Recent Advances in Separation and Analysis of Saponins in Natural Products

: 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 Direct, 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 different detectors to achieve qualitative or quantitative analysis and quality control of saponin compounds in medicinal materials and their preparations. This paper provides the latest valuable insights and references for the analytical methods and continued development and application of saponins. relatively of


Introduction
Saponin is a kind of natural secondary metabolite, which is composed of sapogenin and a sugar chain [1]. They can be divided into triterpenoid or steroidal types according to the sapogenin differences: The triterpenoid saponin's sapogenin is triterpene, whereas the steroidal saponin's is spirostane.
The sapogenin of triterpenoid saponins is a triterpenoid derivative composed of 30 carbon atoms and a basic skeleton of six isoprene units. Again, distinguished by sapogenins differences, the triterpenoid saponins are divided into tetracyclic and pentacyclic triterpenoids. 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 and have the basic skeleton of spirostane. Steroidal saponins are divided into spirostanol, isospirostanol, furostanol, and deformed spirostanol types. Saponins are widely distributed in nature and are commonly found in roots, stems, leaves, flowers, and seeds of terrestrial higher plants. Triterpenoid saponins are mainly distributed in the Leguminosae, Araliaceae, Umbelliferae, Compositae, Polygalaceae, and other plant families. Steroidal saponins are mostly found in monocotyledons, such as Dioscoreaceae, Liliaceae, and Scrophulariaceae plant families. Saponins represent a main effective component in many kinds of Chinese However, most saponins have a similar chemical structure and have no UV absorption 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 quality, reduced biological activity, and limited clinical applications. Given the determination challenges, researchers around the globe have proposed different analytical methods for saponins (Figures 1-3). These methods include spectrophotometry, thin-layer chromatography (TLC), capillary electrophoresis (CE), infrared spectroscopy, high-performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC), quantitative analysis of multicomponents by single marker (QAMS), immunoassay, and metabolomics [17].

Roots and rhizomes, leaves, flowers
Dammar type tetracyclic triterpene saponins, flavonoids, notoginsenosides (amino acids), proteins, volatile oils, acety-Diffuse stasis to stop bleeding, reduce swelling and relieve pain [15,20,21]  However, most saponins have a similar chemical structure and have no UV absorption 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 quality, reduced biological activity, and limited clinical applications. Given the determination challenges, researchers around the globe have proposed different analytical methods for saponins (Figures 1-3). These methods include spectrophotometry, thinlayer chromatography (TLC), capillary electrophoresis (CE), infrared spectroscopy, highperformance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC), quantitative analysis of multicomponents by single marker (QAMS), immunoassay, and metabolomics [17].
In recent years, as significant pharmacological action of saponins has become well known and their general application in various fields has increased, the quality control of saponins has become a global concern (Table 1). Therefore, based on the qualitative and quantitative perspectives, this article discusses the problems and future development trends of these analytical methods by reviewing the literature of the past ten years and provides a valuable reference for choosing appropriate analytical methods to control the quality of saponins (Table 2). It can strengthen the vitality, strengthen the body, nourish blood and blood, nourish the spleen and benefit the lungs, calm the heart, calm the mind and promote wisdom [14,18,19]

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.

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 Separations 2022, 9, 163 6 of 30 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 (K 2 HPO 4 ). 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].

UAE
There are currently some auxiliary techniques combined with ILs, such as microwaveassisted extraction (MAE), ultrasonic-assisted extraction (UAE), which show significant improvements in the field of extraction and separation. Ionic liquid-based ultrasonicassisted 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].

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.

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 chromatography 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].

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 preparative 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 Platycodon 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 four triterpenoid saponins (hederasaponin B, hederacolchiside E, cernuoside 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 chromatography (HSCCC) [46]. Separations 2022, 9, x FOR PEER REVIEW 8 of 31 [45]. Some researchers have isolated two saponins with cytotoxicity to cancer cells from the root of A. chinensis by high-speed countercurrent chromatography (HSCCC) [46]

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

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 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].

Analytical Methods
In addition to the traditional analytical methods, such as spectrophotomet and HPLC, this paper also describes the analytical methods of QAMS, UPLC, im assay, CE, infrared spectroscopy, and metabolomics. This paper reviews the r status 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).

Analytical Methods
In addition to the traditional analytical methods, such as spectrophotometry, TLC and HPLC, this paper also describes the analytical methods of QAMS, UPLC, immunoassay, 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).

Analytical Methods
In addition to the traditional analytical methods, such as spectrophotometry, TLC and HPLC, this paper also describes the analytical methods of QAMS, UPLC, immunoassay, 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).

TLC
TLC is a commonly used method for the analysis of saponins; it has the advantages 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 identification 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.

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 chromatography (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 addition, 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 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.

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 honeyfrying of Cimicifuga foetida and established near-infrared diffuse reflectance spectroscopy 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 steroidal saponins in different species of Paris from Yunnan Province were analyzed, and a fast qualitative analysis method combining Fourier transform infrared spectroscopy and UPLC was adopted. The results showed that linear partial least square regression was more suitable than nonlinear support vector machine regression for the determination of total steroidal saponins in different species of Paris [58]. In addition, another study focused on three saponins (notoginsenoside R 1 , ginsenoside Rg 1 , and ginsenoside Rb 1 ) in Panax notoginseng and established NIRS technology as fast and simple for quantitative analysis. The results showed that this method could accurately predict the total content of three saponins in Panax notoginseng [59]. NIRS has many advantages in the analysis of saponins from natural products such as plants, including its simplicity, its fast analytical speed, its ability to not damage samples, its lack of chemical pollution, and more. Province were analyzed, and a fast qualitative analysis method combining Fourier transform infrared spectroscopy and UPLC was adopted. The results showed that linear partial least square regression was more suitable than nonlinear support vector machine regression for the determination of total steroidal saponins in different species of Paris [58]. In addition, another study focused on three saponins (notoginsenoside R1, ginsenoside Rg1, and ginsenoside Rb1) in Panax notoginseng and established NIRS technology as fast and simple for quantitative analysis. The results showed that this method could accurately predict the total content of three saponins in Panax notoginseng [59]. NIRS has many advantages in the analysis of saponins from natural products such as plants, including its simplicity, its fast analytical speed, its ability to not damage samples, its lack of chemical pollution, and more.

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, evaporative light scattering detector (ELSD), charged aerosol detector (CAD), chromatographic fingerprint, and mass spectrometer detector (MS). LC is connected with these detectors to qualitatively or quantitatively analyze multiple saponins in complex medicinal materials and their preparations.

HPLC-UV/DAD
The UV detector is the detector most commonly paired with HPLC. It has the advantages 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 information 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.

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 determine 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][68][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 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 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-tribution law of flavonoids and saponins in astragalus, with different specifications and different grades [72]. Thus, ELSD can avoid the interference from terminal absorption wavelengths of saponins and overcome the difficulty of traditional analytical methods in the determination of saponins.

HPLC-CAD
As a quality detector, the charged aerosol detector is based on the principle of an aerosol detector, the sample solution is atomized by the atomizing gas (nitrogen) in the atomizer and then hits the collision baffle at a higher flow rate to form solute particles of different sizes. The larger particles are discharged from the waste pipe under the influence of gravity, and the smaller particles flow into the drying pipe with nitrogen; at the same time, the other flow path of the inlet nitrogen passes through the corona device (containing high-voltage platinum wire electrode) to form positively charged nitrogen particles, which collide with the dried solute particles in the collision cell. The charge is then transferred to the particles-the larger the solute particle, the more charge. The solute particles transfer their charge to the collector, and the charge amount of the solute particles is measured by a highly sensitive electrostatic detector. The resulting signal current is proportional to the content of the solute (Figure 8) [73].
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 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 distribution law of flavonoids and saponins in astragalus, with different specifications and different grades [72]. Thus, ELSD can avoid the interference from terminal absorption wavelengths of saponins and overcome the difficulty of traditional analytical methods in the determination of saponins.

HPLC-CAD
As a quality detector, the charged aerosol detector is based on the principle of an aerosol detector, the sample solution is atomized by the atomizing gas (nitrogen) in the atomizer and then hits the collision baffle at a higher flow rate to form solute particles of different sizes. The larger particles are discharged from the waste pipe under the influence of gravity, and the smaller particles flow into the drying pipe with nitrogen; at the same time, the other flow path of the inlet nitrogen passes through the corona device (containing high-voltage platinum wire electrode) to form positively charged nitrogen particles, which collide with the dried solute particles in the collision cell. The charge is then transferred to the particles-the larger the solute particle, the more charge. The solute particles transfer their charge to the collector, and the charge amount of the solute particles is measured by a highly sensitive electrostatic detector. The resulting signal current is proportional to the content of the solute (Figure 8) [73]. The CAD detector is based on a unique new design principle, which solves some limitations of other detector design principles. Its biggest advantage is that the detection does not depend on the molecular structure of the analyte or ionize the analyte, which achieves the purpose of versatility. It has the same response to different compounds and is not sensitive to external influences. It can carry out gradient elution. At the same time, it can achieve higher sensitivity and lower detection limit, good reproducibility, and wide dynamic detection range. The CAD detector combined with a high-performance liquid chromatography system is simple and convenient to operate and has good stability. The CAD detector is based on a unique new design principle, which solves some limitations of other detector design principles. Its biggest advantage is that the detection does not depend on the molecular structure of the analyte or ionize the analyte, which achieves the purpose of versatility. It has the same response to different compounds and is not sensitive to external influences. It can carry out gradient elution. At the same time, it can achieve higher sensitivity and lower detection limit, good reproducibility, and wide dynamic detection range. The CAD detector combined with a high-performance liquid chromatography system is simple and convenient to operate and has good stability. It is 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 chromatographycharged 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 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].

Chromatographic Fingerprint
The chromatographic fingerprint can comprehensively measure the complex components 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 material [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).

HPLC-MS Detector
HPLC-MS is a powerful tool to identify and quantitatively analyze saponins. Compared 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][90][91][92][93][94][95][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 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].

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 smallparticle-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 (identification of traditional Chinese medicine components [88], determination of the components' 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][115][116][117]. Some studies determined the 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].

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.
, in which f km is the RCF of the internal reference and other components to be measured and f k and f m are the absolute correction factors of the internal reference substance and other components to be measured, respectively. A k and A m are the peak areas of the internal reference and other components to be tested, respectively, and W k and W m 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 determination 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 external 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 rationality 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 of the reference substance. The study assessed the accuracy of the QAMS method and showed that the concentration of the analyte in the sample solution was the main parameter affecting the accuracy of the QAMS method. By calculating and controlling the applicable concentration range of the analyte in the sample, the high accuracy of the QAMS method was ensured [127].

Immunoassay
The immunological analysis method has high sensitivity and specificity, which can be used to analyze saponins. One study established a time-resolved fluorescence immunoassay system to determine the content of saikosaponin a (SSa) in 10 commercial samples of bupleurum. The bupleurum methanol extract and a mouse anti-SSa monoclonal antibody were used as materials, and the Eu 3+ -labeled SSa-human serum albumin conjugate was used as a tracer (Figure 9). This technology had the advantages of high sensitivity, convenience, and speed [128]. [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 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 curacy of the QAMS method was ensured [127].

Immunoassay
The immunological analysis method has high sensitivity and specificity, wh be used to analyze saponins. One study established a time-resolved fluorescen munoassay system to determine the content of saikosaponin a (SSa) in 10 com samples of bupleurum. The bupleurum methanol extract and a mouse anti-SSa clonal antibody were used as materials, and the Eu 3+ -labeled SSa-human serum a conjugate was used as a tracer (Figure 9). This technology had the advantages sensitivity, convenience, and speed [128].

Metabolomics
Metabolomics is a new discipline developed in the mid-1990s to analyze all lowmolecular-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 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). In the analysis of saponins, metabolomics techniques are mainly combined with MS, liquid chromatography, gas chromatography, and NMR spectroscopy. NMR spectroscopy provides valuable details about metabolite characteristics of complex plant extracts and has the advantages of fast detection speed, easy absolute quantification, and clear compound identification; these positive attributes make the method an appropriate technique for analyzing large amounts of metabolites. High-performance TLC has the advantages of universal availability of derivatization reagents and a short running time. In addition, the simplicity of preparing HPTLC provides great potential for enhancing metabolomic studies of saponin-rich medicinal materials. The combination of UPLC and electrospray ionization-Q-TOF-MS can shorten the analysis time, provide accurate quality measurements and more molecular formula information, and facilitate the analysis of known compounds and the interpretation of unknown compounds in complex matrixes. Therefore, in recent years, the combination of UPLC with electrospray ionization-Q-TOF-MS has become the preferred technology for the analysis and identification of chemical components and metabolites in vivo after oral administration. Compared with LC-MS, gas chromatography-MS has better peak resolution and sensitivity. How-ever，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. Figure 10. The targeted and untargeted workflow for LC/MS-based metabolomics [129].
In the analysis of saponins, metabolomics techniques are mainly combined with MS, liquid chromatography, gas chromatography, and NMR spectroscopy. NMR spectroscopy provides valuable details about metabolite characteristics of complex plant extracts and has the advantages of fast detection speed, easy absolute quantification, and clear compound identification; these positive attributes make the method an appropriate technique for analyzing large amounts of metabolites. High-performance TLC has the advantages of universal availability of derivatization reagents and a short running time. In addition, the simplicity of preparing HPTLC provides great potential for enhancing metabolomic studies of saponin-rich medicinal materials. The combination of UPLC and electrospray ionization-Q-TOF-MS can shorten the analysis time, provide accurate quality measurements and more molecular formula information, and facilitate the analysis of known compounds and the interpretation of unknown compounds in complex matrixes. Therefore, in recent years, the combination of UPLC with electrospray ionization-Q-TOF-MS has become the preferred technology for the analysis and identification of chemical components and metabolites in vivo after oral administration. Compared with LC-MS, gas chromatography-MS has better 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 significantly 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 combination 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 HPLCelectrospray 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 highperformance 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 Panax notoginseng, one study carried out quantitative and qualitative analyses and a comparison 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 markers were detected, and seven triterpene saponins were significantly different in different tissues [134].

Discussion
Traditional extraction and separation techniques, such as thermal reflux extraction, Soxhlet extraction and liquid chromatography, have many shortcomings, such as long pretreatment time, cumbersome operation steps, and large consumption of organic solvents. At present, both ionic liquids and supercritical fluid chromatography (SFC) are green chromatographic 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. UPLCelectrospray ionization-tandem MS has the advantages of high sensitivity, high resolution, and high-quality measurement accuracy. It is a powerful tool to comprehensively determine 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 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 chromatographic 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 yield 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 saponin components (Figure 11).

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 Figure 11. Characteristic diagram of the saponin analysis method.

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.

Conflicts of Interest:
The authors confirm that this article content has no conflict of interest.