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Migration time (min)

Fig. 1. CZE profile showing the resolution of variously sulfated intact GAGs and HA. CE was performed on an uncoated fused-silica capillary at reversed polarity using 20 mM phosphate, pH 3.0, as operating buffer and detection of separated GAGs at 190 nm. Peaks: 1 = fragmin, an oversulfated fragment of heparin, bearing on the average 2.5-3 sulfate groups per disaccharide residue; 2 = CSA, CSC, or DS bearing on average 0.8-1 sulfate group per disaccharide unit; 3 = HS containing on average 0.6-0.7 sulfates per unit; 4 = HA containing no sulfate groups. Reprinted from ref. 4.

d. Run the samples for 12 min at 34 kV/m.

e. Before each run, rinse and condition the capillary, as in steps a and b.

4. Change the operating buffer and run a standard mixture after every four injections of the samples to ensure the performance of the separation (see Notes 3 and 4).

3.2. CGE of HA Using Pullulan Matrix

A simple open capillary separation allows determination of the amount and estimation of the molecular mass and polydispersity of high-molecular-size HA preparations (5). The addition of the polysaccharide pullulan to the elution buffer creates a gel matrix in which larger molecules become retarded (see Fig. 2). With a separation buffer at a pH of 4.0, most of the glucuronic carboxyl groups are dissociated while the EOF is low, and the electrophoretic mobility (EM) will make the HA move toward the anode. The molecular size and working concentration of the matrix-forming pullulan are important, and the protocol below describes one setup that permits analysis in the 50-to 2000-kDa range. The detection is achieved with low ultraviolet and the specificity can be improved by repeating the analysis after hyaluronidase digestion.

Fig. 2. Separation of variously sized HA in the presence of 0.10% pullulan (molecular mass 1600 kDa) in the operating buffer as a matrix-forming agent. (a) HA-H (molecular mass 15002100 kDa); (b) HA-M (800-1200 kDa) and (c) HA-L (molecular mass 40-60 kDa). Reprinted from Journal of Chromatography, volume 768, Hayase, S., et al., High perfomrance capillary electrophoresis of hyaluronic acid: determination of its amount and molecular mass, 1997, pp. 290-305, with permission from Elseveir Science.

Fig. 2. Separation of variously sized HA in the presence of 0.10% pullulan (molecular mass 1600 kDa) in the operating buffer as a matrix-forming agent. (a) HA-H (molecular mass 15002100 kDa); (b) HA-M (800-1200 kDa) and (c) HA-L (molecular mass 40-60 kDa). Reprinted from Journal of Chromatography, volume 768, Hayase, S., et al., High perfomrance capillary electrophoresis of hyaluronic acid: determination of its amount and molecular mass, 1997, pp. 290-305, with permission from Elseveir Science.

1. Dissolve the sample in water at an approximate concentration of 0.25 mg/mL and add 0.025 volume of 0.1% w/v of naphthalene trisulfonic acid trisodium salt (NTS) as internal standard (see Note 5). The mixture is kept at 4°C pending use.

2. Start the CE instrument. Adjust the detector to monitor absorbance at 190 nm.

3. Prepare the software of the CE instrument as follows:

a. Rinse the capillary with 0.1 M NaOH for 5 min.

b. Rinse the capillary with 2 x distilled water for 3 min.

c. Rinse the capillary with the operating buffer for 5 min.

d. Inject the sample by pressure (300 mbar x s).

e. Run the separation for 25 min at 34 kV/m (20 kV for a total capillary length of 58 cm).

4. The total amount of HA is calculated from the peak area, comparing with standard HA samples also containing the NTS internal standard. All macromolecular HA should disappear after hyaluronidase digestion (see Note 6). The molecular size and polydispersity are estimated from the shape of the HA peak, although accurate calculation of the Mw and Mn values is difficult (see Note 7).

3.3. CGE of HA Oligomers Using PEG Matrix

When studying shorter fragments of HA, the CGE procedure reported by Kakehi et al. (6) and presented here gives an exceptional separation, permitting the resolution of fragments from 2 to 50 disaccharides (see Fig. 3). Running the separation under the same conditions in a longer capillary (50-cm effective length) and for a longer period allows the identification of individual fragments close to 100 disaccharides. Such size is still far from that of many intact HA preparations, which normally consist of several thousand disaccharides per molecule. Information on the molecular size distribution of longer fragments (> 100 disaccharides, i.e., molecular mass > 40 kDa) can be obtained, using the protocol described under Subheading 3.2. The capillary coating abolishes the EOF and migration is therefore caused only by the EM (see Note 8). The

Fig. 3. Separation of HA oligomers in the presence of PEG70000 as a neutral polymer. The fused capillary is coated with 50% phenyl(methyl)polysiloxane (effective length 20 cm, 100 ^m id). Operating buffer is 0.1 M Tris-0.25 M borate, pH 8.5, containing 10% PEG70000. The numbers indicate the degree of polymerization (number of monosaccharide units). Reprinted from ref. 6, © American Chemical Society.

Migration time (min)

Fig. 3. Separation of HA oligomers in the presence of PEG70000 as a neutral polymer. The fused capillary is coated with 50% phenyl(methyl)polysiloxane (effective length 20 cm, 100 ^m id). Operating buffer is 0.1 M Tris-0.25 M borate, pH 8.5, containing 10% PEG70000. The numbers indicate the degree of polymerization (number of monosaccharide units). Reprinted from ref. 6, © American Chemical Society.

operating buffer also contains high-molecular-weight polyethylene glycol which forms a gel through which the HA fragments migrate.

1. Dissolve the samples (1.0 mg/mL) in an aqueous 15 mM citric buffer, pH 5.3, or in the operating buffer.

2. Start the CE instrument. Adjust the detector to monitor absorbance at 200 nm (see Note 9).

3. Prepare the software of the CE instrument as follows.

a. Rinse the capillary with operating buffer for 5 min.

b. Introduce the sample by the electrokinetic method, using 5 kV for 10 s.

c. Run the separation for 25 min at 7.4 kV/m (10 kV for a total capillary length of 27 cm).

4. Compare elution patterns with external HA standards. The HA oligosaccharide fragments (GlcA p 1-3GlcNAc p 1) n should elute in the following order: n = 4, 3, 5, 6, 2, 7, 8, 9, 10, etc. (see Fig. 3; Note 10).

4. Notes

1. To obtain a homogeneous operating solution, the buffer should be kept at room temperature for at least 12 h after dissolution of the polysaccharide. The buffer is degassed under reduced pressure immediately prior to use.

2. This method employs low-UV detection, and it must be borne in mind that a variety of substances will cause absorption. To avoid some of this uncertainty, the spectrum (190-600 nm) may be monitored. Substances with absorbance maximum at wavelengths higher than 190 nm do not represent GAGs (aromatic amino acids of PGs absorb at 280 nm).

3. The electropherograms monitor the homogeneity of the charge density in the GAG preparations. Homogeneous sulfation of the polysaccharide results in sharp peaks. Broad and split peaks indicate that the GAG in question is contaminated or degraded.

4. For alternative CE analyses of charge density and purity of GAGs (see ref. 7).

5. This method was originally described for the analysis of pure HA preparations. Presence of interacting compounds, such as hyalectans and lectins, may interfere with the electro-phoretic performance of HA. The addition of 0.1% (w/v) SDS to the operating buffer may prevent such interactions (2,8).

6. All polyanions detected at 190 nm are not HA, and this lack of specificity still requires purification of the analyte HA and/or a comparison with hyaluronidase-digested aliquots.

7. For alternative CE analyses of high-molecular-weight HA (see refs. 1,8 and 9).

8. Most fragments migrate according to their molecular size, the largest being more retarded. The smallest fragments (2-4 disaccharides), however, differ in this respect, slower migration being obtained with the smallest ones. This effect is difficult to explain, but could be related to the complex system with alkaline borate that may interact with both the reducing end monosaccharide and the PEG matrix.

9. This determination also employs low UV detection, and the concerns for specificity presented in Notes 2, 4, and 6 should also be carefully thought about here.

10. For alternative CE analyses of HA fragments, (see refs. 8 and 9).

References

1. Karamanos, N. K. and Hjerpe, A. (1998) A survey of methodological challenges for gly-cosaminoglycan/proteoglycan analysis and structural characterization by capillary elec-trophoresis. Electrophoresis 19, 2561-2571.

2. Payan, E., Presle, N., Lapicque, F., Jouzeau, J. Y., Bordji, K., Oerther, S., Miralles, G., and Netter, P. (1998) Separation and quantitation by ion-association capillary zone electrophore-sis of unsaturated disaccharide units of chondroitin sulfates and oligosaccharides derived from hyaluronan. Anal. Chem. 70, 4780-4786.

3. Karamanos, N. K., Axelsson, S., Vanky, P., Tzanakakis, G. N., and Hjerpe, A. (1995) Determination of hyaluronan and galactosaminoglycan-derived disaccharides by high-performance capillary electrophoresis at the attomole level. Applications to analyses of tissue and cell culture proteoglycans. J. Chromatogr. A 696, 295-305.

4. Karamanos, N. K. and Hjerpe, A. (1999) Strategies for analysis and structure characterization of glycan/proteoglycans by capillary electrophoresis. Their diagnostic and biopharmaceutical importance. Biomed. Chromatogr. 13, 507-512.

5. Hayase, S., Oda, Y., Honda, S., and Kakehi, K. (1997) High-performance capillary electrophoresis of hyaluronic acid: determination of its amount and molecular mass. J. Chromatogr. A. 768, 295-305.

6. Kakehi, K., Kinoshita, M., Hayase, S., and Oda, Y. (1999) Capillary electrophoresis of N-acetylneuraminic acid polymers and hyaluronic acid: correlation between migration order reversal and biological functions. Anal. Chem. 71, 1592-1596.

7. Toida, T. and Linhardt, R. J. (1996) Detection of glycosaminoglycans as a copper (II) complex in capillary electrophoresis. Electrophoresis 17, 341-346.

8. Grimshaw, J., Kane, A., Trocha-Grimshaw, J., Douglas, A., Chakravarthy, U., and Archer, D. (1994) Quantitative analysis of hyaluronan in vitreous humor using capillary electrophoresis. Electrophoresis 18, 2408-2414.

9. Hong, M., Sudor, J., Stefansson, M., and Novotny, M. V. (1998) High-resolution studies of hyaluronic acid mixtures through capillary electrophoresis. Anal. Chem. 70, 568-583.

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