Separation and Quantification by Ion-Association Capillary Zone

Oct 8, 1998 - An analytical method has been developed for assay of unsaturated disaccharides of chondroitin sulfates and of oligosaccharides (tetra- a...
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Anal. Chem. 1998, 70, 4780-4786

Separation and Quantification by Ion-Association Capillary Zone Electrophoresis of Unsaturated Disaccharide Units of Chondroitin Sulfates and Oligosaccharides Derived from Hyaluronan E. Payan, N. Presle, F. Lapicque, J. Y. Jouzeau, K. Bordji, S. Oerther, G. Miralles, D. Mainard, and P. Netter*

Laboratoire de Pharmacologie et UMR 7561 CNRS-UHP, Faculte´ de Me´ decine, B.P. 184, 54505 Vandoeuvre le` s Nancy Cedex, France

An analytical method has been developed for assay of unsaturated disaccharides of chondroitin sulfates and of oligosaccharides (tetra- and hexasaccharides) of hyaluronan, using ion-association capillary zone electrophoresis. Samples were applied at the anode (the usual polarity), using a borate buffer modified by an ion-pairing reagent, tetrabutylammonium (TBA) phosphate, and the effect of the concentration of the ion-pairing reagent on various electrophoretic parameters (electroosmotic flow, electrophoretic mobility of products, capacity factors) was observed. Increasing concentrations of the reagent led to a decrease of ζ potential, probably due to specific adsorption of the quaternary ammonium ion onto the capillary wall. The authors propose a mechanism of separation, in which anionic borate complexes are formed and interact with TBA ion inside the capillary tube. The capillary electrophoretic system described is potentially a powerful method for specific measurement of the concentrations in joint tissues of chondroitin 4-sulfate, chondroitin 6-sulfate, and hyaluronan, whose relative abundances may vary in various diseases or after local treatments with, for example, antiinflammatory drugs, chondroprotective agents, or orthopedic implants. The extracellular matrix of connective tissues is a charged, macromolecular network of collagen, glycosaminoglycans (GAGs), and glycoproteins. Covalent binding of some GAGs to a core protein leads to proteoglycans, which can be classified into three groups: (i) versicans and aggrecans, large aggregating proteoglycans interacting with hyaluronic acid (HA); (ii) decorins and biglycans, small proteoglycans that interact with fibrillar collagen; and (iii) perlecans, the basement membrane proteoglycans. The main constituents of types i and ii are chondroitin sulfates (CS), dermatan sulfates (DS), and keratan sulfates (KS), while proteoglycans of type iii contain mainly heparan sulfates (HS).1 Most GAGs have disaccharide sequences of uronic acid (Dglucuronic acid or L-iduronic acid) linked to N-acetylated hex* Corresponding author. Tel.: 03 83 59 26 22. Fax: 03 83 59 26 21. E-mail: [email protected]. (1) Hadad, S. J.; Michelacci, Y. M.; Schor, N. Biochim. Biophys. Acta 1996, 1290, 18-28.

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osamine residues (D-glucosamine for HA and HS and D-galactosamine for CS and DS). All GAGs except HA have sulfate substituents, mainly on the N-acetylated hexosamine residues, in position 4 or position 6 or both, and to a lesser extent on the uronic acid residue. The heterogeneous GAGs CS and DS are especially difficult to analyze because of the random linkages of their various nonsulfated, monosulfated, or polysulfated disaccharide units. A recent issue of the Journal of Chromatography focused on the specific separation of oligosaccharides in various analytical conditions (J. Chromatogr. A 1996, 720). In the past few years, high-performance capillary electrophoresis (HPCE) methods have been developed to analyze GAGs. Some of these methods use UV detection of disaccharide units and oligosaccharides. The separation is achieved by capillary zone electrophoresis, either in the usual polarity conditions (i.e., with the sample applied at the anode and a basic running buffer)2,3 or in reversed polarity conditions, using an acidic running buffer. In these conditions, sensitivity was low because of the direct detection of disaccharides.4-7 A precolumn derivatization has been proposed to improve the selectivity and the resolution,8,9 allowing detection limits in the femto- to attomole range.10 Labeling can then be obtained by pyridylamination reaction with 2-aminopyridine (PA)11 or 2-aminoacridone12 at the reducing end of monosaccharides, disaccharides, or oligosaccharides. Chondroitin sulfate-derived and hyaluronic acid oligosaccharides have been also converted (2) Al-Hakim, A.; Linhardt, R. J. Anal. Biochem. 1991, 195, 68-73. (3) Denuzie`re, A.; Taverna, M.; Ferrier, D.; Domard, A. Electrophoresis 1997, 18, 745-750. (4) Pervin, A.; Al-Hakim, A.; Linhardt, R. J. Anal. Biochem. 1994, 221, 182188. (5) Karamanos, N. K.; Axelsson, S.; Vanky, P.; Tzanakakis, G. N.; Hjerpe, A. J. Chromatogr. A 1995, 696, 295-305. (6) Karamanos, N. K.; Vanky, P.; Syrokou, A.; Hjerpe, A. Anal. Biochem. 1995, 225, 220-230. (7) Hayase, S.; Oda, Y.; Honda, S.; Kakeki, K. J. Chromatogr. A 1997, 768, 295-305. (8) Oefner, P. J.; Chiesa, C. Glycobiology 1994, 4, 397-412. (9) Hase, S. J. Chromatogr. 1996, 720, 173-182. (10) Paulus, A.; Klockow, A. J. Chromatogr. A 1996, 720, 353-376. (11) Honda, S.; Iwase, S.; Makino, A.; Fujwara, S. Anal. Biochem. 1989, 176, 72-77. (12) Kitagawa, H.; Kinoshita, A.; Sugahara, K. Anal. Biochem. 1995, 232, 114121. 10.1021/ac9800558 CCC: $15.00

© 1998 American Chemical Society Published on Web 10/08/1998

respectively into 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives13 and 1-(4-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP) derivatives,14 which absorbed UV light strongly and could be detected as low as 10-6 M. However, the increased sensitivity allowed by derivatization is dampened by the increase in analysis duration. On the other hand, when oligosaccharide concentrations are very low or sample amount is very small, derivatization procedures may lead to inaccurate quantitative result in the case of uncomplete reaction. Another capillary electrophoretic system, micellar electrokinetic capillary chromatography (MEKC), first described by Terabe et al.,15,16 has been used to improve the resolution of disaccharides derived from glycosaminoglycans. This system is based on ionpairing and hydrophobic interactions between analytes and surfactant’s micelles. In fact, when anionic or cationic surfactants are added to the separation buffer above their critical micellar concentration, the analytes may be solubilized into micelles, leading to a modification of their electrophoretic mobility. This electrokinetic chromatography combines the performance of HPCE with the separation principles of chromatography and has been used to separate neutral or ionic substances. Using this system, Holland et al.17 succeeded in separating structural isomers or chiral molecules using suitable surfactant. MEKC is also useful for separation and quantification of various analytes after direct injection of biological fluid, since micelles can solubilize proteins.17 MEKC has been applied for analysis of numerous carbohydrates, because these compounds can form with borate ions negatively charged complexes,10,18-20 which interact with micelles of various surfactants.21-23 In this way, some ionic carbohydrates, including unsaturated oligosaccharides derived from glycosaminoglycans, have been separated in borate buffer systems containing an anionic surfactant such as sodium dodecyl sulfate24,25 or a cationic surfactant such as cetyltrimethylammonium bromide26 above their critical micellar concentration. Another method has been proposed for separation of ionic analytes having very similar electrophoretic mobilities through ion-pair formation with suitable buffer additives: Terabe and Isemura27 performed the separation of isomeric ions by ion exchange electrokinetic chromatography. Electrophoretic mobility of highly charged polysaccharides28 can be controlled by ionpairing reagents as proposed for the separation of highly charged (13) Honda, S.; Ueno, T.; Kakeki, K. J. Chromatogr. 1992, 608, 289-295. (14) Kakehi, K.; Honda, S. Appl. Biochem. Biotechnol. 1993, 43, 55-71. (15) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111-113. (16) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, 834-841. (17) Holland, L. A.; Chetwyn, N. P.; Perkins, M. D.; Lunte, S. M. Pharm. Res. 1997, 14, 372-387. (18) Lees, E. M.; Weigel, H. J. Chromatogr. 1964, 16, 360-364. (19) Hoffstetter-Kuhn, S.; Paulus, A.; Gassmann, E.; Widmer, H. M. Anal. Chem. 1991, 63, 1541-1547. (20) El Rassi, Z. Adv. Chromatogr. 1994, 34, 177-250. (21) Cai, J.; El Rassi, Z. J. Chromatogr. 1992, 608, 31-45. (22) Michaelsen, S.; Moller, P.; Sorensen, H. J. Chromatogr. 1992, 608, 363374. (23) Bjergegaard, C.; Michaelsen, S.; Sorensen, H. J. Chromatogr. 1992, 608, 403-411. (24) Carney, S. L.; Osborne, D. J. Anal. Biochem. 1991, 195, 132-140. (25) Ampofo, S. A.; Wang, H. M.; Linhardt, R. J. Anal. Biochem. 1991, 199, 249-255. (26) Michaelsen, S.; Schroder, M. B.; Sorensen, H. J. Chromatogr. A 1993, 652, 503-515. (27) Terabe, S.; Isemura, T. Anal. Chem. 1990, 62, 650-652. (28) Stefansson, M.; Novotny, M. Anal. Chem. 1994, 66, 3466-3471.

metal chelates under the terminology ion-association capillary zone electrophoresis (IA-CZE).29 In this context, we investigated the separation and quantification of unsaturated isomers of disaccharide units derived from glycosaminoglycans without derivatization procedures. Two separation systems were proposed using new borate buffer modifiers: cholic acid for MEKC and tetrabutylammonium phosphate for IACZE. Disaccharides of HA, nonsulfated chondroitin, chondroitin 4-sulfate, and chondroitin 6-sulfate were used to elucidate the mechanism of separation by IA-CZE. Then the IA-CZE method was calibrated for analysis of unsaturated disaccharides of chondroitin 4- and 6-sulfate and the final products (tetra- and hexasaccharides) of enzymatic hydrolyzis of hyaluronan. The method was developed for analysis of major glycosaminoglycans in articular tissues such as cartilage and synovial fluid and offers a new approach to the study of joint diseases, particularly alterations in cartilage metabolism30-34 and pharmacological effects of local treatment of joints. EXPERIMENTAL SECTION Reagents. Chondroitin sulfate C (chondroitin 6-sulfate) isolated from shark cartilage, hyaluronic acid disaccharide (sodium salt), ∆di-HA, [2-acetamido-2-deoxy-3-O-(8-D-gluco-4-enopyranosyluronic acid)-D-glucose], chondroitin disaccharides (sodium salts), ∆di-0S [2-acetamido-2-deoxy-3-O-(8-D-gluco-4-enopyranosyluronic acid)-D-galactose], ∆di-4S [2-acetamido-2-deoxy-3-O-(8-Dgluco-4-enopyranosyluronic acid)-4-O-sulfo-D-galactose], ∆di-6S [2-acetamido-2-deoxy-3-O-(8-D-gluco-4-enopyranosyluronic acid)-6O-sulfo-D-galactose], hyaluronic acid (HA) purified from rooster combs, chondroitinase ABC from Proteus vulgaris (EC 4.2.2.4.), and hyaluronidase from Streptomyces hyalurolyticus (EC 4.2.2.1) were purchased from Sigma (La Verpillie`re, France). Chondroitin sulfate A (chondroitin 4-sulfate) from ovine nasal cartilage was obtained from Jacques Boy Institut (Reims, France). Benzoyl 1-4 phenyl-2 butyric acid (BPBA), used as internal standard, was a gift from Rhoˆne Poulenc Rorer (Vitry/Seine France). All other chemicals and solvents were of analytical grade. Conductivity water (resistivity greater than 18.2 MS‚cm) was used. Instrumentation. Analyses were done on a Spectraphoresis 1000 capillary electrophoresis system using the integrated method and analysis program Spectrasystem CE 1000, manufactured by Thermo Separation Products (TSP, Les Ulis, France). Fused-silica capillaries (75 µm i.d.) were obtained from TSP. Injections were done hydrodynamically. The sample migration was run at various voltages to get the best resolution in the shortest migration time. Compounds were monitored with a focus UV detector at 232 nm. Electrophoresis was done with the usual polarity (sample applied at the anode). Before each analytical series, the capillary was etched at 60 °C with 1 and 0.1 M NaOH for 15 and 10 min, respectively, and rinsed with water for 5 min at 30 °C. Running (29) Iki, N.; Hoshino, H.; Yotsuyanagi, T. J. Chromatogr. 1993, 652, 539-546. (30) Shinmei, M.; Miyauchi, S.; Machida, A.; Miyazaki, K. Arthritis Rheum. 1992, 35, 1304-1308. (31) Deutsch, A. J.; Midura, R. J.; Plaas, A. H. K. J. Orthop. Res. 1995, 13, 230239. (32) Grimshaw, J.; Trocha-Grimshaw, J.; Fisher, W.; Rice, A.; Smith, S.; Spedding, P.; Duffy, J.; Mollan, R. Electrophoresis 1996, 17, 396-400. (33) Sharif, M.; Osborne, D. J.; Meadows, K.; Woodhouse, S. M.; Colvin, E. M.; Shepstone, L.; Dieppe, P. A. Br. J. Rheumatol. 1996, 35, 951-957. (34) Belcher, C.; Yaqub, R.; Fawthrop, F.; Bayliss, M.; Doherty, M. Ann. Rheum. Dis. 1997, 56, 299-307.

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buffer was then introduced and allowed to equilibrate with the silica capillary for 15 min at 30 °C. Each run was followed by a 2-min rinse with running buffer. All buffers were filtered before use on a Millex HV13 0.45-µm filter (Millipore, Saint Quentin en Yvelines, France). Sample Preparation. Study of Various Electrophoretic Conditions. A standard solution of both tetra- and hexasaccharides of HA was obtained by enzyme hydrolysis for 48 h at 37 °C. HA (10 mg) was mixed with 100 units of hyaluronidase in acetate buffer (20 mM, pH 6). Standards of ∆di-HA, ∆di-0S, ∆di-4S, and ∆di-6S were used alone or mixed with acetate buffer (1 mg/mL) to optimize electrophoretic conditions. Validation of Optimized Electrophoretic Conditions. A calibration curve of HA was prepared by mixing various amounts of HA (5-50 µg) with 2 units of hyaluronidase in 690 µL of acetate buffer (20 mM, pH 6). This mixture was incubated for 48 h at 20 °C and filtered for 1 h at 10 000 rpm on Ultrafree-MC (10 000 NMWL polysulfone) (Millipore). The calibration curves of the ∆di-4S (0.5-2 µg) and ∆di-6S (0.625-2.5 µg) mixtures were prepared by adding the substances for assay to 400 µL of Tris/HCl buffer (50 mM, pH 8) and filtering. Before injection, a fixed volume (10 µL) of BPBA solution [160 µg/mL in Tris/HCl buffer (50 mM, pH 8)] was added to 150 µL of ultrafiltrate, as an internal standard. Calibration curves were calculated by least-squares linear regression of the response (i.e., the ratio of the area of the oligosaccharide peak to the area of the internal standard peak) versus the concentration. The reproducibility was evaluated for nine measurements of calibration points. Electrophoretic Conditions. MEKC Analysis in Normal Polarity Using Bile Salts. Standard samples were injected hydrodynamically using borate buffer (pH 9) (10 mmol/L sodium tetraborate and 50 mmol/L boric acid) alone or with sodium cholate (100 mmol/L) added. Various electrophoretic conditions were studied, using various capillary lengths (44, 70, and 94 cm), voltages (10-24 kV), temperatures (30-60 °C), and durations of injection (3, 5, and 10 s). IA-CZE Analysis in Normal Polarity Using an Ion-Pairing Reagent, TBA Ion. Standard samples were injected hydrodynamically, and electrophoresis was performed at 17 kV and 30 °C using the borate buffer (pH 9) with added tetrabutylammonium (TBA) phosphate. Various concentrations (0.5, 1, 2, 5, 7.5, 10, 15, 20, 25, or 50 mM) of the ion-pairing reagent were applied to identify the optimal separation conditions. Optimization Parameters. Parameters determined to optimize the electrophoretic conditions were as follows: The apparent electrophoretic mobility (µa) and the effective solute mobility (µe) (cm2/V‚s) were calculated for each compound according to the relations

µa ) µe + µEOF ) lL/tV

(1)

µe ) lL/V (t-1 - tEOF-1)

(2)

where µEOF is the electroosmotic flow (EOF) mobility, determined with a neutral marker (acetone) moving at a velocity equal to the EOF (cm2/V‚s), l is the effective capillary length of the detector (here 37, 63, or 87 cm), L is the total capillary length (here 44, 70, or 94 cm), t is the migration time (s) of the compound, tEOF is the migration time (s) of the neutral marker, and V is the applied voltage (V). 4782 Analytical Chemistry, Vol. 70, No. 22, November 15, 1998

The resolution Rs between two adjacent peaks was measured according to the relation

Rs ) 2(t2 - t1)/(w1 + w2)

(3)

where t1 and t2 are the migration times (s) of components 1 and 2, respectively, and w1 and w2 are the baseline widths (s) of the peaks of components 1 and 2, respectively. The capacity factor is defined as

k′ ) (tm - t0)/t0

(4)

where tm is the migration time (s) of retained solute and t0 is the migration time (s) of an unretained solute (neutral marker). RESULTS AND DISCUSSION The separation and quantification of disaccharide and oligosaccharide derivatives of glycosaminoglycans by capillary electrophoresis, as reported here, will make it possible to analyze these derivatives in tissues, such as cartilage or synovial fluid, that are rich in GAGs and hyaluronan. For the two different separation methods presented here, hydrolyzed samples were concentrated by ultrafiltration on a lowbinding polysulfone membrane. This process has two advantages: (i) it avoids solvent extraction of GAG disaccharides which are unstable during evaporation, as mentionned by Michaelsen et al.,26 and (ii) it avoids the presence of impurities due to the usual enzymatic treatment of biological samples that may interfere in the electropherogram. Hydrolysis of glycosaminoglycans with specific enzymes such as chondroitinases yields a mixture of saturated and unsaturated disaccharide derivatives with very similar structures. For example, ionized ∆di-HA and ∆di-0S have the same negative charge, due to the carboxylate group of the glucuronic residue, and differ only by the cis or trans position of the hydroxyl function on carbon 4 of the hexosamine residue. For monosulfated derivatives, such as ∆di-4S and ∆di-6S, the only difference is in the position of the sulfated group on the galactosamine residue, the net charge being similar. To separate these groups of isomers, we used an alkaline borate buffer, as proposed by Al-Hakim et al.2 The borate buffer has the additional advantage that it forms anionic borate complexes with disaccharides that have a higher UV absorbance than the free ionic forms.20,35 A running buffer at pH 9 was used because this pH offers a fair compromise between optimal formation of borate complexes and resolution of peaks.36 Before sodium cholate and tetrabutylammonium phosphate were studied as potential buffer modifiers, tetrahydrofuran or methanol was added at 2-5% to borate buffer (pH 9) as organic modifier (data not shown). The results of these preliminary tests were not decisive, since the retention times of borate complexes were longer than those without modifiers and the nonsulfated disaccharides could not be distinguished from the monosulfated disaccharides. Therefore, MEKC and IA-CZE were carried out with sodium cholate and tetrabutylammonium phosphate, respectively. (35) Bo ¨seken, J. Adv. Carbohydr. Chem. 1949, 4, 189-210. (36) Arentoft, A. M.; Michaelsen, S.; Sorensen, H. J. Chromatogr. A 1993, 652, 517-524.

Table 1. Variation of Resolution (Rs) between Unsulfated and Monosulfated Disaccharides versus Capillary Length and Voltagea capillary length, cm

voltage, kV

94

17

94

70 44

∆di-HA/∆di-0S

Without Cholate 0.014

With 100 mM Cholate 10 0.740 13 0.658 15 0.636 17 0.709 20 0.921 22 0.773 24 0.688 15 15

∆di-6S/∆di-4S 0.157 0.723 0.852 0.655 0.919 0.847 0.903 0.980 0.165 0.015

a Conditions: borate buffer B (pH 9) with or without cholate, 100 mM. Hydrodynamic injection, 10 s. All measurements were done at 30 °C.

MEKC Analysis in Normal Polarity Using Sodium Cholate. Because of their amphipatic property, bile salts are useful in separation of chiral compounds, especially for the chiral nonsulfated molecules ∆di-HA and ∆di-0S (Table 1). These micellar conditions were also adapted to determine corticosteroids in synovial fluid.37,38 Therefore, as corticosteroid treatment in articular pathologies resulted in high levels of proteoglycans in synovial fluid, which indicated joint cartilage degradation,39 it would be interesting to evaluate both corticosteroid and glycosaminoglycan derivatives in the same biological sample. In fact, concentrations of glycosaminoglycans, chondroitin sulfate, and hyaluronan in knee synovial fluid may be used as markers of the cartilage degradation process.34,40 The micellar conditions were the same as for the analysis of methylprednisolone in synovial fluid (capillary length 44 cm, hydrodynamic injection lasting 3 s, 15 kV, 30 °C).38 Even though tetra- and hexasaccharides derived from hyaluronan were rapidly separated in these conditions (Figure 1), disaccharide derivatives could not be separated. In fact, to maintain an optimal analysis of corticosteroids, cholate concentration and electrolyte concentration are not changed. Only capillary length, voltage, temperature, and duration of injection have been modified. Within each isomer group, the resolution between disaccharides was largely better when the capillary length was increased to 94 cm (Table 1) but was not affected by an increase in temperature from 30 to 60 °C. The increased temperature shortened the migration times of all components but impaired the quality of the electropherogram by increasing the noise baseline (data not shown). With a capillary length of 94 cm at 30 °C, the best resolution between adjacent peaks was obtained at 17-20 kV (Table 1); under these conditions, the areas under the peaks of all separated disaccharides varied linearly with the hydrodynamic injection duration between 2 and (37) Nishi, H.; Fukuyama, T.; Matsuo, M.; Terabe, S. J. Chromatogr. 1990, 513, 279-295. (38) Payan, E.; Jouzeau, J. Y.; Lapicque, F.; Bordji, K.; Simon, G.; Gillet, P.; O’Regan, M.; Netter, P. J. Control. Release 1995, 34, 145-153. (39) Roneus, B.; Lindlab, A.; Lindholm, A.; Jones, B. J. Vet. Med. 1993, 40, 1016. (40) Okasaki, J.; Kakudo, K.; Kamada, A.; Utoh, E.; Gonda, Y.; Shirasu, R.; Sakaki, T. Eur. J. Oral Sci. 1997, 105, 440-443.

Figure 1. Separation of unsaturated disaccharidic units of glycosaminoglycans and oligosaccharides of hyaluronan. Capillary, fused silica (75 µm i.d., 44 cm); capillary temperature, 30 °C; carrier, borate buffer B (pH 9) with sodium cholate (100 mM); applied voltage, 15 kV; detection, UV absorption at 232 nm. Peaks: a ) ∆di-HA, b ) ∆di-OS, T ) HA tetrasaccharide, H ) HA hexasaccharide, MPL ) methylprednisolone (corticosteroid), c ) ∆di-6S, d ) ∆di-4S.

10 s. The sensitivity of the method being sufficient to determine HA concentration in synovial fluid, injection duration could be kept below 3 s for a satisfactory reproducibility. CZE Analysis in Normal Polarity Using Ion-Pairing Reagent. In alkaline buffer, acidic molecules are negatively charged and migrate toward the anode, whereas under EOF they move to the cathodic side. To modulate migration and to improve both separation and resolution, it was necessary to reduce or reverse the EOF power. The modification of charges by the addition of a tetraalkylammonium ion was proposed by Jorgenson and coworkers.41,42 The mechanism for migration of neutral organic molecules consists then in a dynamic equilibrium between associated species with a positive charge and dissociated species with no charge. The addition of these buffer modifiers allows reduction in electroosmotic flow with improving resolution between separated compounds. Carbohydrate analysis relies upon the formation of an anionic borate-polyol complexe that migrates toward the cathode by combined effects of electrophoresis and electroosmosis.11,36 For acidic carbohydrates such as disaccharides derivatives of glycosaminoglycans, anionic borate complexes are in equilibrium with the intact carbohydrate. However, because of an additional carboxyl group due to uronic acid of disaccharides derivatives, these complexes were strongly retained at the anodic end.43 In the presence of an ionic reagent of opposite charge, analyte anion is combined with the cationic modifier and forms an ion-pair which migrates toward the cathode (Figure 2). Hence, electrophoretic mobility of charged compounds can be modified by the ion-pair formation between anionic analyte and cationic reagent.27 Huang (41) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (42) Walbroehl, Y.; Jorgenson, J. W. Anal. Chem. 1986, 58, 479-481. (43) Honda, S. J. Chromatogr. 1996, 720, 337-351.

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Figure 2. Illustration of the separation mechanism of in situ formed main ion-paired of borate complexes by IA-CZE with tetrabutylammonium phosphate. (A) After equilibrated fused-silica capillary with running buffer pH 9; (B) after sample injection. EOF ) electroosmotic flow; SiO- ) negatively charged fused-silica surface; B- ) B(OH)4-; N+ ) N(Bu)4+.

Figure 3. Effects of various tetrabutylammonium (TBA) phosphate concentrations on the electrophoretic and electroendosmotic mobility, respectively (µa and µEOF) (A) and capacity factors (k′) (B) of solutes ∆di-AH (a), ∆di-OS (b), ∆di-6S (c), and ∆di-4S (d). The capillary was 75 µm i.d. and 94 cm in length, UV detection was at 232 nm, and injection time duration was 10 s. The run was performed with borate electrolyte (pH 9) (10 mM sodium borate; 50 mM boric acid) at 17 kV and 30 °C.

et al.44 reported a mechanism for tetradecyltrimethylammonium bromide that attached to the inner surface of fused-silica capillaries and influenced the electroosmotic flow in an opposite direction; they suggested that absorbed quaternary ammonium salt might also act as an anion exchanger. Using cetyltrimethylammonium bromide (CTAB) above its critical micellar concentration (2 mM), Michaelsen et al.26 observed the formation of a positively charged layer on the inner wall of the capillary and reversal of EOF, the latter being reduced only with CTAB concentration of 0.2 mM.45 Dodecyltrimethylammonium bromide (DTAB), which is known to interact strongly with hyaluronic acid,46 has been used for separation of various molecules by MEKC, as was done for charged metal chelates.29 In comparison, IA-CZE with a smaller quaternary ammonium salt, TBA bromide, provided a good resolution between analytes.29 Under these conditions, EOF was (44) Huang, X.; Luckey, J. A.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1989, 61, 766-770. (45) Varghese, J.; Cole, R. B. J. Chromatogr. 1993, 652, 369-376. (46) De Fatima Santos, S.; Nome, F.; Zanette, D. J. Colloid Interface Sci. 1994, 164, 260-262.

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unchanged but the value of µEOF was diminished. The authors mentioned a specific adsorption of TBA ion onto the capillary wall, decreasing the ζ potential. The optimum separation was obtained for 25 mM TBA ion, and the decreased µa values of the different metal chelates might originate from the formation of ion associates between each solute and TBA ions, leading to reduction of total charge of the associate species. A similar mechanism could be proposed for the separation of disaccharides derivatives by IACZE with tetrabutylammonium ion. The effect of addition of TBA ions to the electrophoresis buffer on the apparent electrophoretic mobility, µa, calculated according to relation 1, is shown in Figure 3A. With increasing concentrations of TBA ions from 0 to 50 mmol/L, µEOF decreased from 753 × 10-6 to 507 × 10-6 cm2.V‚s. The quaternary ammonium ion is adsorbed onto the negatively charged (with SiO-) capillary wall, and as its concentration increases, the effective charge of the wall, or ζ potential, is decreased. Consequently, the apparent electrophoretic mobilities (µa) of all the solutes are decreased to within a range similar to that observed for a neutral marker such as

Figure 4. Effects of various tetrabutylammonium (TBA) phosphate concentrations on the resolution (Rs) between paired peaks (∆di-AH and ∆di-OS, I; ∆di-6S and ∆di4S, II). The capillary was 75 µm i.d. and 94 cm in length, UV detection was at 232 nm, and injection time duration was 10 s. The run was performed with pH 9 borate electrolyte (10 mM sodium borate; 50 mM boric acid) at 17 kV and 30 °C.

acetone (Figure 3A), and the effective electrophoretic mobilities (µe) are very weakly altered by the presence of TBA ions. These results are in accordance with the work of Stefansson and Novotny,28 who have shown that the electrophoretic mobility of highly charged polysaccharides such as heparins can be controlled by the use of ion-pairing reagents. The capacity factors are not much different for adjacent nonsulfated compounds (Figure 3B, a and b) or for monosulfated products (Figure 3B, c and d). However, the resolution between the paired peaks (a,b or c,d) is significantly improved with increasing concentrations of TBA ions (Figure 4). The separation by IA-CZE in the presence of quaternary ammonium ions could be taken into account for the following mechanism (Figure 2). In alkaline borate at pH 9, anionic disaccharide complexes are repelled by the negatively charged surface of untreated fusedsilica capillaries. This repulsion prevents adsorption on the surface, and separation is then due solely to the distribution of negative charges on the borate complex and to its stability. Borate complexation increases the negative charge of polyol disaccharides, and the complexes formed are more stable when the OH radicals involved in the complex are in the cis position for the nonsulfated disaccharides or, for the monosulfated disaccharides, if a sulfated substituent is present in the molecule (∆di-4S or ∆di6S).8,35 In the presence of a cationic surfactant such as TBA phosphate, a positive double layer is formed at the capillary wall. When the concentration of surfactant increases, the density of positive charge in the capillary increases, mostly near the walls, leading to an increase in the current, which varies linearly with the concentration of surfactant from 16 to 90 µA at a constant voltage (17 kV). The formation of this mobile layer of positive charges in the buffer adjacent to the wall presents two advantages. First, when the voltage is applied across the capillary, the quaternary ammonium cations are attracted toward the cathode, and simultaneously the positive charges draw the negatively charged solutes in the same direction, with a velocity depending on the distribution of the negative charges. Second, a flow of negatively charged borate ions is maintained inside the capillary, and they migrate toward

Figure 5. Separation of unsaturated disaccharidic units of glycosaminoglycans. Capillary, fused silica (75 µm i.d., 94 cm); capillary temperature, 30 °C; carrier, borate buffer B (pH 9) with TBA phosphate (15 mM); applied voltage, 17 kV; detection, UV absorption at 232 nm. Peaks: a ) ∆di-HA, b ) ∆di-OS, BPBA ) internal standard, c ) ∆di-6S, d ) ∆di-4S.

the anode (Figure 2A). When the disaccharides are injected and the voltage is applied, the borate ions give complexes of different stabilities and different negative charge distributions, as explained above. At pH 9, the complexes also have a free ionized carboxylate moiety (COO-) that can interact with TBA ions (Figure 2B), thus modifying the charge environments and the masses of the borate complexes. An equilibrium between the various forms (ionpaired borate complexes, borate complexes, and free disaccharides) is established inside the capillary. The potential neutralization of the charge on the carboxylate moiety by ion-pairing avoids the possible repulsion by the charge in the borate complexes, which become more stable. These ion-paired borate complexes, with their reduced charge, could be subject to two opposite processes. The first one would be due to the reduction in the charge distribution, leading to the complexes’ more rapid migration toward the cathode. The second process consists of a possible interaction of positively charged TBA complexes with the negatively charged inner wall, which would slow their progress through the capillary. This latter process could explain why the effective electrophoretic mobility of the ion-paired borate complexes is so weakly influenced by the presence of tetrabutylammonium ions in the buffer (Figure 3A), even if the resolution of the paired nonsulfated and monosulfated compounds is improved by these conditions (Figure 4). The electrophoretic mobility of the borate complexes is in accordance with published data5 on the weak capacity factor of nonsulfated compounds and their lower binding to the capillary than that of monosulfated compounds (Figure 3B). Under our analytical conditions, the retention times of borate complexes were longer than those currently observed in borate buffer2 and in borate/phosphate buffer.24 Paired nonsulfated compounds were clearly separated from paired monosulfated compounds for a concentration of the surfactant TBA at 15 mmol/L (Figure 5). This IA-CZE procedure was validated with standard solutions of ∆di-4S and ∆di-6S, plotting and tetra- and hexasaccharides of HA obtained after hydrolysis by hyaluronidase lyase. The observed peak area (corrected by the area of the peak of the internal standard) versus the concentration of the sample yields linear calibration curves (r ) 0.999) in the concentration Analytical Chemistry, Vol. 70, No. 22, November 15, 1998

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Table 2. Reproducibility Determination of Calibration Points of HA, ∆di-4S, and ∆di-6Sa HA Calibration sample size

mean SD % RSD

10 µg

20 µg

40 µg

9.85 0.32 3.25

19.96 0.66 3.3

40.30 0.49 1.2

∆di-4S Calibration sample size

mean SD % RSD

0.5 µg

1 µg

2 µg

0.51 0.03 6.3

1.01 0.03 3.0

2.00 0.13 6.4

∆di-6S Calibration sample size

mean SD % RSD

0.625 µg

1.25 µg

2.5 µg

0.64 0.03 4.0

1.26 0.04 3.1

2.50 0.18 7.1

a Results are expressed as mean of nine experimental measures (M) ( standard deviation (SD). Calculation of relative standard deviation, % RSD ) SD/M × 100.

range 1.25-6.25 µg/mL for ∆di-4S and ∆di-6S and 7.2-72 µg/ mL for HA, in the buffer and in the enzyme mixture, respectively. The threshold of detection for these compounds in an hydrodynamic injection lasting 5 s was about 5 and, 25 ng, respectively. Good reproducibility of this method was found for the analysis of ∆di-4S, ∆di-6S, and HA. The reproducibility was evaluated for nine measurements of calibration points. The relative standard deviations were below 7% (Table 2) for all the calibration points. These data show that this methodology can be used for the evaluation of GAGs in biological tissues such as cartilage or synovial fluid (Figure 6). In conclusion, MEKC method with cholate can be used to analyze simultaneously corticoids and HA derivatives obtained after hyaluronidase hydrolysis of biological samples. The determination of chondroitin 4S and 6S disaccharides is not satisfactory in these conditions in comparison with the results obtained by CTAB-MEKC.26 However, Cholate-MEKC was studied, taking into account the presence of HA in biological samples. In fact, ∆diHA is obtained after enzymatic treatment of those by chondroitinase ABC and could be interfered with another chiral compound, such as ∆di-0S. These interferences were not observed with CTAB-MEKC.26 As cholate addition in electrolyte does not improve sufficiently disaccharides analytes’ separation, and to avoid changing the optimal separation of corticosteroids, it seemed

4786 Analytical Chemistry, Vol. 70, No. 22, November 15, 1998

Figure 6. Example of electropherograms obtained from a hydrolyzed synovial fluid by hyaluronidase (top electropherogram) and by chondroitinase ABC (lower electropherogram). Capillary, fused silica (75 µm i.d., 94 cm); capillary temperature, 30 °C; carrier, borate buffer B (pH 9) with TBA phosphate (15 mM); applied voltage, 17 kV; detection, UV absorption at 232 nm; hydrodynamic injection, 5 s. Peaks: a ) ∆di-HA, BPBA ) internal standard, c ) ∆di-6S, d ) ∆di4S, T ) HA tetrasaccharide, H ) HA hexasaccharide.

preferable to use another HPCE method to separate all GAG disaccharides. The proposed method, based on IA-CZE mechanism, allows separation of disaccharidic borate complexes without sample prederivatization. Good resolution between unsulfated and monosulfated disaccharides was obtained in comparison of CZE mechanism with only borate buffer.2 In addition, this method can be applied to the analysis of biological tissues or fluids without interferences of endogenous consituents, since sample treatment by enzymatic hydrolysis and ultrafiltration are sufficient to avoid them. These two methods were both successful for analysis of tetra- and hexasaccharides of HA, but MEKC systems with capillary of 44 cm have advantageously smaller analysis durations. If determination of sulfated GAG is considered, then the IA-CZE method is the most adapted, despite long analysis duration. ACKNOWLEDGMENT We thank G. Simon and M. H. Piet for their technical assistance.

Received for review January 21, 1998. Accepted August 25, 1998. AC9800558