Supprimer filigrane Wondershare

PDFelement
Received: 25 March 2021 | Revised: 13 July 2021 | Accepted: 9 August 2021

DOI: 10.1002/mas.21728

REVIEW ARTICLE

Mass spectrometry analysis of saponins

Philippe Savarino | Marie Demeyer | Corentin Decroo | Emmanuel Colson |

Pascal Gerbaux

Organic Synthesis and Mass Spectrometry Abstract

Laboratory, Biosciences Research
Saponins are amphiphilic molecules of pharmaceutical interest and most of their
Institute, University of Mons—UMONS,
Mons, Belgium biological activities (i.e., cytotoxic, hemolytic, fungicide, etc.) are associated to
their membranolytic properties. These molecules are secondary metabolites pre-
Correspondence
sent in numerous plants and in some marine animals, such as sea cucumbers and
Pascal Gerbaux, Organic Synthesis and
Mass Spectrometry Laboratory, starfishes. Structurally, all saponins correspond to the combination of a hydro-
Biosciences Research Institute, University philic glycan, consisting of sugar chain(s), linked to a hydrophobic triterpenoidic
of Mons—UMONS, 23 Place du Parc, B‐
7000 Mons, Belgium.
or steroidic aglycone, named the sapogenin. Saponins present a high structural
Email: [email protected] diversity and their structural characterization remains extremely challenging.
Ideally, saponin structures are best established using nuclear magnetic resonance
experiments conducted on isolated molecules. However, the extreme structural
diversity of saponins makes them challenging from a structural analysis point of
view since, most of the time, saponin extracts consist in a huge number of con-
geners presenting only subtle structural differences. In the present review, we
wish to offer an overview of the literature related to the development of mass
spectrometry for the study of saponins. This review will demonstrate that most of
the past and current mass spectrometry methods, including electron, electrospray
and matrix‐assisted laser desorption/ionization ionizations, gas/liquid chroma-
tography coupled to (tandem) mass spectrometry, collision‐induced dissociation
including MS3 experiments, multiple reaction monitoring based quantification,
ion mobility experiments, and so forth, have been used for saponin investigations
with great success on enriched extracts but also directly on tissues using imaging
methods.

KEYWORDS
glycan, imaging, ion mobility, natural product, saponin

1 | INTRODUCTION (Yasumoto et al., 1966), and sponges (Stonik, 1986). Sa-

ponins are widespread plant specialized metabolites
Saponins form an important class of natural products (Osbourn et al., 2003). In a systematic study done on
first discovered in plants (Kofler & Adam, 1927) and then 1730 Asian plant species, 76% were shown to contain
in a few marine organisms such as sea stars (asteroids) saponins and many plants and vegetables consumed in
(Yasumoto et al., 1966), sea cucumbers (holothuroids) the human diet contain saponins (Kassem et al., 2014).

In honor of Professor Karoly Vekey for his entire career and his involvement in the development of mass spectrometry in Eastern Europe.R-
emembering our many “scientific discussions” during the “Informal Meetings on Mass Spectrometry.”

Mass Spec Rev. 2021;1–30. wileyonlinelibrary.com/journal/mas © 2021 John Wiley & Sons Ltd. | 1
 Supprimer filigrane Wondershare
2 | PDFelement
SAVARINO .
ET AL

The term “saponin” comes from the Latin word “sapo” (Desai et al., 2009). Paradoxically, their biological roles in the
meaning soap, whereas the suffix “ine” has been added host organisms are still very speculative. Different studies
as the signature of a chemical substance. Saponins are have reported that animal saponins are also involved in
indeed present in different plants, such as soaproot several activities such as chemical defense (Mackie et al.,
(Chlorogalum pomeridianum) and soapberry (Sapindus 1975) and interspecific chemical communication (Caulier,
saponaria), whose extracts have been employed as soap Flammang, Gerbaux, et al., 2013). The role of saponins in
for years (Oleszek & Hamed, 2010). These biomolecules plant is primarily associated to the defense against invading
are glycosides that arise from the condensation of one or harmful organisms (fungi, bacteria) and predators
more oligosaccharide chains, called glycan, onto an (insects) (Arabski et al., 2012; Chaieb, 2017; Mert‐Türk &
aglycone moiety of steroidal or triterpenoid nature. A Osbourn, 2006).
typical saponin structure is presented in Figure 1 and The extreme structural diversity of saponins makes them
corresponds to scabraside A (1) found in Holothuria challenging from a structural analysis point of view since,
scabra, a sea cucumber from Madagascar (Caulier, most of the time, saponin extracts consist in a huge number
Flammang, Rakotorisoa, et al., 2013). Both elements are of congeners presenting only subtle structural differences
mainly connected via a glycosidic linkage; even though (Wei et al., 2005). Saponin analysis by mass spectrometry
some saponins present an ester bond associating the methods is nowadays progressively supplementing other
glycan and the aglycone parts. Other chemical func- analytical methods such as nuclear magnetic resonance
tionalities can be appended on the saponin core‐ (NMR). Indeed, saponin extracts from plant or marine ani-
structure, such as a carboxylate function (–COO−) mals are often constituted by a complex mixture of (slightly)
(Yoshikawa et al., 1996), a sulphate group (–SO4−) different saponin molecules that requires extensive pur-
(Demeyer et al., 2014) or different acylated functions ification and separation steps to meet the requirement for
(Kanchanapoom et al., 2001). A huge amount of different NMR spectroscopy measurements. Based on its intrinsic
saponin molecules has already been characterized, but it features, mass spectrometry represents an inescapable tool to
is suspected that more congeners are still to be detected. access the structures of saponins within extracts by using
These molecules are thus characterized by a large che- liquid chromatography mass spectrometry (LC‐MS), matrix‐
mical diversity and a wide variety of biological activities assisted laser desorption/ionization mass spectrometry
(Desai et al., 2009), making them of high interest for the (MALDI‐MS), electron ionization, high resolution mass
pharmaceutical companies due to their hemolytic, cytotoxic, spectrometry (HRMS), tandem mass spectrometry and ion
antibacterial, antifungal, antiviral, and antitumor properties mobility experiments. In this review, we will not provide a

F I G U R E 1 Structure of Scabraside A (1), a saponin found in Holothuria scabra, composed of a glycan (left) and an aglycone (right)
(Caulier, Flammang, Rakotorisoa, et al., 2013) [Color figure can be viewed at wileyonlinelibrary.com]
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 3

complete review of the literature describing the use of mass respectively exemplified by β‐amyrin (2) and cholestanol (3)
spectrometry for the characterization of saponins. This in Figure 2. Note also that a numeration of the carbon atoms
would be totally unnecessary, if not impossible. Indeed, a specific to the sapogenins has been established by Maier and
quick search on Scopus (March 03, 2021) using as keyword is also presented in Figure 2 (Maier, 2008).
“saponin AND mass spectrometry” generates no less than The main sapogenins retrieved in plants are the tri-
3812 references. We rather intend to combine references terpenoids β‐amyrin (2), α‐amyrin (4), and lupeol (5);
presenting the different techniques of mass spectrometry whereas the steroids furostane (6) and spirostane (7) are
which have been used for the study of saponins. In doing so only scarcely detected (Haralampidis et al., 2002; Yang
we will certainly omit to cite important works of eminent et al., 2014). Steroid alkaloids, amongst which spirosolane
colleagues and we already apologize for it. (8) and solanidane (9) in Figure 2 are typical examples,
constitute a class of genins presenting a nitrogen atom in
the backbone (Ghisalberti, 2006). Specificity in the agly-
2 | SAPONIN MOLECULAR cone structure is also characteristic of the animal sapo-
STRUCTURES nins. For instance, holostanol (10) and lanosterol (11) are
specific to sea cucumbers and cholestanol (3) is detected
2.1 | The apolar part of saponin: The in some sea star species (Maier, 2008).
aglycone

The molecules belonging to the crowded saponin family are 2.2 | The polar part of saponin: The
first classified according to their aglycone structure. Three glycan
classes of (sapo)genins, i.e. triterpenoid, steroid and steroid
alkaloid, have been to date identified and are presented in The monosaccharide residues constituting the glycan part
Figure 2. Triterpenoid aglycones contain 30 carbon atoms, of saponins are extremely diversified and largely con-
whereas steroid aglycones count 27 carbon atoms, as tribute to the saponin diversity and structure complexity.

F I G U R E 2 The aglycone diversity composed of three main classes: triterpenoid, steroid and steroid alkaloid with the Maier numeration
for β‐amyrin (2) and cholestanol (3) (Ghisalberti, 2006; Haralampidis et al., 2002; Maier, 2008; Yang et al., 2014) [Color figure can be viewed
at wileyonlinelibrary.com]
 Supprimer filigrane Wondershare
4 | PDFelement
SAVARINO .
ET AL

In plants, β‐D‐glucose (Glc), β‐D‐glucuronic acid (GluA), β‐ Besides these two families, macrocyclic topologies are
D‐rhamnose (Rha), and β‐D‐arabinose (Ara) and their also known and correspond to molecules with a single
epimeric forms, that is, β‐D‐galactose (Gal) or β‐D‐xylose oligosaccharide chain attached at two sites on the agly-
(Xyl), are abundantly encountered (Vincken et al., 2007; cone. A typical example is presented in Figure 3 and
Augustin et al., 2011). In marine invertebrates, the glycan correspond to sepositoside A (14) detected in the saponin
part includes other monosaccharides such as 3‐O‐ extracts from the sea star Echinaster sepositus (De Simone
methylglucose (3‐OMe‐Glc) in sea cucumbers (Caulier, et al., 1981).
Flammang, Rakotorisoa, et al., 2013) or 6‐deoxy‐xylo‐hex‐
4‐ulose in sea stars (Demeyer et al., 2014). The oligo-
saccharidic chains mostly count from one to six sugar 2.3 | One step further toward the
residues, even if some glycans composed of 10 units have structural complexity: Specific chemical
already been reported in Clematoside mandshurica functions
(Khorlin et al., 1965). Additionally, the oligosaccharide
chain can be linear or branched (Sahu et al., 2008). Saponins are glycosidic molecules associating two dis-
Finally, saponins can be subdivided in different fa- tinct parts and characterized by an extreme structural
milies according to their glycan topology. Monodesmosidic diversity that is further exacerbated by the presence of
saponins are characterized by the condensation of a single substituents presenting specific chemical functions. As a
oligosaccharide onto the aglycone, whereas polydesmosidic typical example, saponins in several marine animals
structures appear when several oligosaccharide chains are present one or several sulfate groups on either the agly-
linked onto the aglycone. Typical examples are presented cone or the glycan parts depending on the animals
in Figure 3 and correspond to desholothurin A (Khorlin), (Demeyer et al., 2014; Kang et al., 2015). In sea stars, this
present in Holothuria forskali (Rodriguez, Castro, & sulfate group is specifically localized on the aglycone
Riguera, 1991), and saponin B (13) found in Chenopodium (Demeyer et al., 2014), while it is present on the first
quinoa Willd. seeds (Kuljanabhagavad et al., 2008) xylose residue of the oligosaccharide chain in several sea

F I G U R E 3 Different saponin topologies including monodesmosidic (12), bidesmosidic (13), and macrocyclic (14) structures
(Kuljanabhagavad et al., 2008; Rodriguez et al., 1991; De Simone et al., 1981) [Color figure can be viewed at wileyonlinelibrary.com]
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 5

cucumbers (Caulier, Flammang, Rakotorisoa, et al., (v/v). The butanolic phase is washed twice with Milli‐Q
2013). In Aesculus hippocastanum seeds, the escin (15) water to remove residual salts and impurities. The organic
molecules compose a family of saponins that are struc- solution containing the saponins is evaporated to dryness.
turally related but present some minute differences such The dry residue obtained after evaporation is weighed and
as the structure of the side chains (Figure 4) on the sa- stored in the fridge at 4°C.
pogenin (Yoshikawa et al., 1996). A symptomatic ex- Solid phase extraction (SPE) protocols are also optimized
ample is related to the aglycone substitution at C21, for biological sample cleanup and saponin preconcentration
where five different acyl groups have been detected: tiglic before the MS analysis. Numerous studies report the use of
acid (Tig), angelic acid (Ang), acetic acid (Ac), isobutyric C18 SPE phases for the pretreatment of saponins, including
acid (A), and 2‐methylbutyric acid (B) (Yoshikawa et al., triterpenoidal saponins contained in rat plasma that are quite
1996). Moreover, a second acetyl function can be loca- complex and present at very low concentrations (Wang et al.,
lized in C22 or C28 (Yoshikawa et al., 1996). 2007; Xu et al., 2011). Microwave‐assisted extraction has
been demonstrated to show outstanding saponin extraction
efficiency compared to other conventional extraction meth-
3 | SAPONIN E XTRACTION ods, such as ultrasonic extraction and heat reflux extraction,
FROM THE B IOMASS on Pulsatilla turczanimovii (Xu et al., 2012).

For isolation of saponins, the majority of the numerous

protocols present in the literature are based on successive 4 | B OT TO M ‐ UP APPROACHES
liquid‐liquid extractions with solvents of increasing po- FO R SA P O N I N I D E N T I F I C A T I O N
larity (Campagnuolo et al., 2001; Garneau et al., 1983). As
for a typical example, our extraction method has been Saponin structural characterization may be performed
adapted from the literature and consists in different li- using a bottom‐up strategy meaning that the saponin
quid/liquid extractions (Van Dyck et al., 2009). The molecules are first decomposed upon hydrolysis into
weighed biomass powder is stirred in methanol for 24 h at their building blocks that are further analyzed using on‐
room temperature followed by centrifugation. The super- line gas chromatography (GC‐MS) or liquid chromato-
natant is diluted to 70% methanol with Milli‐Q water. This graphy (LC‐MS) mass spectrometry experiments.
hydromethanolic extracts is partitioned (v/v) successively Upon acid conditions (aqueous HCl), all the glycosidic
against n‐hexane, chloroform and dichloromethane to linkages, being acetal functions, are hydrolyzed producing
remove impurities such as fatty acids. Finally, the hydro- the sapogenin residues together with all the free mono-
methanolic solution, containing saponins, is evaporated at saccharides. These building blocks are further analyzed upon
low pressure. The dry extract is solubilized in Milli‐Q GC‐MS after derivatization. Trimethylsilyl ether derivatives
water to undergo a last partitioning against isobutanol are usually preferred since (i) they are formed within a few

F I G U R E 4 Molecular structures of Escin (15) molecules extracted from Aesculus hippocastanum seeds where Tig, Ang, Ac, A, and B
correspond to tiglic acid, angelic acid, acetic acid, isobutyric acid and 2‐methylbutyric acid, respectively. Adapted from (Yoshikawa et al.,
1996) [Color figure can be viewed at wileyonlinelibrary.com]
 Supprimer filigrane Wondershare
6 | SAVARINOPDFelement
.
ET AL

minutes at room temperature using reagents such as N,O‐ microwave heating to specifically hydrolyze under basic
bis‐(trimethylsilyl)trifluoroacetamide (Burnouf‐Radosevich conditions the bidesmosidic saponins extracted from C.
et al., 1985) or trimethylsilyl cyanide (Jæger et al., 2017) quinoa into their monodesmosidic counterparts (Colson
and (ii) silylation avoids peak tailing and shortens con- et al., 2020). The microwave‐assisted hydrolysis reaction
siderably the retention time of triterpenes as compared to was optimized to quantitatively produce mono-
methylation and acetylation (Ikekawa et al., 1965). As a ty- desmosidic saponins. The membranolytic activity of the
pical example, the acid hydrolysis of ligulataside A (16) saponins (ability of saponins to interact with the mem-
(Figure 5), extracted from the Australian medicinal and food brane leading to its lysis) was assayed based on their
plant Acacia ligulate, results in the aglycone echinocystic hemolytic activity that was shown to be drastically in-
acid (17) (red structure, Figure 5), and free saccharide units creased upon hydrolysis (Colson et al., 2020).
(Jæger et al., 2017) that are further identified upon GC‐MS Enzymatic hydrolysis and structural determination
analysis. Identification of the aglycone moieties is often by LC‐MS/MS is another elegant procedure for the
achieved on the basis of accurate mass measurements and identification of saponins. Enzymes obtained from var-
comparison with commercially available standards. The ious plants and animals have been used for hydrolyzing
analysis of the fragment ions generated upon electron ioni- polysaccharides, which resulted in the release of free
zation (EI‐MS) mass spectra of triterpenes and related sugar units depending on their specific activities ac-
compounds is also nicely documented in older literature cording to the glycosidic bond positions (Jeong et al.,
(Budzikiewicz et al., 1963). In general, the presence of an 2014). Enzymatic transformation of natural molecules,
endocyclic double bond controls the fragmentation behavior, such as platycosides (19), was introduced to modify the
and characteristic mass spectral features have been noted structures of natural molecules to reach enhanced
which frequently allow assignment of a given triterpene in pharmacological activities (Jeong et al., 2014). Enzymatic
one of the major classes by this criterion. In addition, the transformation by hydrolases was further implemented
location of functional groups can often be narrowed down by to selectively hydrolyze glycosidic linkages in platyco-
consideration of the fragmentation pattern (Budzikiewicz sides (19) to discriminate isomers (Jeong et al., 2014).
et al. 1963). Platycosides (19) are saponins extracted from Platycodi
Alkaline hydrolysis is more specific than its acidic Radix and are used as traditional medicines (Nyakudya
counterpart in the sense that glycosidic linkages are not et al., 2014). There are bidesmosidic saponins with an
hydrolyzed under basic conditions. This is particularly oligosaccharide moiety of arabinose, rhamnose, and xy-
interesting in the case of bidesmosidic saponins, such as lose in sequence being attached onto the pentacyclic
ligulataside A (16), when the second saccharide chain is triterpenoid aglycone through an ester linkage at C‐28
appended onto the aglycone via an ester bond, see and the other being constituted by two or three β‐glucose
Figure 5. Alkaline hydrolysis of 16 produces a prosaponin residues linked through linear glycosidic bonds at the C‐3
(18) (red and pink structure, Figure 5), which can be position. Isomers of platycosides (19) are classified by the
analyzed by LC‐MS/MS (Jæger et al., 2017). In a recent, linkage positions of glycan linked at the C‐3 position of
study, we took advantage of the fast and homogeneous the triterpene aglycone and are constitutional isomers

F I G U R E 5 Molecular structure of ligulataside A (16) extracted from Acacia ligulate (Jæger et al., 2017) [Color figure can be viewed at
wileyonlinelibrary.com]
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 7

with Glc‐(1→6)‐Glc or Glc‐(1→3)‐Glc glycosidic linkages minor components” (Costello, 1996). Hence, hydrolysis
(Nyakudya et al., 2014). Discrimination between those eliminates the possibility to confirm the structure of in-
isomers may be achieved by selective glycosidic linkage tact saponins since, by definition, only building bricks
hydrolysis using selected enzymes. For instance, gluco- are identified. This is even more inextricable that the
sidases have a specific activity toward the Glc‐(1→4)‐Glc hydrolyses are often carried out on mixtures of saponins.
glucan, whereas glucanases predominantly hydrolyze The introduction of fast atom bombardment (FAB)
Glc‐(1→3)‐Glc linkages and laminarinases show suffi- mass spectrometry in 1981 has provided a rapid method for
cient selectivity for hydrolyzing Glc‐(1→6)‐Glc linkages. determining the molecular weights of involatile glycosides
After hydrolyzing a saponin extract by laminarinases, the (Adinolfi et al., 1984), such as saponins. In 1984, Adinolfi
reconstructed total ion chromatogram generated by a et al. used muscaroside A (21) and digitonin (22) as model
chemometric technique sorted peaks of deglycosylated compounds to demonstrate the capabilities of FAB mass
platycosides easily discriminating the platycoside con- spectrometry for the structural analysis of saponins
stitutional isomers without any LC‐MS/MS experiments (Adinolfi et al., 1984). They showed that the molecular
(Jeong et al., 2014). weights of intact saponin molecules can be readily de-
Another approach combining hydrolysis, derivatiza- termined using exact mass measurements on the proto-
tion and GC‐MS analysis has been recently introduced for nated [M + H] +and sodium cationized [M + Na]+
saponin analysis and is inspired by the procedure devel- molecules. Further, they also detected in‐source fragmen-
oped by Pettolino et al. for determining the polysaccharide tation reactions that were shown to occur at every glyco-
composition of plant cell walls based of the preparation of sidic bond by a proton rearrangement from the leaving
partially permethylated alditol acetates (PMMAs) (20) group to the interglycosidic oxygen (Adinolfi et al., 1984).
(Pettolino et al., 2012). The method starts by the per- They concluded that FAB fragment ions can be used to
methylation of the saponin molecules using methyl iodide define the glycoside sequence but remains inefficient to
and sodium hydroxide in dimethyl sulfoxide. Complete differentiate isomeric hexoses or to identify the type of
methylation results in the methylation of all free hydroxyl linkage between sugar residues. However, this study defi-
groups not involved in the glycosidic linkage. After acid nitively paved the way to the soon coming reports on the
hydrolysis using trifluoracetic acid, the partially methy- tandem mass spectrometry analysis of saponin ions.
lated monosaccharides that are released are reduced with
sodium borodeuteride (NaBD4) to simultaneously open
the sugar ring to form alditol derivatives and tag the 5.1 | Tandem mass spectrometry
anomeric C atom (C1) with a deuterium atom. To further analysis of saponin ions
increase the volatility of the derivatives for GC separation,
the partially methylated alditols are acetylated with acetic Tandem mass spectrometry, that is, collision‐induced
anhydride derivatives to produce PMMAs that are further dissociation (CID) experiments, is now largely acknowl-
analyzed using GC‐MS (Jæger et al., 2017). Based on the edged as a powerful structural analysis method for sapo-
MS fragmentation pattern of PMMAs ions and compar- nins. Considerable structural information, indicating the
ison with standard molecules, the monosaccharide present nature of the aglycone and the linkage and position of the
in the sample, together with their glycosidic linkage po- monosaccharide residues, may be deduced from the ana-
sitions, are identified (Jæger et al., 2017). lysis of the CID spectra of [M + H] +, [M‐H]− and [M +
Cat]+ (Cat = Li or Na) ions. As a general rule, the frag-
mentation reactions undergone by activated saponin ions
5 | TO P‐ DOWN APPROACHES almost occur within the glycan part of the saponin ions,
FOR SAPONIN IDENTIFICATION although dissociation reactions specific to the aglycone
part may be sometime detected. Basically, upon CID of
The chemical or enzymatical hydrolysis of saponins has saponin ions, the sequence and the branching of the gly-
been previously demonstrated to afford valuable pieces of can moiety as well as the molecular mass of the intact
information for saponin analysis. However, as stated by aglycone can be obtained when the CID experiments are
Costello in her seminal publication related to the tandem conducted under both low (collision energy (CE) < 100
mass spectrometry analysis of saponins: “Structural de- eV) and high (CE > keV) collision energy conditions (Zehl
terminations of saponins present challenges to the ana- et al., 2007). However, it is important to note that the
lyst because of the variety of potential modification sites, distinction between isomeric aglycones is scarcely
the incorporation of both carbohydrate and non- achieved using CID experiments (Zehl et al., 2007).
carbohydrate moieties, and the occurrence of mixtures In 1988, Domon and Costello introduced their sys-
that may include isomeric species and highly‐active tematic nomenclature for carbohydrate fragmentations
 Supprimer filigrane Wondershare
8 | PDFelement
SAVARINO .
ET AL

F I G U R E 6 Carbohydrate fragmentation pathways where Ai, Bi, and Ci designate fragment ions containing the nonreducing end sugar
unit, Xj, Yj, and Zj represent ions still containing the aglycone or the reducing end sugar unit. Subscripts indicate the position of the
dissociated glycosidic bond relative to the reducing end, whereas superscripts indicate dissociations within carbohydrate rings. Adapted
from (Domon & Costello, 1988) [Color figure can be viewed at wileyonlinelibrary.com]

in (FAB) MS/MS spectra of glycoconjugates (Domon & for the low‐energy CID and MALDI‐ToF/RToF for the
Costello, 1988). They also summarized all the fragment high‐energy CID analysis (Zehl et al., 2007) on [M + H]+
ions generated upon CID of ionized glycoconjugates and [M + Cat]+ (Cat = Na and K) precursor ions. The
(glycosphingolipids, glycopeptides, glycosides, and car- principal outcome of this large investigation is that, upon
bohydrates) in both the positive and negative ion modes. CID, whatever the nature of the precursor ions, low‐
As shown in Figure 6, the simplest fragmentation of energy and high‐energy CID yield abundant B, C, Y, and
the carbohydrate chain in collision activated saponin Z fragment ions together with internal cleavage ions,
ions results from the cleavage of the glycosidic bond, that with a global coverture of the glycosidic bonds. They also
is, Y/B and Z/C pathways, yielding information regard- confirmed that abundant 1,5X fragment ions are gener-
ing the sugar sequences. More complex processes invol- ated upon high‐energy conditions (Zehl et al., 2007).
ving the fragmentation within the sugar ring leading to From a mechanistic point of view, the glycosidic bond
the A fragment ions, are observed. Interestingly, both dissociation generating Bi and Yj ions (see Figure 7), with
positive and negative ions present these fragmentation retention of the glycosidic oxygen atom by the species
routes. As shown in Figure 6, Ai, Bi and Ci labels desig- formed from the reducing end, is most of the time de-
nate fragment ions containing the nonreducing end su- tected from [M + H]+, [M + Cat]+ and [M‐H]− precursor
gar unit, whereas Xj, Yj and Zj represent ions still ions (Domon & Costello, 1988). In the positive ion mode,
containing the aglycone or the reducing end sugar unit. it is proposed that the decomposition reaction of [M + H]+
Subscripts indicate the position of the dissociated glyco- ions involves the protonation of the oxygen atom of the
sidic bond relative to the reducing end (the glycosidic glycosidic bond which is broken affording the Bi oxonium
bond at the aglycone side is numbered 0), whereas su- ion (see Figure 7). Before the transient ion/neutral com-
perscripts indicate cleavages within carbohydrate rings plex decomposition, a proton transfer may occur leading
(Domon & Costello, 1988). Specific nomenclature has to the production of the complementary Yj ions (Figure 7).
also been introduced for branched saccharide chains. Negative ion decomposition mechanisms were also stu-
Low‐energy and high‐energy CID spectra of [M + died and were proposed to follow a more complex pathway,
H] +saponin ions have been compared by Claeys et al. involving deprotonation of a hydroxyl group in position 3 (or
(1996) using a hybrid EBQQ and a four‐sector EBEBE 4), epoxidation, sugar ring opening and glycosidic bond
instruments, respectively. They demonstrated that low‐ rupture to yield Yj fragment ions as shown in Figure 8
energy CID fragmentations of sugar residues generate (Prome et al., 1987). Interestingly, from [M‐H]− precursor
abundant Yn+ ions and some low intensity Zn+ ions, ions, the dissociation of the glycosidic bond with retention of
whereas the high‐energy spectra also exhibit strong the glycosidic oxygen atom on the nonreducing end side is
1,5
Xn+ fragment ions, formed by multiple cleavage of the frequently observed and afford C and Z‐type ions.
sugar ring, and significant Zn+ ions (Claeys et al., 1996). In that case, after deprotonation of a hydroxyl group
Zehl et al. performed a tandem mass spectrometry study adjacent to the glycosidic bond, the resulting alkoxy may
on triterpenoid saponins isolated from Bacopa monnieri. undergo an epoxidation with rupture of the glycosidic
They reported the application of different MS methods by bond, generating C‐type ions, see Figure 8. Again, Z‐type
associating data generated on ESI‐ion trap (IT), atmo- ions can be competitively produced upon proton transfer
spheric pressure MALDI‐ion trap (AP‐MALDI‐IT) and before the dissociation of the transient ion/neutral
MALDI‐IT/reflectron time‐of‐flight (MALDI‐IT‐RToF) complex (Prome et al., 1987).
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 9

F I G U R E 7 Production of Bi and Yj fragment ions in the positive ion mode induced by protonation at the oxygen atom of the glycosidic
bond. Adapted from (Domon & Costello, 1988)

Asterosaponins (23) are sulfated saponins with the (Gevrenova et al., 2018). For other saponin ions extracted
sulfate group being attached on the aglycone moiety, see from the same source, a 42 u loss, that is, neutral ketene,
Figure 9. Consequently, since the negative charge is lo- detected in the MS/MS spectra revealed the presence of
calized on the sulfate group, all the CID processes are an acetyl group. A competitive loss of 204 u further
shown to afford fragment ions containing the aglycone confirmed that those saponins contain an acetylated
moiety or part of the aglycone moiety, that is to say Y‐ glucose moiety (Gevrenova et al., 2018).
type ions (see Figure 6) and the CID reactions (low‐ Saikosaponins (27) (Figure 11) are monodesmosidic
energy conditions) were shown to formally correspond to saponins and are considered to be the major bioactive
the unwinding of the oligosaccharide chains converging ingredients of Bupleurum chinense, also known as Radix
to the aglycone part. However, no mechanistic study has Bupleuri, which is a famous traditional Chinese medi-
been performed to date to decipher the position and role cine (Liu et al., 2019). Liu et al. detected six malonylated
of the negative charge in the dissociating ions. saikosaponins and two acetylated ones in the methanolic
Fragmentations involving the aglycone side‐chain extract of B. chinense using LC‐MS experiments. They
may also be observed and, for instance, the asterosaponin developed a MS/MS‐based protocol to discriminate be-
1 (24) ions in Figure 9 were observed, under low‐energy tween the aglycone‐substituted and the saccharide‐
conditions, to expel 100 u that arises from a McLafferty substituted saponins. They showed that, the Y0 – nH2O
side‐chain loss reaction, specific to the asterosaponin 1 (n = 1–2) fragment ions containing or not the substituent
(24) aglycone (Demeyer et al., 2014). Gevrenova et al. are observed in the CID mass spectra of the [M + H] +
reported a large LC‐MS/MS study related to the profiling ions of aglycone‐substituted or saccharide‐substituted
of the saponins extracted from the roots of Gypsophila saikosaponins, respectively. Interestingly, the B ions
trichoma Wend (Gevrenova et al., 2018). These saponins containing or not the substituent are rather observed
are glucuronide oleanane‐type triterpenoid carboxylic from the [M + Na]+ ions of, respectively, the saccharide‐
acid 3,28‐O‐bidesmosides (GOTCAB) (25) (see substituted or aglycone‐substituted saikosaponins (Liu
Figure 10). These saponins presenting a glucuronic acid et al., 2019). These results also clearly point to the fact
at the C3 position are abundantly detected as [M‐H]− that the CID reactions undergone by the [M + H] +and
ions in the negative ion mode. Interestingly, some con- [M + Na]+ saponin ions may be different due to the
geners present a sulphate group on a sugar residue on the structural differences for the [M + H] + versus [M + Na]+
C28 chain, making a huge difference with the aster- precursor ions.
osaponin 1 (24) previously described. Fragmentation CID mass spectra of the [M + Li]+ saponin ions were
pathways for the identification of those saponins were recorded for standard dioscins (28) (Song et al., 2004) and
proposed in that study. Sulphated saponin ions were ginsenosides (29) from Panax ginseng (Song et al., 2005)
shown to expel 80 u, corresponding to a SO3 neutral loss, and CID experiments were demonstrated to be really
as a primary dissociation from the [M‐H]‐ ions efficient to allow isomeric saponin ion discrimination
 Supprimer filigrane Wondershare
10 | SAVARINO PDFelement
.
ET AL

F I G U R E 8 Production of Bi/Yj ions and Ci/Zj ions in the negative ion mode. Adapted from (Adinolfi et al., 1984; Domon &
Costello, 1988)

since cross‐ring cleavage product ions, i.e. X, A, Y and C 5.2 | Saponin characterization: Selected
type fragment ions, are observed to be abundant. CID examples
experiments conducted on [M‐H]− and [M + Li]+ ions
are often combined to get accurate structural information Nowadays, FAB‐MS and liquid secondary ion mass spec-
on the way to saponin structure identification (Lin et al., trometry (Costello, 1996) experiments are advantageously
2007; Song et al., 2005). replaced by Electrospray ionization (ESI) and Matrix‐assisted
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 11

F I G U R E 9 LC‐MS/MS analysis (negative ion mode) of m/z 1257 ions. The CID activation leads to the loss of 100 u, characteristic of a
McLafferty rearrangement specific to the presence of the aglycone side‐chain of asterosaponin 1 (24). Adapted from (Demeyer et al., 2014)
[Color figure can be viewed at wileyonlinelibrary.com]

F I G U R E 10 Saponin reference A (26), a glucuronide oleanane‐type triterpenoid carboxylic acid 3,28‐O‐bidesmoside extracted from the
roots of Gypsophila trichoma Wend (Gevrenova et al., 2018) [Color figure can be viewed at wileyonlinelibrary.com]
 Supprimer filigrane Wondershare
12 | PDFelement
SAVARINO .
ET AL

F I G U R E 11 Saikosaponins (27) extracted from Bupleurum chinense where R can correspond to acetic acid (Ac) or malonic acid (Mal).
Adapted from (Liu et al., 2019) [Color figure can be viewed at wileyonlinelibrary.com]

Laser desorption/ionization (MALDI) for the efficient and and LC‐MS/MS analyses. MALDI‐MS experiments al-
soft detection of intact saponin ions (Demeyer et al., lowed for a rapid screening of the saponin mixtures, while
2014). CID experiments are also nowadays conducted on all LC–MS techniques were used to achieve chromatographic
the standard mass spectrometers, namely Q3 (Chen et al., separation of isomers. In addition to the seventeen mole-
2019), Q‐ToF (Zheng et al., 2017), FT‐ICR (Zhang et al., cules (twelve compositions) already described in the lit-
2010), ion trap (Wang et al., 2015), Orbitrap (Gevrenova erature (Sandvoss et al., 2000, 2001, 2003), we detected
et al., 2019), and QTrap (Shi et al., 2020). nine new molecules (six compositions). The comparison of
Although unable to discriminate between isomeric the saponin contents from the five different body com-
saponins, MALDI‐MS is often used for a rapid screening of ponents revealed that each organ is characterized by a
the saponin content in an extract and for exact mass specific mixture of saponins and that qualitative and
measurements when associated to a high resolution ana- quantitative variability of the saponin contents are also
lyzer, such as orthogonal acceleration time‐of‐flight (oa‐ linked to the sex or to the collecting season (Demeyer
ToF) (Bahrami & Franco, 2015; Demeyer et al., 2014) and et al., 2014). From a biological point of view, this type of
Fourier transform ion cyclotron resonance (FT‐ICR) in- data is of crucial importance in understanding the biolo-
struments (Bondoc et al., 2013). For instance, Bondoc gical role of these specific metabolites. Similarly, Popov
et al. investigated the saponin contents of semi‐purified et al. (2017) applied LC‐MS/MS on the whole body extract
and high‐performance liquid chromatography (HPLC) and extracts of various body components to get a full de-
fractionated extracts from the body wall of three species of scription of the saponins present in the sea cucumber
Holothuriidae to examine their chemical diversities in Eupentacta fraudalix that is living in the shallow waters of
relation to phylogenetic data. MALDI‐FTICR‐MS and the south Part of the Sea of Japan. They identified 54
nano‐HPLC‐chip Q‐ToF‐MS were used for mass profiling compounds, including 26 sulfated, 18 nonsulfated and 10
and isomer separation, respectively, giving a unique che- disulfated triterpene glycosides. By comparing the saponin
mical saponin fingerprint (Bondoc et al., 2013). Obviously, contents of the different extracts, they demonstrated that
the combination between reversed phase liquid chroma- the profiles of the different body components are quali-
tography (RP‐LC) and ESI‐MS is much more effective for tatively similar and that only small quantitative vari-
the analysis of saponins enabling the possibility of separ- abilities are observed (Popov et al., 2017).
ating the isomers before MS and MS/MS analysis (Kite Bahrami et al. also used the efficient combination be-
et al., 2004; Li et al., 2012; Lin et al., 2007; Sandvoss et al., tween MALDI‐MS/MS and LC‐MS/MS for identifying the
2000, 2001, 2003)). As for a typical example from our lab, saponins present in the viscera of Holothuria lessoni, an
we used LC‐MS and LC‐MS/MS to highlight the molecular Australian sea cucumber. The saponins were obtained by
diversity and the body distribution of saponins in the sea successive liquid/liquid extractions and further purified by
star Asterias rubens (Demeyer et al., 2014). Asterosaponins high performance centrifugal partition chromatography.
(23) are well‐known secondary metabolites of the com- They detected 75 saponins, including 39 new congeners
mon sea star A. rubens in which they would be involved in with a high structural diversity; revealing that the viscera
chemical defense, digestion, and reproduction. In this samples possess a higher diversity and content than the
study, we analyzed separately the saponin content of dif- body wall (Bahrami, Zhang, Franco, et al., 2014). In a
ferent organs using the combination between MALDI‐MS second investigation, they used MALDI‐MS and LC‐MS/MS
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 13

to elucidate the structure of five novel isomeric saponins C3 and C28. Bankefors et al. (2011) therefore developed a
(Bahrami, Zhang, Chataway, et al., 2014). multistep MS3 strategy on an ion‐trap instrument to re-
Quillaja saponaria saponins possess extremely com- cover the information about the structures of the C3 and
plexed structures that consist of the triterpene quillaic C28 glycan groups, including regioisomer distinction
acid aglycone substituted at C3 with a di‐ or trisaccharide Beside structural characterization, CID experiments
and at C28 with a complex oligosaccharide (Nord & may also be of great interest for reaching higher sensi-
Kenne, 1999). The first monosaccharidic residue of the tivity and selectivity for saponin detection on wide dy-
latter glycan is an acyl‐substituted fucosyl residue, linked namic ranges by using multiple reaction monitoring
via an ester bond at C28 of the aglycone (see Figure 12). (MRM) experiments. As for a striking example, Chen
As for an example, Quillaja saponin B1 (30) is presented et al. (2016) developed an MRM‐based strategy for the
in Figure 12 and is characterized by a complexed fattyl detection of ginsenosides (29) extracted from Panax no-
acyl residue on the fucosyl residue. The CID spectrum— toginseng, the so‐called Notoginseng total saponin
MS2—of the [M + Na]+ ions is totally dominated by two (NGTS) extract, using a Sciex Q‐trap 4500 mass spectro-
signals, annotated [A + Na]+ and [B + Na]+ in Figure 12. meter. [M + HCOO]− anions are usually detected as the
The corresponding fragment ions arise from the cleavage dominant ions for gingenosides (29) under LC‐MS(‐)
of the glycosidic bonds linking the C3 and C28 oligo- conditions using formic acid as the mobile phase addic-
saccharides to the triterpene, respectively. The dissocia- tive. By monitoring the formate anion‐to‐deprotonated
tion of the glycosidic bond at C3 affords [A + Na]+ Y ion transitions, that is, [M + HCOO]− → [M‐H]− reac-
ions, whereas the rupture of the glycosidic bond at C28 tion, they detected 112 saponins, including 107 trace
yields [B + Na]+ ions consisting of only the C28 oligo- components and 12 potential new gingenosides (29).
saccharide (including a formed insaturation in the fucose A mass spectrometry‐based database for rapid iden-
residue), see Figure 12. In other words, such a MS2 mass tification of saponins has been recently developed by
spectrum does not contain any information regarding the Huang et al. (2020) and is referred to as saponin mass
detailed structures of the oligosaccharide chains at both spectrometry database. The freely available database

F I G U R E 12 ESI‐IT‐MS/MS analysis (positive ion mode) of m/z 2055 ions, Quillaja saponin B1 (30) [M + Na]+. The CID activation leads
to the production of two major signals annotated [A + Na]+ and [B + Na]+, respectively arising from the cleavage of the glycosidic bonds
linking the C3 and C28 oligosaccharides to the triterpene. Adapted from (Bankefors et al., 2011) [Color figure can be viewed at
wileyonlinelibrary.com]
 Supprimer filigrane Wondershare
14 | PDFelement
SAVARINO .
ET AL

(http://cpu-smsd.com) is based on HR/MS and high‐ extracts from ginseng, were directly analyzed using
energy CID analysis in the negative ionization mode. The DART‐MS and [M + H] + or [M + NH4] + ions of per-
database is to date constituted by more than 4.000 methylated saponins were detected. Extensive fragmen-
saponins. tations were observed during the ionization and mostly
concerned glycosidic bond cleavages, cross‐ring cleavages
and methanol neutral losses (He et al., 2017).
5.3 | Direct analysis in real time
(DART) mass spectrometry
6 | ION MOBILITY
Beside MALDI‐MS and LC‐MS analyses, DART mass S P E C T R O M ET R Y F O R SA P O N I N
spectrometry has also been used to analyze saponins CHARACTERIZATION
directly from raw materials such as plant tissues. One of
the major issues when using DART for the screening of Ion mobility mass spectrometry (IMS) is nowadays lar-
phytochemicals is the low ionization efficiency of DART gely implemented amongst the MS‐based methods for the
for hydrophilic glycosides since the hydrophilic interac- structural analysis of biomolecules, with a special inter-
tion between glycosides and the plant matrix molecules est paid at large molecules such as proteins (Uetrecht
hinders the vaporization of ionized glycosides (Kim et al., et al., 2010) and nucleotides (Quinn et al., 2013). This
2014). In situ permethylation has been successfully ap- methodology consists in the temporal separation of gas-
plied by Kim et al. for the direct analysis of timosaponin eous ions based on their mobility in a cell filled with a
AIII (31) from Anemarrhena asphodeloides Bunge rhi- buffer gas under the influence of an electric field. IMS
zome. The powder was subjected to permethylation with separates gaseous ions according to their ion mobility K,
tetramethylammonium hydroxide (25 wt.% in methanol) a physical quantity related to the ion charge (z) and
simultaneously with DART ionization. The detection of collisional cross section (CCS) (Mesleh et al., 1996;
unfragmented permethylated timosaponin AIII (31), ca- Shvartsburg & Jarrold, 1996). As a first approximation,
tionized by a trimethylammonium ion (Figure 13), was the CCS directly reflects the ion spatial structure. The
explained by the enhancement of volatility of per- drift time (tD) of the ions across the mobility cell, that is,
methylated compounds requiring ionization in less harsh the measurand, is directly proportional to their CCS
DART conditions (Kim et al., 2014). which reflects the three‐dimensional shape of the ions in
He et al. developed a solid‐phase methylation of sa- the gas phase (Gabelica & Marklund, 2018). IMS is then
ponins using a stainless‐steel methylation column (He used to obtain direct information about the spatial
et al., 2017). A stainless‐steel column was filled with structure of gaseous ions. To identify the 3D structure of
powdered NaOH in acetonitrile and DMSO saponin so- gaseous ions, theoretical estimates of the CCSs (CCSth)
lutions containing 10% of methyl iodide were passed are computed based on atomistic computational simu-
through the reaction column and collected in centrifuge lations and directly compared to the experimental values
tubes. The standard solutions, as well as the saponin (Gabelica & Marklund, 2018).

F I G U R E 13 Molecular structures of the timosaponin AIII (31) and its permethylated homologue that appear as tetramethylammonium
adduct upon DART analysis in presence of TMAH. Adapted from (Kim et al., 2014). DART, Direct analysis in real time; TMAH,
tetramethylammonium hydroxide [Color figure can be viewed at wileyonlinelibrary.com]
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 15

We introduced IMS, using a traveling wave ion mo- presenting closely related structures, which is often the
bility mass spectrometer (TWIMS), in combination with case when considering the extract from a natural source.
molecular dynamics (MD) simulations for the structural We further applied IMS together with MD simulations
analysis of saponins in 2017 and we focused on the sa- on a broad selection of saponin molecules covering a wide
ponins extracted from H. forskali, a sea cucumber abun- range of different structures in terms of the number of sac-
dantly observed around the Atlantic coasts of northwest charide units and their topology, including linear and
Europe and in the Mediterranean Sea (Decroo et al., 2017). branched as well as monodesmosidic and bidesmosidic sa-
H. forskali contains 26 saponins in its Cuvierian tubules ponins (Decroo et al., 2019). We also investigated whether
and 12 in its body wall (Decroo et al., 2017). Such a high the selection of the cationization reaction, that is, yielding
diversity is assumed to be linked to a chemical defense [M + H]+, [M + Na]+, and [M + K]+, may affect the IMS
mechanism, that is, the most commonly accepted biolo- results in terms of isomer separations. We showed that IMS
gical role for these specialized metabolites. Holothuroid may efficiently contribute to the structural characterization
saponins are triterpene glycosides. The structure of the of saponins, since different saponin ions can present sig-
aglycone moiety, a holostanol (10), is derived from the nificantly distinct CCSs. Depending on the nature of the
tetracyclic triterpene lanosterol (11) in which the D‐ring cation (in the positive ion mode), the differences in CCS can
contains a γ‐18(20)lactone (Decroo et al., 2017). The car- also be exacerbated, optimizing the gas‐phase separation.
bohydrate moiety is bound to the C3 of the aglycone and However, we also concluded that the structural diversity and
may include xylose (Xyl), glucose (Glc), quinovose (Qui), complexity of the saponins can definitively not be unraveled
and 3‐O‐methylglucose (3‐OMe‐Glc) residues, as well as a using IMS. The structural characterization of unknown sa-
sulphate group (Decroo et al., 2017). ponins will be difficult to achieve based on IMS alone, even
As a typical example, isomeric holothurinoside C2 (32) in combination with MD simulations.
and holothurinoside C (33) are linear 4‐sugar saponins, re- We nevertheless obtained a nice result for the dis-
spectively presenting the methylquinovose‐glucose‐glucose‐ crimination between monodesmosidic and bidesmosidic sa-
xylose and the methylglucose‐glucose‐quinovose‐xylose oli- ponins. Actually, as shown in Figure 17, for the same
gosaccharidic chains (Figure 14). These molecules are nicely number of monosaccharides, the [M + Na]+ bidesmosidic
separated by liquid chromatography with an elution time ions are always significantly more compact than their
difference around 2 min and are readily distinguished using [M + H] +homologues (about 10% reduction in CCS),
CID experiments (Decroo et al., 2017). However, in the IMS whereas for the monodesmosidic molecules, the CCS are
experiments, the corresponding [M + Na]+ ions are char- almost identical for the [M + H] + and [M + Na]+ ions
acterized by quasi identical arrival times due to really similar (Decroo et al., 2019). This difference is considered as a
CCS = 256 Å2. Actually, these two isomeric saponins adopt characterization criterion to distinguish monodesmosidic
quite similar gas phase structures upon ionization due to the and bidesmosidic saponins. This was recently exploited for
collapse of the glycan and aglycone parts around the Na+ the follow‐up of the microwave‐assisted hydrolysis of the
ion, as shown in Figure 15. bidesmosidic saponins extracted from C. quinoa that pro-
Another striking observation made in that seminal duces monodesmosidic saponins by the specific hydrolysis of
study is that different gas phase ion structures may be the ester bond at C28, see saponin B (13) in Figure 3 (Colson
created from a single molecular structure, i.e. a unique et al., 2020).
molecule, during the ionization process (Decroo et al., Regioisomeric and stereoisomeric saponins were re-
2017). This is typically the case for the [M + Na]+ ions of cently successfully discriminated using ion mobility ex-
holothurinoside F (34) (Figure 14) that are detected with periments, using a cyclic ion mobility (cIMS) setup with
two different arrival times upon IMS, see Figure 16. high ion mobility resolution (Colson et al., 2019). The
Actually, holothurinoside F (34) is a branched 6‐sugar cyclic TWIMS device is characterized by a scalable re-
saponin (Figure 14) with three and two monosaccharidic solution, ranging from 65 (1 pass) upward depending on
residues attached on the xylose residue. Upon Na+ the number of passes/cycles (Giles et al., 2019). The IMS
complexation, two different ion structures are base‐line resolution increases with the square root of the number
separated using IMS experiments as shown in Figure 16. of passes (Giles et al., 2019).
In the most extended structure, only one part of the As a model system, we selected the escin 1 (35) mo-
oligosaccharidic chain is in interaction with the sodium lecules extracted from the horse chestnut (HC) seeds that
ion, whereas, in the most folded structure, the aglycone comprise isomeric saponins containing subtle differences
and both arms of the oligosaccharidic chain are inter- such as cis‐trans ethylenic configuration (stereoisomers) of
acting with the sodium ion (see Figure 16). a side chain or distinct positions of an acetyl group (re-
The principal conclusion of this initial study was that gioisomers) on the aglycone, see Figure 18 for the escin 1
IMS experiments fail in discriminating saponin isomers (35) family of isomers. For the [M + Na]+ escin 1 (35)
 Supprimer filigrane Wondershare
16 | PDFelement
SAVARINO .
ET AL

F I G U R E 14 Molecular structures of isomeric four‐sugar saponins holothurinoside C2 (32) and holothurinoside C (33) and six‐sugar
saponin holothurinoside F (34) extracted from Holothuria forskali (Decroo et al., 2017) [Color figure can be viewed at
wileyonlinelibrary.com]

isomeric ions, small differences in the arrival time dis-

tribution (ATD) are monitored using a TWIMS when the
isomeric saponins are first separated with liquid chroma-
tography (Colson et al., 2019). The HC extract was further
analyzed by direct infusion (no LC separation) on the
cyclic ion mobility system. Whereas the [M + Na]+ ions of
the four escin 1 (35) isomers are not separated by cIMS
with one pass, after fifteen passes, the ATD recorded upon
direct infusion can be nicely deconvoluted into four dif-
ferent contributions, as presented in Figure 19.

7 | ON ‐T I S S U E L O C A L I Z A T I O N
F I G U R E 15 Molecular dynamics simulation of OF SAPONINS BY IMAGING
holothurinoside C (33) sodium adduct (m/z 1125) with the ME T H OD S
aglycone in orange, the glycan in green, the glycosidic
bonds in red, and the Na + cation in purple. Adapted from Direct visualization of saponins present in the plant or
(Decroo et al., 2017) [Color figure can be viewed at animal tissues represents an elegant method for assaying
wileyonlinelibrary.com] the biological activities of these glycosides. Matrix‐assisted
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 17
F I G U R E 16 LC‐IMS analysis and
molecular dynamic simulations of
Holothurinoside F (34) sodium adducts (m/z
1433) with the aglycone in orange, the glycan in
green, the glycosidic bonds in red, and the Na+
cation in purple. Adapted from (Decroo et al.,
2017) [Color figure can be viewed at
wileyonlinelibrary.com]

F I G U R E 17 Comparison between
measured CCSexp of [M + H]+ and [M + Na]+
ions of monodesmosidic (left) and bidesmosidic
(right) saponins with three and four saccharide
units. Adapted from (Decroo et al., 2019) [Color
figure can be viewed at wileyonlinelibrary.com]

laser desorption/ionization‐mass spectrometry imaging difficult based only on the morphological appearance.
(MALDI‐MSI), also called MALDI Imaging, has under- Ginsenoside content increases with root age (Bai et al.,
gone so many developments in the recent years (Am- 2016) and environmental variations (Bai et al., 2016),
stalden van Hove et al., 2010), allowing it to be now used leading to higher prizes in the market. Also, ginseno-
in many domains like clinical proteomics (Amstalden van sides (29) are classified into three common types de-
Hove et al., 2010) or pharmaceutical fields (Amstalden van pending on their aglycone, namely the 20(S)‐
Hove et al., 2010). This procedure permits to detect and protopanaxadiols (40), the 20(S)‐protopanaxatriols
localize ions of interest directly on tissue sections and (41), and the oleanolic acids (42) (Bai et al., 2016), see
nearly without any preparation. MALDI imaging experi- Figure 20. The pharmacological effects of different
ments performed on specialized metabolites start to be ginsenosides on the human body are significantly dif-
reported in the literature (Gadea et al., 2020), since the ferent depending on the molecular structures (Bai et al.,
great added value of using MALDI‐MS direct tissue ana- 2016). LC‐MS profiling of ginsenosides in root extract is
lysis is to detect specific molecules in a spatially‐resolved the standard method to access the ginsenoside content
analysis. By this way, MALDI‐MSI or MALDI profiling is of the roots (Bai et al., 2016).
recognized to open the door for important breakthroughs Direct profiling of ginsenosides (29) on root cross sec-
in the field of chemical ecology (Le Pogam et al., 2016). tions definitively appears as a less time‐consuming and more
The localization of specialized secondary metabolites in an informative alternative of the standard extraction‐based
organism depends on their functional roles, as stated by procedure. Using MALDI‐ToF‐MSI, Bai et al. detected 31
McKey et al. who postulated that specialized metabolites ginsenosides (29) with distinct localizations across the root
are primarily allocated to organism parts of high fitness cross sections. All the three types of ginsenosides (29) were
value, high risk of predation, or both (McKey, 1974). successfully detected and visualized in images that nicely
MALDI‐ToF‐MSI has been used by Bai et al. to ex- correlate with anatomical features. The P. ginseng at different
plore the distribution of ginsenosides (29) in the roots of ages were shown to be distinguishable based on the spatial
P. ginseng with a special attention paid at the age of the distribution of the ginsenosides (29) (Bai et al., 2016). The
plants (Bai et al., 2016). Actually, the establishment of different localization of protopanaxadiol‐type (40) and
P. ginseng age is economically relevant but remains protopanaxatriol‐type ginsenosides (41) in the root was also
 Supprimer filigrane Wondershare
18 | SAVARINOPDFelement
.
ET AL

F I G U R E 18 Nature of the isomeric relations between escin 1a (36), escin 1b (37), isoescin 1a (38), and isoescin 1b (39). Adapted from
(Colson et al., 2019) [Color figure can be viewed at wileyonlinelibrary.com]

F I G U R E 19 ESI‐cIMS analyses (positive ion mode) by direct infusion of the HC extract—arrival time distribution for the [M + Na]+
ions of the escin 1 (35) isomers (m/z 1153.5): comparison between one pass (left) and fifteen passes (right, deconvolution applied). Adapted
from (Colson et al., 2019) [Color figure can be viewed at wileyonlinelibrary.com]

previously observed using MALDI‐MSI by Taira et al. (2010) has also be used for studying the ginsenosides (29) locali-
at a 100 µm resolution. Their analysis revealed that the zation in the ginseng main and branch roots (Yang et al.,
ginsenosides are more concentrated in the lateral roots and 2021), indicating a tissue specific localization of the ginse-
are preferentially located toward the outer portion and the noside (29) congeners (Yang et al., 2021).
tip of the lateral roots (Taira et al., 2010). Desorption elec- The rhizome of Glycyrrhiza glabra (licorice) was
trospray ionization mass spectrometry imaging (DESI‐MSI) analyzed by high‐resolution mass spectrometry imaging
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 19

F I G U R E 20 Molecular structures of the three common aglycones of the ginsenosides (29): 20(S)‐protopanaxadiols (40), the 20(S)‐
protopanaxatriols (41), and oleanolic acids (42) (Bai et al., 2016) [Color figure can be viewed at wileyonlinelibrary.com]

and tandem mass spectrometry imaging using atmo- MALDI direct tissue analysis of a body wall tissue sec-
spheric pressure matrix‐assisted laser desorption/ioniza- tion, we identified 8 major saponin ions, namely ho-
tion coupled to a high resolution orbitrap analyzer (Li lothurinoside C (33) (m/z 1125), desholothurin A (12)
et al., 2014). (m/z 1141), holothurinoside E (47) (m/z 1287), ho-
In this study, Li et al. focused on different families of lothurinoside A (48) (m/z 1303), holothurinoside F (34)
active compounds, including flavonoids, chalcones, iso- (m/z 1433), holothurinoside G (49) (m/z 1449), ho-
flavanes and triterpene saponins. Glycyrrhizic acid (43) lothurinoside H (50) (m/z 1463), and holothurinoside I
and licorice saponins G2 (44), B2 (45), and J2 (46) are the (51) (m/z 1479) as [M + Na]+ ions (Van Dyck et al.,
major saponins found in licorice (Figure 21) (Li et al., 2011), see Figure 14 for selected molecules. As shown in
2014). Interestingly, the [M + Na]+ ions of 44 (m/z Figure 23, these saponins showed specific localizations
861.38793) and the [M + K]+ ions of 43 (m/z 861.36695) within the body wall cross section with the saponins
are isobaric with a mass difference of only 0.021 Da being mainly localized in the mesothelium (inner part of
(Figure 21), requiring a high resolution analyzer for their the body wall) for the relaxed animals. When the living
discrimination. animals were submitted to a smooth stress period
Figure 22 presents a mass spectrum from a rhizome (turning soft balls) before euthanasia, the MALDI‐MSI
cross section in the corresponding mass window reveal- profile was observed to be significantly affected, see
ing the coexistence of the [LS G2 + Na]+ ions and the Figure 23. The saponins have migrated toward the ex-
[GA + K]+ ions. Figure 22 also reveals that the spatial ternal part of the body wall, the so‐called epidermis, and
distributions of licorice saponin G2 (44) and glycyrrhizic some of them, typically m/z 1287 and 1303, were even no
acid (43) are strongly different across the rhizome (Li longer detected, suggesting their release in the sur-
et al., 2014) (Figure 22). rounding water for chemical defense (Van Dyck
Back to the marine animal saponins, we investigated et al., 2011).
using MALDI‐MSI the role of the holothuroid triterpene Several species of sea cucumbers of the family
glycosides in the chemical defense mechanisms of the sea Holothuriidae, particularly H. forskali, possess a par-
cucumber H. forskali (Van Dyck et al., 2011). Using ticular mechanical defense system, called the
 Supprimer filigrane Wondershare
20 | PDFelement
SAVARINO .
ET AL

F I G U R E 21 Molecular structures of the four major saponins found in licorice: glycyrrhizic acid (43) and licorice saponin G2 (44),
licorice saponin J2 (45), and licorice saponin B2 (46) (Li et al., 2014) [Color figure can be viewed at wileyonlinelibrary.com]

Cuvierian tubules (Van Dyck, Gerbaux, et al., 2010). It

was demonstrated that these organs are characterized
by a highly concentrated cocktail of specific saponins
(Van Dyck, Gerbaux, et al., 2010). Using MALDI‐MSI,
the precise localization of these saponins across the
Cuvierian tubules has been investigated with a special
attention paid at the stress/unstress state of the ani-
mals, before the euthanasia. Again, a differential
composition in saponins depending on the condition of
the animal has been detected and detection of varia-
tions in the saponin content at the molecular level was
made possible using MALDI‐imaging (Van Dyck,
Gerbaux, et al., 2010). The same type of analysis was
carried out on the sea star Asteria rubens and allowed
F I G U R E 22 MALDI‐MSI analysis (positive ion mode) of the us to obtain information on the distribution of sapo-
rhizome cross section showing the coexistence and the spatial nins within different organs. MALDI‐MSI performed at
distributions of [LS G2 + Na]+ and [GA + K] +ions. Adapted from different spatial resolutions revealed that the inter‐ and
(Li et al., 2014) [Color figure can be viewed at intra‐organ distributions of saponin congeners are not
wileyonlinelibrary.com] homogeneous (Demeyer et al., 2015).
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 21

F I G U R E 23 Molecular images pointing the localization of saponin ions (in green) in the body wall sections (epidermis on the top,
dermis in the middle and mesothelium on the bottom) obtained by MALDI‐MSI on relaxed (above) and stressed (below) Holothuria forskali:
holothurinoside C (33), desholothurin A (12), holothurinoside E (47), holothurinoside A (48), holothurinoside F (34), holothurinoside G
(49), holothurinoside H (50), and holothurinoside I (51). Adapted from (Van Dyck et al., 2011) [Color figure can be viewed at
wileyonlinelibrary.com]

8 | QUANTITATIVE ANALYSIS OF brown chromophore that is quantified using UV‐vis

SAPONINS spectrophotometry (450 nm). However, this method can
only measure the total saponin content and is not specific
The previous sections reveal that mass spectrometry enough. For the orcinol reaction, upon acidic hydrolysis
methods have nowadays reached a sufficiently high de- in harsh conditions, the released monosaccharides are
gree of maturity to allow identifying saponins in plant ultimately converted by dehydration in furfural deriva-
and animals extracts, as well as directly on tissue by tives that react with the Bial reagent (orcinol, HCl and
imaging methods. This is all the more remarkable that FeCl3) generating a blue chromophore whose con-
most of the time saponins occur as a multicomponent centration is determined using spectrophotometry
mixture with similar structures and polarities. As an methods (540 nm) (Dai et al., 2020). Interestingly, the
additional typical example, more than fifty oleanane‐type vanillin‐sulfuric acid and the orcinol assays may appear
triterpene (52) saponins were detected and identified in complementary since they target the aglycone and the
the Camellia sinensis seed extracts (Wu et al., 2019). glycan parts of the saponin molecules, respectively.
Although saponin extracts have been shown to pre- However, discrepancies may be encountered since both
sent a broad spectrum of biological properties and ap- assays are affected by bias such as the non‐specificity of
plication potentials, validated methods are lacking to the vanillin assay and the coexistence of hexose and
provide full quantitative analysis for saponins in extract. pentose in the polysaccharidic chains whose dehydration
Actually, saponin quantification remains challenging due products are different (Le et al., 2018; Dai et al., 2020).
to the lack of reference molecules such as isotopic la- Also, the orcinol assay do not considered the coexistence
belled internal standards (ISs) or even pure saponins. of saponin molecules presenting a different number of
This is often the case for many phytochemicals whose monosaccharidic residues (Dai et al., 2020; Van Dyck,
quantification remains challenging. Flammang, et al., 2010).
Generally, the total saponin content within an extract The total saponin content is often estimated based on
is determined using indirect methods such as the the determination of the hemolytic activity of the ex-
vanillin‐sulfuric acid assay (Le et al., 2018), the orcinol tracts, that is, the propensity to cleave the erythrocyte cell
reaction assay (Dai et al., 2020) or the measure of the membranes releasing hemoglobin in the extracellular
hemolytic activity (Habicht et al., 2011). The vanillin‐ medium (Kalinin et al., 1996; Mackie et al., 1968). The
sulfuric acid assay is the most common spectro- measurement of the hemolytic activity of the saponin
photometric method for saponin quantification since it is extracts thus reflects the effectiveness of the saponin
simple and fast to operate (Le et al., 2018). This proce- mixture to lyse erythrocytes. The quantification of the
dure starts from an acidic hydrolysis to release the free hemolytic activity is based on the determination of the
aglycones that further react with vanillin to produce a free hemoglobin concentration by spectrophotometry at
 Supprimer filigrane Wondershare
22 | PDFelement
SAVARINO .
ET AL

540 nm (Mackie et al., 1968). The spectrophotometric is an important medical herb used in traditional Chinese
measurements are converted to milligram equivalents of medicine (Kafle et al., 2020). Due to the availability of
plant saponins by gram of extract, using a standard curve astragaloside IV (55), Kafle et al. succeeded in developing
prepared with a reference saponin extract. This method a robust quantitative method based on the standard ad-
neglects that differences in saponin structures may affect dition method. Quantification by standard addition pro-
their hemolytic activity (Van Dyck, Flammang, vides an intrinsic correction of ion suppression or ion
et al., 2010). enhancement upon LC‐MS. This method provides better
Coupling liquid chromatography to mass spectrometry accuracy and can be used in the absence of an isotopic
and photo‐diode array detector respectively affords quali- labelled IS (Colson et al., 2020; Kafle et al., 2020). Ha et al.
tative and quantitative data for the characterization of developed a quantitative and qualitative method for the
phytochemicals such as flavonoids, for instance (Chen determination of saponins in Platycodi Radix by coupling
et al., 2012). At variance with saponins, numerous flavo- liquid chromatography to mass spectrometry for the
noids are commercially available and standard solutions qualitative dimension and to evaporative light scattering
can then be prepared for quantitative measurements. detection for the quantitative aspect (Ha et al., 2006). The
Moreover, saponins show very weak absorbance in the method has been validated for linearity, accuracy and
short wavelength range (<210 nm) rendering sensitive de- limit of detection (LOD). The linearity was established for
tection by ultraviolet spectroscopy almost impossible. Li- the ten analyzed saponins on the 0.3–20 µg range with a
quid chromatography ultraviolet analysis of saponins result LOD around 0.15 µg (Ha et al., 2006).
in a high level of baseline, limiting the choice of solvents Zhang et al. (2015) used LC‐MS/MS to simultaneously
and the use of mobile phase modifiers for improved se- analyze 5 steroid saponins in rat plasma. The quantifica-
paration (Ha et al., 2006). As a striking example, Wu et al. tion was achieved using MRM experiments in the positive
used ultra‐performance liquid chromatography diode array ion mode and selecting gingenoside RB1 (56) as the IS.
detection mass spectrometry (UPLC‐DAD‐MS) for the The selection of the IS was made based on its high ex-
qualitative and quantitative analysis of the saponins ex- traction efficiency and its chromatographic retention time
tracted from the C. sinensis seeds (Wu et al., 2019). They similar to the analytes. The MRM strategy was built on the
first isolated theasaponin E1 (53) from tea seeds and used it a priori determined major sensitive transitions for each of
as reference standard for their UV detection at 210 nm (Wu the [M + H]+ precursor ions and all correspond to sugar
et al., 2019). They compared the LC‐MS and LC‐DAD losses. MRM experiments were also advantageously im-
profiles of the crude extract, obtained by refluxing the plemented by Liu et al. (2015) to investigate the tissue
biomass in 70% methanol, and a saponin‐enriched extract, distribution of six major saponins present in rats after oral
obtained by consecutive extractions with different polar administration of Zhenqi Fuzheng capsules. Again, the
solvents (petroleum ether, ethyl acetate, 1‐butanol). Their quantification was performed using low‐energy collision
analysis reveals that, whereas the LC‐MS profile is almost CID in MRM scan modes using precursor ion→product
insensitive to the purity of the extracts, the LC‐DAD mea- ion transitions established based on CID experiments
surement is more applicable for detecting saponins at performed on standard molecules. Hesperedin (57)
higher concentrations. The second disadvantage of this was used as the IS. This study reveals that the
method is that all saponins are quantified by a unique saponin tissue concentrations decrease from spleen>-
standard substance that is moreover used as an external stomach>thymus>lung>liver>kidney>heart>testicle.
standard since it belongs to the extracted saponins. Re- Quantification of saponins within an extract remains
cently, we have qualitatively and quantitively characterized challenging and when analyzing saponin extracts with
the saponins extracted from the C. quinoa husks (Quinoa MS‐based methods, handling the data remains proble-
extract [QE]) only using LC‐MS experiments (Colson et al., matic for the comprehensive report of the results, but
2020). We introduced hederacoside C (54) (Sigma‐Aldrich) also for their efficient comparison. Often, identified sa-
as an IS to determine the mass proportions of all the sa- ponins are compiled in tables presenting all molecules
ponins detected in the quinoa extract. hederacoside C (54) together with, at least, their molecular masses, their
is extracted from the Hedera helix leaf and therefore is not chemical compositions, and the mass‐to‐charge ratios of
present in the QE (Havlíková et al., 2015). The molar pro- the corresponding ions. These tables, however, are
portion (% in the quinoa husk extract) and the mass frac- usually difficult to read and interpret. We therefore in-
tion (mg/g of the quinoa husk powder) of each saponin was troduced a representation in sector diagrams to combine,
estimated based on the ion signal ratios as determined by in a single graph, the data generated by MALDI, LC‐MS
LC‐MS experiments. and ion mobility experiments (Decroo et al., 2017). For a
Astragaloside IV (55) is one of the major ingredients given extract, MALDI‐ToF or LC‐MS analyses (together
present in the Astragali Radix, the root of Astragalus, that with HRMS measurements) are used to identify the
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 23

different saponin elemental compositions that are char- for a direct and fast comparison, both in terms of com-
acterized by their m/z ratio and their relative abundances position and relative proportion of the saponin contents
(as measured on the basis of peak intensities or LC signal in different extracts (Decroo et al., 2017).
integration). For the calculation of relative abundances, As a direct application of this representation, we de-
all isotopic signals are to be considered. If a saponin monstrated that the microwave‐assisted hydrolysis of the
molecule appears with different cationization, that is, bidesmosidic saponins extracted from the Quinoa Husk
[M + H] +and [M + Na]+, the ion intensities are added. is quantitative by the direct comparison between the
At a second level of description, the LC‐MS data are used sector diagrams built for the natural extracts and the
to characterize within each elemental composition (m/z hydrolyzed one (Colson et al., 2020).
ratio), the isomeric diversity. This is done by using the Indeed, as shown in Figure 25, saponin O (58) is a
retention time and the relative peak integration. Finally, [3 + 1] bidesmosidic saponin presenting a hydrolyzable
for each of the retention time, the ion mobility data ester function at C28, whereas the principal trisacchari-
(including the relative peak integration measured from dic chain is appended at C3 via an acetal function. Upon
the ATD) is added to identify coeluting isomeric saponins microwave heating at pH 10, 150°C for 5 min, saponin Oh
or catiomers. All those data are compiled within sector (59), the monodesmosidic counterpart molecule, is gen-
diagrams presenting different areas corresponding to the erated as demonstrated using mass spectrometry. The
different identified elemental compositions. The surfaces sector diagram presented in Figure 26a reveals that four
are labeled according to the m/z ratio of the detected [3 + 1] saponins are detected at m/z 1113, 1127, 1141, and
ions. In the diagram, the inner concentric circle re- 1157 from the natural extract, with the m/z 1157 saponin
presents the LC‐MS data, whereas the outer circle pre- being the most abundant. After the microwave‐assisted
sents the ion mobility experiment data (Decroo et al., hydrolysis, the [3 + 1] saponins have totally disappeared
2017). As for a typical example, we analyzed the saponins and are quantitively converted into their [3 + 0] coun-
extracted from different organs of H. forskali, namely the terparts as shown in the second sector diagram of
body wall, the gonads and the Cuvierian tubules, using Figure 26B. It is important to note that the relative pro-
MALDI‐MS, LC‐MS/MS, and LC‐IMS (Decroo et al., portions between the two sets of saponins, natural and
2017). We detected 10, 16, and 22 different saponins, that hydrolyzed, have remained constant, indicating that the
are four‐, five‐, and six‐sugar saponins, within the body hydrolysis is quantitative for the four molecules (Colson
wall, gonads, and Cuvierian tubules. Each organ was et al., 2020).
shown to contain a specific cocktail of saponins as ex-
emplified for the four‐sugar saponins in Figure 24. A
quick look at the comparison between the three sector 9 | MISCELLANEOUS
diagrams reveals that (i) two different saponin composi-
tions are present and detected at m/z 1125 and 1141 for Saponin‐rich plant extracts are often prescribed in traditional
the [M + Na]+ ions, (ii) that the relative abundances of medicine preparations due to their anti‐inflammatory, anti-
these compositions is organ dependent, and (iii) that the hepatotoxic, and immunostimulant activities (Moghimipour
m/z 1125 saponin is unique in the body wall, whereas & Handali, 2015). Based on the concept that protein‐ligand
one and two additional isomers are detected in the go- interactions are involved in numerous biological processes,
nads and the Cuvierian tubules, respectively. This data including drug‐related medicinal activities, the investigation
integration is useful for data interpretation since it allows of ex situ protein‐ligand complexes are of extreme

F I G U R E 24 Compilation of MALDI‐ToF‐MS, LC‐MS/MS and LC‐IMS analyses permitting to establish the distribution of the four‐sugar
saponins (m/z 1125 and 1141) in body wall (A), gonads (B), and Cuvierian tubules (C) with the retention times (a, min) and drift times
(b, ms; n.a., not attributed). Adapted from (Decroo et al., 2017) [Color figure can be viewed at wileyonlinelibrary.com]
 Supprimer filigrane Wondershare
24 | PDFelement
SAVARINO .
ET AL

F I G U R E 25 Microwave‐assisted hydrolysis of saponin O (58), extracted from Chenopodium quinoa, to saponin Oh (59). Adapted from
(Colson et al., 2020) [Color figure can be viewed at wileyonlinelibrary.com]

F I G U R E 26 Compilation of MALDI‐ToF‐MS, LC‐MS/MS and LC‐IMS analyses permitting to establish the composition of the [3 + 1]
saponins from Chenopodium quinoa (A) and the composition of the [3 + 0] saponins generated by the microwave‐assisted hydrolysis (B)
with retention times (a, min) and collisional cross section (b, Å2), showing a quantitative and nonspecific hydrolysis (pH 10, 150°C, 5 min).
Adapted from (Colson et al., 2020) [Color figure can be viewed at wileyonlinelibrary.com]

importance to decipher the role of small therapeutic mole- 2007). They further evaluated the interactions of ginse-
cules against different pathologies (Barczyk et al., 2005). nosides (29) and amino acids (AA) using ESI‐MS and 1:1
Cytochrome c (Cyt) is one of the deeply characterized pro- and 2:1 noncovalent complexes of ginsenosides (29) and
teins and often serves as a model protein for the study of amino acids (AA) were observed in the mass spectra.
ligand‐protein complexes (Barczyk et al., 2005). Based on the determined dissociation constants, they
The noncovalent complexes of cytochrome c (Cyt) concluded that the acidic and the basic amino acids,
and ginsenosides (29) have been studied using electro- more strongly bind to cytochrome c (Cyt) than other
spray Ionization mass spectrometry (ESI‐MS) and 1:1 and amino acids. This was further corroborated using theo-
1:2 complexes between cytochrome c (Cyt) and several retical calculations that reveal that H bond interactions
ginsenosides (29) have been detected (Zhang et al., 2007). are responsible for the stabilization of the detected
The dissociation constants of the 1:1 and 1:2 complexes complexes (Qu et al., 2009).
have been determined based on titration experiments by In 2012, Liu et al. investigated the noncovalent interac-
ascribing the ion abundances to the ion solution con- tions between cytochrome c (Cyt) and several saikosaponins
centrations, as it is often performed when studying a (60) and c (61) (Figure 27) extracted from Bupleurum fal-
ligand‐protein interactions using ESI‐MS (Zhang et al., catum. They first explored the binding propensity of
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 25

F I G U R E 27 Molecular structures of saikosaponin a (60) and saikosaponin c (61) (Liu et al., 2012) [Color figure can be viewed at
wileyonlinelibrary.com]

F I G U R E 28 ESI‐MS analysis (positive ion mode) showing the multiply charged complexes of cytochrome C (Cyt) with Saikosaponin c
(61) where ● represents the 1:1 noncovalent complexes and ○ represents the 1:2 noncovalent complexes, each with their respective state of
charge. Adapted from (Liu et al., 2012) [Color figure can be viewed at wileyonlinelibrary.com]

saikosaponins a (60) or c (61) with cytochrome c (Cyt) and formally arising from the condensation of a saccharide
detected 1:1 and 1:2 noncovalent complexes (Liu et al., 2012), chain—the glycan—onto a lipophilic triterpene—the
see Figure 28. Interestingly, they also separately monitored aglycone. The saponin family of molecules is char-
the noncovalent interactions between saponin fragments, acterized by such an extreme structural diversity that
that is, oligosaccharide and aglycone, to get insight into the their structural characterization remains difficult.
structure‐binding relationship of saponins with Cyt (Liu Mass spectrometry methods are constantly being de-
et al., 2012). Doing so, no complex ions between triterpenes veloped to allow the study of biological systems at the
and Cyt were detected, whereas similar dissociation constant molecular level. The study of saponins obviously benefits
(Kd) values were obtained for the Cyt complexes of saiko- from all these developments which allow for a possible
saponins or their fragment oligosaccharides. They hypothe- characterization of saponins within enriched extracts and
sized that the saponin glycosyl moiety represents the directly on tissues. Basically, most of the structural char-
effective interaction group with Cyt and they proposed that acterization MS‐based methods have been applied on sa-
saikosaponins and saccharides interact with Cyt by hydro- ponin ions, including GC‐MS, LC‐MS, MALDI‐MS,
gen bonds (Liu et al., 2012) (Figure 28). accurate mass measurements, CID, ion mobility experi-
ments, MALDI‐imaging, DESI‐MS, DART‐MS and native
mass spectrometry for the investigation of saponin/protein
10 | C ONC LUS I O NS interactions.
Whatever the current successes of the characterization
In the present review, we explored the many different of saponins by mass spectrometry, several aspects remain
possibilities offered by mass spectrometry for the in- problematic. First of all, from a structural point of view,
vestigation of saponins. Saponins are specific metabo- one of the current challenges concerns the establishment
lites present in plants and animals that mostly fulfill of the stereochemistry of the different stereogenic centers
defensive roles, even if they are nowadays presumed to present in saponins, including the glycosidic bond con-
play significant roles in the intra and interspecies figurations and the discrimination between epimeric su-
communications. Saponins are natural glycosides gars. It is likely that the development of the capabilities of
 Supprimer filigrane Wondershare
26 | PDFelement
SAVARINO .
ET AL

ion mobility experiments, in combination with multistep congeners from three tropical holothurian sea cucumbers.
CID experiments may afford nice pieces of information. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 2013; 166:
The complementarity between IMS and CID represents 182–193.
Budzikiewicz H, Wilson JM, Djerassi C. Mass spectrometry in
one of our research subjects nowadays, in conjunction
structural and stereochemical problems. XXXII. Pentacyclic
with theoretical chemistry.
triterpenes. J. Am. Chem. Soc. 1963; 85: 3688–3699.
The development of efficient and sensitive methods Burnouf-Radosevich M, Delfel NE, England R. Gas chromatography-
for the study of saponins and specific metabolites in mass spectrometry of oleanane- and ursane-type triterpenes—
general is a prerequisite for the establishment of their Aapplication to Chenopodium quinoa triterpenes. Phytochemistry.
structure/activity relationship for understanding their 1985; 24: 2063–2066.
biological in vivo roles, but also for securing their ap- Campagnuolo C, Fattorusso E, Taglialatela-Scafati O. Feroxosides A
plications in pharmaceutical applications. -B, two norlanostane tetraglycosides from the Caribbean sponge
Ectyoplasia ferox. Tetrahedron. 2001; 57: 4049–4055.
Caulier G, Flammang P, Gerbaux P, Eeckhaut I. When a repellent
ACKNOWLEDGMENTS
becomes an attractant: Harmful saponins are kairomones at-
We acknowledge the “Fonds National de la Recherche tracting the symbiotic Harlequin crab. Sci. Rep. 2013; 3: 1–5.
Scientifique” (FRS‐FNRS) for its contribution to the ac- Caulier G, Flammang P, Rakotorisoa P, Gerbaux P, Demeyer M,
quisition of the Waters QToF Premier and the Waters Eeckhaut I. Preservation of the bioactive saponins of Ho-
Synapt G2‐Si mass spectrometers that are extensively lothuria scabra through the processing of trepang. Cah. Biol.
used in our saponin investigations. PS, MD, CD and EC Mar. 2013; 54: 685–690.
thank the FRIA for their PhD fellowships. Chaieb I. Saponins as Insecticides: A Review. J. plant. Prot. 2017; 5:
39–50.
Chen J, Guo X, Song Y, Zhao M, Tu P, Jiang Y. MRM-based
REFERENCES strategy for the homolog-focused detection of minor ginse-
Adinolfi M, Mangoni L, Marino G, Parrilli M, Self R. Fast atom nosides from notoginseng total saponins by ultra-
bombardment mass spectrometry of the muscarosides. An aid performance liquid chromatography coupled with hybrid
to the glycoside sequence determination. Biol. Mass Spectrom. triple quadrupole-linear ion trap mass spectrometry. RSC
1984; 11: 310–314. Adv. 2016; 6: 96376–96388.
Amstalden van Hove ER, Smith DF, Heeren RMA. A concise re- Chen J, Tan M, Zou L, Liu X, Chen S, Shi J, Chen C, Wang C,
view of mass spectrometry imaging. J. Chromatogr. A. 2010; Mei Y. Qualitative and Quantitative Analysis of the Saponins
1217: 3946–3954. in Panacis Japonici Rhizoma using ultra-fast liquid chroma-
Arabski M, Wȩgierek-Ciuk A, Czerwonka G, Lankoff A, Kaca W. tography coupled with triple quadrupole-time of flight tandem
Effects of saponins against clinical E. coli strains and eu- mass spectrometry and ultra-fast liquid chromatography
karyotic cell line. J. Biomed. Biotechnol. 2012; : 1–6. coupled with triple quadrupole-linear ion trap tandem mass
Augustin JM, Kuzina V, Andersen SB, Bak S. Molecular activities, spectrometry. Chem. Pharm. Bull. 2019; 67: 839–848.
biosynthesis and evolution of triterpenoid saponins. Chen S, Wu BH, Fang JB, Liu YL, Zhang HH, Fang LC, Guan L,
Phytochemistry 2011; 72: 435–457. Li SH. Analysis of flavonoids from lotus (Nelumbo nucifera)
Bahrami Y, Franco C. Structure elucidation of new acetylated sa- leaves using high performance liquid chromatography/pho-
ponins, lessoniosides A, B, C, D, and E, and non-acetylated todiode array detector tandem electrospray ionization mass
saponins, lessoniosides F and G, from the viscera of the sea spectrometry and an extraction method optimized by ortho-
cucumber Holothuria lessoni. Mar. Drugs. 2015; 13: 597–617. gonal design. J. Chromatogr. A. 2012; 1227: 145–153.
Bahrami Y, Zhang W, Chataway T, Franco C. Structural elucida- Claeys M, Van den Heuvel H, Chen S, Derrick PJ, Mellon F,
tion of novel saponins in the sea cucumber Holothuria. Mar. Price KR. Comparison of high- and low energy collision-
Drugs. 2014; 12: 4439–4473. induced dissociation tandem mass spectrometry in the ana-
Bahrami Y, Zhang W, Franco C. Discovery of novel saponins from lysis of glycoalkaloids and their aglycons. J. Am. Soc. Mass
the viscera of the sea cucumber Holothuria lessoni. Mar. Spectrom. 1996; 7: 173–181.
Drugs. 2014; 12: 2633–2667. Colson E, Decroo C, Cooper-Shepherd D, Caulier G, Henoumont C,
Bai H, Wang S, Liu J, Gao D, Jiang Y, Liu H, Cai Z. Localization of Laurent S, De Winter J, Flammang P, Palmer M,
ginsenosides in Panax ginseng with different age by matrix- Claereboudt J, Gerbaux P. Discrimination of regioisomeric
assisted laser-desorption/ionization time-of-flight mass spec- and stereoisomeric saponins from Aesculus hippocastanum
trometry imaging. J. Chromatogr. B. 2016; 1026: 263–271. seeds by ion mobility mass spectrometry. J. Am. Soc. Mass
Bankefors J, Broberg S, Nord LI, Kenne L. Electrospray ionization Spectrom. 2019; 30: 2228–2237.
ion-trap multiple-stage mass spectrometry of Quillaja sapo- Colson E, Savarino P, Claereboudt E, Cabrera-Barjas G, Deleu M,
nins. J. Mass Spectrom. 2011; 46: 658–665. Lins L, Eeckhaut I, Flammang P, Gerbaux P. Enhancing the
Barczyk K, Kreuter M, Pryjma J, Booy EP, Maddika S, Ghavami S, membranolytic activity of Chenopodium quinoa saponins by
Berdel WE, Roth J, Los M. Serum cytochrome c indicatesin fast microwave hydrolysis. Molecules. 2020; 25: 1731.
vivo apoptosis and can serve as a prognostic marker during Costello CE. 1996. Application of tandem mass spectral approaches
cancer therapy. Int. J. Cancer. 2005; 116: 167–173. to structural determinations of saponins. In: Waller GR,
Bondoc KGV, Lee H, Cruz LJ, Lebrilla CB, Juinio-Meñez MA. Yamasaki K, editors. Saponins used in food and agriculture.
Chemical fingerprinting and phylogenetic mapping of saponin Boston: Springer. p 317–330.
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 27

Dai YL, Kim EA, Luo HM, Jiang YF, Oh JY, Heo SJ, Jeon YJ. and compounds in bitter gourd varieties. Food Chem. 2011;
Characterization and anti-tumor activity of saponin-rich 126: 172–176.
fractions of South Korean sea cucumbers (Apostichopus japo- Haralampidis K, Trojanowska M, Osbourn AE. Biosynthesis of
nicus). J. Food Sci. Technol. 2020; 57: 2283–2292. triterpenoid saponins in plants. Adv. Biochem. Eng. Biotechnol.
Decroo C, Colson E, Demeyer M, Lemaur V, Caulier G, Eeckhaut I, 2002; 75: 31–49.
Cornil J, Flammang P, Gerbaux P. Tackling saponin diversity Havlíková L, Macáková K, Opletal L, Solich P. Rapid determination
in marine animals by mass spectrometry: data acquisition and of α-hederin and hederacoside C in extracts of Hedera helix
integration. Anal. Bioanal. Chem. 2017; 409: 3115–3126. leaves available in the Czech Republic and Poland. Nat. Prod.
Decroo C, Colson E, Lemaur V, Caulier G, De Winter J, Cabrera‐ Commun. 2015; 10: 1529–1531.
Barjas G, Cornil J, Flammang P, Gerbaux P. Ion mobility mass He Y, Liu W, Su R, Xiu Y, Pei J. Detection of saponins and oligo-
spectrometry of saponin ions. Rapid Commun. Mass Spectrom. saccharides in herbs using direct analysis in real-time mass
2019; 33: 22–33. spectrometry. Chem. Res. Chinese Univ. 2017; 33: 172–178.
Demeyer M, De Winter J, Caulier G, Eeckhaut I, Flammang P, Huang FQ, Dong X, Yin X, Fan Y, Fan Y, Mao C, Zhou W. A mass
Gerbaux P. Molecular diversity and body distribution of sa- spectrometry database for identification of saponins in plants.
ponins in the sea star Asterias rubens by mass spectrometry. J. Chrom. A. 2020; 1625: 461296.
Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 2014; 168: Ikekawa N, Natori S, Itokawa H, Tobinaga S, Matsui M. Gas
1–11. chromatography of triterpenes. I. Ursanane, oleanane, and
Demeyer M, Wisztorski M, Decroo C, De Winter J, Caulier G, lupane groups. Chem. Pharm. Bull. 1965; 13: 316–319.
Hennebert E, Eeckhaut I, Fournier I, Flammang P, Gerbaux P. Jeong EK, Ha IJ, Kim YS, Na YC. Glycosylated platycosides:
Inter- and intra-organ spatial distributions of sea star saponins Identification by enzymatic hydrolysis and structural de-
by MALDI imaging. Anal. Bioanal. Chem. 2015; 407: termination by LC-MS/MS. J. Sep. Sci. 2014; 37: 61–68.
8813–8824. Jæger D, Ndi CP, Crocoll C, Simpson BS, Khakimov B, Guzman‐
Desai SD, Desai DG, Kaur H. Saponins and their biological activ- Genuino RM, Hayball JD, Xing X, Bulone V, Weinstein P,
ities. Pharma Times. 2009; 41: 13–16. Møller BL, Semple SJ. Isolation and structural characteriza-
Domon B, Costello CE. A systematic nomenclature for carbohy- tion of echinocystic acid triterpenoid saponins from the aus-
drate fragmentations in FAB-MS/MS spectra of glycoconju- tralian medicinal and food plant Acacia ligulata. J. Nat. Prod.
gates. Glycoconj. J. 1988; 5: 397–409. 2017; 80: 2692–2698.
Gabelica V, Marklund E. Fundamentals of ion mobility spectro- Kafle B, Baak J, Brede C. Quantification by LC–MS/MS of as-
metry. Curr. Opin. Chem. Biol. 2018; 42: 51–59. tragaloside IV and isoflavones in Astragali radix can be more
Gadea A, Fanuel M, Le Lamer AC, Boustie J, Rogniaux H, accurate by using standard addition. Phytochem. Anal. 2020;
Charrier M, Lohézic-Le Devehat F. Mass spectrometry ima- 32: 466–473.
ging of specialized metabolites for predicting lichen fitness Kalinin VI, Prokofieva NG, Likhatskaya GN, Schentsova EB,
and snail foraging. Plants. 2020; 9: 70. Agafonova IG, Avilov SA, Drozdova OA. Hemolytic activities
Garneau FX, Simard JL, Harvey O, Apsimon JW, Girard M. The of triterpene glycosides from the holothurian order den-
structure of psoluthurin A, the major triterpene glycoside of drochirotida: Some trends in the evolution of this group of
the sea cucumber Psolus fabricii. Can. J. Chem. 1983; 61: toxins. Toxicon. 1996; 34: 475–483.
1465–1471. Kanchanapoom T, Kasai R, Yamasaki K. Acetylated triterpene sa-
Gevrenova R, Bardarov V, Bardarov K, Voutquenne-Nazabadioko ponins from the Thai medicinal plant, Sapindus emarginatus.
L, Henry M. Selective profiling of saponins from Gypsophila Chem. Pharm. Bull. 2001; 49: 1195–1197.
trichotoma Wend. by HILIC separation and HRMS detection. Kang HK, Seo C, Park Y. The effects of marine carbohydrates and
Phytochem. Anal. 2018; 29: 250–274. glycosylated compounds on human health. Int. J. Mol. Sci.
Gevrenova R, Doytchinova I, Kołodziej B, Henry M. In-depth 2015; 16: 6018–6056.
characterization of the GOTCAB saponins in seven cultivated Kassem AS, Ahmed AM, Tariq MR. Study of saponins in methanol
Gypsophila L. species (Caryophyllaceae) by liquid chromato- extract of the leaves of Acacia etbaica subspecies etbaica. Res.
graphy coupled with quadrupole-Orbitrap mass spectrometer. J. Pharm. Biol. Chem. Sci. 2014; 5: 803–810.
Biochem. Syst. Ecol. 2019; 83: 91–102. Khorlin AY, Chirva VY, Kochetkov NK. Triterpenoid saponins
Ghisalberti EL. Steroidal glycoalkaloids: IsolatiOn, Structure, communication 15. Clematoside C-A triterpenoid oligoside
Analysis, And Biosynthesis. Nat. Prod. Commun. 2006; 1: from the roots of the manchurian clematis (Clematis man-
859–884. shurica RUPR.). Bull. Acad. Sci. USSR Div. Chem. Sci. 1965; 14:
Giles K, Ujma J, Wildgoose J, Pringle S, Richardson K, 790–795.
Langridge D, Green M. A cyclic ion mobility-mass spectro- Kim HJ, Park SR, Jang YP. Extraction-free in situ derivatisation of
metry system. Anal. Chem. 2019; 91: 8564–8573. timosaponin aiii using direct analysis in real time TOF/MS.
Ha YW, Na YC, Seo JJ, Kim SN, Linhardt RJ, Kim YS. Qualitative Phytochem. Anal. 2014; 25: 373–377.
and quantitative determination of ten major saponins in Pla- Kite GC, Howes MJR, Simmonds MSJ. Metabolomic analysis of
tycodi Radix by high performance liquid chromatography with saponins in crude extracts of Quillaja saponaria by liquid
evaporative light scattering detection and mass spectrometry. chromatography/mass spectrometry for product authentica-
J. Chromatogr. A. 2006; 1135: 27–35. tion. Rapid Commun. Mass Spectrom. 2004; 18: 2859–2870.
Habicht SD, Kind V, Rudloff S, Borsch C, Mueller AS, Pallauf J, Kofler L, Adam PA. Die Wertbestimmung der Saponindrogen.
Yang R, Krawinkel MB. Quantification of antidiabetic extracts Arch. Pharm. (Weinheim). 1927; 265: 624–652.
 Supprimer filigrane Wondershare
28 | PDFelement
SAVARINO .
ET AL

Kuljanabhagavad T, Thongphasuk P, Chamulitrat W, Wink M. Oleszek W, Hamed A. 2010. Saponin-based surfactants. In:
Triterpene saponins from Chenopodium quinoa Willd. Kjellin M, Johansson I, editors. Surfactants from renewable
Phytochemistry. 2008; 69: 1919–1926. resources. New Jersey: John Wiley & Sons. p 239–249.
Le AV, Parks SE, Nguyen MH, Roach PD. Improving the vanillin- Osbourn AE, Qi X, Townsend B, Qin B. Dissecting plant secondary
sulphuric acid method for quantifying total saponins. metabolism—Constitutive chemical defences in cereals. New
Technologies 2018; 6: 84. Phytol. 2003; 159: 101–108.
Li B, Bhandari DR, Janfelt C, Römpp A, Spengler B. 2014. Natural Pettolino FA, Walsh C, Fincher GB, Bacic A. Determining the
products in Glycyrrhiza glabra (licorice) rhizome imaged at the polysaccharide composition of plant cell walls. Nat. Protoc.
cellular level by atmospheric pressure matrix-assisted laser 2012; 7: 1590–1607.
desorption/ionization tandem mass spectrometry imaging. Plant Le Pogam P, Legouin B, Geairon A, Rogniaux H, Lohézic-Le Dé-
J. 80:161–171. véhat F, Obermayer W, Boustie J, Le Lamer AC. Spatial
Li W, Sun Y, Wang Z, Zheng Y. Isolation and purification of sa- mapping of lichen specialized metabolites using LDI-MSI:
ponins from Platycodon grandiflorum by semi-preparative Chemical ecology issues for Ophioparma ventosa. Sci. Rep.
high performance liquid chromatography and LC-ESI/MS. 2016; 6: 37807.
J. Liq. Chromatogr. Relat. Technol. 2012; 35: 547–557. Popov RS, Ivanchina NV, Silchenko AS, Avilov SA, Kalinin VI,
Lin S, Wang D, Yang D, Yao J, Tong Y, Chen J. Characterization of Dolmatov IY, Stonik VA, Dmitrenok PS. Metabolite profiling of
steroidal saponins in crude extract from Dioscorea nipponica triterpene glycosides of the far eastern sea cucumber Eupentacta
Makino by liquid chromatography tandem multi-stage mass fraudatrix and their distribution in various body components
spectrometry. Anal. Chim. Acta. 2007; 599: 98–106. using LC-ESI QTOF-MS. Mar. Drugs. 2017; 15: 302.
Liu G, Zhang Z, Lv X, Zhan S, Ding B, Yang X, Zhu Q. Localization Prome J-C, Aurelle H, Prome D, Savagnac A. Gas phase glycosidic
of malonyl and acetyl on substituted saikosaponins according cleavage of oxynions from alkyl glycosides. Org. Mass
to the full-scan mass spectra and the fragmentation of sodium- Spectrom. 1987; 22: 6–12.
adduct ions in the positive mode. Rapid Commun. Mass Qu C, Yang L, Yu S, Wang S, Bai Y, Zhang H. Investigation of the
Spectrom. 2019; 33: 883–893. interactions between ginsenosides and amino acids by mass
Liu XH, Zhu RJ, Hu F, Guo L, Yang YL, feng SI. Tissue distribution spectrometry and theoretical chemistry. Spectrochim. Acta
of six major bio-active components after oral administration of Part A Mol. Biomol. Spectrosc. 2009; 74: 478–483.
Zhenqi Fuzheng capsules to rats using ultra-pressure liquid Quinn R, Basanta-Sanchez M, Rose RE, Fabris D. Direct infusion
chromatography-tandem mass spectrometry. J.Chrom.B. 2015; analysis of nucleotide mixtures of very similar or identical
986-987: 44–53. elemental composition. J. Mass Spectrom. 2013; 48: 703–712.
Liu Y, Su B, Wang X. Study on the noncovalent interactions of saiko- Rodriguez J, Castro R, Riguera R. Holothurinosides: New antitumour
saponins and cytochrome c by electrospray ionization mass non sulphated triterpenoid glycosides from the sea cucumber
spectrometry. Rapid Commun. Mass Spectrom. 2012; 26: 719–727. Holothuria forskalii. Tetrahedron. 1991; 47: 4753–4762.
Mackie AM, Grant PT, Lasker R. Avoidance reactions of a mollusc Sahu NP, Banerjee S, Mondal NB, Mandal D. Steroidal saponins.
Buccinum undatum to saponin-like surface-active substances Fortschr. Chem. Org. Naturst. 2008; 89: 45–141.
in extracts of the starfish Asterias rubens and Marthasterias Sandvoss M, Pham LH, Levsen K, Preiss A, Mügge C, Wünsch G.
glacialis. Comp. Biochem. Physiol. 1968; 26: 415–428. Isolation and structural elucidation of steroid oligoglycosides
Mackie AM, Singh HT, Fletcher TC. Studies on the cytolytic effects from the starfish Asteria rubens by means of direct online LC-
of seastar (Marthasterias glacialis) saponins and synthetic NMR-MS hyphenation and one- and two-dimensional NMR
surfactants in the plaice Pleuronectes platessa. Mar. Biol. 1975; investigations. Eur. J. Org. Chem. 2000; 2000: 1253–1262.
29: 307–314. Sandvoss M, Preiss A, Levsen K, Weisemann R, Spraul M. Two new
Maier MS. 2008. Biological activities of sulfated glycosides from asterosaponins from the starfish Asteria rubens: Application of a
echinoderms. In: Rahman AU, editor. Studies in natural cryogenic NMR probe head. Magn. Reson. Chem. 2003; 41:
products chemistry. Amsterdam: Elsevier B.V. p 311–354. 949–954.
McKey D. Adaptive patterns in alkaloid physiology. Am. Nat. 1974; Sandvoss M, Weltring A, Preiss A, Levsen K, Wuensch G. Combi-
108: 305–320. nation of matrix solid-phase dispersion extraction and direct
Mert-Türk F, Osbourn A. Saponins versus plant fungal pathogens. on-line liquid chromatography–nuclear magnetic resonance
Trends Plant Sci. 2006; 5: 13–17. spectroscopy–tandem mass spectrometry as a new efficient
Mesleh MF, Hunter JM, Shvartsburg AA, Schatz GC, Jarrold MF. approach for the rapid screening of natural products:
Structural information from ion mobility measurements: Ef- J. Chromatogr. A. 2001; 917: 75–86.
fects of the long-range potential. J. Phys. Chem. 1996; 100: Shi J, Cai Z, Chen S, Zou L, Liu X, Tang R, Ma J, Wang C, Chen J,
16082–16086. Tan M. Qualitative and quantitative analysis of saponins in
Moghimipour E, Handali S. Saponin: properties, methods of eva- the flower bud of Panax ginseng (Ginseng Flos) by UFLC-
luation and applications. Annu. Res. Rev. Biol. 2015; 5: 207–220. Triple TOF-MS/MS and UFLC-QTRAP-MS/MS. Phytochem.
Nord LI, Kenne L. Separation and structural analysis of saponins in Anal. 2020; 31: 287–296.
a bark extract of Quillaja saponaria Molina. Carbohydr. Res. Shvartsburg AA, Jarrold MF. An exact hard-spheres scattering
1999; 320: 70- 81. model for the mobilities of polyatomic ions. Chem. Phys. Lett.
Nyakudya E, Jeong JH, Lee NK, Jeong YS. Platycosides from the 1996; 261: 86–91.
roots of platycodon grandiflorum and their health benefits. De Simone F, Dini A, Finamore E, Minale L, Pizza C, Riccio R,
Prev. Nutr. Food Sci. 2014; 19: 59–68. Zollo F. Starfish saponins. Part 5. Structure of sepositoside A, a
 Supprimer filigrane Wondershare
SAVARINO ET AL. PDFelement
| 29

novel steroidal cyclic glycoside from the starfish Echinaster extraction and high-performance liquid chromatography tandem
sepositus. J. Chem. Soc. Perkin Trans. 1981; 1: 1855–1862. mass spectrometry. Anal. Biochem. 2011; 419: 323–332.
Song F, Cui M, Liu Z, Yu B, Liu S. Multiple-stage tandem mass Xu HJ, Shi XW, Ji X, Du YF, Zhu H, Zhang LT. A rapid method for
spectrometry for differentiation of isomeric saponins. Rapid simultaneous determination of triterpenoid saponins in Pul-
Commun. Mass Spectrom. 2004; 18: 2241–2248. satilla turczaninovii using microwave-assisted extraction and
Song F, Liu Z, Liu S, Cai Z. Differentiation and identification of high performance liquid chromatography–tandem mass
ginsenoside isomers by electrospray ionization tandem mass spectrometry. Food Chemistry. 2012; 135: 251–258.
spectrometry. Anal. Chim. Acta. 2005; 531: 69–77. Yang Y, Laval S, Yu B. Chemical synthesis of saponins. Adv.
Stonik VA. Some terpenoid and steroid derivatives from echi- Carbohydr. Chem. Biochem. 2014; 71: 137–226.
noderms and sponges. Pure Appl. Chem. 1986; 58: 423–436. Yang Y, Yang Y, Qiu H, Ju Z, Shi Y, Wang Z, Yang L. Localization
Taira S, Ikeda R, Yokota N, Osaka I, Sakamoto M, Kato M, Sahashi Y. of constituents for determining the age and parts of ginseng
Mass Spectrometric imaging of ginsenosides localization in Panax through ultraperfomance liquid chromatography quadrupole/
ginseng Root. Am. J. Chin. Med. 2010; 38: 485–493. time of flight-mass spectrometry combined with desorption
Uetrecht C, Rose RJ, van Duijn E, Lorenzen K, Heck AJR. Ion electrospray ionization mass spectrometry imaging. J. Pharm.
mobility mass spectrometry of proteins and proteinassemblies. Biomed. Anal. 2021; 193: 113722.
Chem. Soc. Rev. 2010; 39: 1633–1655. Yasumoto T, Tanaka M, Hashimo Y. Distribution of saponin in
Van Dyck S, Caulier G, Todesco M, Gerbaux P, Fournier I, echinoderms. Nippon Suisan Gakkaishi. 1966; 32: 673–676.
Wisztorski M, Flammang P. The triterpene glycosides of Ho- Yoshikawa M, Murakami T, Matsuda H, Yamahara J, Murakami N,
lothuria forskali: Usefulness and efficiency as a chemical defense Kitagawa I. Bioactive saponins and glycosides. III. Horse
mechanism against predatory fish. J. Exp. Biol. 2011; 214: chestnut. (1): The structures, inhibitory effects on ethanol
1347–1356. absorption, and hypoglycemic activity of escins Ia, Ib, IIa, IIb,
Van Dyck S, Flammang P, Meriaux C, Bonnel D, Salzet M, and IIIa from the seeds of Aesculus hippocastanum L. Chem.
Fournier I, Wisztorski M. Localization of secondary metabo- Pharm. Bull. 1996; 44: 1454–1464.
lites in marine invertebrates: Contribution of MALDI MSI for Zehl M, Pittenauer E, Jirovertz L, Bandhari P, Singh B, kaul VK,
the study of saponins in cuvierian tubules of H. forskali. PLOS Rizzi A, Allmaier G. Multistage and tandem mass spectrometry
One. 2010; 5: e13923. of glycosylated triterpenoid saponins isolated from Bacopa
Van Dyck S, Gerbaux P, Flammang P. Elucidation of molecular monnieri: Comparison of the information content provided by
diversity and body distribution of saponins in the sea cu- different techniques. Anal. Chem. 2007; 79: 8214–8221.
cumber Holothuria forskali (Echinodermata) by mass spec- Zhang H, Ding L, Qu C, Li D, Zhang H. Study on the noncovalent
trometry. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. complexes of ginsenoside and cytochrome c by electrospray
2009; 152: 124–134. ionization mass spectrometry. Spectrochim. Acta Part A Mol.
Van Dyck S, Gerbaux P, Flammang P. Qualitative and quantitative Biomol. Spectrosc. 2007; 68: 312–316.
saponin contents in five sea cucumbers from the Indian ocean. Zhang PC, Zheng CF, Wu CS, Sheng YX, Zhang JL. Mass spectral
Mar. Drugs. 2010; 8: 173–189. fragmentation analysis of triterpene saponins from Ardisia
Vincken JP, Heng L, de Groot A, Gruppen H. Saponins, classifi- crenata Sims by electrospray ionization Fourier transform ion
cation and occurrence in the plant kingdom. Phytochemistry. cyclotron resonance mass spectrometry. J. Asian Nat. Prod.
2007; 68: 275–297. Res. 2010; 12: 64–69.
Wang W, Wang GJ, Xie HT, Sun JG, Zhao S, Jiang XL, Li H, Zhang X, Li J, Ito Y, Sun W. Simultaneous quantification of five
Xu HLMJ, Wang R. Determination of ginsenoside Rd in steroid saponins from Dioscorea zingiberensis C.H. Wright in
dog plasma by liquid chromatography–mass spectrometry rat plasma by HPLC–MS/MS and its application to the phar-
after solid-phase extraction and its application in dog macokinetic studies. Steroids. 2015; 93: 16–24.
pharmacokinetics studies. J. Chromatogr. B. 2007; 852: Zheng W, Wang F, Zhao Y, Sun X, Kang L, Fan Z, Qiao L, Yan R,
8–14. Liu S, Ma B. Rapid characterization of constituents in tribulus
Wang W, Zhao Y, Jing W, Zhang J, Xiao H, Zha Q, Liu A. terrestris from different habitats by UHPLC/Q-TOF MS. J. Am.
Ultrahigh-performance liquid chromatography-ion trap mass Soc. Mass Spectrom. 2017; 28: 2302–2318.
spectrometry characterization of the steroidal saponins of
Dioscorea panthaica Prain et Burkill and its application for AU T HO R B I O G R A P HI E S
accelerating the isolation and structural elucidation of ster-
oidal saponins. Steroids. 2015; 95: 51–65.
Wei F, Ma LY, Cheng XL, Lin RC, Jin WT, Khan IA, Lu JQ. Pre- Philippe Savarino obtained his MSc degree
parative HPLC for purification of four isomeric bioactive sa- in Chemistry from the University of Mons,
ponins from the seeds of Aesculus chinensis. J. Liq. Belgium, in 2019. He is currently PhD
Chromatogr. Relat. Technol. 2005; 28: 763–773. student in the Laboratory of Organic
Wu X, Jia L, Wu J, Liu Y, Kang H, Liu X, Li P, He P, Tu Y, Li B. Synthesis and Mass Spectrometry (S2MOs)
Simultaneous Determination and quantification of triterpene at the University of Mons and the Labora-
saponins from Camellia sinensis seeds using UPLC-PDA-
tory of Phytopathology, Microbial and Molecular Farm-
QTOF-MS/MS. Molecules. 2019; 24: 3794.
Xu H, Ji X, Shi X, Du Y, Zhu H, Zhang L. Development of a novel
ing (PMMF) at the “Haute Ecole Provinciale du Hainaut
method for triterpenoidal saponins in rat plasma by solid-phase Condorcet”, under the supervision of Prof Pascal
 Supprimer filigrane Wondershare
30 | PDFelement
SAVARINO .
ET AL

Gerbaux and Dr Nicolas Desoignies. His work focuses on

the evaluation of saponins as bio‐based fungicides for Emmanuel Colson graduated from the
organic farming via a structure/activity approach by mass University of MONS (UMONS), Belgium
spectrometry. in 2020. He realized his PhD thesis
within the Laboratory of Organic Synth-
esis and Mass Spectrometry (S2MOs), in
Marie Demeyer obtained her PhD in
partnership with the Biology of Marine
Chemistry from the University of Mons,
Organisms and Biomimetics laboratory (BOMB) at
Belgium, in 2015. Her thesis focused on
UMONS under the supervision of Prof Pascal
the study of the saponins of the starfish
Gerbaux and Prof Patrick Flammang. His thesis
Asterias rubens by mass spectrometry
focused on green waste valorization by tuning the
under the supervision of Prof Pascal
biological properties of horse chestnut and quinoa
Gerbaux (Laboratory of Organic Synthesis and Mass
husk saponins. He actually works as scientist at
Spectrometry ‐ S2MOs) and Prof Patrick Flammang
Quality Assistance (Thuin), a company offering
(Biology of Marine Organisms and Biomimetism –
product‐dedicated expertise in analytical sciences.
BOMB). She is currently Project Associate at Certech
(Seneffe) in the materials and environment group.
Her work consists in evaluating the VOC (volatil Pascal Gerbaux has obtained his PhD in
organic compounds) emissions emitted by automotive Science from the University of Mons‐
interior materials by GC‐MS analyses. Hainaut in 1999 under the supervision of
Prof Robert Flammang. After having
completed several postdoctoral stays
Decroo Corentin obtained his PhD in
abroad, i.e. Ecole Polytechnique de
Sciences from the University of Mons –
Palaiseau, University of Washington and University
Belgium – in 2018. The thesis subject
of Utrecht, he got a permanent position at the
consisted in the application of ion
University of Mons as a F.N.R.S. Senior Research
mobility mass spectrometry to achieve
Associate. For years, his research topics are related to
the full structural characterization of
the study of gas phase ions using mass spectrometry
saponins extracted from plants and marine animals.
methods. He is now Full Professor of organic
This thesis was conducted under the supervision of
chemistry and mass spectrometry and head of the
Prof Pascal Gerbaux (Laboratory of Organic Synthesis
Organic Synthesis and Mass Spectrometry Laboratory
and Mass Spectrometry ‐ S2MOs) and Prof Patrick
at UMONS.
Flammang (Biology of Marine Organisms and Biomi-
metism – BOMB) in collaboration with Dr. Jérôme
Cornil (Laboratory for Chemistry of Novel Materials –
CMN). He is currently Scientific Coordinator in the How to cite this article: Savarino P, Demeyer M,
Bioprofiling platform (UMONS‐ULB) ‐ part of MS Decroo C, Colson E, Gerbaux P. Mass spectrometry
QUANTA (UMONS‐ULIEGE). His work consists in analysis of saponins. Mass Spectrometry Reviews,
MRM method development for biomarkers (Peptides/ (2021);1‐30. https://doi.org/10.1002/mas.21728
Proteins) found in bacteria, tissues, or cells.