Complete Monosaccharide Analysis by High-Performance Anion

Mar 24, 2012 - The sensitivity, linearity, and RFs reported here are specific to the instrument used in this work and may be slightly different on oth...
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Complete Monosaccharide Analysis by High-Performance AnionExchange Chromatography with Pulsed Amperometric Detection Zhenqing Zhang,* Nazeer M. Khan, Karen M. Nunez, Edward K. Chess, and Christina M. Szabo Baxter Healthcare Corporation, Round Lake, Illinois 60073, United States S Supporting Information *

ABSTRACT: Monosaccharide analysis is a critical way to profile the composition of complex carbohydrates. Methods to analyze neutral and amino sugars have been established for a long time, but methods for acidic sugars are rare. The acidic sugars, including uronic acids and sialic acids, are also important components in some complex carbohydrates. In this report, a highperformance anion-exchange chromatography method with pulsed amperometric detection was initially developed to analyze acidic sugars including different uronic acids and sialic acids. Subsequently, a method to profile complete monosaccharides, including most neutral, amino, and acidic sugars, was developed. This method has a limit of quantitation of ∼12.5 × 10−3 nmol for each sugar as well as good linearity over a wide range. This is a convenient procedure because it avoids additional derivatization of monosaccharides and has a broad application to a wide range of complex carbohydrates. The monosaccharide compositions of a variety of complex carbohydrates such as different glycosaminoglycans, alginate, fucoidan, and glycans were profiled by this comprehensive method. In addition, the hydrolysis patterns of these complex carbohydrates are discussed.

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precision, sensitivity, and efficiency. Some examples of such methods are as follows: high-performance liquid chromatography (HPLC),12 gas chromatography (GC),13 gas chromatography with mass spectrometry (GC/MS),14 and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).15 Both HPLC and GC methods require derivatization of the monosaccharide to obtain sensitive detection and better separation.12−14 The HPAEC-PAD method was developed in the 1990s.15 This method has high sensitivity, has baseline separation, and avoids the derivatization of sugars.16,17 Most of these monosaccharide analyses are only focused on profiling neutral and amino sugars, however, not the acidic sugars. Some complex carbohydrates are composed of acidic sugars or both neutral/amino and acidic sugars, which are described below. Glycosaminoglycans (GAGs) are a family of linear polysaccharides composed of repeating disaccharides, aminoglucose (GlcN)/amino-galactose (GalN) and glucuronic (GlcA)/iduronic acid (IdoA). GAGs participate in and regulate many cellular events and physiological and pathophysiological

he complexity of carbohydrate structures arises from many things: different monosaccharide compositions including neutral, amino, and acidic sugars; complex linkage patterns including linkage positions and patterns such as linear or branched; and various functional and positional substitutions on the sugar rings.1−3 Polysaccharides also have high molecular weight (Mw) and wide polydispersity.4 Structure elucidation has been an analytical challenge in this area for a long time. Monosaccharide analysis is still one of the most important ways to profile the composition of complex carbohydrates. It is applied widely to characterize various polysaccharides or glycans employed in bioscience, medicine, food, and material research areas.5−8 The challenges in monosaccharide analysis are separation and detection. Monosaccharides are highly hydrophilic molecules with multiple hydroxyl groups. They have similar structures, and many of them are epimers of each other. Different methods have been applied to solve these problems over the last 70 years. Monosaccharide analysis originated with paper chromatography in the 1940s.9,10 Subsequently, a thin layer chromatography (TLC) method was developed.11 These two methods have relatively low sensitivity and resolution. Also, these two methods could not quantify monosaccharides properly.9,11 Afterward, other chromatographic methods were developed to analyze monosaccharides with higher accuracy, © 2012 American Chemical Society

Received: January 23, 2012 Accepted: March 24, 2012 Published: March 24, 2012 4104

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describes the development of a single method to analyze neutral sugars, amino sugars, uronic acids, and sialic acids. The latter method is applicable to quantitative analysis to complete monosaccharide profile from different samples, such as Hp, HS, CS, DS, HA, alginate, fucoidan, and N-glycans.

processes, such as cell proliferation and differentiation, cell−cell and cell−matrix interactions, and viral infection through their interaction with different proteins.18,19 On the basis of the different compositions, they are classified as heparin (Hp, mainly 4 GlcN α 1−4 IdoA α1), heparan sulfate (HS, mainly 4 GlcN α 1−4 GlcA β/IdoA α 1), chondroitin sulfate (CS, 3 GalN β 1−4 GlcA β 1), dermatan sulfate (DS, 3 GalN β 1−4 IdoA α/GlcA β 1), and hyaluronic acid (HA, 3 GlcA β 1−4 GlcA β 1).20,21 Various GAG functions result from their structural diversity including monosaccharide composition.18,19 Analysis of only amino sugars is not sufficient to identify these GAGs,22,23 and the uronic acids need to be confirmed. However, in the typical disaccharide analysis of GAGs, uronic acids are cleaved and converted to the same unsaturated uronic acid after digestion by GAG lyases so that the uronic acid composition information is lost.24 Nuclear magnetic resonance (NMR) was used to characterize different GAGs, including identification and relative quantitation of the different uronic acids.25 However, NMR requires relatively high amounts of sample and poorly resolves epimers. Thus, it is important to develop a method that can classify different GAGs and characterize their structural properties effectively and with high sensitivity. Alginate, a linear polysaccharide, is extracted from brown algae and is composed of mannuronic (ManA) and guluronic acid (GulA).26,27 The ratio (M/G) and sequence of ManA and GulA in alginate depend on the algae species, and algae harvest time and locations.28 M/G determines the different properties of alginate and leads to different applications, for example, as a food additive, as a gel additive, and even for drug delivery.28,29 NMR was used to characterize M/G, but the large sample requirement and low resolution limited its application.30 Fucoidan is another polysaccharide that is extracted from brown algae. It is always coextracted with alginate from the cell wall of the brown algae. Thus, alginate and fucoidan are often impurities of each other. Glycans in the glycocalyx on the cell surface, such as glycoproteins and glycolipids, are another source of complex carbohydrates. Neutral sugars, amino sugars, and sialic acids are always observed in these glycans. Sialic acid is typically the outermost monosaccharide unit on the glycan chains. It is one of the most important factors to characterize these glycoconjugates because of its significant role in the signaling in many physiological and pathobiological procedures.31 The HPLC method using 1,2-diamino-4,5-methylenedioxybenzene (DMB) labeling has been used for sialic acid analysis for the last 20 years.32 In the 1990s, a HPAEC-PAD method was developed to analyze sialic acids and avoided labeling.32 However, this method differs significantly from the conventional HPAEC-PAD neutral sugar analysis method, which is another part of glycan analysis for glycoproteins or glycolipids.33 Thus, the hydrolysate has to be analyzed for neutral/amino sugars and sialic acids separately to profile the monosaccharide composition of glycans, consuming much time, labor, and sample. Natural complex carbohydrates including poly- and oligosaccharides are composed of different monosaccharides. They could be uniform neutral, amino, or acidic sugars, but they also could be heterogeneous, like the examples above. It is important to establish the complete sugar component profile of different carbohydrates to clarify their structural properties and classification. This report describes a method to analyze acidic monosaccharides with HPAEC-PAD and, furthermore,

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MATERIALS AND EXPERIMENTS

Monosaccharide Standards. Neutral and amino sugars [glucose (Glc), galactose (Gal), arabinose (Ara), mannose (Man), rhamnose (Rha), fucose (Fuc), xylose (Xyl), glucosamine (GlcN) hydrochloride, and galactosamine (GalN) hydrochloride] were purchased from Sigma-Aldrich LLC (St. Louis). Acidic sugars [glucuronic acid (GlcA), galacturonic acid (GalA), and N-acetylneuraminic acid (Neu5Ac)] were also purchased from Sigma-Aldrich LLC; iduronic acid (IdoA) was purchased from Santa Cruz Biotech, Inc. (Santa Cruz, CA) and N-glycolylneuraminic acid (Neu5GC) was purchased from Toronto Research Chemicals (North York, Ontario, Canada). Acidic sugars, sodium mannuronic acid (ManA) and guluronic acid (GulA), were prepared by the hydrolysis of polymannuronate and polyguluronate. Polymannuronate and polyguluronate, also called M and G blocks, are from alginate. They were purchased from Elicityl (Crolles, France). Specific amounts of M and G blocks were dissolved in 2 M trifluoroacetic acid (TFA) to afford 2.5 mg/mL solutions, respectively, and incubated at 100 °C in an oven for 5 h. The hydrolysates were monitored by TLC34 to confirm the completeness of hydrolysis. The excess TFA was removed; and the hydrolysates were dried using a SpeedVac concentrator (Thermo, Waltham, MA). The hydrolysate was redissolved in the same volume of water and the pH was adjusted with dilute NaOH to neutral. Carbazole assay was applied to quantify ManA and GulA.35 Hydrolysis of Polysaccharides and Glycans. GAGs (Hp, HS, and DS) were purchased from Celsus (Cincinnati, OH); CS-A and HA were purchased from Sigma. Other polysaccharides, alginate and Fucus vesiculosus (F.v.) fucoidan, were also purchased from Sigma. Two N-glycan standards were purchased from QABio LLC (Palm Desert, CA). One is monosialo-fucosylated biantennary oligosaccharide (A1F), and the other is trisialo-galactosylated triantennary oligosaccharide (A3). All polysaccharide structures are listed in Table 1. Table 1. Complex Polysaccharide and Glycan Structures

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The hydrolysis and sample preparations are similar to those used to prepare ManA and GulA. Each of the GAGs was hydrolyzed at 120 and 100 °C for 5, 12, 24, and 30 h. Alginate and fucoidan were hydrolyzed at 100 °C for 5 and 12 h, respectively. N-Glycans were hydrolyzed at 100 °C for 1 h. The details are described in the Supporting Information. The samples were diluted 50-fold prior to HPAEC-PAD analysis. HPAEC Methods. A Dionex HPAEC (ICS 3000, Dionex, Sunnyvale, CA) with dual pumps and pulse amperometric (Au) detector was used. The Carbopac guard column (4 × 50 mm, Dionex) and Carbopac PA1 analytical column (4 × 250 mm, Dionex) were used at 1 mL/min and 30 °C. Different isocratic schemes with various concentrations of NaOH and NaOAc were used to analyze acidic sugars. A multiple step gradient method using isocratic 15 mM NaOH for 10 min followed by a linear NaOAc gradient with fixed 15 mM NaOH for the next 30 min was used to analyze all monosaccharides including neutral, amino, and acidic sugars. The waveform of PAD was the Dionex default program for carbohydrates. The injection volume was set to 25 μL. The retention time of each monosaccharide standard in the mixtures with different methods was confirmed by the analysis of each monosaccharide individually.

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RESULTS AND DISCUSSION Gradient Optimization for Acidic Sugar Analysis and Complete Sugar Analysis. Different isocratic elutions with various concentrations of NaOH and NaOAc were applied to analyze the acidic sugar mixture including GalA, GlcA, GulA, ManA, IdoA, Neu5Ac, and Neu5Gc. The different pKa values of each of these sugars led to their separation by HPAEC. The first series of experiments was set up to optimize the NaOH concentration with a fixed NaOAc concentration. The chromatograms with different NaOH concentrations (from 100 to 5 mM) are shown in Figure 1. In general, all acidic sugars eluted faster with the lower concentration of NaOH. In Figure 1A−E, the retention time of Neu5Gc is most affected by the NaOH concentration. It eluted as the sixth peak at ∼8.6 min in the chromatogram with 100 mM NaOH, while it eluted as the second peak at 3.7 and 2.7 min when the NaOH decreased to 15 and 5 mM, respectively. The retention time of Neu5Ac was not affected significantly by NaOH concentration until it was dropped to 50 mM. Neu5Ac eluted even faster with the 15 and 5 mM NaOH. The decreasing of NaOH concentration did not affect the elution of uronic acids until it dropped below 50 mM. All uronic acids eluted faster with 15 and 5 mM NaOH than with 50 mM NaOH and above. The separation pattern of all acidic sugars stayed the same, except Neu5Gc. The carboxyl groups in these acidic sugars are less ionized in the lower concentration of NaOH. Subsequently, their binding to quaternary ammonium groups on the PA1 column was weaker, and they eluted more rapidly. The optimal NaOH concentrations appeared to be 15 mM for several reasons: it resulted in baseline separation for the acidic sugar mixture in 10 min, compared to the relatively long separation time and low resolution with higher concentrations of NaOH (50, 75, and 100 mM); there was a risk of peak overlap with the lower NaOH (5 mM) concentration; and 15 mM NaOH was in the range to separate neutral and amino sugars using a Carbopac PA1 column.15,33 The second series of experiments was set up to optimize the NaOAc concentrations with a fixed NaOH concentration. The chromatograms with different NaOAc concentrations (from

Figure 1. Chromatograms of acidic sugar mixture with a fixed NaOAc concentration of 150 mM and different NaOH concentrations. (A) 100 mM NaOH; (B) 75 mM NaOH; (C) 50 mM NaOH; (D) 15 mM NaOH; (E) 5 mM NaOH.

150 to 50 mM) are shown in Figure 2. Like regular anionexchange chromatography, all acidic sugars eluted faster at high concentrations of NaOAc, while at low concentration of salt they eluted slowly with relatively broad peaks. Afterward, a multiple-step gradient was set up based on the optimization to analyze the complete set of sugars. The method uses 15 mM NaOH to separate neutral and amino sugars, and subsequently, a linear NaOAc gradient with fixed 15 mM NaOH to separate acidic sugars. The 16 monosaccharide standards were well-separated in 40 min. Sensitivity, Linearity, and Relative Response Factor. The PAD with the gold electrode was developed for testing carbohydrates. The current produced from the oxidation reaction makes the detection of carbohydrate highly sensitive without derivatization.15 In this study, the monosaccharide standards from 50 to 0.5 nmol/mL were analyzed with the gradient method. The signal-to-noise (S/N) of all standards at 0.5 nmol/mL was ∼10, indicating that the method has good sensitivity for quantitation at this level. The calibration curves of all these standards were plotted by the peak areas as a function of their concentrations from 0.5 to 50 nmol/mL. All the equations and the correlation coefficients of these monosaccharide standards are listed in Table 1, Supporting 4106

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Figure 2. Chromatograms of acidic sugar mixture with a fixed NaOH concentration of 100 mM and different NaOAc concentrations. (A) 150 mM NaOAc; (B) 100 mM NaOAc; (C) 50 mM NaOAc.

Information. The correlation coefficients for Neu5Ac and Neu5Gc are 99.38% and 99.61%, respectively, and all others are greater than 99.8%. Two representative chromatograms and four representative standard curves are shown in Figure 1, Supporting Information. As the correlation coefficient and the standard curve showed, the highest concentration of Neu5Ac at 50 nmol/mL affected the linearity slightly. Thus, the quantitation range of Neu5Ac is defined at 0.5−25 nmol/mL. All other monosaccharide standards have good linearity over a wide range (0.5−50 nmol/mL). The injection volume was 25 μL; the lowest amount to be quantified was 12.5 × 10−3 nmol. The monosaccharide standards have different responses to the PAD due to their differences in their structures and configurations, resulting in different currents being produced from their oxidation reactions. The relative response factors (RF) of these monosaccharide standards were calculated by setting the peak area of glucose to 1 in the experiment with 50 nmol/mL standards, as listed in Table 1, Supporting Information. These factors could be used to calibrate the peak areas of each sugar to determine their relative amounts. The sensitivity, linearity, and RFs reported here are specific to the instrument used in this work and may be slightly different on other instruments. Applications. Complete monosaccharide analysis is still a challenge for complex carbohydrates, especially those having acidic sugar components. The ideal method would profile all potential sugar units in one experiment. This method is a universal procedure to analyze monosaccharides for many types of complex carbohydrates. GAGs. Different GAGs were analyzed by this gradient method. The monosaccharide profiling chromatograms of different GAGs are shown in Figure 3. 1. Figure 3B shows the monosaccharide profile of Hp after hydrolysis at 100 °C for 24 h. The GlcN, GlcA, and IdoA are observed based on the assignment of standards in Figure 3A. These monosaccharides are the major components of Hp. Besides the monosaccharides, several unknown peaks were observed between 26 and 29 min. These unknown peaks are observed at higher levels in the chromatograms of Hp

Figure 3. Chromatograms of complete monosaccharide analysis of different GAGs. (A) Standards; (B) Hp; (C) HS; (D) DS; (E) CS; (F) HA. Peaks labeled with asterisks are corresponding to di- or oligosaccharides.

hydrolysate from relatively weak hydrolysis conditions, such as 100 °C for 12 h (Figure 2, Supporting Information). They were observed at lower levels in the chromatograms of Hp hydrolysate from relatively harsh hydrolysis conditions, such as 120 °C for 12 h (Figure 2, Supporting Information). It suggests the unknown peaks between 26 and 29 min are disaccharides or oligosaccharides of Hp. This is consistent with TLC results (data not shown). The peak areas of monosaccharides were calibrated using the corresponding RF. The sugar compositions were calculated and are listed in Table 2. Since the structure of Hp is a repeating disaccharide, which is composed of GlcN and IdoA/GlcA, the ratio of GlcN to IdoA/GlcA should be 1:1. However, the levels of GlcA and IdoA to GlcN were not detected in this ratio. The ratio may be affected by partial hydrolysis in which the released monosaccharide pool was not reflective of the entire Hp backbone. It also could be affected by the degradation of uronic acid, especially IdoA, from the relatively harsh hydrolysis. The peak areas of uronic acids showed no significant change between the hydrolysates generated at 100 °C for 12 and 24 h, but it decreased in the hydrolysates generated under the harsher condition (100 °C for 30 h), especially for IdoA (data not shown). This suggests the uronic acids are released, and subsequently degraded, from 12 h hydrolysis to 24 h hydrolysis. After 24 h, degradation dominated, especially for IdoA. 2. Figure 3C shows the monosaccharide profiling of HS after hydrolysis at 100 °C for 30 h. HS contains GlcN coupled with GlcA (major) and IdoA (minor). The β1−4 linkage between GlcA and GlcN in HS is stronger than the α1−4 linkage 4107

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Table 2. Hydrolysis Conditions and Complete Monosaccharide Compositions compositions (mol %) stds Fuc Rha GalN Ara GlcN Gal Glc Man Xyl Neu5Ac Neu5Gc GalA GulA GlcA ManA IdoA

Hp (100 °C 24 h)a

HS (100 °C 30 h)a

DS (100 °C 12 h)a

CS (100 °C 24 h)a

HA (100 °C 24 h)a

alginate (100 °C 5 h)a 6.0

52.4 89.0

fucoidan (100 °C 5 h)a

A1F (100 °C 1 h)a

82.6

85.2b

13.3

1c

6.8

7.0b

27.2 22.8

2c 2c

28.6 32.1

3c 3c

0.7 5.0

0.7b 5.1b

34.3

3c

32.1

3c

7.2

1c

43.9

83.6

46.9 2.6

2.5

38.1 4.9

16.4

5.2

53.5

53.1 55.9

6.2

A3 (100 °C 1 h)a

0.4 1.9 2.6

0.2c

1.9b

42.4

a Hydrolysis conditions in 2 M TFA. bThe monosaccharide composition (mol %) of the fucoidan excluding alginate. cThe monosaccharide ratio of glycans calculated based on the composition in the previous column.

between IdoA and GlcN, the major structure of Hp.36 The hydrolysis of HS takes a longer time than Hp. However, similar to Hp, the harsher hydrolysis conditions did not break all glycosidic bonds as the di- and oligosaccharides peaks were still identified, but it did degrade the uronic acid. The uronic acid levels are too low to match that of GlcN, and the IdoA is not even observed in Figure 3C. 3. Figure 3D shows the monosaccharide profile of DS after hydrolysis at 100 °C for 12 h. Most of the components in this hydrolysate were monosaccharides with an exception of a small peak observed as a disaccharide of DS at ∼28.5 min. This was consistent with the TLC results (data not shown). Unlike Hp or HS, DS has a relatively homogeneous structure and a relatively weak α1−3 linkage between IdoA and GalN.36 There were lower levels of resistant di- or oligosaccharides in DS than in Hp or HS. In addition, the uronic acid content matched that of GalN, which reflects the repeating disaccharide structure of DS. All these observations imply that DS was hydrolyzed efficiently and properly with the intact monosaccharide products. Also, it is not surprising that a higher amount of disaccharide is observed when the hydrolysis was carried out over a shorter period; and degradation of IdoA was observed when the hydrolysis was carried out under harsher conditions (data not shown). The compositions of monosaccharides are listed in Table 2. There are 5.2% GlcA and 42.4% IdoA vs 52.4% GalN in this DS. 4. Figure 3E shows the monosaccharide profile of CS after hydrolysis at 100 °C for 24 h. Most of the components in this hydrolysate were monosaccharides with an exception of a peak observed as a disaccharide of CS at ∼28.5 min, which had the same retention time as that of DS disaccharide. The β1−3 linkage between GlcA and GalN is stronger than that in DS, and it required a longer time to be cleaved.36 The content of GlcA matched with that of GalN plus Gal, which reflects the repeating disaccharide structure of CS. All these observations imply that the CS was hydrolyzed and the released monosaccharide was stable after 24 h hydrolysis. The disaccharide peak was observed at higher levels in the hydrolysate carried out over a shorter period. Under this mild

condition, IdoA, which was shown to be easily hydrolyzed in Hp and HS, was still not observed (data not shown), suggesting that there is no IdoA in the CS. 5. Figure 3F shows the monosaccharide profile of HA after hydrolysis at 100 °C for 24 h. Most of the components in this hydrolysate were monosaccharides with an exception of a small peak observed as a dimer of CS at ∼31.9 min. This was a disaccharide peak, which was confirmed by TLC (data not shown). The hydrolysis pattern of HA was similar to that of CS. After 24 h hydrolysis at 100 °C, most monosaccharides of HA were released and remained intact. The monosaccharide analysis reflected the repeating disaccharide structure of HA, GlcN-GlcA. Alginate and Fucoidan. Alginate and fucoidan were also analyzed with this method. Alginate was reported as an impurity of fucoidan.37 However, there is no efficient method to identify and quantify alginate at low levels in fucoidan. NMR analysis has low resolution and sensitivity. Carbazole assay is sensitive for all uronic acids, but not specific to alginate.35 In addition, the high content of neutral sugars in fucoidan would be carbonized by the concentrated sulfuric acid and affect this colorimetric method. Their monosaccharide profile chromatograms are shown in Figure 4. 1. Figure 4B showed the monosaccharide profile of alginate after hydrolysis at 100 °C for 5 h. GulA and ManA were observed as expected, and M/G is ∼1.5. Besides these two major components, a small peak was observed at ∼5.5 min corresponding to fucose. The fucose should belong to fucoidan, which is always coextracted with alginate from the cell wall of brown algae. Thus, the fucoidan is an impurity of the commercial alginate. 2. Figure 4C shows the monosaccharide profile of fucoidan after hydrolysis at 100 °C for 5 h. Fucose was the major component, and the minor Gal, Man, and Xyl were also observed as well. Besides these common components, GulA and ManA are observed, suggesting the presence of impurity of alginate in the F.v., fucoidan. Interestingly, another uronic acid, GlcA, was also observed, though it is at low levels (1.9%). GlcA was observed in other fucoidans, but not F.v. fucoidan.38 As it 4108

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under the hydrolysis conditions at 100 °C for 1 h. Alternatively, degradation could be avoided by controlled acid hydrolysis conditions such as microwave irradiation.39 2. Figure 5C shows the complete monosaccharide profiling chromatograms of A3 N-glycan standard. The measured ratio of GlcN:Gal:Man:Neu5Ac was 3:3:3:1 (Table 2) instead of its theoretical ratio of 5:3:3:3, based on its structure (Table 1). Similar to the result of A1F, the GlcN−GlcN was resistant to the hydrolysis and GlcN was observed at lower response. Meanwhile, all Neu5Ac was released and partially degraded.39

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CONCLUSION The isocratic method with 150 mM NaOAc and 15 mM NaOH can be used to analyze acidic sugars. This method has baseline separation for the seven acidic sugars in 10 min and does not require any derivatization after hydrolysis. Subsequently, the gradient method was developed for complete monosaccharide profiling with high sensitivity (limit of quantitation ∼12.5 × 10−3 nmol). This method is linear over a wide range, is a convenient procedure without additional derivatization, and shows a broad application for complex carbohydrates. The gradient method was applied to qualitatively analyze monosaccharide compositions of different complex carbohydrates. GAGs including Hp, HS, DS, CS, and HA are some of the most complicated molecules in biochemistry. The monosaccharides of DS, CS, and HA were profiled by this method after hydrolysis. The results generally reflected their structural compositions. However, the results of Hp and HS did not reflect their real structures due to the partial hydrolysis and the degradation of uronic acids, especially IdoA. Alginate and fucoidan were confirmed to be impurities to each other using this method, even though the impurities were present in small amounts. The hydrolysis of N-glycans had similar problems to that of Hp and HS. The GlcN−GlcN did not break down to monosaccharide during the hydrolysis, while the sialic acid began to degrade. In this case, sialic acid and other monosaccharides could potentially be released separately and analyzed by this method. In summary, the established chromatographic method is universal, but the hydrolysis conditions for specific cabohydrates need to be optimized. With an optimized hydrolysis step that releases all monosaccharides without degradation, this method can provide a complete monosaccharide profiling in different types of complex carbohydrates efficiently, comprehensively, and sensitively.

Figure 4. Chromatograms of complete monosaccharide analysis of alginate and fucoidan. (A) Standards; (B) alginate; (C) fucoidan.

was not observed in alginate, GlcA could be a part of the fucoidan. The monosaccharide compositions of fucoidan with and without the alginate impurity are shown in Table 2. Glycans. Finally, two N-glycan standards were analyzed with this method. Many glycans contain neutral sugars, amino sugars, and sialic acid. They are always analyzed by two different methods for neutral/amino sugars and sialic acid separately, and derivatization is also involved. In this study, the N-glycan was hydrolyzed and all sugar units were analyzed in one injection. The chromatograms are shown in Figure 5.

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Figure 5. Chromatograms of complete monosaccharide analysis of Nglycans. (A) Standards; (B) A1F; (C) A3.

ASSOCIATED CONTENT

S Supporting Information *

1. Figure 5B shows the complete monosaccharide profiles of A1F N-glycan standard. The measured ratio of Fuc:GlcN:Gal:Man:Neu5Ac was 1:2:2:3:0.2 (Table 2) instead of its theoretical ratio of 1:4:2:3:1, based on its structure (Table 1). The ratios of Fuc, Gal, and Man are consistent with the theoretical values, but not for those of GlcN and Neu5Ac. Only two units of GlcN sugars were observed instead of four. This was due to the stronger glycosidic bond between GlcN−GlcN, which is a part of the structure of A1F. It was resistant to the hydrolysis at 100 °C for 1 h. The shoulder peak before Man at 15.1 min in Figure 5B may be due to GlcN−GlcN. Sialic acid (Neu5Ac) was also measured at lower levels than expected. The glycosidic bond of sialic acid is too weak to be resistant to the hydrolysis and is easily degraded.39,40 This suggests that the sialic acid was released from the glycans and partially degraded

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 847-270-2609. Fax: 847-270-5897. E-mail: zhenqing_ [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Bush, C. A.; Martin-Pastor, M. Annu. Rev. Biophys. Biomol. Struct. 1999, 28, 269−293. 4109

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Analytical Chemistry

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(2) Xia, J.; Margulis, C. J.; Case, D. A. J. Am. Chem. Soc. 2011, 133, 15252−15255. (3) Chronakies, I. S. Crit. Rev. Food Sci. Nutr. 1998, 38 (7), 599−637. (4) Venturoli, D.; Rippe, B. Am. J. Physiol. Renal Physiol. 2005, 288 (4), F605−F613. (5) Shur, B. D. Curr. Biol. 1994, 4 (11), 996−999. (6) Schweda, E. K.; Twelknever, B.; Li, J. Innate Immun. 2008, 14 (4), 199−211. (7) Asp, N. G. Mol. Aspects Med. 1987, 9 (1), 17−29. (8) Jørgensen, T. E.; Sletmoen, M.; Draget, K. I.; Stokke, B. T. Biomacromolecules 2007, 8 (8), 2388−2397. (9) Partridge, S. M. Nature 1946, 158, 270−271. (10) Trevelyan, W. E.; Procter, D. P.; Harrison, J. S. Nature 1950, 166 (4219), 444−445. (11) Baker, N.; Huebotter, R. J.; Schotz, M. C. Anal. Biochem. 1965, 10, 227−235. (12) Takeuchi, M.; Takasaki, S.; Inoue, N.; Kobata, A. J. Chromatogr. 1987, 400, 207−213. (13) Richey, J. M.; Richey, H. G., Jr.; Schraer, R. Anal. Biochem. 1964, 9, 272−280. (14) Reineccius, G. A.; Kavanagh, T. E.; Keeney, P. G. J. Dairy Sci. 1970, 53 (8), 1018−1022. (15) Lee, Y. C. Anal. Biochem. 1990, 189, 151−162. (16) Bruggink, C.; Maurer, R.; Herrmann, H.; Cavallli, S.; Hoefler, F. J. Chromatogr. A 2005, 1085, 104−109. (17) Harazono, A.; Kobayashi, T.; Kawasaki, N.; Itoh, S.; Tada, M.; Hashii, N.; Ishii, A.; Arato, T.; Yanagihara, S.; Yagi, Y.; Koga, A.; Tsuda, Y.; Kimura, M.; Sakita, M.; Kitamura, S.; Yamaguchi, H.; Mimura, H.; Murata, Y.; Hamazume, Y.; Sato, T.; Natsuka, S.; Kakehi, K.; Kinoshita, M.; Watanabe, S.; Yamaguchi, T. Biologicals 2011, 39 (3), 171−180. (18) Bernfield, M.; Gotte, M.; Park, P. W.; Reizes, O.; Fitzgerald, M. L; Lincecum, J.; Zako, M. Annu. Rev. Biochem. 1999, 68, 729−777. (19) Lin, X. Development 2004, 131 (24), 6009−6021. (20) Taylor, K. R.; Gallo, R. L. FASEB J. 2006, 20, 9−22. (21) Sasisekharan, R.; Raman, R.; Prabhakar, V. Annu. Rev. Biomed. Eng. 2006, 8, 181−231. (22) Pan, J.; Qian, Y.; Zhou, X.; Pazandak, A.; Frazier, S. B.; Weiser, P.; Lu, H.; Zhang, L. Glycobiol. Insights 2010, 2010 (2), 1−12. (23) Pan, J.; Qian, Y.; Zhou, X.; Pazandak, A.; Frazier, S. B.; Weiser, P.; Lu, H.; Zhang, L. Nat. Biotechnol. 2010, 28 (3), 203−207 author reply 207−211. (24) Koseki, M.; Kimura, A.; Tsurumi, K. J. Biochem. 1978, 83 (2), 553−558. (25) Li, F.; Yamada, S.; Basappa; Shetty, A. K.; Sugiura, M.; Sugahara, K. Glycoconj. J. 2008, 25 (7), 603−610. (26) Haug, A.; Larsen, B.; Smidsrød, O. Acta Chem. Scand. 1967, 21, 691−704. (27) Smidsrød, O.; Haug, A.; Larsen, B. Acta Chem. Scand. 1966, 20, 1026−1034. (28) De Vos, P.; De Haan, B.; Van Schilfgaarde, R. Biomaterials 1997, 18 (3), 273−278. (29) Mancini, F.; McHugh, T. H. Nahrung 2000, 44 (3), 152−157. (30) Vilén, E. M.; Klinger, M.; Sandström, C. Magn. Reson. Chem. 2011, 49, 584−591. (31) Varki, N. M.; Varki, A. Lab. Invest. 2007, 87, 851−857. (32) Stanton, P. G.; Shen, Z.; Kecorius, E. A.; Burgon, P. G.; Robertson, D. M.; Hearn, M. T. J. Biochem. Biophys. Methods 1995, 30 (1), 37−48. (33) Hardy, M. R.; Townsend, R. R. Methods Enzymol. 1994, 230, 208−225. (34) Zhang, Z.; Xie, J.; Zhang, F.; Linhardt, R. J. Anal. Biochem. 2007, 371 (1), 118−120. (35) Bitter, T.; Muir, M. Anal. Biochem. 1962, 4, 330−334. (36) Yu, G.; Zhang, Y.; Zhang, Z.; Song, L.; Wang, P.; Chai, W. Anal. Chem. 2010, 82 (22), 9534−9542. (37) Berteau, O.; Mulloy, B. Glycobiology 2003, 13, 29R−40R. (38) Li, B.; Lu, F.; Wei, X.; Zhao, R. Molecules 2008, 13 (8), 1671− 1695.

(39) Cheng, M. C.; Wang, K. T.; Inoue, S.; Khoo, K. H.; Wu, S. H. Anal. Biochem. 1999, 267 (2), 287−293. (40) Cheng, M. C.; Lin, S. L.; Wu, S. H.; Inoue, S.; Inoue, Y. Anal. Biochem. 1998, 260 (2), 154−159.

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dx.doi.org/10.1021/ac300176z | Anal. Chem. 2012, 84, 4104−4110