**2.4. CE-MS**

320 The Complex World of Polysaccharides

most sensitive response for the ulosonic acids.

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

**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

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

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,

The fast atom bombardment (FAB) ionization is less and less used due to the fact that the MS suppliers no more produce this type of sources. Nevertheless, this ionization process is of interest, because it is significantly more energetic than MALDI and ESI. In consequence, fragmentation can be observed in the ion source for natively charged glycans and be applied well to positive or ions. In the positive mode sodium cationized species are easily analyzed,

acid (Kdo), galacturonic acid (GalA) and glucuronic acid (GlcA).

200 to 2μg/mL with an injection volume of 5 μl.

unlike those obtained for all other saccharides.

**2.3. FAB MS and FAB MS/MS of carbohydrates** 

### *2.4.1. Capillary electrophoresis and laser induced fluorescence.*

Capillary electrophoresis, due to its high resolving power and sensitivity, has been useful in the analysis of intact and derived oligosaccharides and polysaccharides affording concentration and structural characterization data essential for understanding their biological functions. As most glycans do not have any strong chromophores on their structures and have low extinction coefficient they are difficult to detect using UV absorbance. Indirect UV has been used, it is based on using a chromophore in the electrolyte resulting in negative peaks but because the chromophore must have a mobility very near that of the sugars it is often difficult to ensure an adequate detection limit [77, 78]. Labelling with a chromophore is very useful, because Laser induced fluorescence (LIF) [79,80] can be used on monosaccharides as well as on polysaccharides. CE experiments are comparable to GC experiments to identify monosaccharides. Acetylation or sylilalion of alditol is replaced by an amino reduction step using an amino fluorescent dye and NaBH3CN. Most of the time 1-aminopyrene-3,6,8-trisulfonate (APTS) is used for monosaccharide, their migration time allow the identification of the different derivatized alditols, it is very convenient for small quantities of natural polysaccharides and specially polysaccharides extracted from DOC PAGE. An acidic compound is more easily prepared than with classical alditol strategies for GC analysis [81].

For polysaccharides the same labeling reaction can be used and the different compounds can be easily separated. Recently, optimization of the boric acid concentration (which increases the charge of the polysaccharide by complexation) and linear polyacrylamide concentration in the buffer yielded a separation of 12 polysaccharides of very similar structures, most with baseline separation in less than six minutes [85]. Using this technique, high throughput glycosylation can be performed [82]. Thirty-two IgG N-glycans were analyzed using a combination of weak anion exchange enrichment and exoglycosidase digestion. Aminobenzamide and Aminopyrentrisulfonate labeling of polysaccharide followed by a UPLC on 1.7 μm HILIC or CE-LIF respectively have been compared. Combination of these data demonstrated that complete structural annotation is possible within a total analysis time of 20 min due to the advantageous orthogonality of the separation mechanisms [83]. This work confirms the use of glycosidase in CE-LIF studies for the determination of the structure of polysaccharides. Significant labeling improvements were made by replacing the conventionally used acetic acid catalyst for NaBH3CN reduction with citric acid. Using a 1:10 glycan to fluorophore molar ratio resulted in a 95% derivatization yield at 55°C with only 50 min reaction time and negligible terminal sialic acid loss [84].

#### *2.4.2. Capillary electrophoresis and mass spectrometry.*

A large number of CE-MS and CE-MS/MS experiments were driven for polysaccharide structural determination and have been extensively reviewed recently by Y. Mechref [85]. Different interfaces between the CE-LIF instrument and MS are commercially available. Most works use APTS labeling and ESI ionization, although MALDI is also used but with lab-made instruments [86]. A very interesting study concerning polysaccharides from IgG was realized. Figure 4 presents a schema of the CE-MS system with on-line LIF detection adjusted 20 cm from the ESI tip. A sheath liquid consisting of a 50:50 isopropanol:water (0.2% ammonia) was added coaxially to the separation capillary at flow-rate 4 μL/min. CE was performed with PVA coated capillaries (60 cm total length x 50 cm effective length × 50 *μ*m ID) and a running buffer of 40 mM *alpha*-aminocaproic acid, pH 4.5 (adjusted with acetic acid) + 0.02% hydroxypropylmethylcellulose (HPMC) with an applied voltage of -30 kV were used [87]. The inherent mass accuracy of the MS permitted the identification of major and minor glycans using their (M-H) ions. This study demonstrated for the first time the ability to attain CE-MS separation efficiency somewhat comparable to that commonly observed in CE-LIF analysis. As seen in figure 4 the four early migrating species are clearly visible in both the online LIF and MS electropherograms. The MS electropherograms suffer from a shift in migration time and some loss in separation efficiency, which is due to dead volumes originating from the addition of the MS detector.

Neutral markers to label acidic polysaccharide can be used. Their main advantage will be to allow the separation of the acidic polysaccharide following the number of acid residues [88], 2-aminoacridone was proposed in place of APTS. Nakano *et al* [89] used 9-fluorenylmethyl chloroformate (Fmoc-Cl), during the digestion of a glycoprotein by PNGase which can label the released 1-amino function of the first GlcNac residue. It prevents reductive amination.

acid loss [84].

*2.4.2. Capillary electrophoresis and mass spectrometry.* 

volumes originating from the addition of the MS detector.

and minor glycans using their (M-H)-

For polysaccharides the same labeling reaction can be used and the different compounds can be easily separated. Recently, optimization of the boric acid concentration (which increases the charge of the polysaccharide by complexation) and linear polyacrylamide concentration in the buffer yielded a separation of 12 polysaccharides of very similar structures, most with baseline separation in less than six minutes [85]. Using this technique, high throughput glycosylation can be performed [82]. Thirty-two IgG N-glycans were analyzed using a combination of weak anion exchange enrichment and exoglycosidase digestion. Aminobenzamide and Aminopyrentrisulfonate labeling of polysaccharide followed by a UPLC on 1.7 μm HILIC or CE-LIF respectively have been compared. Combination of these data demonstrated that complete structural annotation is possible within a total analysis time of 20 min due to the advantageous orthogonality of the separation mechanisms [83]. This work confirms the use of glycosidase in CE-LIF studies for the determination of the structure of polysaccharides. Significant labeling improvements were made by replacing the conventionally used acetic acid catalyst for NaBH3CN reduction with citric acid. Using a 1:10 glycan to fluorophore molar ratio resulted in a 95% derivatization yield at 55°C with only 50 min reaction time and negligible terminal sialic

A large number of CE-MS and CE-MS/MS experiments were driven for polysaccharide structural determination and have been extensively reviewed recently by Y. Mechref [85]. Different interfaces between the CE-LIF instrument and MS are commercially available. Most works use APTS labeling and ESI ionization, although MALDI is also used but with lab-made instruments [86]. A very interesting study concerning polysaccharides from IgG was realized. Figure 4 presents a schema of the CE-MS system with on-line LIF detection adjusted 20 cm from the ESI tip. A sheath liquid consisting of a 50:50 isopropanol:water (0.2% ammonia) was added coaxially to the separation capillary at flow-rate 4 μL/min. CE was performed with PVA coated capillaries (60 cm total length x 50 cm effective length × 50 *μ*m ID) and a running buffer of 40 mM *alpha*-aminocaproic acid, pH 4.5 (adjusted with acetic acid) + 0.02% hydroxypropylmethylcellulose (HPMC) with an applied voltage of -30 kV were used [87]. The inherent mass accuracy of the MS permitted the identification of major

ability to attain CE-MS separation efficiency somewhat comparable to that commonly observed in CE-LIF analysis. As seen in figure 4 the four early migrating species are clearly visible in both the online LIF and MS electropherograms. The MS electropherograms suffer from a shift in migration time and some loss in separation efficiency, which is due to dead

Neutral markers to label acidic polysaccharide can be used. Their main advantage will be to allow the separation of the acidic polysaccharide following the number of acid residues [88], 2-aminoacridone was proposed in place of APTS. Nakano *et al* [89] used 9-fluorenylmethyl chloroformate (Fmoc-Cl), during the digestion of a glycoprotein by PNGase which can label the released 1-amino function of the first GlcNac residue. It prevents reductive amination.

ions. This study demonstrated for the first time the

**Figure 4.** Expanded-scale electropherograms of rMAb 1: A) standard CE−LIF electropherogram using a 60 cm capillary, B) CE−LIF trace obtained on-line with MS detection and C) CE−MS base peak electropherogram. D) Scheme of the CE−MS system with on-line LIF detection.

Using a very simple 20 mM phosphate buffer (pH 8.5), the authors separated the different polysaccharides following the number of sialic acids, then MS and MS/MS spectra identified the composition of each polysaccharide thanks to (M+H)2+ or (M+H)3+ ions. An example of the mass electropherogram and the different mass spectra are presented in figure 4 which concerns the glycans from bovine fetuin. In this study the authors showed that this method can be used to identify polysaccharides from glycoprotein extracted from an SDS PAGE separation.
