**2. Structural analyses using mass spectrometry**

### **2.1. Gas chromatography coupled to mass spectrometry**

The analysis of polysaccharides is relatively easy to run using ESI-MS and ESI-MS/MS to identify the concatenation of polysaccharide in hexose, deoxy-hexose, pentose, acidic sugars (polyhydroxy carboxylic acids), but it does not provide the polysaccharide composition (glucose, mannose, fructose…) mainly because many monosaccharides have isobaric masses, and often lack an ion (X and A) to identify how the monosaccharides are branched. GC-MS using electron ionization (EI) and chemical ionization (CI) is well suited for such a purpose. Some recent reviews have been published which gather and summarize the different numerous strategies developed in the literature for sugar analysis [4, 5, 6].

Because polysaccharides are mainly composed of aldoses, ketoses, deoxymonosaccharide, amino-monosaccharide and acidic monosaccharide, GC-MS analysis is based on the hydrolysis of the polysaccharides followed by derivatization and GC/MS analysis to identify the different sugar units through their migration times and/or mass spectra. Among the hydrolyzed monosaccharides some cannot be observed like uronic acids, ulosonic acids (*e.g.* Kdo), 4-amino-monosaccharide, and other charged species. Acids cannot be observed because their sodium salts are not volatile. Their treatment must be adapted for study by GC-MS. We describe below the main treatments for the study of polysaccharides: hydrolysis, derivatization, and we evoke the case of the acidic sugars.

306 The Complex World of Polysaccharides

matrix at low concentrations precluding study by MS/MS.

**2. Structural analyses using mass spectrometry** 

**2.1. Gas chromatography coupled to mass spectrometry** 

to couple chromatographic and MS systems on-line.

*meliloti.* 

Resonance (NMR) and cryoprobes have opened new avenues. Unfortunately, NMR presents major drawbacks such as sample purity, sample complexity and concentration requirements. However, in the last decade, the development of highly sensitive, highthroughput Mass Spectrometers (MS) and software able to make global approaches ("omics") on very complex mixtures, have induced a new interest for the MS analyses (especially for the study of protein glycosylation). This time, the instruments mostly use soft ionization techniques associated to powerful MS2 or n capacity analyzers experiments on electrospray ionization coupled to tandem mass spectrometers (ESI-MS/MS) or matrix assisted laser desorption and ionization coupled to a time of flight analyser (MALDI-ToF) [3]. Finally, the recent appearance of ToF and FTICR (Fourier transform ion cyclotron resonance) analyzers on the market allowed access to high resolution measurements enabling the determination of the exact molecular masses (with ToF error of less than 5 ppm and FTICR less than 0.5 ppm). Exact masses give access to the elementary composition of the molecule that is extremely precious when the analyte is present in a complex biological

Beside the evolution of structural analysis, separations techniques have also been enhanced especially by using higher resolution techniques (narrow bore columns, high pressure tolerant or acido-basic tolerant chromatographic phases). This progress has made it possible

In this chapter, we will describe the classical MS couplings like GC-MS or HPLC-MS, but also focus on unusual but useful polysaccharide analysis systems like capillary electrophoresis coupled with mass spectrometry (CE-MS) or high performance anion exchange chromatography coupled with mass spectrometry (HPAEC-MS) that we have developed. Finally, we will present the practical uses of these techniques in a fundamental application: the analysis of the exo-, lipo- and capsular polysaccharides of *Sinorhizobium* 

The analysis of polysaccharides is relatively easy to run using ESI-MS and ESI-MS/MS to identify the concatenation of polysaccharide in hexose, deoxy-hexose, pentose, acidic sugars (polyhydroxy carboxylic acids), but it does not provide the polysaccharide composition (glucose, mannose, fructose…) mainly because many monosaccharides have isobaric masses, and often lack an ion (X and A) to identify how the monosaccharides are branched. GC-MS using electron ionization (EI) and chemical ionization (CI) is well suited for such a purpose. Some recent reviews have been published which gather and summarize the

Because polysaccharides are mainly composed of aldoses, ketoses, deoxymonosaccharide, amino-monosaccharide and acidic monosaccharide, GC-MS analysis is based on the hydrolysis of the polysaccharides followed by derivatization and GC/MS analysis to identify

different numerous strategies developed in the literature for sugar analysis [4, 5, 6].

Scheme 1 presents the general strategy which is used in GC-MS to analyze polysaccharides by determining the nature of the sugars, the relative quantities and the mode of attachment.

**Scheme 1.** General strategy used in GC-MS to analyze polysaccharides

#### *2.1.1. Quality and relative quantities of monosaccharides: alditol acetate*

To identify the nature of the monosaccharide, hydrolysis using H2SO4, HCl or trifluoroacetic acid (weaker than the others but easy to evaporate) is performed. After a reduction step using sodium borohydride [7], acetylation with acetic anhydride [8] results in alditol acetates which can be identified using GC retention time. GC-MS with chemical ionization using ammonia confirms the mass of the pseudo molecular ions (M+H)+ and (M+NH4)+ of the alditol acetates. The chromatographic peaks help to identify the relative number of monomers composing the polysaccharide. Reduction with NaBD4 allows easy identification of the anomeric carbon.

The difference in the hydrolysis rate between the sugars results in certain difficulties i.e. uronic acids being partially hydrolyzed, the monosaccharide linked to it is underrepresented. The same difficulties also exist for 2-acetamidohexoses which are partially *N*-deacetylated under excessive acid concentrations. The 2-aminohexoses obtained are not hydrolyzed and the carbohydrates linked to them are underrepresented [9].

For the acidic sugars, a modified strategy can be used, it consists first in methyl esterification of the uronic acids using DMSO and methanol or diazomethane. The methoxy group (or any other substitution) is a good leaving group for reduction of esters with sodium borohydride to yield aldehydes and, subsequently, their respective neutral sugars. This way, the reduction of the esters by NaBD4 prior to hydrolysis yields a neutral polysaccharide allowing total hydrolysis and, after acetylation, identification by GC-EI-MS of the CD2OCOCH3 moiety bearing the monosaccharides [10,11,12] characteristic of the initial acidic sugars. Alternatively, methanolysis is also used to release monosaccharides as methylglycosides with esterified carboxyl groups. Conditions of methanolysis are presented in table 1.


Table 1 presents different hydrolysis conditions, reduction and acetylation on different kinds of polysaccharides.

in table 1.

kinds of polysaccharides.

100 μL sample, 50 μL of 0.7 M 4-methylmorpholine borane (MMB) and 100 μL of 6 M TFA , 80 °C, 30min.Then,

evaporation and addition of 200 μL 2 M TFA and 500 μL of acetonitrile. Dried again.

Sample was suspended in 2 N H2SO4 heated under vacuum at 100 °C for 3 h. 50 μL of water containing 500 ng monosaccharide internal standard and1 mL H2O was added. Then,2 mL 50% *N*,*N*dioctylmethylamine are added to neutralize the remaining acid. After centrifugation, the top layer was purified on a C18

50 μL of 0.7M MMB.

column.

**Hydrolysis conditions Reduction and acetylation** 

10 M HCl (80 °C, 30 min) Excess of NaBH4 (2 h, 20 °C),

**conditions** 

partially *N*-deacetylated under excessive acid concentrations. The 2-aminohexoses obtained

For the acidic sugars, a modified strategy can be used, it consists first in methyl esterification of the uronic acids using DMSO and methanol or diazomethane. The methoxy group (or any other substitution) is a good leaving group for reduction of esters with sodium borohydride to yield aldehydes and, subsequently, their respective neutral sugars. This way, the reduction of the esters by NaBD4 prior to hydrolysis yields a neutral polysaccharide allowing total hydrolysis and, after acetylation, identification by GC-EI-MS of the CD2OCOCH3 moiety bearing the monosaccharides [10,11,12] characteristic of the initial acidic sugars. Alternatively, methanolysis is also used to release monosaccharides as methylglycosides with esterified carboxyl groups. Conditions of methanolysis are presented

Table 1 presents different hydrolysis conditions, reduction and acetylation on different

acetylated with a 1:1 Ac2O– pyridine mixture (100 °C, 1 h)

Dried sample +100 μL acetic anhydride and 100 μL TFA, reaction at 50 °C for 10 min. Then 1mL toluene to each dried sample . After dissolution in 1.5 mL CH2Cl2 and wash with 0.5M Na2CO3 and water. Sample are then dissolved in CH2Cl2 spiked

with internal standards

Add 50 μL of 100 mg/mL NaBD4 and store at 4 °C overnight. Evaporate with methanol/acetic acid under vacuum. Then add 300 μL of acetic anhydride, react at 100 °C for 13–16 h. Quench with 750 μL H2O for 1 h at room temperature. Extract with 1 mL of chloroform (CHCl3)Remove aqueous phase, add 0.8mL of cold 80% NaOH, mix, elute with CHCl3 on a Chem-Elut column.

**Polysaccharide ref** 

[13]

[14]

[14]

LPS of *Providencia alcalifaciens* O12

Agar containing 3,6 anhydrogalactose

Agar containing 3,6 anhydrogalactose

are not hydrolyzed and the carbohydrates linked to them are underrepresented [9].



#### **Table 1.** asd

Scheme 2 presents different ions obtained for alditol acetate in EI. Generally, no molecular ion and only very low abundance of (M-CH3COO. )+ are observed in the spectrum. Stereoisomers give very near spectra and the base peak is the acetylium ion (CH3CO+) at m/z 43. The primary fragments are formed by cleavage of the carbon chain and then these odd mass number ions may lose an acetic acid (loss of 60 mass units) or a ketene (loss of 42 mass units) or an acetic anhydride (loss of 102 mass units) [23].

If deoxycarbons are present, alfa cleavage resulting in methyl radical loss is insignificant [24]. Some even mass number ions come from rearrangements resulting in the loss of acetaldehyde (loss of 44 mass units), acetic acid, acetic anhydride and ketene from the same mass number molecular ions [25].

When an acetamido group is present, the main ions are due to alfa cleavages induced by the N atom [26].

Consequently comparison of the mass spectrum (EI or CI) and the migration time to standards allows the monosaccharides to be identified.

Trimethylsilyl derivatives can also be used for alditol derivatives [27, 28]. No molecular peaks can be detected. As for most TMS derivatives in EI-MS, small M-15 ions are identified and m/z 103 ((CH3)3SiOCH2+) and m/z (nx102)+1 (n being the number of carbon atoms in the Coupled Mass Spectrometric Strategies for the Determination of Carbohydrates at Very Low Concentrations: The Case of Polysaccharides Involved in the Molecular Dialogue Between Plants and Rhizobia 311

chain of the alditol fragment) are recorded with their corresponding ion coming from a loss of 90 amu ((CH3)3SiOH).

**Scheme 2.** Main fragmentation of hexitol peracetate.

310 The Complex World of Polysaccharides

Add MeOH 0.5M HCl (acetyl chloride 140mL + 4mL MeOH) at 80 ◦C for 16 h. dry under a stream of nitrogen

2mL of 0.01, 0.125, 0.25 or 0.5M HCl in MeOH added to 10mg sample, heated at85°C. Neutralize with Ag2CO3, dry

Add 1.5mg sample to 0.5mL of MeOH/HCL (15mL MeOH and 0.4mL acetyl chloride). Heat at 80°C for 24h and dry.

Add 2mL 0.5M HCl/MeOH to 5mg sample at 80°C. Neutralize to pH 6.

mass number molecular ions [25].

the supernatant

**Table 1.** asd

N atom [26].

gas.

**Hydrolysis conditions Reduction and acetylation** 

**conditions** 

300 μl of TriSilR reagent are added to dried material, at 80 ◦C for 20min and the reagents removed with a nitrogen

stream.1mL hexane extraction of the sample, dry and add hexane.

Add to the dry sample 0.5mL of pyridine, hexamethyldisilazane , trimethylchlorosilane (9:3:1, v/v/v), 80°C 2h. Dry and add

30μL/30μLpyridine and *BSTFA*

trifluoroacetamide) added to dry

Scheme 2 presents different ions obtained for alditol acetate in EI. Generally, no molecular

Stereoisomers give very near spectra and the base peak is the acetylium ion (CH3CO+) at m/z 43. The primary fragments are formed by cleavage of the carbon chain and then these odd mass number ions may lose an acetic acid (loss of 60 mass units) or a ketene (loss of 42 mass

If deoxycarbons are present, alfa cleavage resulting in methyl radical loss is insignificant [24]. Some even mass number ions come from rearrangements resulting in the loss of acetaldehyde (loss of 44 mass units), acetic acid, acetic anhydride and ketene from the same

When an acetamido group is present, the main ions are due to alfa cleavages induced by the

Consequently comparison of the mass spectrum (EI or CI) and the migration time to

Trimethylsilyl derivatives can also be used for alditol derivatives [27, 28]. No molecular peaks can be detected. As for most TMS derivatives in EI-MS, small M-15 ions are identified and m/z 103 ((CH3)3SiOCH2+) and m/z (nx102)+1 (n being the number of carbon atoms in the

(N,O-bis(trimethylsilyl)

0.5mL hexane.

sample, 2h 20°C.

ion and only very low abundance of (M-CH3COO.

units) or an acetic anhydride (loss of 102 mass units) [23].

standards allows the monosaccharides to be identified.

Gelling

**Polysaccharide ref** 

polysaccharides [20]

[21]

[12]

Major grape

carrageenans and

Polysaccharides containing uronic

)+ are observed in the spectrum.

acids.

Plant gums [22]

agarose

Monosaccharides can also be analyzed prior to reduction by NaBH4 or NaBD4, under their cyclic forms, but the chromatograms are more complicated due to the hemiacetal group in sugars which leads to multiple structures [29].

## *2.1.2. Quality and relative quantities of monosaccharides: derivatives of cyclic forms of monosaccharides*

Most of time the polysaccharide is submitted to a methanolysis and after the sample is silylated (see Table 1). With ammonia, CI can be used to identify the (M+H)+ and (M+NH4)+ ions and can be identified by comparison of the migration times of standard the sugar. It is known that the relative intensity of the (M+NH4-CH3OH)+ ion allows the assignment of pyranoside and furanoside structure [30]. Other fragmentation ions exist resulting from the loss by (M+NH4)+ or (M+H)+ of CH3OH or a single or two (CH3)3SiOH molecules.

EI is frequently used and the resulting fragmentation has been extensively reported [30, 31]. If only very rare molecular ions are present in the spectrum, pentose and hexose are characterized by (M-CH3)+ ions and losses of CH3OH and (CH3)3SiOH from this last ion. Deoxyhexoses give M+. and (M-CH3O. )+. Glucuronic and galacturonic acids also exhibit (M-CH3)+ ions in the spectrum and losses of CH3OH and (CH3)3SiOH but also an *m/z* 234 ion is

characteristic of acidic derivatives resulting from a complex McLafferty-type rearrangement of trimethylsilyl group to the carboxyl function. Finally, an *m/z* 204 ion is closely related to ring size, being favored by a six-atom cyclic structure [32].

For pyranose forms the relative intensity of this ion (compared to base peak) varies between 30 and 100% while for furanose it is lower (5%), while *m/z* 217 is very intense for furanose forms.

These fragmentations and migration times of the TMS derivatives will help to determine the species that are difficult to characterize owing to their migration times.

## *2.1.3. The attachment mode*

#### **2.1.3.1. Synthesis of partially methylated alditol acetate**

The use of methylated alditol acetate is a convenient protocol to identify the mode of attachment, it has been well reviewed by Hellerqvist and Sweetman [33]. The first step of this study consists in a permethylation of the polysaccharide, using finely powdered sodium hydroxide and methyl-iodide. A recent article shows that presolubilization of 100-300μL of a solution of 1mg/mL polysaccharide with 5 μL of anhydrous glycerol dried prior to derivatization, offers better methylation yields [34]. Recently, solid-phase spin column permethylation and solid phase capillary permethylation were described [35, 36] and are presented in Table 2. These permethylation processes are derived from the classical method described by Costello [37, 38]. After the permethylation, the polysaccharide is hydrolyzed. For example: the polysaccharide is dried, then 1.8mL glacial acetic acid is added and the sample is briefly sonicated; 0.20mL of 2M sulfuric acid are added and heated at 100°C for 9h. Then acetic acid can be removed in a rotary evaporator several times by adding water and keeping the temperature below 40°C. An equimolar amount of BaCO3 is added to remove the sulfuric acid, and the precipitate is filtered and washed in a minimum volume of water. Then washings and filtrates are combined and about 5mg of NaBH4 (or NaBD4) are added, the reaction is driven for at least 1h. Finally, per-acetylation is driven as reported in table 1.

GC/MS using ammonia CI and then EI are performed, to determine the molecular mass and the structure of the partially methylated acetate alditol respectively. Following this protocol, the positions of the *O*-methyl functions reflect the initial position of the free OH functions of the polysaccharides.

#### **2.1.3.2. Fragmentation rules of partially methylated alditol acetate in EI**

In EI, no molecular ions can be observed. The rules include amino sugars and predict the formation of ions formed by cleavage of the C-C chain. i) The major ion corresponds to the cleavage between acetyl-N-methylaminated carbon and *O*-methylated carbon, with charge retention predominantly on the aminylated carbon, ii) an intense ion comes from two *O*methylated carbons with charge retention on either carbon (the intensities of both ions are inversely proportional to the size of the fragment), iii) weaker ions are due to the cleavage between two C atoms one acetylated the other methoxylated which keeps the charge. These ions may lose ketene, acetic acid or methanol molecules. Table 3 gives the different masses of the different possible ions. With the knowledge of the (M+H)+ or/and (M+NH4)+ ion, obtained in GC:CI/MS and the fragment ions from the EI spectrum, the position of the methyl and the attachment of the different sugars can be identified. Combining these data with ESI-MS/MS gives the correct sequence of the polysaccharide.


**Table 2.** Permethylation of polysaccharides.

312 The Complex World of Polysaccharides

*2.1.3. The attachment mode* 

the polysaccharides.

forms.

ring size, being favored by a six-atom cyclic structure [32].

**2.1.3.1. Synthesis of partially methylated alditol acetate** 

species that are difficult to characterize owing to their migration times.

characteristic of acidic derivatives resulting from a complex McLafferty-type rearrangement of trimethylsilyl group to the carboxyl function. Finally, an *m/z* 204 ion is closely related to

For pyranose forms the relative intensity of this ion (compared to base peak) varies between 30 and 100% while for furanose it is lower (5%), while *m/z* 217 is very intense for furanose

These fragmentations and migration times of the TMS derivatives will help to determine the

The use of methylated alditol acetate is a convenient protocol to identify the mode of attachment, it has been well reviewed by Hellerqvist and Sweetman [33]. The first step of this study consists in a permethylation of the polysaccharide, using finely powdered sodium hydroxide and methyl-iodide. A recent article shows that presolubilization of 100-300μL of a solution of 1mg/mL polysaccharide with 5 μL of anhydrous glycerol dried prior to derivatization, offers better methylation yields [34]. Recently, solid-phase spin column permethylation and solid phase capillary permethylation were described [35, 36] and are presented in Table 2. These permethylation processes are derived from the classical method described by Costello [37, 38]. After the permethylation, the polysaccharide is hydrolyzed. For example: the polysaccharide is dried, then 1.8mL glacial acetic acid is added and the sample is briefly sonicated; 0.20mL of 2M sulfuric acid are added and heated at 100°C for 9h. Then acetic acid can be removed in a rotary evaporator several times by adding water and keeping the temperature below 40°C. An equimolar amount of BaCO3 is added to remove the sulfuric acid, and the precipitate is filtered and washed in a minimum volume of water. Then washings and filtrates are combined and about 5mg of NaBH4 (or NaBD4) are added, the reaction is driven for at least 1h. Finally, per-acetylation is driven as reported in table 1.

GC/MS using ammonia CI and then EI are performed, to determine the molecular mass and the structure of the partially methylated acetate alditol respectively. Following this protocol, the positions of the *O*-methyl functions reflect the initial position of the free OH functions of

In EI, no molecular ions can be observed. The rules include amino sugars and predict the formation of ions formed by cleavage of the C-C chain. i) The major ion corresponds to the cleavage between acetyl-N-methylaminated carbon and *O*-methylated carbon, with charge retention predominantly on the aminylated carbon, ii) an intense ion comes from two *O*methylated carbons with charge retention on either carbon (the intensities of both ions are inversely proportional to the size of the fragment), iii) weaker ions are due to the cleavage between two C atoms one acetylated the other methoxylated which keeps the charge. These

**2.1.3.2. Fragmentation rules of partially methylated alditol acetate in EI** 


**Table 3.** Principal fragmentations in EI/MS of the C-C chain of permethylated alditol acetate We do not present the ions which can lose CH3OH, CH2CO, CH3COOH.

#### **2.2. ElectroSpray ionisation mass spectrometry (ESI-MS) analyses**

Using electrospray technology, the analyte is introduced into the mass spectrometer as a solution delivered by a syringe pump (direct input) or as a fraction eluted from a liquid chromatography (HPLC/UPLC). The analyte solution passes through a needle on which a high potential difference is applied (classically 3 kV). This produces a spray of droplets with a surface charge of the same polarity as the needle. The charged droplets shrink as the solvent evaporates. Charge promiscuity then produces continual explosions of the droplets into smaller ones until they reach the gas phase. During this process, the molecules present in the solution are concentrated and this often produces a suppression effect. This effect consists in the masking of one compound by another present in a mixture. The suppression effect is minimal when all the compounds together are chemically equivalent in terms of hydrophobicity or acido-basicity. As hydrophilic compounds stay longer in the dissolved form during the ESI process, the more hydrophobic compounds will generally suppress the more hydrophilic compounds. In the positive ion mode, basic compounds will easily suppress more acidic compounds. As carbohydrates are very hydrophilic and often acidic, only two possibilities exist to analyze them without suppression: a separation of the sugars based on their acidity (ion exchange chromatography) or derivatization of the hydroxyls to generate more hydrophobic compounds. The latter techniques include derivatization such as permethylation [44;45] or metal adduction [46;47] which has been extensively studied. Structural analysis of underivatized saccharides using negative mode ion electrospray ionization has also been investigated [48].

A systematic nomenclature for labeling fragment ions observed in MS/MS has been introduced by Domon and Costello [49] (Figure 1).

**Figure 1.** Systematic nomenclature for labeling fragment ions observed in MS/MS following Domon and Costello.

Acidic moieties strongly influence the fragmentation energetics and patterns of tandem mass spectrometric daughter ion formation. Non-sulfated compounds dissociate in the positive mode into abundant ions through B and Y type fragmentation resulting from the cationisation of the glycosidic oxygen [50], these fragmentations being facilitated on 4- and 6-linked reducing terminal residues [51]. In the negative mode deprotonated neutral carbohydrates produce C- and Z-type ion fragments [52], when the acidic sugars undergo Band Y-type fragmentation [53]. Therefore, interpretation of the CID spectra implies knowledge of the fragmentation pathway, and the use of mass tables of the common monosaccharide building blocks is often essential to build the sequence of unknown structures. Of course, understanding the biosynthetic pathway of oligosaccharides helps to avoid making mistakes in the sequence [54].

## *2.2.1. Direct Input (DI/MS)*

314 The Complex World of Polysaccharides

ionization has also been investigated [48].

and Costello.

introduced by Domon and Costello [49] (Figure 1).

**2.2. ElectroSpray ionisation mass spectrometry (ESI-MS) analyses** 

Using electrospray technology, the analyte is introduced into the mass spectrometer as a solution delivered by a syringe pump (direct input) or as a fraction eluted from a liquid chromatography (HPLC/UPLC). The analyte solution passes through a needle on which a high potential difference is applied (classically 3 kV). This produces a spray of droplets with a surface charge of the same polarity as the needle. The charged droplets shrink as the solvent evaporates. Charge promiscuity then produces continual explosions of the droplets into smaller ones until they reach the gas phase. During this process, the molecules present in the solution are concentrated and this often produces a suppression effect. This effect consists in the masking of one compound by another present in a mixture. The suppression effect is minimal when all the compounds together are chemically equivalent in terms of hydrophobicity or acido-basicity. As hydrophilic compounds stay longer in the dissolved form during the ESI process, the more hydrophobic compounds will generally suppress the more hydrophilic compounds. In the positive ion mode, basic compounds will easily suppress more acidic compounds. As carbohydrates are very hydrophilic and often acidic, only two possibilities exist to analyze them without suppression: a separation of the sugars based on their acidity (ion exchange chromatography) or derivatization of the hydroxyls to generate more hydrophobic compounds. The latter techniques include derivatization such as permethylation [44;45] or metal adduction [46;47] which has been extensively studied. Structural analysis of underivatized saccharides using negative mode ion electrospray

A systematic nomenclature for labeling fragment ions observed in MS/MS has been

**Figure 1.** Systematic nomenclature for labeling fragment ions observed in MS/MS following Domon

Acidic moieties strongly influence the fragmentation energetics and patterns of tandem mass spectrometric daughter ion formation. Non-sulfated compounds dissociate in the As explained just before, analyzing carbohydrate mixtures by direct introduction is a challenge due to the easy suppression of these compounds by numerous other natural compounds like proteins, aminoacids, lipids or other differently charged or decorated saccharides.

In comparison to MALDI, ESI produces less in-source fragmentation of acidic glucans and other fragile ions and is easily coupled on one side to tandem MS allowing structural investigations and on the other side on-line to liquid chromatography. As this ionization technique is very soft, true MS (without fragmentation) can be obtained. However, MALDI produces less complex charge state patterns and less multiple cation adducts and suppression effects. Actually, the analyte is not in solution with the salts and the deposit is quite heterogenic indicating places where suppression effects could be less important [55]. The polymeric complexity of carbohydrates often produces an overlap of the ESI charge state patterns making them extremely difficult to analyze [56] even with software such as MaxEnt (1 and 3). As a result, except for acidic glucans, ESI analysis of glycans requires chromatographic pre-separation of the sample.

#### *2.2.2. High-resolution liquid chromatography coupled to Mass Spectrometry (HPLC-MS)*

Normal phase HPLC on naked inorganic oxides was the technique first developed but it exhibited numerous drawbacks (peak tailing, retention time shifts, etc.), therefore, reversed phase HPLC was developed and proved to be suitable for most all bioanalytical solutes. However, a problem remained: how to create retention in RP-HPLC for polar compounds without dewetting the C18 phase [57]. Lack of retention of hydrophilic compounds is almost always due to solvophilicity. Actually, the polar functions enter more favorable dipolar interactions with the polar RP-HPLC eluent, than with the stationary phase [58]. When the lack of retention is due to charge, retention can easily be achieved on ion-exchange chromatography (see next paragraph) or by using ion pairing that does not require specific apparatus. Using this technique, glycosaminoglycans polymers up to a polymerization degree of 40 can be observed on reversed phase ion pairing HPLC [59]. Unfortunately, ion pairing has been shown to reduce ESI-MS sensitivity [60].

**Figure 2.** HILIC/LIF/ESI-MS analysis of 2-AB labeled glucose ladder. At the top: improved LIF detection of the neutral saccharides, in the middle: positive mode ESI-MS total ion current, at the bottom: spectrum obtained by summing all the spectra of the compounds eluting between 15 and 50 minutes.

minutes.

**Figure 2.** HILIC/LIF/ESI-MS analysis of 2-AB labeled glucose ladder. At the top: improved LIF detection of the neutral saccharides, in the middle: positive mode ESI-MS total ion current, at the bottom: spectrum obtained by summing all the spectra of the compounds eluting between 15 and 50 The greatest problem concerns the highly hydrophilic but still neutral analytes. The classical way to analyze them is to convert them into more hydrophobic compounds by a chemical reaction [61]. In addition to the retention of the compounds, such organic derivatization improves their detection (UV absorption or fluorescence emission) (figure 2). The main drawbacks of this approach are that the separation of all the compounds is only based on the same hydrophobic tail, derivatization is time-consuming, and quantitative labeling cannot be systematically achieved. Therefore, a new strategy has been developed and is based on a new stationary phase. In practice, the stationary phase is polar and attracts the more polar part of the eluent that will act as the retentive phase. The complete eluent is relatively hydrophobic but is a sufficiently good solvent to allow distribution between the stationary and mobile phases. This technology is named hydrophilic interaction chromatography (HILIC). The quite neutral hydrophobic/hydrophilic balance of the eluent allows an easy interfacing with ESI-MS unlike the normal phase HPLC (NP-HPLC) working with totally organic solvents which are not compatible with the nebulization process [62].

## *2.2.3. High Performance Anion Exchange Chromatography coupled to Mass Spectrometry (HPAEC-MS)*

High performance anion exchange chromatography (HPAEC) using sodium hydroxide or acetate based eluents is a well-established technique for determining underivatized carbohydrates [63;64] and high-performance separation of alditols, mono- and oligosaccharides ranging from 2 to 60 mers have been described using pulsed amperometric detection (PAD) [65]. The identification of individual carbohydrates is usually performed by comparison of their retention times with those of reference samples. In biological samples complex overlapping occurs: acidic sugars are eluted in the "oligosaccharide domain" making this approach very uncertain. Moreover, the nature of sugar moieties and the variability of glycosidic linkages makes the retention times of the oligosaccharides unpredictable. For these reasons, coupling of HPAEC-PAD with ESI-Q-ToF (ESI coupled to a hybrid tandem mass spectrometer constituted of a quadripole and a ToF) MS was required to collect structural information on the PS sequences. Two detections are necessary. PAD allows, from retention time, the nature of the saccharide (neutral, acidic, oligosaccharidic) to be determined while ESI-Q-ToF MS gives access to the composition. Coupling HPAEC with MS presents a technological challenge, due to the non-volatility and high conductance of the sodium hydroxide or acetate dissolved in water, used as eluent. To avoid this limitation, a commercial desalting device using a selective cation exchange membrane and a regenerant can be installed on-line between the column and the MS [71,66]. The use of this on-line desalter with oligosaccharide separations with MS has only been rarely described [67, 68, 69, 70, 71].

We have developed HPAEC and ESI-Q-ToF MS conditions to perform efficient on-line coupling of the two techniques [72]. PA1 ion exchange columns have been used for oligosaccharide analysis even though they are designed for monosaccharides, because the PA 200 column is not available in the narrowbore size. Two different gradients have been used at a flow rate of 0.3 mL/min. For neutral carbohydrates: during the first 15min NaOH concentration increased from 10 to 50mM, and during the following 5 min up to 80mM,

finally, during the last 15 min the concentration of NaOAc increased from 0 to 90 mM while NaOH remains constant, return to the initial conditions was done over 10 min. A wait of 35 min between two injections is necessary to equilibrate the column. To separate acidic sugars, the gradient started with a NaOAc concentration ramp increasing from 0 to 90 mM in 15 min while the concentration of NaOH was kept constant at 80 mM, the concentration of NaOAc (90 mM) and NaOH (80 mM) were then held constant for 5 min then 5 min to return to initial conditions. A wait of 15 min between two injections was necessary to equilibrate the column.

Due to the low amounts of polysaccharide that can be dissolved, no post-column split could be used to couple ESI-Q-ToF with an analytical sized column. This methodology was never used for the analysis of heteropolysaccharides.

Performing HPAEC for carbohydrate analyses implies the use of a NaOH and NaOAc concentration gradient. At high pH (>13.5), carbohydrates are in the anionic form allowing their retention on the phase. Electrocatalytic oxidation mediated by NaOH at the surface of the gold electrode occurs by application of a positive potential. The current generated is then proportional to the carbohydrate concentration. Therefore obtaining a PAD signal, implies placing the suppressor after the PAD but before the MS (Scheme 3). Since a high ionic content is not compatible with electrospray ionization mode, the salt level was reduced using an on-line 4 mm ASRS (anion self-regenerating suppressor) desalter. The desalting efficiency and the pH of the mobile phase after desalting were checked. The peak broadening due to the additional void volume of the ASRS and the reference electrode cavity was then investigated. Desalting efficiency was followed through a conductometer as a second detector. The pH was followed each minute (using the combination pH reference electrode in the electrochemical cell of the PAD unit) to be sure that the residual conductivity was not due to acetic acid. When the regenerant used in the ASRS is pure water at a flow rate of 2 mL/min, it is possible to suppress NaOH up to 100 mM (at a flow rate 0.3 mL/min), but it does not provide the suppression of 100 mM NaOAc (the pH increased up to 13 at 25 min). In contrast, the use of 0.25% H2SO4 in water at a flow rate of 2 mL/min can maintain the conductivity below 300 μS for the entire analysis. Desalting of the NaOH eluent resulted in the measurement of a stable pH of 7; during the NaOAc gradient, the pH decreased regularly to pH 3. During conductimetric detection, a current is continuously applied to the membrane (50 mA). No effect of regeneration current on the membrane regeneration efficiency was observed.

The coupling of both PAD and MS detectors offers two advantages. First, the safety of the mass spectrometer, intrusion of salt in the source and in the mass analyzer can dramatically affect the performance of the mass spectrometer, because only a small quantity of salt produces signal suppression. Therefore, when signal is present on PAD but disappears on MS, it is an indication that desalting is no longer sufficient. The second advantage, as discussed in the next paragraphs, is that the MS and PAD sensitivity are complementary.

As we performed on line PAD and MS analyses, the first before and the second after desalting, we measured peak broadening, and separation efficiency. A 120 μL void volume was measured between the PAD and the ESI source with 30% peak broadening but without loss of efficiency. Since the flow rate of 0.3 mL/min is compatible with the ESI source, no split was necessary.

318 The Complex World of Polysaccharides

used for the analysis of heteropolysaccharides.

regeneration efficiency was observed.

the column.

finally, during the last 15 min the concentration of NaOAc increased from 0 to 90 mM while NaOH remains constant, return to the initial conditions was done over 10 min. A wait of 35 min between two injections is necessary to equilibrate the column. To separate acidic sugars, the gradient started with a NaOAc concentration ramp increasing from 0 to 90 mM in 15 min while the concentration of NaOH was kept constant at 80 mM, the concentration of NaOAc (90 mM) and NaOH (80 mM) were then held constant for 5 min then 5 min to return to initial conditions. A wait of 15 min between two injections was necessary to equilibrate

Due to the low amounts of polysaccharide that can be dissolved, no post-column split could be used to couple ESI-Q-ToF with an analytical sized column. This methodology was never

Performing HPAEC for carbohydrate analyses implies the use of a NaOH and NaOAc concentration gradient. At high pH (>13.5), carbohydrates are in the anionic form allowing their retention on the phase. Electrocatalytic oxidation mediated by NaOH at the surface of the gold electrode occurs by application of a positive potential. The current generated is then proportional to the carbohydrate concentration. Therefore obtaining a PAD signal, implies placing the suppressor after the PAD but before the MS (Scheme 3). Since a high ionic content is not compatible with electrospray ionization mode, the salt level was reduced using an on-line 4 mm ASRS (anion self-regenerating suppressor) desalter. The desalting efficiency and the pH of the mobile phase after desalting were checked. The peak broadening due to the additional void volume of the ASRS and the reference electrode cavity was then investigated. Desalting efficiency was followed through a conductometer as a second detector. The pH was followed each minute (using the combination pH reference electrode in the electrochemical cell of the PAD unit) to be sure that the residual conductivity was not due to acetic acid. When the regenerant used in the ASRS is pure water at a flow rate of 2 mL/min, it is possible to suppress NaOH up to 100 mM (at a flow rate 0.3 mL/min), but it does not provide the suppression of 100 mM NaOAc (the pH increased up to 13 at 25 min). In contrast, the use of 0.25% H2SO4 in water at a flow rate of 2 mL/min can maintain the conductivity below 300 μS for the entire analysis. Desalting of the NaOH eluent resulted in the measurement of a stable pH of 7; during the NaOAc gradient, the pH decreased regularly to pH 3. During conductimetric detection, a current is continuously applied to the membrane (50 mA). No effect of regeneration current on the membrane

The coupling of both PAD and MS detectors offers two advantages. First, the safety of the mass spectrometer, intrusion of salt in the source and in the mass analyzer can dramatically affect the performance of the mass spectrometer, because only a small quantity of salt produces signal suppression. Therefore, when signal is present on PAD but disappears on MS, it is an indication that desalting is no longer sufficient. The second advantage, as discussed in the next paragraphs, is that the MS and PAD sensitivity are complementary.

As we performed on line PAD and MS analyses, the first before and the second after desalting, we measured peak broadening, and separation efficiency. A 120 μL void volume

**Scheme 3.** Coupling scheme. The PAD is placed on-line with the MS, but before the desalting device to ensure the electrocatalytic process.

Analysis of acidic sugars opens up the possibility of using the positive or negative mode for MS detection. Theoretically, detection in the negative mode decreases the noise due to the increased specificity, and therefore increases the signal-to-noise ratio (S/N) and the sensitivity. The sugars we are looking for contain a carboxylic acid function and are easily ionisable in the negative mode. We studied the response factor of a commercial acidic oligosaccharide: 6'-sialyl-lactose; surprisingly, the TIC intensities were almost identical for ESI+ and ESI-. Only a sensitivity factor difference of two was measured at 5 μg/mL, and the positive ionization mode did not exhibit significantly more noise. In the positive mode, the presence of at most one sodium adduct was observed (as counter-ion of the acid). As both MS modes gave similar results, the behavior using MS/MS was investigated in both modes. However, collision energy is lower in the positive mode, which could be an advantage to obtain a better sensitivity, since high energy ions are difficult to refocus.

The detection sensitivity using HPAEC-PAD or HPAEC-ESI-Q-ToF MS is dependent on the nature of the sugar. The first fast oxidation step occurring on the gold electrode (PAD) involves the aldehyde of the carbohydrate, resulting in the formation of a carboxylic acid and the production of two electrons. The second fast oxidation reaction is the cleavage of the C1-C2 bond, followed by conversion of C2 and C6 to the corresponding carboxylates, resulting in the production of 6 electrons (most efficient response) [72]. Therefore the predictive response factors are: Hex>6-desoxyHex>HexA>2-desoxyHex>ulosonic acids (Figure 3). For ESI-Q-ToF MS, the ionization occurs on the glycosidic linkage and is often facilitated through the presence of acid functions close to the ionization site, implying the most sensitive response for the ulosonic acids.

**Figure 3.** Chromatogram obtained by PAD detection of common sugars : Rhamnose (Rha), Galactose (Gal), Glucose (Glc), Mannose (Man), N-Acetyl Neuraminic acid (NANA), 2-Keto,3-deoxyoctuolosonic acid (Kdo), galacturonic acid (GalA) and glucuronic acid (GlcA).

The concentration response of ESI-MS is often not linear and is very variable from one sugar to the other. For this reason, a quantification curve and the limit of detection (LOD) for each type of saccharide standard were measured for the five standards in solutions ranging from 200 to 2μg/mL with an injection volume of 5 μl.

The HPAEC-ESI-Q-ToF MS response measurements surprisingly indicate that uronic acids respond weaker than expected, even less than hexoses. The LOD of GalA was not satisfying, unlike those obtained for all other saccharides.
