, and studies have shown that
HSCCC is an effective method for the separation and purification of two different steroidal
saponins from plant extracts [44]. Some studies have used a linear gradient elution method
to separate
Separations 2022, 9, xfour triterpenoid
FOR PEER REVIEW saponins (hederasaponin B, hederacolchiside E, cernuoside 8 of 31 A,
cernuoside B) from Pulsatilla officinalis [45]. Some researchers have isolated two saponins
with cytotoxicity to cancer cells from the root of A. chinensis by high-speed countercurrent
[45]. Some researchers have isolated two saponins with cytotoxicity to cancer cells from
chromatography (HSCCC) [46].
the root of A. chinensis by high-speed countercurrent chromatography (HSCCC) [46]
Figure 4. Schematic diagram of different types of HSCCC [47]. (A) The synchronous planetary
Figure 4. Schematic diagram
motion of aof different
multilayer types of
coil separation HSCCC
column; [47].
(B) design of the(A) The
coiled synchronous
column for dual HSCCC;planetary
(C)
motion of a multilayer Design of the coiled column
coil separation for foam(B)
column; HSCCC; (D) mechanism
design of pH-zone-refining
of the coiled column for HSCCC.
dual HSCCC;
(C) Design of the coiled2.2.3.
columnFoamfor foam HSCCC; (D) mechanism of pH-zone-refining HSCCC.
Fractionation
It is a physical adsorption and separation technology that concentrates sur-
face-active substances according to their different surface activities. This technology has
the advantages of simple equipment, small investment, low energy consumption, and
strong environmental adaptability, and is a “solvent-free” substitute for solvent extrac-
tion (Figure 5). Since saponins are typical biosurfactants, they have good foaming prop-
erties. Therefore, saponins can be enriched from the leachate by foam fractionation. Some
researchers have developed a new process for the separation of Achyranthes saponins
2.2.3. Foam Fractionation

It is a physical adsorption and separation technology that concentrates surface-active
substances according to their different surface activities. This technology has the advantages
of simple equipment, small investment, low energy consumption, and strong environmental
adaptability, and is a “solvent-free” substitute for solvent extraction (Figure 5). Since saponins
are typical biosurfactants, they have good foaming properties. Therefore, saponins can be
enriched from the leachate by foam fractionation. Some researchers have developed a new
process for the separation of Achyranthes saponins combined with extraction and foam
fractionation. The main compounds in the concentrated and purified foam salts obtained
Separations 2022, 9, x FOR PEER REVIEW
by the developed technology are triterpenoid saponins [48]. Some researchers use foam
separation and resin adsorption technology to separate soybean saponins in soybean meal [49].
Figure5.5.
Figure Experimental
Experimental apparatus
apparatus of foamof foam fractionation
fractionation [48]. [48].
3. Analytical Methods
In addition to the traditional analytical methods, such as spectrophotometry, TLC and
HPLC,Inthisaddition todescribes
paper also the traditional analytical
the analytical methodsmethods,
of QAMS, such
UPLC,as spectrophotomet
immunoassay,
CE, infrared spectroscopy, and metabolomics. This paper reviews
and HPLC, this paper also describes the analytical methods of QAMS, the research status of
UPLC, im
different analytical methods of saponins during the past 10 years to provide the latest
assay, CE, infrared spectroscopy, and metabolomics. This paper reviews the r
valuable insights and references for quality control and clinical application of saponins
status 6).
(Figure of different analytical methods of saponins during the past 10 years to pro
latest valuable insights and references for quality control and clinical applic
saponins (Figure 6).
In addition to the traditional analytical methods, such as spectrophotometry, TLC
and HPLC, this paper also describes the analytical methods of QAMS, UPLC, immuno-
assay, CE, infrared spectroscopy, and metabolomics. This paper reviews the research
status of different analytical methods of saponins during the past 10 years to provide the
latest valuable insights and references for quality control and clinical application of
saponins (Figure 6).
Figure
Figure 6.
6. Structural
Structural diagram
diagram of
of analytical methods for
analytical methods for Saponins.
Saponins.
3.1. TLC
TLC is a commonly used method for the analysis of saponins; it has the advan-
tages of simple operation, strong separation ability, low cost, and fast detection speed. A
study on the quantitative analysis and comparison of diosgenin in Rhizoma Paridis by
reversed-phase HPLC and TLC showed that the two methods had good separation effects
on diosgenin in Rhizoma Paridis. There was no significant difference in the determination
results [50]. Another study examined the simultaneous detection and quantitative analysis
of diosgenin and sea buckthorn diosgenin in the extract of Cornus officinalis. This study
established a sensitive, fast, and effective test (TLC). The results showed that the retention
coefficients of diosgenin and sea buckthorn diosgenin on TLC plates were 0.49 and 0.6,
respectively [51]. TLC can also be used for simultaneous quantitative analysis and identifi-
cation of several saponins in medicinal preparations. One study carried out a quantitative
analysis of ginsenoside Rb1 and Rg1 in Sanqi shangyao capsules by a TLC scanning method;
it combined this assessment with the determination of naringin to jointly control the quality
of the preparation [52]. Another study carried out a qualitative analysis of saikosaponin in
Hugan tablets, as identified by TLC. The TLC method was fast, simple, easy to operate, and
low cost [53]. TLC has the characteristics of simple operation as well as rapid qualitative
and quantitative determination of various compounds, so it has become one of the main
methods for the determination of saponins in natural drugs and preparations.
3.2. CE
CE is a powerful separation and quantitative analysis technology, which has become a
standard tool for the analysis of saponins in many plant extracts. A study screened licorice
extracts by affinity CE to identify active anti-HIV components. Then, solid-phase extraction
technology was used to separate and purify the effective parts. The research explored
a simple, fast, and effective method for combining CE-electrospray MS and liquid chro-
matography (LC)-electrospray MS. The results showed that glycyrrhizin and glycyrrhizin
G2 were the main components providing anti-HIV activity [54]. In another study, rapid
separation and quantitative determination of bupleurum saponins a, c, and d in Chinese
herbal extracts from different regions (Nacalai Tesque, Kyoto, Japan; Toray, Siga, Japan)
were performed by capillary zone electrophoresis. This method has become a powerful
technology for the analysis of complex extracts in Chinese herbal medicines [55]. In addi-
tion, another study achieved simultaneous separation and quantitative determination of
diastereomers of triterpene saponins in Alexandrium algae (soybean saponin I methyl ester
and azukisaponin V methyl ester or bersimoside I methyl ester and bersimoside II methyl
Separations 2022, 9, 163 10 of 30
ester). In this study, β-cyclodextrin was selected as a stereoselective reagent, underwent

borate complexation, and was assessed by CE. The method had the characteristics of high
resolution, high sensitivity, good repeatability, and high detection limit [56]. CE has become
an ideal analytical method for the separation and quantitative analysis of saponins because
of its advantages of high selectivity, short analysis time, low sample consumption, high
efficiency, and high resolution. Of course, CE also has some disadvantages, such as low
detection sensitivity, poor reproducibility, and small injection volume, etc.
3.3. NIRS
Near-infrared spectroscopy (NIRS) is a rapid analytical technique developed recently
and is widely used to detect saponins in medicinal materials and preparations (Figure 7).
One study described the quantitative analysis of shengmaxinside I in the process of honey-
frying of Cimicifuga foetida and established near-infrared diffuse reflectance spectroscopy
Separations 2022, 9, x FOR PEER REVIEW 11 of 31
as a simple and effective analytical method. The study also used the partial least square
method to establish the near-infrared quantitative model. The research showed that the
experimental model had better prediction ability [57]. In another study, the content of total
Province were analyzed, and a fast qualitative analysis method combining Fourier
steroidal saponins in different species of Paris from Yunnan Province were analyzed, and a
transform infrared spectroscopy and UPLC was adopted. The results showed that linear
fast qualitative analysis method combining Fourier transform infrared spectroscopy and
partial least square regression was more suitable than nonlinear support vector machine
UPLC was adopted. The results showed that linear partial least square regression was
regression for the determination of total steroidal saponins in different species of Paris
more suitable than nonlinear support vector machine regression for the determination of
[58]. In addition, another study focused on three saponins (notoginsenoside R1, ginseno-
total steroidal saponins in different species of Paris [58]. In addition, another study focused
side Rg1, and ginsenoside Rb1) in Panax notoginseng and established NIRS technology as
on three saponins (notoginsenoside R1 , ginsenoside Rg1 , and ginsenoside Rb1 ) in Panax
fast and simple for quantitative analysis. The results showed that this method could ac-
notoginseng and established NIRS technology as fast and simple for quantitative analysis.
curately
The results predict the that
showed totalthis
content
methodof three
couldsaponins in Panax
accurately predictnotoginseng [59]. NIRS
the total content has
of three
many advantages in the analysis of saponins from natural products such as
saponins in Panax notoginseng [59]. NIRS has many advantages in the analysis of saponins plants, in-
cluding its simplicity, its fast analytical speed, its ability to not damage samples, its
from natural products such as plants, including its simplicity, its fast analytical speed, its lack
of chemical
ability to notpollution, and more.
damage samples, its lack of chemical pollution, and more.
NIRS spectra
Figure 7. NIRS spectra of
of 150
150 batches
batches of Rhizoma Cimicifugae [57].
3.4. HPLC
In recent years, HPLC has been widely used to identify and quantitatively analyze
saponins and their preparations because of its high resolution, high selectivity, and high
sensitivity. HPLC combined with various detectors has become the mainstream method
for saponin analysis; potential combinations include the UV/diode array detector, evapo-
rative light scattering detector (ELSD), charged aerosol detector (CAD), chromatographic
fingerprint, and mass spectrometer detector (MS). LC is connected with these detectors to
Separations 2022, 9, 163 11 of 30
qualitatively or quantitatively analyze multiple saponins in complex medicinal materials

and their preparations.
3.4.1. HPLC-UV/DAD
The UV detector is the detector most commonly paired with HPLC. It has the ad-
vantages of wide application range, high sensitivity, wide linear range, and compatibility
with gradient elution. In addition, HPLC diode array detection is a classic method of
natural product analysis. The diode array approach is widely used to analyze complex
samples of natural products. Its repeatability and, similar to UV detection, its high linearity
in the determination of saponins are positive features. Compared with the UV detector,
the diode array detector can detect many saponins simultaneously through a segmented
monitoring strategy based on variable wavelength detection. Although the diode array
detector provides a multi-wavelength spectrum, its sensitivity is lower than that of the
UV detector.
HPLC diode array detection provides a potential analytical platform for the quality
control and pharmacodynamic evaluation of various saponins with medicinal potential [60].
Compared with the colorimetric method, this analytical method can provide more infor-
mation about the chemical composition of herbal extracts and their preparations [61]. One
Study used ilexoside II as the external standard to verify the validity of the determination of
total saponins in the immature fruits of Ilex paraguariensis by HPLC-UV spectrophotometry.
This method had a high saponin yield and good reproducibility [62]. Kwon and Park
separated astragalin and astragaloside from the head, main root, and lateral root of 4-,
5-, and 6-year-old astragalus by reversed-phase chromatography and detected them with
high sensitivity by pulsed amperometric detection under alkaline conditions. The research
showed that standardized cultivation and an appropriate storage technology are key to
producing high-quality astragalus extracts rich in bioactive substances [63]. Extending
this research, Lee realized the simultaneous quantitative analysis of isoflavones (calyx
glycosides, formalonin) and triterpenoid saponins (AST-I-IV) in astragalus by increasing
the detection potential [64]. In addition, another study used HPLC diode array detection
technology to rapidly and simultaneously determine the four effective components of a
Panax notoginseng injection: notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, and
ginsenoside Rb1 [65]. HPLC combined with photodiode array detection can provide a
three-dimensional fingerprint, making this method not only easy to understand but also
effective for obtaining quantitative information about target components and accurate
results. Therefore, this method has broad application prospects in the rapid determination
of complex samples.
3.4.2. HPLC-ELSD
As a general detection method for saponin compounds, ELSD has been successfully
applied to the quantitative analysis of saponins. Many studies have explored a fast and
accurate HPLC approach combined with ELSD to simultaneously and quantitatively deter-
mine the content of saponins in traditional Chinese medicines from different origins and
harvest periods [66]. Combining this approach with the methods of principal component
analysis (PCA) and cluster analysis to classify and identify saponins of different samples
evaluates the quality of medicinal materials even more thoroughly [67–69]. To compare
the differences between medicinal materials from different areas, one study established a
simple and reliable HPLC method combined with ELSD to compare the main saponins
in the samples of Platycodon grandiflorus from southern and northern China [70] and to
compare the contents of Ophiopogonin D0 , Ophiopogonin D, Ophiopogonin B in the tubers
and fibrous roots of Ophiopogonin japonicas in Cixi City, Zhejiang Province, and Santai
County, Sichuan Province [71]. In addition, a study used HPLC diode array detection ELSD
to determine the content of seven kinds of flavonoids and five kinds of saponins in the
roots of Astragalus membranaceus var. mongholicus from Shanxi, Hunyuan, with different
specifications and grades. The study found obvious differences in the concentration dis-
tablished a simple and reliable HPLC method combined with ELSD to compare the main
saponins in the samples of Platycodon grandiflorus from southern and northern China [70]
and to compare the contents of Ophiopogonin D′, Ophiopogonin D, Ophiopogonin B in
the tubers and fibrous roots of Ophiopogonin japonicas in Cixi City, Zhejiang Province, and
Santai County, Sichuan Province [71]. In addition, a study used HPLC diode array de-
Separations 2022, 9, 163 tection ELSD to determine the content of seven kinds of flavonoids and five kinds of30
12 of
saponins in the roots of Astragalus membranaceus var. mongholicus from Shanxi, Hunyuan,
with different specifications and grades. The study found obvious differences in the
concentration
tribution lawdistribution
of flavonoids law ofsaponins
and flavonoids and saponins
in astragalus, in different
with astragalus, with different
specifications and
specifications and different grades [72]. Thus, ELSD can avoid the interference
different grades [72]. Thus, ELSD can avoid the interference from terminal absorption from
terminal absorption
wavelengths wavelengths
of saponins of saponins
and overcome and overcome
the difficulty the difficulty
of traditional of traditional
analytical methods in
analytical methods in
the determination of the determination of saponins.
saponins.
3.4.3.HPLC-CAD
3.4.3. HPLC-CAD
AsAsa aquality
qualitydetector,
detector,the
thecharged
chargedaerosol
aerosoldetector
detectorisisbased
basedon onthe
theprinciple
principleofofanan
aerosoldetector,
aerosol detector,the thesample
samplesolution
solutionisisatomized
atomizedby bythe
theatomizing
atomizinggas gas(nitrogen)
(nitrogen)ininthe
the
atomizerand
atomizer andthen
thenhitshitsthe
thecollision
collisionbaffle
baffleatata ahigher
higherflow
flowrate
ratetotoform
formsolute
soluteparticles
particlesofof
different sizes. The larger particles are discharged from the waste
different sizes. The larger particles are discharged from the waste pipe under the pipe under the influence
influ-
of gravity, and the smaller particles flow into the drying pipe with
ence of gravity, and the smaller particles flow into the drying pipe with nitrogen; nitrogen; at theatsame
the
time,time,
same the other flow path
the other flowofpath
the inlet nitrogen
of the passes through
inlet nitrogen passes the coronathe
through device (containing
corona device
high-voltage
(containing platinum wire
high-voltage electrode)
platinum wire to electrode)
form positively charged
to form nitrogen
positively particles,
charged which
nitrogen
collide with
particles, whichthecollide
dried solute particles
with the driedin the collision
solute particlescell. Thecollision
in the charge iscell.
thenThe
transferred
charge isto
the particles—the larger the solute particle, the more charge. The
then transferred to the particles—the larger the solute particle, the more charge. Thesolute particles transfer
so-
their charge to the collector, and the charge amount of the solute particles
lute particles transfer their charge to the collector, and the charge amount of the solute is measured by a
highly sensitive electrostatic detector. The resulting signal current
particles is measured by a highly sensitive electrostatic detector. The resulting signalis proportional to the
content of the solute (Figure 8) [73].
current is proportional to the content of the solute (Figure 8) [73].
Figure 8. The structure and working diagram of the CAD [74]. 1 HPLC eluent inlet. 2 Nitrogen
Figure 8. The structure and working diagram of the CAD [74]. ① HPLC eluent inlet. ② Nitrogen
inlet.
3 Spray chamber: droplets form a spray. 4 Waste pipe: It attaches to large droplets.
inlet. ③ Spray chamber: droplets form a spray. ④ Waste pipe: It attaches to large droplets.
5 Drying
⑤
tube: dry particles are left after evaporation.
6 Corona electrodes: the gas is positively
Drying tube: dry particles are left after evaporation. ⑥ Corona electrodes: the gas is positively charged after
charged after
ionization. Collision⑦
7ionization. cell: positivecell:
Collision charges migrate
positive to the
charges surface
migrate of the
to the of the
particle.
surface 8particle. ⑧it
Ion trap:
Ion trap: it removes charged particles with high mobility. ⑨ Collector: the electrometer measures
removes charged particles with high mobility.
9 Collector: the electrometer measures the surface
the surface
charge. 10charge.
⑩ Electrometer:
Electrometer: signalisstrength
signal strength is proportional
proportional to the
to the amount of amount of the analyte.
the analyte.
TheCAD
The CADdetector
detectorisisbased
basedononaaunique
uniquenewnewdesign
designprinciple,
principle,which
whichsolves
solvessome
some
limitations of other detector design principles. Its biggest advantage is that
limitations of other detector design principles. Its biggest advantage is that the detection the detection
doesnot
does notdepend
dependon onthe
themolecular
molecularstructure
structureofofthe
theanalyte
analyteororionize
ionizethe
theanalyte,
analyte,which
which
achievesthe
achieves thepurpose
purposeof ofversatility.
versatility. ItIt has
has the
the same
same response
response toto different
differentcompounds
compoundsand andis
isnot
notsensitive
sensitivetotoexternal
externalinfluences.
influences.ItItcancancarry
carryoutoutgradient
gradientelution.
elution.At
Atthe
thesame
sametime,
time,it
itcan
canachieve
achievehigher
highersensitivity and
sensitivity and lower detection
lower detectionlimit, good
limit, reproducibility,
good and wide
reproducibility, and
dynamic detection range. The CAD detector combined with a
wide dynamic detection range. The CAD detector combined with a high-performance high-performance liquid
chromatography
liquid chromatography system is simple
system and convenient
is simple and convenientto operate andand
to operate has has
good stability.
good It is
stability.
widely used to detect most semi-volatile and non-volatile organic compounds, especially
suitable for the analysis and detection of saponin compounds. One study simultaneously
detected and quantitatively analyzed 15 triterpenoid saponins in the leaves, stems, root
bark, and fruits of Acanthopanax senticosus. High-performance liquid chromatography-
charged aerosol detection-electric spray mass spectrometry technology proved to be a
simple and accurate method for the detection of triterpenoid saponins. The results showed
that the baseline was stable, the sensitivity was high, and the reproducibility was good,
which was significantly better than HPLC-UV [75]. Another study combined UPLC with
Separations 2022, 9, 163 13 of 30
CAD to quantitatively analyze marker composition in ginseng. The results showed that the
contents of ginsenoside Re, Rd, and Rg1 as well as compound K comprise approximately
22% of the ginseng plant. The total saponin content was determined by vanillin sulfuric
acid system colorimetry and computer-aided design reaction [76].
3.4.4. Chromatographic Fingerprint

The chromatographic fingerprint can comprehensively measure the complex compo-
nents of traditional Chinese medicine and provide an effective means for quality control of
this medicine. The purposes of this method are to identify species, evaluate quality, and
develop the consistency and stability of traditional Chinese medicine. One study used
a simple method of HPLC with a UV light scattering detector to quantitatively analyze
10 batches of saponins from a pair of medicines—Anemarrhena and Phellodendron—and
combined the method with the chromatographic fingerprint, for the first time in 2014 [77].
Tian et al. used HPLC-ELSD and high-performance TLC simultaneously to analyze the
fingerprint of saikosaponin in bupleurum; the study found that the roots of different species
of bupleurum can be easily distinguished with this technology, making it convenient for the
classification of commercial samples [78]. Another study used HPLC with a UV fingerprint
to analyze the Xuesaitong injection. The results showed that the fingerprints of 10 batches
of samples had 27 characteristic peaks [79]. Qi et al. compared the changes of the liquid
phase characteristic map of saponins in 13 batches of samples of astragalus before and after
alkalization and determined that astragalus saponins I, II, III, and IV were the characteristic
components. This method monitored the content and proportion of the four components at
the same time, which could more truly reflect the quality of the original medicinal mate-
rial [23]. In addition, another study combined HPLC-MS and nuclear magnetic resonance
(NMR) techniques to characterize the structure of two isoflavones and four saponins. The
study found that the combination of HPLC-MS and nuclear magnetic resonance (NMR)
techniques could achieve an accurate description of the structure of these compounds [80].
Thus, chromatographic fingerprint analysis is a comprehensive qualitative method that
simultaneously provides determination of multiple components of traditional Chinese
medicine and comprehensive measurement of the complex component information of
traditional Chinese medicine (Tables 3 and 4).
Table 3. Detailed conditions for the analysis of saponins in natural medicines and foods by high-
performance liquid chromatography.
Detection Compound Stationary Phase Mobile Phase The Detector Ref.

996 Photodiode array
Luna C18 (2) column water (0.1% acetic acid) (A)
4 triterpenoid saponins detector (Waters Corp.),
(150 × 4.6 mm; 5 µ and acetonitrile [66]
from Sophora flavescens 75 ELS detector Sedex
particle size) (0.1% acetic acid) (B)
(SEDERE) And MS: ESI: TOF
water (0.02% formic) (A)
3 steroidal saponins Tigerkin C18 column and acetonitrile Mass spectrometry [81]
(0.02% formic) (B)
acetonitrile (A) and formic
RP-18e monolithic column
6 steroidal saponins acid aqueous solution Mass spectrometry [82]
(50 × 2 mm)
(0.1%, v/v) (B)
SPDM10AVP Photodiode
volatile ion pair
11 saponins of Inertsil PREP-ODS column Array Detector and
reagent (dihexyl [83]
Achyranthes bidentate (20 × 250 mm) Shimadzu LC-MS-2020
ammonium acetate)
Mass spectrometry
diosgenin methyl: water
Rich Alorich Ascentis C8
12 diosgenin in six batches (A) and methanol (B) High Resolution Mass
column (100 mm × 4.6 mm, [84]
of polygala samples 11 other saponins: water Spectrometry: (-):HESI(+/−)
3 µm)
(A) and acetonitrile (B)
Separations 2022, 9, 163 14 of 30
Table 3. Cont.
Detection Compound Stationary Phase Mobile Phase The Detector Ref.

Dikma Diamonsil C18
4 triterpene saponins in the acetonitrile (A) and
column (4.6 mm × 250 mm, 2000ES Diode array detector [67]
Asparagus leaves water (B)
5 µm)
SPD-M20A DAD detector
water (0.1% formic acid)
Kromasil 100-5 C18 column and LTQ Orbitrap Velos Pro
9 oleic acid saponins (A) and acetonitrile [60]
(250 mm × 4.6 mm, 5 µm) mass spectrometer:
(0.1% formic acid) (B)
(-): electrospray
Shiseido CapCell PAK C18
5 triterpenoidal saponins water (A) and
analytical column MS: (-): ESI [68]
In Pulsatilla koreana acetonitrile (B)
(4.6 mm × 150 mm, 5 µm)
15 triterpenoid saponins
Charged Aerosol Detection
from the leaves, stems, root Kinetex XB-C18 column acetonitril (A) and
and Agilent 6530q-TOF mass [75]
skins and fruits of (100 mm × 4.6 mm, 2.6 µm) water (B)
spectrometry: (+)
Acanthopanax quiculata
Diamonsil C18 column acetonitrile (A) and UV detector, Sedex75 ELSD
4 steroidal saponins [77]
(4.6 mm × 250 mm, 5 µm) water (B) system and DAD
YMC Hydrosphere C18
6 components in extract of acetonitrile (A) and G4212A UV-Visible Diode
analytical column [61]
ivy leaf 0.1% phosphoric acid (B) Array Detector
(150 × 4.6 mm, 5 µm)
triterpene saponins in Inertsil ODS-3 column methanol (A) and 5 mM
UV-visible light detector [85]
Camellia plants (2.1 mm × 100 mm) trifluoroacetic acid (B)
Agilent 6460 Triple
Reverse phase Zorbax
Arachnoside F in ammonium acetate (A) and Quadrupole Mass
SB-C18 column [86]
rat plasma acetonitrile (B) Spectrometer:
(150 × 4.6 mm, 5 µm)
Electrospray (+)
Agilent Zorbax SB-AQ
5 saponins in 10 batches of deionized water (A) and
analytical column Diode array detector [87]
Panax notoginseng acetonitrile (B)
(4.6 mm × 50 mm, 3.5 µm)
Panax notoginseng saponin
Zorbax ODS C8 column water (A) and Waters 2996 photodiode
Fc and Ginsenoside Rc in [88]
(250 mm × 4.6 mm, 5 µm) acetonitrile (B) array detector
Notoginseng leaf
3.4.5. HPLC-MS Detector

HPLC-MS is a powerful tool to identify and quantitatively analyze saponins. Com-
pared with other detectors, it has higher sensitivity and offers more complete structural
information, and it has the advantages of short analysis time, low sample consumption,
and online access. For this method, common ion sources include electron bombardment
ionization sources and chemical ionization sources for gas samples; electrospray ionization
sources, an atmospheric pressure chemical ionization source, and an atmospheric pressure
photoionization source for liquid samples; and a matrix-assisted laser desorption ionization
source for solid sample analysis.
HPLC-MS has been used widely to identify and quantitatively analyze saponins and
to assess the quality control of medicinal extracts [69,75,84,89–96]. One study has carried
out qualitative and quantitative analyses of bitterness from fresh and processed asparagus
components, specifically the Bidens saponin 1a/β-6, using a sensitive method of HPLC-
MS/MS. The study found that the single bridged saponin 5a/β was the main factor causing
the bitterness of fresh asparagus, whereas the saponins 1a/β and 2a/β were the main factors
contributing to the bitter taste of processed asparagus [97]. Another study used the multiple
reaction monitoring of positive and negative ion switching in a quantitative analysis of
15 active ingredients of Chaihu Guizhi decoction. The study in rats after oral administration
of the decoction established a simple, sensitive, and selective HPLC-electrospray tandem
Separations 2022, 9, 163 15 of 30
MS approach [98]. In another study, qualitative and quantitative analyses of the effective
components of raw and processed licorice were used to explore the in vitro metabolism of
the two decoctions in the gastrointestinal tract. The study used two HPLC methods, one
paired with a diode array detector and one paired with an electrospray mass spectrometer.
The results showed that the processing of licorice could change the content of the main
components and affect its GI metabolism (Table 4) [99].
Table 4. Application of high-performance liquid chromatography-mass spectrometry in the determi-

nation of medicinal materials and their preparations containing saponin.
Qualitative/ Chromatographic
Name Analytical Method Test Results Ref.
Quantitative Conditions
stationary phase: Alorich 12 diosgenin:Dioscin, Gracillin,
Ascentis C8 column Deltonin, Trillin, Prosapogenin A,
(10 cm × 4.6 mm, 3 µm), zingiberensis New Saponin,
Polygonti Qualitative UHPLC-Q-Exactive
mobile phases: water (A) Protodioscin, Protogracillin, [84]
Rhizome and quantitative Orbitrap HRMS
and acetonitrile (B), Protodeltonin, Pseudoprotodioscin,
column temperature: Pseudoprotogracillin,
25 ◦ C Methyl protodioscin
stationary phase: Gemini
C18 column
(150 × 4.6 mm, 5 µm),
Qualitative SPE-HPLC-MALDI-
Soybeans mobile phase: water soyasaponins I and βg [95]
and quantitative TOF-MS
(0.25% acetic acid) (A)
and methanol
(0.25% acetic acid) (B)
stationary phase:Inertsil
PREP-ODS column chikuset-susaponins IVa and V,
(20 × 250 mm), mobile achyranthosides B, C, D, E and G,
Achyranthes Quantitative LC-MS [83]
phase: UPW (5 mM sulfachyranthosides B and D, and
DHAA) (A) and MeCN betavulgarosides II and IV
(5 mM DHAA) (B)
12 steroidal saponins: Dichotomin,
Protosaponin3Glc-Rha-Ara,
stationary phase: Methyldichotomin,
Kromasil RP-C18 column Methyl protodioscin,
Paris (4.6 mm × 250 mm, Methylprotosaponin2Glc-2Rha-Ara,
Qualitative HPLC-ESI (+/−)-MSn [69]
and Trillium 5 µm), mobile phase: Diosgenin 2Glc-3Rha, PolyPhyllin H,
water (A) and Methylprotogracillin,
acetonitrile (B) Diosgenin2Glc-Rha-Ara,
PennogeninGlc-2Rha, Pennogenin
2Glc-Rha, Diosgenin2Ara-Rha-Glc
stationary phase:
Tigerkin C18 column,
three steroidal saponins:
Ophiopogon mobile phase: water
Quantitative HPLC-MS cixi-ophiopogon A, cixi-ophiopogon [81]
japonicus (0.02% formic acid ) (A)
B, cixi-ophiopogon C
and acetonitrile
stationary phase: Agilent
Zorbax SB-C18 column
anionic adducts-based
(100 × 3.0 mm, 3.0 µm),
liquid chromatography saikosaponin a, saikosaponin c,
Chaihu Quantitative mobile phase: water [94]
tandem mass saikosaponin d and saikosaponin b2
(0.06% formic acid) (A),
spectrometry method
acetonitrile (B) and
methanol (C)
stationary phase: Halo® 15 active compounds: Saikosaponin A,
C18 column Baicalin, Wogonin, Glycyrrhizic acid,
(2.1 × 100 mm, 2.7 µm), Glycyrrhetinic acid, Albiflorin,
Chaihu-Guizhi
Quantitative HPLC-ESI- MS/MS mobile phase: water Paeoniflorin, Liquiritin, Isoliquiritin, [98]
decoction
(0.1% formic acid) (A) Liquiritigenin, Isoliquiritigenin,
and acetonitrile (B), flow Cinnamic acid, Gallic acid,
rate: 0.3 mL/min Wogonoside and Oroxylin A
Separations 2022, 9, 163 16 of 30
Table 4. Cont.
24 saponins, 3 xanthones, 1
anthraquinone and 2 alkaloids:
Neomangiferin, Mangiferin,
Isomangiferin, Timosaponin B-V,
Timosaponin B-VI, Timosaponin H1,
stationary phase: Zorbax Timosaponin I1, Timosaponin B-IV,
XDB-C18 analytical Timosaponin I2; Timosaponin H2,
high-performance liquid
column (2.1 × 50 mm, Neohyacinthoside, Timosaponin E1,
Zhimu-Baihe chromatography and
Qualitative 1.8 µm), mobile phase: Timosaponin E, Timosaponin N, [100]
herb-pair time-of-flight
water (0.1% formic acid) Timosaponin E2, Macrostemonoside
mass spectrometry
(A) and acetonitrile (B), K, Timosaponin B-II, Timosaponin D,
flow rate: 0.2 mL/min Timosaponin B-I, Timosaponin B-III,
Brownioside 1, Brownioside 2,
Timosaponin F, Anemarrhenasaponin
I, Anemarrhenasaponin Ia,
Timosaponin G, Timosaponin AIII,
Timosaponin A-I, Colchicine, Emodin
stationary phase: Gemini
C18 column
Radix (4.6 mm × 250 mm,
Qualitative HPLC-Q- TOF/MS 22 types of astragaloside IV [90]
Astragali 5 µm), mobile phase:
(A) and ACN (B)
8 steroidal saponins: ophiogenin
3-O-α-L-rhanose-(1→2)-β-D-xylose-
(1→3)-β-D-glucose-(1→4)-β-D-
glucose, ophiogenin
3-O-α-L-rhanose-(1→2)-β-D-glucose-
stationary phase: β-D-glucose, ophiogenin
Tigerkin C-18 column 3-O-α-L-rhanose-(1→2)-β-D-xylose-β-
(4.6 × 250 mm, 5.0 µm), D-glucose, pennogenin
mobile phase: water 3-O-α-L-rhanose-(1→2)-β-D-xylose-
Ophiopogon High-Performance Liquid
(0.05% formic acid) (A) (1→3)-β-D-glucose, ruscogenin
Japonicus Qualitative Chromatography with Ion [91]
and acetonitrile 3-O-α-L-rhanose-(1→2)-β-D-xylose-
Ker-Gawler Trap Mass Spectrometry
(0.05% formic acid) (B), (1→3)-α-L-araβinose, ruscogenin
flow rate: 0.5 mL/min, 3-O-α-L-rhanose-(1→2)-β-D-xylose-
detection wavelength: (1→3)-β-D-fucose, pennogenin
203 nm 3-O-α-L-Rha-(1→2)-O-β-D-Xyl-
(1→3)-O-β-D-Xyl-(1→4)-O-β-D-Glc,
pennogenin 3-O-α-L-Rha-(1→2)-O-β-
D-Glc-(1→3)-O-β-D-Glc or ruscogenin
3-O-α-L-Rha-(1→2)
-O-β-D-Glc-(1→3)-O-β-D-Glc
19 oleic acid alkanestype triterpene
saponins: uralsaponin C, uralsaponin
stationary phase: Agilent D, uralsaponin F, uralsaponin E,
rapid-resolution liquid
ZorBax SB-C18 column 24-hydroxyl-licorice E2 ,
chromatography with
Glycyrrhiza (4.6 × 50 mm, 1.8 µm), licorice-saponin A3 ,
Qualitative time-of-flight mass [93]
uralensis mobile phase: water 22-acetoxyl-glycyrrhizin,
spectrometry
(0.2% formic acid) (A) licorice-saponin E2 ,
(RRLC/TOF-MS)
and acetonitrile (B) 22-acetoxyl-Glycyrrhaldehyde,
licorice-saponin G2 , glycyrrhizin,
18a-glycyrrhizin and uralsaponin B
stationary phase: Zorbax
XDB-C18 column Albiflorin, oxypaeoniflorin,
Shaoyao- (2.1 mm × 50 mm, paeoniflorin, liquiritin, isoliquiritin,
Gancao- Quantitative HPLC–MS/MS 3.5 µm), mobile phase: liquiritigenin, isoliquiritigenin, [101]
Decoction water (0.1% formic acid) ononin, glycyrrhizin
(A) and methanol andglycyrrhetinic acid
Separations 2022, 9, 163 17 of 30
Table 4. Cont.
stationary phase:
YMC-Pack
ODS-A column
(4.6 mm × 250 mm,
Glycyrrhiza 5 µm), mobile phase: glyyunnansapogenin I, yunganosides
Qualitative HPLC–MS/MS [92]
yunnanensis acetonitrile (A) and water E3 , L, M, N1 ,O, P and N2
(0.1% formic acid)
(B), column temperature:
35 °C, flow rate:
1 mL/min
stationary phase: RP-18e
high-performance liquid monolithic column six steroid saponins: HSY-14, HSY-10,
Dioscorea
chromatography- (50 mm × 2 mm), mobile dioscin (DS), gracillin (GC),
panthaica Prain Quantitative [82]
electrospray tandem phase: acetonitrile (A) pseudoprotodioscin (PDD),
et Burk
mass spectrometry and water (0.1% formic pseudoprotogracillin (PDG)
acid) (B)
Eclipse Plus C18 column
Ultra fast liquid
(100 mm × 2.1 mm,
chromatography-
Qualitative 1.8 µm), mobile phase: 13,28-epoxy-oleanane-type
Ardisia Crenata electrospray quadrupole [96]
and quantitative water (0.1% formic acid) triterpenoid saponins
mass
(A) and acetonitrile
spectrometry (UFLC-MS)
(0.1% formic acid) (B),
flow rate: 0.2 mL/min
stationary phase: Kinetex
XB-C18 column
Acanthopanax (100 mm × 4.6 mm,
Qualitative HPLC-ESI-TOF-MS 15 triterpenoid saponins [75]
henryi 2.6 µm), mobile phase:
acetonitrile (A) and
water (B)
stationary phase: agilent
Eclipse XDB-C18 column
(250 mm × 4.6 mm, 5 µm)
Panax mobile phase: water
Qualitative HPLC-QTOF/MS 234 ginsenosides [89]
notoginseng (0.1% formic acid) (A)
and acetonitrile
(0.1% formic acid) (B),
flow rate: 0.8 mL/min
stationary phase:
reversephase analytical
column (25 cm × 4.6 mm,
Triguero Qualitative saponins (HTSAP1 to HTSAP8) and
HPLC-MS 5 µm), mobile phase: [102]
asparagus and quantitative protodioscin
(A) and acetonitrile
Eclipse XDB-C18 column
fresh and (150 × 2.1 mm, 5 µm),
Qualitative and the monodesmosidic saponins 5a/b,
cooked HPLC-MS/MS mobile phase: acetonitrile [97]
quantitative bidesmosides 1a/b and 2a/b
white asparagus (0.1% formic acid) (A)
and water (0.1% formic
acid) (B)
stationary phase:
eleven constituents: liquiritin
Kromasil 100-5 C18
apioside, liquiritin, licuraside,
crude column (4.6 × 250 mm,
isoliquiritin, ononin, glycyrrhizin,
Glycyrrhizae radix 5 µm), mobile phase:
Qualitative HPLC-ESI/MS liquiritigenin-7,4’-diglucoside, [99]
and processed water (0.1% formic acid)
licorice saponin A3 ,
Glycyrrhizae radix (A) and acetonitrile (B),
22β-acetoxylglycyrrhizic acid, licorice
detection wavelength:
saponin G2 , and yunganoside E2
254 nm
Separations 2022, 9, 163 18 of 30
3.5. UPLC
In 1996, the Waters Corporation launched Alliance HPLC. With the progress of science
and technology and the development of industry, the requirements for LC in various
fields are increasing day by day. In 2004, the Waters Corporation launched the world’s
first ultra-high-performance liquid chromatograph, Acquity UPLC, which uses a small-
particle-packed column (less than 2 µm) and an ultra-high pressure system (more than
105 kpa) and is suitable for the separation of trace complex mixtures and high-throughput
research [103]. Compared with the HPLC system, UPLC can significantly improve the
separation degree of the chromatographic peaks and the detection sensitivity and, at the
same time, greatly shorten the analysis time and reduce the solvent consumption. However,
there are some limitations to UPLC, such as the lengthy sample pretreatment time and the
system’s ultra-high pressure resulting from the small particle size packing. Because of these
limitations, higher requirements are placed on the sealing of the instrument, the injection
of the injection valve, the infusion of the pump, and the performance of the detector.
UPLC and its combination technology have developed rapidly, especially with MS.
Examples of its use include analysis of components of traditional Chinese medicine (iden-
tification of traditional Chinese medicine components [88], determination of the com-
ponents’ content [104,105], fingerprint study of traditional Chinese medicine [106]) and
metabonomics. In one study, UPLC-quadrupole time-of-flight (Q-TOF)-MS was used to
qualitatively and quantitatively analyze the chemical structure of the main saponins in
quinoa seeds and assessed the contents of two quinolone saponin components, FQ70 and
FQ90. The study found that both quinolone saponin components significantly improved
the humoral and cellular immune responses to ovalbumin (OVA) in mice, with obvious
immune adjuvant properties [107]. In another study, the sources of 12 kinds of ginseng
were assessed to analyze the development trend of ginseng varieties; assessments were
completed with fast and accurate UPLC-tandem MS [108]. Another study identified and
determined the content of six steroidal glycosides and one aglycone in pangolin and yam
using the efficient and reliable UPLC-Q-TOF-MS. Then, the researchers compared the
chemical composition of pangolin and yam by chromatographic fingerprint similarity
evaluation, using a significance test (t test) and PCA. The study results demonstrated that
the chemical composition of all samples of pangolin and yam showed a high degree of
overall similarity [109]. An additional study on the distribution and quantitative analysis
of the main active saponins in different tissues of Panax notoginseng (cork, cortex, phloem,
and xylem) used a simple, sensitive, and accurate UPLC-Q-TOF-MS combined with a
fluorescence microscope and laser microdissection technology. The research revealed the
distribution of the main saponins of Panax notoginseng in tissues [110].
For UPLC techniques, the differentiation among the type of detector and ionization
could be better described, and so this technique could be used in the rapid separation,
structure identification, and content determination of several saponins in complex natural
products. In one study, astragalus was used as an example to explore simple, economic,
and effective quality control methods. The astragaloside content was determined by UPLC,
and the fingerprints of astragaloside and total flavonoids of astragalus were established
by full scan mode, which met the requirements of product quality supervision in the
production process [111]. In addition, the contents of 25 compounds in different parts (roots,
rhizomes, stems, leaves, and flowers) of two species of astragalus have been analyzed and
compared in another study [112], and 14 main chemical components (five flavonoids and
nine triterpenoid saponins) in 94 batches of astragalus from different places (China, Korea,
and Germany) had been determined simultaneously [113]. Astragaloside III was not only an
important chemical marker for the identification of astragalus and membranous astragalus,
but it was also a potential chemical marker for the classification of cultivated astragalus and
semi-wild astragalus, as determined with UPLC. Many studies focus on the identification
and quantitative analysis of triterpenoid saponins in Glycyrrhiza plants by UPLC-MS
with the simultaneous determination and rapid screening of several effective components,
including saponins and flavonoids in licorice [114–117]. Some studies determined the
Separations 2022, 9, 163 19 of 30
eight triterpenoid saponins in dog plasma after oral administration of total saponins in
Glycyrrhiza [118], and another performed preliminary identification of the active ingredients
of licorice in Wutou decoction [119]. Another study applied a UPLC-MS method based
on a standard addition to quantitative analysis of 14 compounds in Glycyrrhiza. Using
this method, G. glabra, G. uralensis and G. inflata in a variety of forms, including root
powders and extracts, as well as complex dietary supplements, could be differentiated and
chemically standardized [120].
3.6. QAMS
QAMS was first proposed by Zhimin Wang et al. in 2006 [121]. By studying the
internal functional proportional relationship between the active ingredients of traditional
Chinese medicine and introducing a relative correction factor on the basis of the internal
standard method, this study achieved for the first time the simultaneous determination
of the contents of multiple components tested from traditional Chinese medicine and the
preparations with a reference substance. The basic research supporting this method relies
on the principle that the amount of component (mass or concentration) in a certain linear
range is proportional to the response of the detector, which is represented by W = f × A. In
the multi-index quality evaluation, a representative component of the medicinal material
(the stable and easily obtained reference substance) is used as an internal reference, and
the relative correction factor (RCF) between this internal reference and other components
is established, without providing reference products of other components, by the RCF to
calculate the number of other components.
Suppose a sample contains i components, and fi = [(Wi ) ÷ (Ai )] (i = 1, 2...k,...m),
in which Wi is the component concentration and Ai is the component peak area. Se-
lect one of the components k as the internal reference, and establish the RCF between
the component k and the other components m. The quantitative calculation formula
Wm = [(Wk ÷ fkm ) × (Am ÷ Ak )], in which fkm is the RCF of the internal reference and other
components to be measured and fk and fm are the absolute correction factors of the internal
reference substance and other components to be measured, respectively. Ak and Am are the
peak areas of the internal reference and other components to be tested, respectively, and
Wk and Wm are the concentration (or mass) of the internal reference and other components
to be tested, respectively [87]. This method is suitable for the simultaneous qualitative
and quantitative determination of the same kind of multiple components when a reference
substance is rare [122] and preparation cost is high [123].
QAMS uses a single index to quantitatively analyze multiple components [124,125];
this approach not only reduces the analysis time and cost but also improves the analysis
efficiency to provide a more comprehensive quality evaluation of medicinal materials
and prescription preparations. One study established a QAMS method for the determina-
tion of the active components of treatments for rheumatoid arthritis (using astragaloside
IV as the internal standard). There was no significant difference between the content of
active components in Astragalus membranaceus and the content determined by the ex-
ternal standard method (RSD < 0.05), and the RCF established had good reliability [106].
The latest research evaluated and discussed the volatility and stability of RCFs by using
19 kinds of ginsenosides as reference standards under different MS conditions (different
HPLC-MS instruments and different de-aggregation potentials). This study found that
the RCF had enough reproducibility under a wide range of changes to verify the ratio-
nality of simultaneous determination of 19 ginsenosides using a single test and multiple
evaluation methods [126]. Another study used a relative response factor method and
a UV-MS gradient elution method to determine the content of ginsenosides in ginseng
extract and ginseng products. Compared with the external standard method, the QAMS
method that was based on the relative response factor has a smaller difference [125]. In
addition, another study established a QAMS method for the simultaneous determination of
11 saponins in Panax notoginseng and identification of the effects of the chemical structure
of the internal standard, the concentration of the quantitative components, and the purity
[125]. In addition, another study established a QAMS method for the simultaneo
termination of 11 saponins in Panax notoginseng and identification of the effects
chemical structure of the internal standard, the concentration of the quantitativ
ponents, and the purity of the reference substance. The study assessed the accuracy
Separations 2022, 9, 163 20 of 30
QAMS method and showed that the concentration of the analyte in the sample s
was the main parameter affecting the accuracy of the QAMS method. By calculati
controlling the applicable concentration range of the analyte in the sample, the h
of the reference substance. The study assessed the accuracy of the QAMS method and
curacy
showed ofthatthe
theQAMS method
concentration of thewas ensured
analyte [127]. solution was the main parameter
in the sample
affecting the accuracy of the QAMS method. By calculating and controlling the applicable
concentration
3.7. Immunoassayrange of the analyte in the sample, the high accuracy of the QAMS method
was ensured [127].
The immunological analysis method has high sensitivity and specificity, wh
be
3.7.used to analyze saponins. One study established a time-resolved fluorescen
Immunoassay
munoassay system toanalysis
The immunological determine
methodthe has content of saikosaponin
high sensitivity and specificity,awhich
(SSa)caninbe10 com
samples of bupleurum. The bupleurum methanol extract and a immunoas-
used to analyze saponins. One study established a time-resolved fluorescence mouse anti-SSa
say system to determine the content of saikosaponin a (SSa)3+in 10 commercial samples
clonal antibody were used as materials, and the Eu -labeled SSa-human serum a
of bupleurum. The bupleurum methanol extract and a mouse anti-SSa monoclonal anti-
conjugate wasasused
body were used as a and
materials, tracer (Figure
the Eu 9). SSa-human
3+ -labeled This technology had the
serum albumin advantages
conjugate
sensitivity,
was used as aconvenience,
tracer (Figure and speed
9). This [128]. had the advantages of high sensitivity,
technology
convenience, and speed [128].
Figure
Figure 9.9.Schematic
Schematic diagram
diagram of theof the TRFIA
TRFIA system
system for for SSa[128].
SSa detection detection [128].
3.8. Metabolomics
Metabolomics is a new discipline developed in the mid-1990s to analyze all low-
molecular-weight metabolites of a certain organism or cell qualitatively and quantitatively.
Its core approach is to take the physiological and pathological process of the human
body as a dynamic system and study the types, quantity, and changes in endogenous
metabolites after the organism is disturbed by internal and external environmental factors.
Metabolomics can be divided into nontargeted and targeted metabolomics according to
different research purposes. Nontargeted metabolomics is a systematic and comprehensive
analysis of endogenous metabolites, whereas targeted metabolomics is the analysis of
specific metabolites. Targeted metabolomics is accurate in qualitative and quantitative
analyses, but its coverage of substances is limited. Although the coverage of nontargeted
metabolomics across substances is extensive, this approach lacks absolute qualitative and
quantitative data (Figure 10).
tatively. Its core approach is to take the physiological and pathological process of the
human body as a dynamic system and study the types, quantity, and changes in endog-
enous metabolites after the organism is disturbed by internal and external environmental
factors. Metabolomics can be divided into nontargeted and targeted metabolomics ac-
cording to different research purposes. Nontargeted metabolomics is a systematic and
comprehensive analysis of endogenous metabolites, whereas targeted metabolomics is
Separations 2022, 9, 163 the analysis of specific metabolites. Targeted metabolomics is accurate in qualitative and21 of 30
quantitative analyses, but its coverage of substances is limited. Although the coverage of
nontargeted metabolomics across substances is extensive, this approach lacks absolute
qualitative and quantitative data (Figure 10).
Figure 10. The targetedFigure

and untargeted
10. The targeted workflow for
and untargeted LC/MS-based
workflow metabolomics
for LC/MS-based [129].
metabolomics [129].
In the analysis of saponins, metabolomics techniques are mainly combined with MS,
In the analysis ofliquid
saponins, metabolomics
chromatography, techniques
gas chromatography, are mainly
and NMR combined
spectroscopy. with MS,
NMR spectros-
liquid chromatography, copygas chromatography,
provides and
valuable details about NMRcharacteristics
metabolite spectroscopy. NMR
of complex spectroscopy
plant extracts
and has the advantages of fast detection speed, easy absolute quantification, and clear
provides valuable details about metabolite characteristics of complex plant extracts and has
compound identification; these positive attributes make the method an appropriate
the advantages of fasttechnique
detection speed,large
for analyzing easyamounts
absolute quantification,
of metabolites. and clear
High-performance TLC compound
has the
advantages of
identification; these positive universal availability
attributes make of derivatization
the method reagents and a short running
an appropriate time.
technique for
In addition, the simplicity of preparing HPTLC provides great potential for enhancing
analyzing large amounts of metabolites. High-performance TLC has the advantages
metabolomic studies of saponin-rich medicinal materials. The combination of UPLC and of
universal availabilityelectrospray
of derivatization reagentscanand
ionization-Q-TOF-MS a short
shorten running
the analysis time. accurate
time, provide In addition,
qual- the
ity measurements and more molecular formula information, and facilitate the analysis of
simplicity of preparing HPTLC provides great potential for enhancing metabolomic studies
known compounds and the interpretation of unknown compounds in complex matrixes.
of saponin-rich medicinal materials.
Therefore, in recentThe combination
years, the combination of UPLC
of UPLC and electrospray
with ionization-
electrospray ioniza-
Q-TOF-MS can shorten tion-Q-TOF-MS
the analysis has time,
becomeprovide
the preferred technology
accurate for the analysis
quality and identification
measurements and more
of chemical components and metabolites in vivo after oral administration. Compared
molecular formula information, and facilitate the analysis of known compounds
with LC-MS, gas chromatography-MS has better peak resolution and sensitivity. How- and the
interpretation of unknown
ever，thiscompounds
technology alsoinhas
complex matrixes.such
some disadvantages, Therefore,
as relativelyinlowrecent years, the
signal repro-
combination of UPLCducibility and measurement of volatile compounds, and thus gas chromatography is not
with electrospray ionization-Q-TOF-MS has become the preferred
suitable for the determination of saponins.
technology for the analysis and identification of chemical components and metabolites
in vivo after oral administration. Compared with LC-MS, gas chromatography-MS has bet-
ter peak resolution and sensitivity. However, this technology also has some disadvantages,
such as relatively low signal reproducibility and measurement of volatile compounds, and
thus gas chromatography is not suitable for the determination of saponins.
The metabolic spectrum analysis technology can help researchers understand the
biochemical composition of an organism in more detail. Many medicinal plants use
saponins as their main metabolites, and the changes in metabolic components are sig-
nificantly related to genetics (per species or in varieties within species) and environmental
factors (geographic location and planting time). One study revealed the composition
differences of primary and secondary metabolites in Glycyrrhiza through the combina-
tion of NMR and MS technologies combined with multivariable data analysis. It was
found that the glycoside conjugates of glycyrrhizic glycoside, 4-hydroxyphenylacetic
acid, and glycyrrhizin/isoglycyrrhizin are the main spectral peaks to distinguish species
in the 1 H NMR and MS spectra [130]. In another study, a fast and sensitive HPLC-
electrospray ionization-tandem MS was used to identify the structure of a novel composite
bellflower glycoside metabolite transformed by human intestinal bacteria. The study
showed that under chromatographic conditions, eleven main peaks were detected in the
metabolites of Platycodon grandiflorum. Through the comparison of spectra in positive
and negative ion modes, clear information about the molecular weight of metabolites
was found [131]. Another study identified and studied the whole metabolic process of
Ophiopogon japonicus roots at different ages (1 to 3 years old) collected from two producing
areas (Zhejiang Province and Sichuan Province, China) by coupling 1 H NMR and high-
performance TLC. It was found that Ophiopogon saponin, Ophiopogon saponin C and
Ophiopogon saponin D were the marker metabolites in Ophiopogon japonicus roots [132]. In
view of the significant differences in chemical components in different parts of
Separations 2022, 9, 163 22 of 30
Panax notoginseng, one study carried out quantitative and qualitative analyses and a com-
parison of different parts of Panax notoginseng (rhizome, main root, lateral root, and fibrous
root) in its main production area of Wenshan City, Yunnan Province, through the proven
UPLC-Q-TOF-MS method and nontargeted metabolomics. The study showed significant
differences between rhizome and other parts, and it identified the content of monomer
saponins and total saponins as the highest in the rhizome. The results showed that this
part was suitable to use as the raw material for ginsenoside products [133]. A study
used UPLC-Q-TOF-MS metabolomics to identify and quantitatively analyze the different
chemical constituents of the roots, stems, leaves, and seeds in Polygala. A total of 22 mark-
ers were detected, and seven triterpene saponins were significantly different in different
tissues [134].
4. Discussion
Traditional extraction and separation techniques, such as thermal reflux extraction,
Soxhlet extraction and liquid chromatography, have many shortcomings, such as long pre-
treatment time, cumbersome operation steps, and large consumption of organic solvents.
At present, both ionic liquids and supercritical fluid chromatography (SFC) are green chro-
matographic extraction and separation technologies, which show great potential to replace
traditional organic solvents in many fields. High-speed countercurrent chromatography
(HSCCC) is an all-liquid-partition chromatography method that eliminates the irreversible
adsorption loss of samples on solid support matrix columns and has been widely used for
the separation of saponins from natural products due to its superior separation ability.
Currently, the main analytical methods of saponins are thin-layer chromatography,
CE, NIRS, HPLC, UPLC, QAMS, immunoassay, and metabonomics. HPLC has been
combined with a variety of detectors, such as UV/diode array detector, ELSD, CAD, MS,
and chromatographic fingerprint.
These analytical methods have their own advantages and disadvantages; for example,
spectrophotometry is a simple and reliable operation but can only provide the content
of total saponins. TLC has the advantages of simple operation, strong separation ability,
low cost, and fast detection speed, so it can be used for the analysis of several saponins.
CE is an effective analytical technique with a short analysis time, high resolution, small
sample size needs, and high selectivity, but it has disadvantages of low sensitivity and
poor reproducibility. NIRS has many significant advantages in the analysis of saponins in
natural products, including its simple use, fast analysis speed, lack of damage to samples,
lack of chemical pollution, and more. The immunoassay approach has high sensitivity
and specificity, which can rapidly determine saponin content to support quality control
assessments of drugs and their preparations; this method also contributes to the exploration
of mechanisms of action in saponin-rich drugs and helps identify the active substances.
However, the disadvantage of metabolomics is the need for a large number of samples.
This analysis also must be combined with other analytical instruments. A single-test
multi-evaluation method comprehensively evaluates the quality of medicinal materials on
the basis of several indexes. It is not only easy to operate but also can reduce the cost of
detection, so it has been widely used. UPLC has many advantages, such as a fast analysis
speed, high resolution, and less solvent consumption. However, because the particle size of
the packing in the UPLC column is small, the sample must be pretreated carefully. UPLC-
electrospray ionization-tandem MS has the advantages of high sensitivity, high resolution,
and high-quality measurement accuracy. It is a powerful tool to comprehensively deter-
mine a variety of saponins in complex Chinese medicinal materials, but it has numerous
requirements regarding the types and acquisition of reference substances.
HPLC combined with a variety of detectors also has obvious advantages and inevitable
limitations. For example, a UV detector has the advantages of a wide application range,
high repeatability, wide linear range, and compatibility with the gradient elution. Because
UV detection is limited to analytes with suitable chromophores, some problems occur in the
determination of saponins—namely, low sensitivity and low accuracy—so the utilization
Separations 2022, 9, 163 23 of 30
rate is gradually reduced. As a general detection method of saponins, ELSD overcomes the
difficulty of determination and avoids the interference of a terminal absorption wavelength.
Even when it is used in gradient elution analysis of nonchromophores and nonvolatile
compounds, ELSD has a stable baseline and has been successfully applied in saponin
extraction and quantitative analysis. However, ELSD has the disadvantages of its complex
sample pretreatment, narrow linear range, low sensitivity, and inability to quantitatively
determine tracesaponins. CAD is a new detection technology, which is suitable for the
analysis of weak or non-UV absorption compounds. It has the advantages of a suitable
gradient elution, a stable baseline, high sensitivity, and simple operation. The chromato-
graphic fingerprint can comprehensively reflect the complex components of traditional
Chinese medicine, and this method effectively assesses the quality control of traditional
Chinese medicines. The ion trap mass spectrometer can be used for multi-stage tandem
MS to provide chemical structure information. Q-TOF-MS has the advantages of accurate
quality information and high sensitivity, but it also has the limitation of great expense.
The combination of multiple analytical methods to achieve high sensitivity, high
selectivity, and high accuracy for simultaneous qualitative and quantitative analyses of
multiple saponins in medicinal materials and their preparations has become a trend. HPLC
and MS detectors combine the rapid separation ability of LC with the high sensitivity,
high specificity, and good selectivity of MS detectors. This combination can produce more
accurate and specific analysis results. Therefore, HPLC-MS has become the preferred
method for the rapid determination of complex saponins in medicinal materials. As
a new, efficient, and low-cost method for evaluating the overall quality of traditional
Chinese medicine, QAMS can effectively overcome the difficulties in preparing reference
materials with complex structures and the instability of saponins under acidic conditions.
These obvious advantages will increase the role of QAMS in saponin analysis, and QAMS
may become a powerful tool for assessing the quality control of saponin compounds.
Conversely, the combination of metabolomics and gene expression analysis has become a
hot research topic. The combination of these two analyses can clarify the mechanism of
saponin biosynthesis, find key enzyme genes, improve the 25yield
Separations 2022, 9, x FOR PEER REVIEW of 31 of saponin-rich medicinal
materials by controlling gene expression, and guide the cultivation of excellent plant
varieties through the best aspects of biosynthesis to enhance the development and use of
excellent plant varieties through the best aspects of biosynthesis to enhance the devel-
saponin components (Figure 11).
opment and use of saponin components (Figure 11).
Figure 11. Characteristic diagram of the saponin analysis method.

Figure 11. Characteristic diagram of the saponin analysis method.
5. Conclusions
In conclusion, this article provided a systematic and comprehensive review of
methods for the separation and analysis of saponins over the past 10 years. The collected
data provide the latest valuable insights and references for separation, quality control
and for continued development and application of saponins.
Author Contributions: Reviewing the literature, collating documents, discussing the layout, writ-
ing the manuscript and finalizing the paper, Y.W.; finishing the artworks (figures and tables), and
Separations 2022, 9, 163 24 of 30
5. Conclusions
In conclusion, this article provided a systematic and comprehensive review of methods
for the separation and analysis of saponins over the past 10 years. The collected data provide
the latest valuable insights and references for separation, quality control and for continued
development and application of saponins.
Author Contributions: Reviewing the literature, collating documents, discussing the layout, writing
the manuscript and finalizing the paper, Y.W.; finishing the artworks (figures and tables), and
finalizing the paper, Y.M.; retrieving the relevant literature, discussing the layout, L.T., X.Z., F.H. and
S.Z.; designing this manuscript, L.H. and C.B. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by Class A: “Western Light” and “Western Young Scholars” of the
Chinese Academy of Sciences in 2019 [grant number 2009A-6], Ningxia Natural Science Foundation
[grant number 2020 A0564], Ningxia Natural Science Foundation [grant number 2020 A0450].
Conflicts of Interest: The authors confirm that this article content has no conflict of interest.
Abbreviations
ILs ionic liquids

SFC supercritical fluid chromatography
UAE ultrasonic-assisted extraction
MAE microwave-assisted extraction
HSCCC high-speed counter-current chromatography
DAD diode array detector
MSD mass spectrometry detector
ELSD evaporative light-scattering detector
CAD charged aerosol detector
QAMS quantitative analysis of multi-components by single-marker
NIRS near-infrared spectroscopy
TLC thin-layer chromatography
CE capillary electrophoresis
HPLC high-performance liquid chromatography
UPLC ultra-high-performance liquid chromatography
UV/DAD ultraviolet/diode array detection
SMD standard method difference
PLS partial least square
PCA principal component analysis
RSD relative standard deviation
CG calycosin-7-o-β-d-glucoside
NMR nuclear magnetic resonance
HPTLC high-performance thin-layer chromatography
IT ion-trap
PR polygala radi
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Separations 2022, 9, 163. https://doi.org/10.3390/separations9070163 https://www.mdpi.com/journal/separations

Separations 2022, 9, x FOR PEER REVIEW 3 of 31

No. Drug Name Medication Site Main Ingredient Efficacy Ref.

No. Drug Name Medication Site Main Ingredient Efficacy Ref.

No. Drug Name Medication Site Main Ingredient Efficacy Ref.

No. Drug Name Content Determination

2. Extraction and Separation Methods

2.1. Extraction of Saponins

2.2. Isolation of Saponins

2.2.3. Foam Fractionation

ester). In this study, β-cyclodextrin was selected as a stereoselective reagent, underwent

qualitatively or quantitatively analyze multiple saponins in complex medicinal materials

3.4.4. Chromatographic Fingerprint

Detection Compound Stationary Phase Mobile Phase The Detector Ref.

Detection Compound Stationary Phase Mobile Phase The Detector Ref.

3.4.5. HPLC-MS Detector

Table 4. Application of high-performance liquid chromatography-mass spectrometry in the determi-

Figure 10. The targetedFigure

Figure 11. Characteristic diagram of the saponin analysis method.

ILs ionic liquids

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