2.1.3. Hydrogenation of the Exocyclic Double Bond

Catalytic hydrogenation of the exocyclic olefinic bond has also been studied in detail for some DGs and the sensory evaluation of their derivatives has also been reported. In this reaction, both isomers, α-CH3 and β-CH3 at position C-16 of the aglycone have been obtained and characterized Figure 2.

**Figure 2.** Catalytic hydrogenation of the exocyclic double bond in some steviol glycosides.

Some diterpene glycoside compounds have been prepared from rebaudiosides A, B, C and D using different catalysts and reaction conditions [43,44]. The organoleptic properties of the mixture of the reduced α-CH3 and β-CH3 diterpene glycoside isomers have been assayed. The reduction of the exocyclic olefinic bond at C-16 decreased the sweet taste of the natural DGs from 25 to 75%, hence the reductive modification of this site of the aglycone steviol is not a good target for improving the organoleptic properties of natural DGs.

#### 2.1.4. Enzymatic Reactions

Steviol glycosides and gibberellins share the first steps of the biosynthetic pathway where the pathway splits after the formation of *ent*-kaurenoic acid to yield gibberellins and steviol. Steviol glycosides are formed after glycosylation by means of the Uridine 5- -diphospho-glycosylltransferase enzymes producing glucosylation, xylosylation, rhamnosylation, etc. in different positions contributing to the formation of wide spectrum in monosaccharide arrangements [45,46].

The preparation of known and new DGs by using glycosyltransferase enzymes and/or genetically modified microorganisms (e.g., *Saccharomyces cerevisiae* strains) have been used as an approach for producing DGs with improved taste. Glycosyltransferase enzymes transfer glycosyl moieties from an activated nucleoside monosaccharide into mono or oligosaccharide acceptor containing molecules. Some glycosyltransferase enzymes retain or invert the stereochemistry of the substrates. Some scientific articles have been recently published and various patents have been granted.

Some of the examples found in the literature retaining the stereochemistry of the substrate are the bioconversion of rebaudioside A into rebaudioside I and M2 (rebaudioside M isomer with Glcβ(1-6)[Glcβ(1-2)]Glcβ1- at C-19) and the bioconversion of rebaudioside D3 into (Rebaudioside D isomer with Glcβ(1-6)[Glcβ(1-2)]-Glcβ1- at C-13) from rebaudioside E employing UDP-glycosyltransferases using uridine 5-diphosphoglucose (UDP-glucose) as a donor of the sugar moiety.

In the same way, some new minor DGs with inversion of the stereochemistry of some anomeric protons were isolated from a cyclodextrin glycosyltransferase glucosylated *Stevia* extract containing a high percentage of DGs. Interestingly, α-glucosyl linkages were mainly detected at position 4 of some sugar monosaccharide of moieties attached at position C-13 and/or C-19 of the steviol aglycone: 13-[(2-*O*-β-D-glucopyranosyl-3-*O*-(4-*O*-α-D-glucopyranosyl)-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-*O*-α-D-glucopyranosyl-β-D-glucopyranosyl) ester]; 13-[(2-*O*-β-Dglucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-*O*-(4-*O*-(4-*O*-α-Dglucopyranosyl)-α-D-glucopyranosyl)-α-D-glucopyranosyl)-β-D-glucopyranosyl ester]; 13- [(2-*O*-β-D-glucopyranosyl-3-*O*-(4-*O*-(4-*O*-(4-*O*-α-D-glucopyranosyl)-α-D-glucopyranosyl) α-D-glucopyranosyl)-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur16-en-19-oic acid β-D-glucopyranosyl ester and 13-[(2-*O*-β-D-glucopyranosyl-3-*O*-(4-*O*- (4-*O*-(4-*O*-α-Dglucopyranosyl)-α-D-glucopyranosyl)-α-D-glucopyranosyl)-β-D-glucopyranosyl-β-Dglucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-*O*-α-D-glucopyranosyl-β-D-glucopyranosyl) ester]. The different enzymes, cyclodextrin glycosyl transferase, α and β-Glucosidases, α and β-Galactosidase and β-fructosidase transglycosylation systems together with β-Glycosyltransferase glycosylation systems using UDP-sugars used in the biotransformation of steviol glycosides have been summarized [47].

Additionally, enzymatic hydrolysis seems to be the most efficient method to produce steviol in quantities. Several enzymes have been used for this purpose e.g., juices of the snail *Helix pomatia*, pectinase or hesperidinase affords the aglycone, steviol [6,48,49].

#### **3. Methods of Separation for Diterpene Glycosides**

#### *3.1. Analytical Methods*

#### 3.1.1. High-Performance Thin-Layer Chromatography

High-Performance Thin-Layer Chromatography (HPTLC) is a standardized methodology that has been widely used in the Botanical Industry [50] but has also found multiple

applications in different fields [51–53]. Several HPTLC methods have been developed to identify or quantify selected steviol glycosides (stevioside, steviolbioside, dulcoside A and rebaudiosides A–D) in food and Stevia formulations, and also to detect degradation products like steviol and isosteviol [54]. The main stationary phases used have been silica gel 60, silica gel 60 diol, LiChrospher silica gel 60 F254 S and Proteochrom although the best separation has been achieved on HPTLC silica gel 60 plates. Multiple mobile phases have been assessed to optimize the separation of selected steviol glycosides but ethyl acetate, methanol and formic acid (93:40:1 *v*/*v*/*v*) and ethyl acetate, methanol and acetic acid (3:1:1, *v*/*v*/*v*) seem to be the mixtures where the best separation has been achieved so far. However, with these mobile phases the migration of very polar steviol glycosides like rebaudioside M, N and O is not guaranteed and the separation of the endocyclic steviol isomer, a degradation product formed under acidic conditions from steviol glycosides, needs to be further studied. Steviol glycosides have been visualized on the silica plates by derivatization mainly with a solution of 2-naphthol or primuline for aglycones. Hence, densitometric analysis using absorption and fluorescence modes have been used [54,55] for quantitation. Steviol glycosides can be identified by HPTLC-ESI-MS using a TLC-MS interface and specific structural information can be obtained if collision energy is carefully studied as discussed in 4.3. In Figure 3 the HPTLC separation of selected steviol glycosides is presented.

**Figure 3.** Silica gel HPTLC plate for rebaudiosides D, A, C, stevioside, rebaudioside B, dulcoside A and steviolbioside (tracks 1 to 7). Developed with ethyl acetate, methanol and formic acid (93:40:1 *v*/*v*/*v*) with no saturation but activated with MgCl2 at a relative humidity of 33%. The detection was performed after derivatization with a solution of 2-naphthol under white light.

#### 3.1.2. High-Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is the usual methodology for the analyses of DGs with UV, PDA and MS as the most common detectors [56,57]. Several methods have been developed to separate DGs from complex *S. rebaudiana* extract mixtures. Since the most common aglycone is steviol, novel potential sugar-substitutes could be found based on the number of monosaccharides, type of sugar units and their arrangements. These slight structural differences often cause co-elution of DGs in a single HPLC method.

An overview of the advantages and disadvantages of some HPLC methods using different stationary phases (HILIC, NH2, RP-C18, Sepaxdiol, Synergi, silica gel) is given herein, although detailed information can be found [40,58]. Several purified DGs containing different numbers of sugars and linkage arrangements to steviol were analyzed for this purpose. Interestingly, in all the methods reported, acetonitrile: water acidified elution gradients were described for the analyses of DGs [40,58]. The use of a common binary mobile phase is a practical methodology for the analyses of DGs with simple changes in

the column adsorbent chemistry (Figure 4). There is no change in selectivity between the silica gel and the amino method. In silica gel, stevioside and rebaudioside C coelute while in an amino column, a good separation is achieved, but rubusoside and steviolbioside are better resolved in silica than in amino column. When a Synergi column is utilized, there is a big room for the separation of very polar steviol glycosides (more polar than rebaudioside O) and also between rebaudiosides A and N. There is no change in selectivity if HILIC column is compared with Sepax-diol one, although sharper peaks are gotten with a Sepax-diol column. The appropriate combination of HPLC methods which best resolved components in the range of retention times of interest give a good understanding of the DGs in the extract and chromatography fractions and provide guidance for further preparative methods to be used.

**Figure 4.** *Cont*.

**Figure 4.** Selected HPLC methods used for the analysis of steviol glycosides: silica method (**A**), RP-C18 (**B**), Synergi (**C**), amino (**D**), HILIC (**E**) and Sepax-diol (**F**).

#### *3.2. Preparative Methods*

Several approaches have been described in the literature to isolate and purify known and novel DGs. However, most of the preparative methods described allowed the purification of some few milligrams of DGs. Since novel DGs with refined organoleptic properties need to be found to enhance *Stevia* DGs consumption as a non-caloric sweetener and thus improve human health, the purification process of DGs needs to be scaled-up to access for assay the very minor glycosides as potential sugar-substitutes still undiscovered. Herein we describe some strategies using our own technology to get known compounds in gram to kilogram quantities. Using this technology [59], several novel DGs were purified in hundreds of milligrams and gram quantities [40]. This technology also provides an economic and convenient approach to the isolation and purification of larger quantities of potentially interesting natural products for confirmation of their bioactivities.

Two main approaches have been used for scaling-up the purification process of DGs: utilization of reverse phase (C-18) and silica gel stationary phases. Several sized columns were utilized for the isolation and purification of DGs: (1 × 25 cm, 5 μm), preparative (2.1 × 40 cm, 10 μm) and large-scale high-performance chromatography (7.5 × 50 cm, 10 μm) [59]. Reverse phase chromatography is perceived to have economic advantages over normal phase chromatography due to the usual practice of replacement of the normal phase adsorbent after one or at most a few uses whereas the reverse phase adsorbent can be used for hundreds of separations. The most used mobile phase for purifying DGs in reverse phase chromatography is acidified mixtures of acetonitrile: water in different ratios depending on the analytes. The solvent mixtures after processing the fractions are recovered (c.a 70% in acetonitrile as an aqueous azeotrope) and re-used in the next chromatography after adjusting the ratios based on the density of the mixtures. Thus, we can save solvents and make the purification process of these compounds at large scale more economic.

On the other hand, silica gel stationary phase for chromatography is perceived to not be reusable or of a very limited useful life. Consequently, less expensive poor-quality normal phase adsorbents are typically employed in preparative column packings. The poorer quality normal phase adsorbent is usually of irregular shaped particles and possesses a wide particle size distribution which together provides overall poor chromatographic performance for the packed bed. High quality normal phase adsorbents are available with

spherical particles and narrow particle size distributions. As quality normal phase adsorbent costs about \$5000 or more per kilogram and as the adsorbent is perceived not to be reusable, development of silica gel preparative chromatographic processes has largely been avoided or not considered. In our own experience a high-performance preparative silica gel column (10 μm, spherical silica gel) was packed in 2009 and hundreds of separations have been performed until now without re-packing. Appropriate column regeneration and re-equilibration to the next mobile phase composition increases the lifetime of the stationary phase and restores the performance qualities of the column [60].

With the technologies outlined above, silica gel chromatographic processes can be developed providing cost savings to users through better performance, higher capacity, easier product recovery, less costly solvent recovery, and less costly solvent disposal. Compound recovery from the organic solvent of the mobile phase used in silica gel chromatography is easier and less expensive than from the water containing mobile phase used in reverse phase chromatography. At production scale, the energy required to recycle the normal phase organic solvents is significantly less than that of reversed phase aqueous solvents. The waste disposal costs are reduced for the normal phase organic solvents because of their usually higher BTU content.

One of the most common mobile phases for separating non-glycosylated steviol and related aglycones using this technology are mixtures of *n*-heptane: wet acidified EtOAc and/or *n*-heptane: *wa*EtOAc: MeOH [40]. However, the detection of these compounds is generally set at 205 or 210 nm, below the UV absorbance cut off wavelength of ethyl acetate. An alternative mobile phase to solve this problem is the use of methyl tert-butyl ether (MTBE). This mobile phase was useful for the separation of isosteviol from a mixture of steviol and other isomers [40].

Obviously, normal phase separation of the glycosides requires a more polar mobile phase. Mixtures of EtOAc: MeOH: H2O with 0.1% AcOH or MTBE: MeOH: H2O with 0.1% AcOH in different ratios have been widely used in our experience with good separation of several DGs. Both organic mobile phases are easily recovered, and the solvent composition ratios could be adjusted by scouting by TLC for subsequent chromatography.

All methodologies have been useful to purify several steviol glycosides most of them in gram quantities and could be applied for any natural product research. In Table 2 are shown all the tetracyclic diterpene reported as natural compounds from *S. rebaudiana*.

#### **4. Structure Elucidation for Diterpene Glycosides**

The diversity of DGs has been considerably enhanced with the discovery of several new compounds in the last decade. Herein, we summarize all the DGs isolated from *S. rebaudiana* and reported as natural compounds from 1931 to 2021 (Table 2). The main structural differences have been found in the number of sugar units linked to the aglycone at positions C-13 and C-19 and the different arrangements described, thus some rebaudioside families have been described. Nevertheless, some other DGs with an aglycone core different from steviol have also been reported, and chemical structures are compiled in Figure 1.

The isolation and structure elucidation of minor DGs in quantities not only requires appropriate instrumentation and several chromatographic steps but also high-quality spectroscopic and spectrometric equipment. NMR analysis is the most extensively used technique to elucidate unambiguously natural compounds. However, as an aid for structure elucidation of new steviol glycosides some alternative methods have been reported in the literature and described herein.



**Table 2.** *Cont.*


**Table 2.** *Cont.*


**Table**

**2.**

*Cont.*

### *4.1. Reversed-Phase High-Performance Liquid Chromatography*

High-performance liquid chromatograph is a very common instrument in most of the laboratories today. Thus, a method based on using reversed-phase C18 column retention times of several previously characterized natural DGs and others with no sugar at position C-19 was described as an aid for structure elucidation of new and known DGs [26]. Specifically, the method is helpful to identify known and detect novel aglycone-C13 oligosaccharide moieties and give some hints about C-19 linkages. Elution order of several DGs and their aglycone-C13 oligosaccharide substituted with different sugar arrangements are summarized in Table 3.

**Table 3.** Diterpene glycosides organized by increasing retention times in an RP-C18 HPLC method.


Steviolmonoside (Stev-mono); Iso-steviolbioside (Iso-Stevbio); Rubusoside (Rub); Steviolbioside (Stev-bio), Stevioside (Stev); Iso-Steviolbioside (Iso-Stevbio); Iso-Stevioside (Iso-Stev); Rebaudioside G1 (Reb G1); Rebaudioside G (Reb G); Dulcoside A1 (Dulc A1); Dulcoside A (Dulc A); Rebaudioside B (Reb B); Rebaudioside A (Reb A); Iso-Rebaudioside A1 (Iso-Reb A1); Iso-Rebaudioside A (Iso-Reb A); Dulcoside B (Dulc B); Rebaudioside C (Reb C); Rebaudioside F1 (Reb F1); Rebaudioside F (Reb F); Rebaudioside L1 (Reb L1); Rebaudioside L (Reb L); Rebaudioside H (Reb H); Rebaudioside H1 (Reb H1); **a:** 13-[(2-*O*-β-Dglucopyranosyl-3-*O*-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]*ent*-hydroxyatis-16-en-19-oic acid -β-D-glucopyranosy ester; **b:** 13-[(2-*O*-*β*-D-xylopyranosyl-*β*-D-glucopyranosyl- )oxy]*ent*-kaur-16-en-19-oic acid *β*-D-glucopyranosyl ester; **c:** 13-[(2-*O*-β-D-glucopyranosyl-3-*O*-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]*ent*-hydroxyatis-16-en-19-oic acid; **d:** 13- [(2-*O*-6-deoxy-*β*-D-glucopyranosyl-3-*O*-*β*-D-glucopyranosyl-*β*-D-glucopyranosyl)oxy]*ent*kaur-16-en-19-oic acid *β*-D-glucopyranosyl ester; **e:** 13-[(2-*O*-β-D-xylopyranosyl-β-Dglucopyranosyl-)oxy]kaur-16-en-19-oic acid; **f:** 13-[(2-*O*-6-deoxy-β-D-glucopyranosyl-3-*O*β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaur-16-en-19-oic acid.

The number of sugar components may be inferred by retention time ranges: *DGs* with retention times < 7 min (oligosaccharide with five to seven units), although rebaudioside E or similar isomers, with two monosaccharide units at C-13 and C-19, four glucose units with a 1-6 linkage in C-13 or four glucose units attached to a an *ent*-atisene core show retention times under 7 min, *DGs* with retention times between 7 to 11.5 min (with a di or trisaccharide at C-13 and a monosaccharide at C-19, although three glucose units at C-13 with a 1-6 linkage or linked to an *ent*-atisene core also elute in this range), *DGs* with retention times between 11.6- 12.5 min (a tetrasaccharide oligomer is attached at C-13 and no sugar at C-19) while rubusoside, the only steviol glycosides reported with one sugar unit at both C-13 and C-19, elutes at 12.5 min and *DGs* with retention times greater than 1*2.5 min* (a mono, di or trisaccharide at C-13 and a free carboxylic acid at C-19). However, to confirm the identity of highly substituted DGs, a normal phase chromatographic method should be run.

The method is based on comparing retention times of a pure natural DG and its saponification product with those reported in Table 3. If the retention time of a natural DG and its saponification product match with one of those reported in Table 3, most probably the DG has previously been identified. During the analyses of novel DGs few possibilities may be found: (1) DGs with a novel C-13 and known C-19 arrangement; (2) DGs with known C-13 and new C-19 arrangement; (3) DGs with unknown C-13 and C-19 arrangements or (4) DGs with known C-13 and C-19 moieties but with a unique combination that makes the DGs become an undescribed structure e.g., rebaudioside V [26].

For identifying the sugar component numbers and arrangement at the C-13 moiety, the C-19 portion is cleaved under alkaline hydrolysis and clean up following acidification. HPLC retention time of the aglycone-C13 oligosaccharide moiety is obtained using specific concentration and chromatographic conditions [26]. Comparing retention times with those reported in Table 3 will provide rapid information about the C-13 oligosaccharide arrangement or its novelty.

The strength of the alkaline hydrolysis is also important to get hints about the number of saccharide units and infer their positions of the C-19 oligomer. Mild alkaline hydrolysis over 30 min easily cleaves the monosaccharide linked at position C-19 (rebaudioside A, stevioside, rebaudioside F, etc.). Ready cleavage also occurs when less hindered oligomers are attached at position C-19 e.g., rebaudioside I (Glcβ(1-3)Glcβ-) and rebaudioside U (Araα(1-6)Glc-). However, the same easy cleavage with a disaccharide hexose/pentose (1- 4) hexose is expected. We also believe that an easy cleavage could happen in a trisaccharide or a tetrasaccharide with position 2 of the sugar directly linked at C-19 unsubstituted [26]. DGs with hindered disaccharide Glcβ(1-2)Glcβ- at position C-19 (Rebaudiosides D, E, J) and also trisaccharides and tetrasaccharides (with position 2 of the sugar directly bonded

to C-19 substituent) as rebaudiosides M and O, respectively, only cleave completely using strong alkaline conditions (reflux, 1 h).

#### *4.2. High-Resolution Electrospray Ionization Tandem Mass Spectrometry*

As glycosidic compounds, DGs give greater ionization efficiencies in negative ion mode. The dissociation pattern of DGs has been carefully described by ranging collision energies (CE) using high-resolution electrospray ionization tandem mass spectrometry (HRESIMS) [73,74]. The understanding of the HRESIMS dissociation pattern of DGs is of great importance since this allows the rapid detection and identification of known and novel DGs. Obviously, chemical structures of new DGs should be confirmed by NMR experiments.

*Stevia* DGs are a good model to study the sequence of cleavage, by tandem mass spectrometry, of each sugar monomer from the parent ions due to the variety of types of sugar monomers and their arrangement in the oligosaccharide.

#### 4.2.1. Cleavage of the C-19 Moiety

It is well-known that monosaccharides and oligomers attached at position C-19 (ester portion) is the first moiety cleaved from DGs parent ions. Only glycosides with glucose directly attached at C-19 have been found so far. Ranging of collision energies (CE) allows one to infer important structural information about the C-19 moiety: number of sugar units at C-19 and position of sugar linkages. DGs containing a monosaccharide at C-19 position cleave easily at CE of 10 eV supported with the presence of two major ions in the MS spectrum, the deprotonated molecule [M-H]− and the major product ion [M-H-162]- Da. All the DGs containing a monosaccharide at position C-19 analyzed by HRESIMS showed the same trend e.g., rebaudiosides A, G, H, L and stevioside among others. In the same way, DGs containing less hindered disaccharides at position C-19 also cleave at low collision energy. Rebaudiosides I (Glc(1-3)Glc-) and U (Ara(1-6)Glc-) are a couple of DGs with a less hindered disaccharide attached at C-19. Both compounds show cleavage of the C-19 moiety at 10 eV but produce lower product ion intensities: rebaudioside I-steviol-C13−, 14.4% intensity and rebaudioside U-steviol-C13−, 54% intensity. Product ion intensities of these less hindered C-19 disaccharides should be studied carefully since most probably the position of the second monosaccharide could be also inferred.

However, DGs with two sugars at both positions, C-13 and C-19, show different product ion intensities of the steviol with the C-13 moiety [steviol-C13]− at 10 eV. Rebaudiosides E and S, the only examples at hand, showed both the deprotonated molecule ion [M-H]− and the product ion [steviol-C13]−. Nevertheless, significantly lower product ion intensities were observed: rebaudioside E, [M-H-324]− Da with *m*/*z* = 641 Da and intensity of 5.9% due to the loss of two glucoses and rebaudioside S, [M-H-308]− Da with *m*/*z* = 641 Da and intensity of 7.5% with loss of one glucose and one rhamnose.

On the other hand, DGs containing hindered disaccharides (Rebaudioside D, E, K, etc.) and longer oligomer chains (Rebaudioside s M, N, O, W, etc.) at position C-19 with a C-13 moiety of more than two sugar units do not cleave at low CE. Due to this highly hindered ester, 40 eV needed to be applied to cleave the C-19 portion and produce detectable [steviol-C13 glycoside]− product ions. We also expect a relatively easy cleavage at low CE of the C-19 moiety in DGs with a tri and tetrasaccharide oligomer at C-19 with the C-2 of the glucose directly linked to C-19 unsubstituted.

#### 4.2.2. Cleavage of the C-13 Moiety

In contrast to the C-19 moiety, a broad cleavage of the C-13 moieties in DGs occurs at 70 eV. In branched trisaccharides at C-13, sugars at C-3 cleave first, followed by sugars at C-2 and finally sugars directly linked at C-13. The dissociation pattern of three tetraglycosides with a glucose attached at C-19 and deoxyGlc*β*(1-2)[Glc*β*(1-3)]Glc*β*1- (13-[(2-*O*-6-deoxy-*β*-D-glucopyranosyl-3-*O*-*β*-D-glucopyranosyl*-β*-D-glucopyranosyl) oxy]*ent*-kaur-16-en-19 oic acid *β*-D-glucopyranosyl ester), Xyl*β*(1-2)[Glc*β*(1-3)]Glc*β*1- (rebaudioside F) and Glc*β*(12)[Glc*β*(1-3)]Xyl*β*1- (rebaudioside R) are three DG examples that allow the understanding of the sequential loss of each sugar unit from C-13 (Figure 5).

Rebaudiosides H, L, VIII, IX and IXd are the only reported examples of natural DGs with more than three monosaccharide units at C-13. Rebaudiosides L, VIII and series IX contain only glucoses which makes it difficult to understand the sequential cleavage of C-13 moieties. However, it was found that rebaudioside H [Glc(1-3)Rha*α*(1-2)[Glc*β*(1-3)]Glc*β*1-] showed a sequential loss of four sugar units at CE (70 eV) following the C-19 cleavage as follows: first two glucoses, *m*/*z* = 787.3729 Da and 625.3197 Da, followed by a rhamnose *m*/*z* = 479.2637 Da and a further glucose loss *m*/*z* = 317.2106 Da. Probably, the glucose attached at C-3 of the rhamnose is the first cleaved followed by glucose at C-3 of the glucose directly attached at C-13. DGs have been grouped into 11 groups by molecular weights or structure similarities for an ease of understanding of the different dissociation pattern of several DGs and isomers. MS data of several natural and prepared DGs have been recently reported [74].

**Figure 5.** Fragmentation pattern of steviol glycosides.
