Development of an Analytical Method for Unsaturated Disaccharides from Heparan Sulfate Using Dansylhydrazine Derivatization and High-Performance Liquid Chromatography with Fluorescence Detection

Abstract

Glycosaminoglycans (GAGs), such as heparan sulfate (HS), play essential roles in living organisms. Understanding the functionality of HS and its involvement in disease progression necessitates the sensitive and quantitative detection of HS-derived unsaturated disaccharides. Conventionally, fluorescence derivatization precedes the HPLC analysis of these disaccharides. However, the presence of excess unreacted derivatization reagents can inhibit rapid and sensitive analysis in chromatographic determinations. In this study, we describe analytical methods that use dansylhydrazine as a derivatization agent for the detection and determination of HS-derived unsaturated disaccharides using HPLC. In addition, we have developed a straightforward method for removing excess unreacted reagent using a MonoSpin NH₂ column. This method may be employed to remove excess pre-labeling reagents, thereby facilitating the analysis of HS-derived unsaturated disaccharides with satisfactory reproducibility.

INTRODUCTION

Glycosaminoglycans (GAGs), such as heparan sulfate (HS), play an important role in living organisms. For example, HS proteoglycans are involved in the development, homeostasis, and different aspects of disease progression in humans.¹⁾ However, the function of HS and the changes in its synthesis induced by disease have not been thoroughly elucidated. To clarify the function of HS in humans, there is need for sensitive analytical methods that can be used to detect HS-derived unsaturated disaccharides.

Conventionally, the separation of HS-derived unsaturated disaccharides by HPLC is preceded by fluorescence derivatization, following enzymatic digestion of HS into unsaturated disaccharides using heparinase. Previous studies have typically used 2-aminobenzamide (2-AB) or 2-aminoacridone (AMAC) as pre-labeling reagents, and 2-cyanoacetamide as a post-labeling reagent to detect the disaccharides using HPLC.^2–6) While obviating the need for a derivatization step, the post-labeling approach generally requires a more complicated HPLC system configuration that incorporates a reaction bath and mixing and cooling coils.

In the pre-labeling process, the analysis of analytes can be used to increase sensitivity. For example, urine, an abundantly available biological sample, typically contains low levels of GAGs; nevertheless, these can be detected with heightened sensitivity through pre-labeling techniques. However, the presence of excess unreacted reagent can impede the rapid and sensitive analysis of GAGs, and cause ion suppression during mass spectrometry (MS) analysis. Consequently, removing excess reagents before analysis by HPLC or LC-MS is an essential step, and methods such as gel permeation chromatography using a G-10 column or solid phase extraction techniques are typically used to achieve this aim.^2,3,7,8) However, despite their effectiveness, these steps are time consuming. In a previous study on the analysis of neutral sugars, it was reported that excess reagents could be removed using a commercially available MonoSpin NH₂ column.⁹⁾ We hypothesized that this method could be applied to remove excess unreacted reagents, specifically dansylhydrazine, for the analysis of reducing disaccharides.

In this study, we developed and tested a method to remove excess unreacted derivatization reagents using a MonoSpin NH₂ column easily and rapidly, and achieved satisfactory reproducibility with HS-derived unsaturated disaccharides. We also developed analytical methods to detect and identify six kinds of HS-derived unsaturated disaccharides using dansylhydrazine as a derivatization reagent in HPLC with a hydrophilic interaction chromatography (HILIC) column. These methods were applied to the analysis of HS in urine as a biological sample.

MATERIALS AND METHODS

Ethics Statement

This study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Hoshi University.

Materials and Reagents

Standards for HS-derived unsaturated disaccharides, 2-acetamido-2-deoxy-4-O-(4-deoxy-α-L-threo-hex-enepyranosyluronic acid)-D-glucose (ΔUA-GlcNAc), 2-acetamido-2-deoxy-4-O-(4-deoxy-α-L-threo-hex-enepyranosyluronic acid)-6-O-sulfo-D-glucose (ΔUA-GlcNAc6S), 2-deoxy-2-sulfamino-4-O-(4-deoxy-α-L-threo-hex-enepyranosyluronic acid)-D-glucose (ΔUA-GlcNS), 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-α-L-threo-hex-enepyranosyluronic acid)-D-glucose (ΔUA2S-GlcNS), 2-deoxy-2-sulfamino-4-O-(4-deoxy-α-L-threo-hex-enepyranosyluronic acid)-6-O-sulfo-D-glucose (ΔUA-GlcNS6S), 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-α-L-threo-hex-enepyranosyluronic acid)-6-O-sulfo-D-glucose (ΔUA2S-GlcNS6S), heparinase I, heparinase II, and heparinase III were purchased from Iduron (Manchester, U.K.). Dansylhydrazine was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Acetonitrile (HPLC grade) and trichloroacetic acid (TCA) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Ultrapure water was prepared using a Milli-Q direct water purification system (Merck, Darmstadt, Germany). MonoSpin NH₂ columns were purchased from GL Sciences (Tokyo, Japan). All other chemicals used were of analytical reagent grade.

Derivatization Procedure with Dansylhydrazine

The derivatization of HS-derived unsaturated disaccharides with dansylhydrazine was performed based on methods previously reported for the detection of hyaluronic acid, chondroitin sulfate, and dermatan sulfate using dansylhydrazine.^10,11) Briefly, 20 µL of the aqueous sample containing unsaturated disaccharides derived from HS was vaporized by a rotary evaporator. The dried sample was then redissolved in 20 µL of 90% methanol. Next, 20 µL of 1.0% (w/v) TCA in acetonitrile and 20 µL of 3.0% (w/v) dansylhydrazine in acetonitrile were successively added and mixed well. The sample was then placed in a water bath at 50 °C for 2.5 h to complete the derivatization reaction.

Removal of Excess Unreacted Reagent

After dansylhydrazine labeling, excess reagents were removed using a MonoSpin NH₂ column. The solvents used were as follows: solvent A, acetonitrile–ultrapure water–acetic acid (8.95 : 0.95 : 0.10 (v/v/v)), was prepared for conditioning and rinsing; and solvent B, acetonitrile–methanol–ammonia solution (5 : 2 : 3 (v/v/v)), was prepared for conditioning and elution. After attaching the spin column to the waste fluid tube, 400 µL of solvent B was added to the spin column and the column was centrifuged at 7500 × g for 1 min (Conditioning step 1). Next, 400 µL of solvent A was added and the column was centrifuged at 7500 × g for 1 min (Conditioning step 2). A 50 µL volume of the sample containing the disaccharides labeled with dansylhydrazine was then added, followed by centrifugation at 7500 × g for 1 min (Adsorption step). Then, 50 µL of solvent A was added to the column, which was centrifuged at 7500 × g for 1 min (Rinsing step). After putting the spin column into the recovery tube, 50 µL of solvent B was added, and the tube was then centrifuged at 7500 × g for 1 min (Elution). A 10 µL aliquot of the elution sample was then used for chromatographic analysis.

HPLC Analysis

The HPLC system consisted of a double plunger pump (LC-20AD, Shimadzu, Kyoto, Japan), a degasser (DGU-20A_3R, Shimadzu), a column oven (CO-965, Jasco, Tokyo, Japan), and a fluorescence detector (RF-20A, Shimadzu). The column used for the separation analysis was an XBridge™ Amide column (2.1 × 150 mm, 3.5 µm, Waters; Milford, MA, U.S.A.) at 50 °C. Eluent A consisted of 0.10 M ammonium formate buffer (pH 9.0); the pH was adjusted to 9.0 with ammonia solution. Eluent B consisted of acetonitrile. The mobile phase was passed through the column at a flow rate of 0.3 mL/min with a 30 min linear gradient of 90–60% of eluent B. The derivatized sample was detected by fluorescence with excitation and emission wavelengths at 350 nm and 530 nm, respectively.

Preparation of Human Urinary HS

The procedure used to recover human urinary HS was based on that employed in a previous study with minor modifications.⁵⁾ Briefly, 1.8 mL of 5% cetylpyridinium chloride (CPC) was added to 27 mL of human urine and kept at 4 °C for 4 h. After centrifugation at 2300 × g for 15 min, the precipitate was washed twice with 1.5 mL of 0.1% CPC. To the precipitate, 3 mL of 2.5 M sodium chloride was added and mixed well, and the mixture was centrifuged at 2300 × g for 15 min. To the supernatant, 11 mL of 85% ethanol was added, after which the mixture was kept at 4 °C overnight. After recovering the crude GAGs by centrifugation at 2300 × g for 15 min at 4 °C, the resulting precipitate was lyophilized and then redissolved in 1 mL of ultrapure water. To a 200 µL aliquot of the sample, 1 mU each of heparinase I, heparinase II, and heparinase III in 0.10 M sodium acetate buffer containing 10 mM calcium acetate (pH 7.0) was added, and the mixture was incubated at 37 °C overnight. A 20 µL portion of the enzymatically treated sample was then derivatized with dansylhydrazine as described above.

RESULTS AND DISCUSSION

Analysis of HS-Derived Unsaturated Disaccharides

We developed a method to sensitively and quantitatively analyze dansylhydrazone (Dns) HS-derived unsaturated disaccharides. First, we established whether dansylhydrazine derivatization could be performed without the need for dehydration using a rotary evaporator. Since the derivatization reaction did not proceed favorably without dehydration, derivatization was conducted after 20 µL of the sample was vaporized using a rotary evaporator, as described by Volpi.¹²⁾ The results showed that derivatization was successful and that a dehydration step was necessary for the derivatization of HS-derived unsaturated disaccharides using dansylhydrazine.

Next, we examined the separation of six kinds of disaccharides. Initially, the standard mixture was analyzed with 0.10 M ammonium formate buffer (without pH adjustment) as eluent A and acetonitrile as eluent B. However, since Dns-ΔUA2S-GlcNS and Dns-ΔUA-GlcNAc6S could not be separated, we examined whether satisfactory separation could be achieved by modifying the pH of eluent A (0.10 M ammonium formate buffer) to pH 9.0 with ammonia solution. Thus, we analyzed the standard mixture using a linear gradient consisting of 0.10 M ammonium formate buffer (pH 9.0) as eluent A and acetonitrile as eluent B, which changed from 90–60% eluent B over a period of 30 min. As a result, the satisfactory separation of two disaccharides was achieved.

Removal of Excess Unreacted Reagent

The presence of excess unreacted derivatization reagents can prevent rapid and sensitive chromatographic analysis.¹¹⁾ Since dansylhydrazine also exhibits fluorescence, the presence of this excess reagent adversely affected the reproducibility of the analysis. Additionally, excess reagents could lead to ion suppression in the MS analysis, particularly if analyzed with LC-MS. Therefore, we investigated the removal of excess regents and fluorescence dye using MonoSpin NH₂ columns.

Firstly, we examined the effect of solvent composition on elution efficiency. An acidic substance was added to both solvent A and solvent B following the methods employed in a previous study.⁹⁾ The results showed that only one of several target peaks were detected, suggesting that the derivatives exhibited strong retention on the spin column. This phenomenon was attributed to the expectation that the NH₂ column would facilitate both hydrophilic interactions and anion-exchange modes under low pH conditions. However, the efficacy of anion-exchange interactions could decrease under high pH conditions. Thus, we considered that adding an acidic substance to solvent A could potentially enhance the retention of derivatives, while adding a basic substance to solvent B might improve the elution of these derivatives.

We therefore investigated the addition of ammonia solution, a basic modifier, to solvent B. As a result, six types of unsaturated disaccharide derivatives were detected, implying that the addition of a basic substance to solvent B improved the elution efficacy. We then examined various concentrations of ammonia solution, which effectively improved the elution of these derivatives as the concentration increased. However, when we used a mixture of acetonitrile, methanol, and ammonia solution (5 : 2 : 3 (v/v/v)) as solvent B, six kinds of derivatives were detected with satisfactory reproducibility, so we employed this ratio for the composition of solvent B. Next, we added acidic substances to solvent A to improve the chromatogram. After the addition of acetic acid, all of the peak areas were increased compared to that observed after the addition of formic acid. Therefore, we selected the addition of acetic acid to solvent A as an acidic modifier.

The results of chromatographic analysis of standard HS-derived unsaturated disaccharides treated with and without MonoSpin NH₂ columns is shown in Fig. 1. The results obtained using a MonoSpin NH₂ column (Fig. 1b) showed that all six disaccharides were detected without interference from excess unreacted reagents, and even highly polar substances such as Dns-ΔUA2S-GlcNS6S were effectively eluted. Furthermore, the removal of excess unreacted reagents using a MonoSpin NH₂ column facilitated stabilization of the baseline of the chromatogram.

Fig. 1. HPLC Chromatograms of Standard Unsaturated Disaccharides Derived from Heparan Sulfate

HPLC conditions: column, XBridge™ Amide (2.1 × 150 mm, 3.5 µm); column temperature, 50 °C; eluent A, 0.10 M ammonium formate buffer (pH 9.0); eluent B, acetonitrile at a flow rate of 0.3 mL/min with 30 min linear gradient of 90–60% eluent B; excitation wavelength, 350 nm; emission wavelength, 530 nm. Injection volume, 10 µL. Peaks correspond to: 1, Dns-ΔUA-GlcNAc; 2, Dns-ΔUA-GlcNS; 3, Dns-ΔUA2S-GlcNS; 4, Dns-ΔUA-GlcNAc6S; 5, Dns-ΔUA-GlcNS6S; and 6, Dns-ΔUA2S-GlcNS6S. (a) Solution prepared without a MonoSpin NH₂ column, and (b) Solution prepared with a MonoSpin NH₂ column.

As demonstrated above, only centrifugation is sufficient to remove unreacted excess reagents and the fluorescence dye derived from dansylhydrazine, ensuring reproducible outcomes.

Calibration Curve

We obtained a calibration curve using five different concentrations (1, 2.5, 5, 10, 20 µM), which yielded a correlation coefficient (r) greater than 0.996. The limit of quantification (LOQ) for the six HS-derived unsaturated disaccharides was estimated to be 0.5 µM (signal-to-noise ratio = 10). As shown in Table 1, the coefficient of variation (CV) was less than 7.1% (n = 5).

Table 1. Coefficient of Variation (CV) of Six HS-Derived Unsaturated Disaccharides (n = 5)

Disaccharides type	Area CV (%)
Dns-ΔUA-GlcNAc	6.3%
Dns-ΔUA-GlcNS	5.4%
Dns-ΔUA2S-GlcNS	4.6%
Dns-ΔUA-GlcNAc6S	5.4%
Dns-ΔUA-GlcNS6S	4.7%
Dns-ΔUA2S-GlcNS6S	7.1%

Analysis of Human Urinary HS

We applied the developed method to the analysis of human urinary HS. A typical chromatogram of human urinary HS is shown in Fig. 2. The chromatogram shows four of the most commonly detected disaccharides as well as several unknown peaks. Similar unknown peaks were detected even when the HS standard underwent enzymatic digestion, suggesting the possible presence of impurities or other reducing sugars. We estimated that the disaccharide concentrations in urine to be as follows: ΔUA-GlcNAc at 0.169 µg/mL, ΔUA-GlcNS at 0.032 µg/mL, ΔUA2S-GlcNS at 0.003 µg/mL, and ΔUA-GlcNAc6S at 0.136 µg/mL, respectively. Although the concentrations of ΔUA-GlcNS6S and ΔUA2S-GlcNS6S were less than 1 µM, the proportion and composition of unsaturated disaccharides were largely in agreement with previous reports.^5,13) Since the proportion and composition of human urinary HS may vary due to differences in age or sex, further analyses are considered necessary.

Fig. 2. HPLC Chromatogram of Unsaturated Disaccharides Derived from Human Urinary Heparan Sulfate

Peaks: 1, Dns-ΔUA-GlcNAc; 2, Dns-ΔUA-GlcNS; 3, Dns-ΔUA2S-GlcNS; 4, Dns-ΔUA-GlcNAc6S; 5, Dns-ΔUA-GlcNS6S; 6, Dns-ΔUA2S-GlcNS6S.

CONCLUSION

In this study, we developed analytical methods for six types of HS-derived unsaturated disaccharides using dansylhydrazine in conjunction with HPLC equipped with a HILIC column. We consider that the procedure developed for removing excess dansylhydrazine using a MonoSpin NH₂ column offers a simpler alternative to purification by gel chromatography. Specifically, the MonoSpin NH₂ column facilitates the straightforward and rapid removal of excess unreacted reagents with satisfactory reproducibility. This technique could potentially be applied to the removal of other excess unreacted derivatization reagents, such as 2-AB or AMAC, in the analysis of unsaturated disaccharides derived from GAGs or other acidic reducing sugars prior to column analysis. We plan to apply the developed methods to the detection and quantification of HS-derived unsaturated disaccharides in a variety of biological samples.

Acknowledgments

This work was supported in part by the Japan Food Chemical Research Foundation.

Conflict of Interest

The authors declare no conflict of interest.

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