CHINESE JOURNAL OF ANALYTICAL CHEMISTRYVolume 37, Issue 11, November 2009 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2009, 37(11), 1590–1595.

Received 3 April 2009; accepted 13 June 2009 * Corresponding author. E-mail: yanxiaojun@nbu.edu.cn This work was supported by the National Natural Science Foundation of China (No. 30800860), the Program for Changjiang Scholars, Innovative Research Team in University of China (No. IRT0734), the Ningbo Municipal Science Project of China (No. 2006C100030), and the K.C. Wong Magna Fund in Ningbo University. Copyright © 2009, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(08)60143-7

RESEARCH PAPER

Analysis of Oligo- -carrageenan by Reversed Phase Ion-pair Ultra Performance Liquid Chromatography Coupled with Electrospray Ionization-Time of Flight-Mass Spectrometry GAO Yang, CHEN Hai-Min, XU Ji-Lin, CHEN De-Ying, YAN Xiao-Jun*Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China

Abstract: The reverse phase ion-pair ultra performance liquid chromatography coupled with electrospray ionization time of flight mass spectrometry (RPIP-UPLC-ESI-Q-TOF-MS) method was developed for the analyses and elucidation of sulfated oligosaccharides. Mass spectra were obtained by ESI-Q-TOF-MS in both positive and negative ionization modes. Oligo- - carrageenans were separated on BEH C18 column using heptylamine (20 mM, pH 4) as the ion-pairing reagent and A (H2O with 25% heptylammonium formate) and B (MeOH with 25% heptylammonium formate) as gradient eluent. Thus, oligo- -carrageenans were separated until pentatetrasaccharides were obtained. In addition, abundant structural information was obtained through the MS spectrometric analysis, and all acid hydrolyzed oligo- -carrageenans were identified as odd-numbered oligosaccharides. To further confirm these compounds, polyacrylamide gel electrophoresis (PAGE) was carried out. Furthermore, the described method has wide applicability to rapidly screen and provide structural information of oligo- -carrageenans. Key Words: -Carrageenan; Reversed-phased ion-pairing chromatography; Ultra performance liquid chromatography; Time-of-flight mass spectrometry; Polyacrylamide gel electrophoresis

1 Introduction

Carrageenan is the generic name for a family of linear, sulfated galactans, obtained by extraction from a certain species of marine red algae[1]. They are composed of alternating 3-linked -D-galactopyranose (G-units) and 4-linked -dgalactopyranose (D-units) or 4-linked 3,6-anhydro- -D-galactopyranose (DA-units), forming the disaccharide repeating unit of carrageenans. Depending on the number and position of anionic O-sulfo (sulfate) groups, several types of carrageenans can be recognized. For example, the three most commercially exploited carrageenans, namely kappa- ( , DA-G4S), iota- ( , DA2S-G4S), and lambda- ( , D2S, 6S-G2S) carrageenans, differ by the presence of one, two, and three sulfate ester groups per repeating disaccharide unit, respectively[2]. Carrageenans are widely used as food

additives and have been found to posses biological activities such as anticoagulant activity, antitumor activity, and antiviral activity. However, based on recent studies, compared with sulfated polysaccharides, sulfated oligosaccharides have more advantages as they are structurally more homogeneous and exhibit less toxicity because of the reduced anticoagulant activity and easy excretion. In addition, oral delivery may be more feasible[3–7].

The relation between molecular structure and functional properties is a major research theme within the area of carrageenan research[8]. However, sulfated oligosaccharides, such as carrageenans and glycosaminoglycans, are difficult to analyze because of the lability of the sulfate substituents and sequence heterogeneity. The first successful mass spectrometric (MS) analyses of heparin oligosaccharides reported were performed using fast atom bombardment ionization or 252Cf

GAO Yang et al. / Chinese Journal of Analytical Chemistry, 2009, 37(11): 1590–1595

plasma desorption. However, considerable sulfate loss was observed in both the techniques[9–11]. The use of soft ionization techniques, such as MALDI and ESI, in the positive and negative modes, together with MS/MS fragmentation, has provided important strategies for determination of sulfate substitution patterns. However, because the sulfate substituent is labile, it can be lost by expulsion as HSO4

–, NaSO3 or NaHSO4; thus, it could result in the loss of structural information[12]. Recently, Aristotelis reported the use of ESI-MS with ion-pair reversed-phase liquid chromatography (IP-RPLC) for the analysis of oligo- - carrageenans in the positive mode. IP-RPLC achieved the separation of oligo- - carrageenans with a degree of polymerizetion (DP) from 4 to 18[13]. Then, IP-RPLC-ESIMS has been used to analyze heparin oligosaccharides[14]. In this study, the separation of oligo- -carrageenan with a degree of polymerization (DP 3 to 45) and the structural analysis of oligo- -carrageenan standards (DP 3 to 27) by the positive and negative modes using RPIP-UPLC-ESI-Q-TOF-MS are described.

2 Experimental

2.1 Materials

-Carrageenan (80 mesh size) and Sephadex G10 were purchased from Lvbao Biochemistry Co. Ltd (Quanzhou, China) and Pharmacia, respectively. The standard oligo- - carrageenans, methanol (chromatographic grade), formic acid (chromatographic grads), and heptylamine were purchased from SIGMA-ALDRICH (UK). Deionized water (18 M ) was obtained from a Mili-Q water purification system.

2.2 Preparation of oligo- -carrageenan Approximately 1 g of -carrageenan was placed in 100 ml of

water and was boiled to solubilize the solid carrageenan and then was cooled to 40 °C for using in the experiment. The 1% (w/V) carrageenan solution was added to 0.2 M HCl and was incubated at 60 °C for 30 min. The hydrolysate was neutralized by 1 M NaCl, concentrated by vacuum evaporation, purified by passing through Sephadex G-10 resin to remove salts, freeze-dried, and stored at 4 °C before starting the experiments.

2.3 Chromatographic conditions The UPLC analysis was carried out on a Waters

ACQUITY Ultra Performance LC system (UPLC) using an ACQUITY UPLC BEH C18 analytical column (100 mm × 2.1 mm, 1.7 m). The flow rate was 0.35 ml min–1, and the column temperature was set at 45 °C. For the preparation of ion-pairing reagent (20 mM of heptylammonium ion, pH 4), 0.238 ml formic acid and 0.745 ml heptylamin were diluted

using 250 ml water. A linear gradient elution of A (H2O with 25% heptylammonium formate ater) and B (MeOH with 25% heptylammonium formate) was used with the gradient procedure as follows: 0 min, 25% B to 1 min, 50% B; followed by a linear gradient from 50% B to 65% B for more than 3 min; during the next 12 min, the percentage of B was increased from 65% to 80%; then the proportion of B was increased from 80% to 100% over the next 0.5 min and held for 3.5 min; finally, the percentage of B was changed from 100% to 25% B in 0.5 min; the mobile phase was then held isocratically for 4.5 min.

2.4 Mass spectrometric conditions The RPIP-UPLC-ESI-Q-TOF-MS analyses was performed

by UPLC system that is described above in combination with a Waters Q-TOF Premier mass spectrometer operating in the negative and positive ion electrospray mode. The spray conditions were optimized to achieve minimal fragmentation in the spray interface resulting in the following conditions: the mass scan range was 300–4000 amu with scan duration of 0.1 s, and high-purity nitrogen was used as nebulizer and drying gas. Nitrogen was used as desolvation gas at flow rate of 400 l h–1, and the desolvation temperature was 250 ºC. The source temperature was 120 ºC. For the positive mode, the electrospray capillary voltage was set at 2.5 kV, the sampling cone voltage was set at 40 V, the collision energy was set at 5 V; for the negative-ion mode, the electrospray capillary voltage was set at 2.8 kV, the sampling cone voltage was set at 45 V, and the collision energy was set at 5 V. The lock mass spray for precise mass determination was set by 0.5 ng μl–1 leucine enkephalin at m/z 556.2771 in the positive-ion mode, while the leucine enkephalin at m/z 554.2615 in the negative-ion mode.

2.5 Gel electrophoresis Polyacrylamide gel electrophoresis (PAGE) was performed

as mentioned earlier[15]. Briefly, a polyacrylamide min-gel (8 cm × 6 cm, 25%, total acrylamide, Tris-Boric acid-EDTA system) was prepared, and 5 μg of each carrageenan-derived oligosaccharide was subjected to electrophoresis at a constant current of 20 mA for 180 min. Oligosaccharides were visualized by Alcian Blue staining. If the band is not clear, we repeat staining by the silver-staining method.

3 Results and discussion 3.1 Separation of oligo- -carrageenan

The total ion current (TIC) chromatogram of the

oligo- -carrageenan is shown in Fig.1.

GAO Yang et al. / Chinese Journal of Analytical Chemistry, 2009, 37(11): 1590–1595

Fig.1 Positive and negative total ion current chromatogram (TIC) of oligo- -carrageenan Numbers above the peaks represent oligo- -carrageenan of monosaccharide unit

Oligo- -carrageenans with different degree of

polymerization have been well separated by BEH C18. In addition, the chromatographic signal increased based on the retention time. By comparing the results of the negative ionization mode with that of the positive ionization mode, the former was able to separate oligo- -carrageenans from trisaccharide ( -3) up to pentatetrasaccharide ( -45), whereas the latter separated only up to heptacosasccharide ( -27). Hence, in the negative ionization mode, the peak shape was better and the impurity was less than that in the positive ionization mode. Furthermore, the mass information of oligo- -carrageenans was more abundant in negative ionization mode. In this study, MeOH/H2O with 25% heptylammonium formate was used as eluent in linear gradient mode. The concentration of the ion-paring reagent was maintained constant because a variation in the content of the mobile phase in organic modifier would create a variation in the concentration of the ion-pairing reagent in the stationary phase.

According to the principles of ion-pair chromatography[16], heptylamine (Hep), which was added into the eluent, changed the charged polarity oligo- -carrageenan to the neutral molecule with hydrophobic characteristics. Hence, the retention capacity was enhanced in C18 stationary phase. Because the hydrophobicity of molecules was in direct proportion with the quantity of ion-pair reagents, the quantity of ion-pair reagents combined with the oligosaccharide molecules and hydrophobic property enhanced along with the increase of the degree of polymerization of oligosaccharides. Furthermore, with the advantage of strong separation efficiency and relatively short analysis time, the oligosaccharides with continuous degree of polymerization were well separated by UPLC. In the previous reports, UPLC/(+)-ESI analysis for oligo- -carrageenans has been carried out only up to octadecasaccharide ( -18)[13], and in negative ionization mode, it has been carried out only up to dodecasaccharide ( -12)[17]. In this study, the separation efficiency and detection sensitivity of oligosaccharides were improved by optimizing the experimental conditions.

3.2 Characterization of oligomers

High-resolution MS analyses for oligo- -carrageenans utilizing the electrospray ionization (ESI) source in the positive and negative ionization modes were performed. In both the ionization modes, the exact mass data of oligo- -carrageenans from trisaccharide ( -3) up to heptacosasaccharide ( -27) were obtained, which were consistent with the ideal structures of acid hydrolyzed oligo- -carrageenans. In addition, they all were odd-numbered oligosaccharides (Table 1).

In the mass spectra analysis, heptylamine adducts of all the oligosaccharides were yielded. This phenomenon could be explained by the fact that the heptylamine molecules are connected to the sulfate groups to form ion-pairs, which will not break during the ionization. In the positive ionization mode, the base peaks of oligo- -carrageenans were observed as single charge ions ( -3 to -5) (Fig.2). When the oligo- -carrageenans was up to -7, the double charge ions were be obtained, but the single charge ions were still the base peak; However, from -9 to -17 (Fig.3), the double charge ions had the highest abundance. While from -19 to -27, the triple charge ions were generated (Fig.4); but from -21 to -27, the triple charge ions become the base perks.

Thus, according to these phenomena, it could be concluded that multiply charged ions can be obtained readily along with the increase in the degree of polymerization. In the negative ion mode, the mass spectra of -5 and -17 contained double and triple charged ions, respectively. In addition, many sodium adduct ions were observed rather than heptylamine adduct ions, suggesting that the affinities of heptylamine molecule and the sulfate constituent are weaker than that in the positive ionization mode. Otherwise, under the same experimental conditions as described above in the negative ionization mode, legible structural information of the oligo- -carrageenans ( 27) was obtained. With the increase in the degree of polymerization, the abundance of quasi-molecular ion peak decreased and the other fragment ions increased (Figs.3 and 4). It can be elucidated that the higher the degree of polymerization of oligosaccharides, the lower the stability of the ion. In addition, when the concentration of MeOH of gradient eluent increased, the ionization of oligosaccharide molecule weakened.

GAO Yang et al. / Chinese Journal of Analytical Chemistry, 2009, 37(11): 1590–1595

Table 1 Ions identified for oligo- -carrageenans in the positive and negative ionization modes

Oligosaccharide (A-G4S) Positive ionization mode m/z Negative ionization mode m/z Trisaccharide [(A-G4S)G4S + SO3 + (C7H15NH3)4 – H2O]+ 1168 [(A-G4S)G4S + SO3 + (C7H15NH3)2 – H2O]– 936 -3 [(A-G4S)G4S + (C7H15NH3)3]+ 992

[(A-G4S)G4S – SO3 + (C7H15NH3)2 + H2O]+ 797 [(A-G4S)G4S – SO3 + H2O] – 565 Pentasaccharide [(A-G4S)2G4S + SO3 + (C7H15NH3)5 – H2O]+ 1669 [(A-G4S)2G4S + SO3 + (C7H15NH3)3 – H2O] – 1437 -5 [(A-G4S)2G4S + (C7H15NH3)4]+ 1493 [(A-G4S)2G4S + (C7H15NH3)2] – 1261

[(A-G4S)2G4S + (C7H15NH3)3 + Na]+ 1400 [(A-G4S)2G4S + (C7H15NH3)2 + 2Na]+ 1307 [(A-G4S)2G4S-SO3 + (C7H15NH3)] – 1066 [(A-G4S)2G4S + (C7H15NH3)-H2O]2– 563 [(A-G4S)2G4S – SO3]2– 475 Heptasaccharide [(A-G4S)3G4S + SO3 + (C7H15NH3)6 – H2O]+ 2171 [(A-G4S)3G4S + SO3 + (C7H15NH3)4 – H2O] – 1938 -7 [(A-G4S)3G4S + (C7H15NH3)5]+ 1994 [(A-G4S)3G4S + (C7H15NH3)3] – 1762

[(A-G4S)3G4S + (C7H15NH3)4 + Na]+ 1901 [(A-G4S)3G4S + (C7H15NH3)6]2+ 1056 [(A-G4S)3G4S + SO3 + (C7H15NH3)7 – H2O]2+ 1144 [(A-G4S)3G4S-SO3 + (C7H15NH3)]2– 725 [(A-G4S)3G4S – 2SO3]2– 628 [(A-G4S)3G4S-SO3 + Na – H2O]2– 668 Nonasaccharide [(A-G4S)4G4S + SO3 + (C7H15NH3)7 – H2O]+ 2673 [(A-G4S)4G4S + SO3 + (C7H15NH3)5 – H2O] – 2440 -9 [(A-G4S)4G4S + (C7H15NH3)6]+ 2497 [(A-G4S)4G4S + (C7H15NH3)4] – 2264

[(A-G4S)4G4S + (C7H15NH3)5 + Na]+ 2403 [(A-G4S)4G4S + SO3+(C7H15NH3)8 – H2O]2+ 1394 [(A-G4S)4G4S + (C7H15NH3)7]2+ 1306 [(A-G4S)4G4S – SO3 + (C7H15NH3)2]2– 976 [(A-G4S)4G4S – 2SO3 + (C7H15NH3)]2– 878 Hendesaccharide [(A-G4S)5G4S + SO3 + (C7H15NH3)9 – H2O]2+ 1645 [(A-G4S)5G4S + SO3 + (C7H15NH3)5 – H2O]2– 1412 -11 [(A-G4S)5G4S + (C7H15NH3)8]2+ 1557 [(A-G4S)5G4S + (C7H15NH3)4]2– 1324

[(A-G4S)5G4S + (C7H15NH3)7 + Na]2+ 1511 [(A-G4S)5G4S – 2SO3 + (C7H15NH3)2]2– 1129 Tridesaccharide [(A-G4S)6G4S + SO3 + (C7H15NH3)10 – H2O]2+ 1896 [(A-G4S)6G4S + SO3 + (C7H15NH3)6 – H2O]2– 1664 -13 [(A-G4S)6G4S + (C7H15NH3)9]2+ 1808 [(A-G4S)6G4S + (C7H15NH3)5]2– 1576

[(A-G4S)6G4S + (C7H15NH3)8 + Na]2+ 1761 [(A-G4S)6G4S – SO3 + (C7H15NH3)4]2- 1477 Pentadesaccharide [(A-G4S)7G4S + SO3+(C7H15NH3)11 – H2O]2+ 2147 [(A-G4S)7G4S + SO3 + (C7H15NH3)7 – H2O]2- 1914 -15 [(A-G4S)7G4S + (C7H15NH3)10]2+ 2058 [(A-G4S)7G4S + (C7H15NH3)6]2- 1826

[(A-G4S)7G4S + (C7H15NH3)9 + Na]2+ 2012 [(A-G4S)7G4S + (C7H15NH3)3 + 2Na + H2O]3– 1123 Heptadesaccharide [(A-G4S)8G4S + SO3 + (C7H15NH3)12 – H2O]2+ 2398 [(A-G4S)8G4S + SO3 + (C7H15NH3)8 – H2O]2– 2165 -17 [(A-G4S)8G4S + (C7H15NH3)11]2+ 2309 [(A-G4S)8G4S + (C7H15NH3)7]2– 2077

[(A-G4S)8G4S + (C7H15NH3)10 + Na]2+ 2263 [(A-G4S)8G4S + (C7H15NH3)4 + 2Na + H2O]3– 1290 Nonadesaccharide [(A-G4S)9G4S + SO3 + (C7H15NH3)13 – H2O]2+ 2648 [(A-G4S)9G4S + SO3 + (C7H15NH3)9 – H2O]2– 2416 -19 [(A-G4S)9G4S + (C7H15NH3)12]2+ 2560 [(A-G4S)9G4S + (C7H15NH3)8]2– 2328

[(A-G4S)9G4S + (C7H15NH3)13]3+ 1745 [(A-G4S)9G4S + (C7H15NH3)6 + Na]3– 1484 [(A-G4S)9G4S + SO3 + (C7H15NH3)14 – H2O]3+ 1804 Heneicosaccharide [(A-G4S)10G4S + SO3 + (C7H15NH3)14 – H2O]2+ 2899 [(A-G4S)10G4S + SO3 + (C7H15NH3)10 – H2O]2– 2667 -21 [(A-G4S)10G4S + (C7H15NH3)13]2+ 2811 [(A-G4S)10G4S + (C7H15NH3)9]2– 2578

[(A-G4S)10G4S + (C7H15NH3)12 + Na]2+ 2764 [(A-G4S)10G4S + SO3 + (C7H15NH3)15 – H2O]3+ 1971 [(A-G4S)10G4S + (C7H15NH3)14]3+ 1912 Tricosasccharide [(A-G4S)11G4S + SO3 + (C7H15NH3)15 – H2O]2+ 3149 -23 [(A-G4S)11G4S + (C7H15NH3)14]2+ 3061

[(A-G4S)11G4S + (C7H15NH3)13 + Na]2+ 3015 [(A-G4S)11G4S + (C7H15NH3)15]3+ 2080 [(A-G4S)11G4S + (C7H15NH3)9]3– 1848 [(A-G4S)11G4S + SO3 + (C7H15NH3)16 – H2O]3+ 2138 [(A-G4S)11G4S + SO3 + (C7H15NH3)10 – H2O]3– 1906 Pentacosasccharid [(A-G4S)12G4S + SO3 + (C7H15NH3)16 – H2O]2+ 3400 -25 [(A-G4S)12G4S + (C7H15NH3)15]2+ 3313

[(A-G4S)12G4S + (C7H15NH3)16]3+ 2247 [(A-G4S)12G4S + (C7H15NH3)10]3– 2014 [(A-G4S)12G4S + SO3+(C7H15NH3)17 – H2O]3+ 2306 [(A-G4S)12G4S + SO3 + (C7H15NH3)11 – H2O]3– 2073 Heptacosasccharid [(A-G4S)13G4S + SO3+(C7H15NH3)18 – H2O]3+ 2473 [(A-G4S)13G4S + SO3 + (C7H15NH3)12 – H2O]3– 2241 -27 [(A-G4S)13G4S + (C7H15NH3)17]3+ 2414 [(A-G4S)13G4S + (C7H15NH3)11]3– 2182

Fig.2 Positive and negative ESIMS spectra of pentasaccharide ( -5)

GAO Yang et al. / Chinese Journal of Analytical Chemistry, 2009, 37(11): 1590–1595

Fig.3 Positive and negative ESIMS spectra of Heptadesaccharide ( -17)

Fig.4 Positive and negative ESIMS spectra of Heptacosasaccharide ( -27) 3.3 Analysis of oligo- -carrageenan by PAGE

In this study, the separated oligosaccharides were all

odd-numbered oligosaccharide, which was inconsistent with the even-numbered oligosccharides. However, the same phenomenon has been described for the odd-number oligosaccharides by acid hydrolysis. To further confirm that odd/even number of the degree of polymerization of oligosccharides, electrophoresis analyses of oligo- -carrageenans ( -CO) were utilized by PAGE. Octasaccharide ( -8) (Fig.5, lane 1) and dodecasaccharide ( -12) (Fig.5, lane 2) were used as standards. As shown in Fig.5, there was an obvious single band of -12, and there were several bands in the range from nonasaccharide ( -9) to nonadesaccharide ( -19) of -CO in the PAGE analysis, whereas -8 had no band. Above all, the odd-numbered oligosccharides generated by acid hydrolysis was identified by PAGE analysis.

Fig.5 PAGE analysis of oligo- -carrageenan

It was also demonstrated that the combination of electrophoresis and MS was helpful for the structure analysis of oligosaccharide. On the one hand, due to the differences of mobile phase between the ion-pair chromatography technology and traditional RP-HPLC, there occurred a necessity to change the solvent and clean the system constantly during the experiment process, which was inconvenient for operation. In this study, electrophoresis used as a conventional approach was available for rapid determination of the degree of polymerization of oligosaccharides. On the other hand, information on the structural characterization cannot be obtained in electrophoresis. However, LC-MS has wide applicability to rapidly screen and provide structural information through optimizing the experimental conditions.

3.4 Fragmentations rule of carrageenan for ESIMS Because sulfate group in the carrageenan molecule was

subject to cleave at high voltage ionization state, the insource-CID was happened through high cone voltage to form complex mass spectra, which was not good for the rapid identification of molecular weight. However, if the cone voltage was too low, the sensitivity would reduce. As mentioned above, it is important to characterize the structure of oligo- -carrageenan by elucidating each peak and the fragmentation pathways of oligosaccharides. Through the ESI-MS analyses of 13 kinds of isolated oligo- -carrageenans ( -3 to the -27), the characteristic fragment was observed by

GAO Yang et al. / Chinese Journal of Analytical Chemistry, 2009, 37(11): 1590–1595

Table 2 Fragmentations rule of carrageenan for ESIMS Mass

differenceCharacteristic fragmentation pattern

75 116 – 23 – 18 C7H15NH3+ not replace Na+, and a lose of

H2O93 116 – 23 C7H15NH3

+ not replace Na+

176 116 + 80 – 18 – 2 add [SO3 – C7H15NH3], a lose of H2O and 2H+

195 116 + 80 – 1 lose of [SO3 – C7H15NH3], and add H+

213 195 + 18 lose of [SO3 – C7H15NH3], add H+, then a lose of H2O

in-source CID (Table 2), which was used for structural identification of oligo-carrageenans. References

[1] Ji M H. Seaweed Chemistry. Beijing: Science Press, 1997: 117–182

[2] Hu Y Q, Zhu M. Transactions of Oceanology, 2005, 1: 94–102 [3] Gefei Z, Hua X, Wenxu S, Yueping S, Zhien L, Zuhong X.

Pharmacological Research, 2006, 53:129–134 [4] Luescher-Mattli M. Curr. Med. Chem-Anti-infect Ag., 2003, 2:

219–225 [5] Haimin C, Xiaojun Y, Jing L, Feng W, Weifeng X. J. Agric.

Food Chem. 2007, 55: 6910–6917 [6] Mi T Y, Yan X J, Chen H M, Lin J, Feng W, Wang F, Xu W F.

Acta Pharmaceutica Sinica, 2008, 43(5): 474–479 [7] Huamao Y, Weiwei Z, Xuegang L, Xiaoxia L, Ning L, Xuelu

G, Jinming S. Carbohydrate Research, 2005, 340: 685–692 [8] van de Velde F. Food Hydrocolloids, 2008, 22: 727–734 [9] Rice K G, Kim Y S, Grant A C, Merchant Z M, Linhardt R J.

Analytical Biochemistry, 1985, 150(2): 325–331 [10] Reinhold V N, Carr S A, Green B N, Maurice P, Jean C,

Pierre S. Carbohydrate Research, 1987, 161(2): 305–313 [11] McNeal C J, Macfarlane R D. J. Am. Chem. Soc., 1986,

108(9): 2132–2139 [12] Jennifer T A, Fabian M D. J Am Soc Mass Spectrom., 2006,

17: 96–103 [13] Aristotelis A, Patrick F, William H, Michel L. Carbohydrate

Research, 2004, 339: 1301–1309 [14] Jens H, Peter R, Lene H R. Carbohydrate Research, 2006,

341: 382–387 [15] LU Z H, Zhao X, Yu G L, Wang Y H, Xu J M. Chinese

Journal of Biochemical Pharmaceutics, 2002, 23(l): 17–18 [16] Da S L. Introduction to Chromatography, Wuhan: Wuhan

University Press, 1988: 380–395 [17] Yuko F, Marina C, Hiroshi N, Alberto S C, Rosa E, Mar a C

M. Carbohydrate Research, 2002, 337: 1553–1562