Carbohydrate Polymers 104 (2014) 23–28

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Determination of M/G ratio of propylene glycol alginate sodium

sulfate by HPLC with pre-column derivatization
Jian Wu a , Xia Zhao a,b,∗ , Li Ren c , Yiting Xue a , Chunxia Li a,b , Guangli Yu a,b , Huashi Guan a,b
a
Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China
b
Shandong Provincial Key Laboratory of Glycoscience and Glycoengineering, Ocean University of China, Qingdao 266003, China
c
Qingdao Chiatai Haier Pharmaceutical Co., Ltd, Qingdao 266103, China

a r t i c l e i n f o a b s t r a c t

Article history: A reliable high performance liquid chromatography with pre-column derivatization method was devel-
Received 1 November 2013 oped for the determination of the mannuronic acid (M)/guluronic acid (G) ratio of propylene glycol
Received in revised form alginate sodium sulfate (PSS). The hydrolysis conditions of PSS were investigated by four degradation
18 December 2013
methods based on the degree of destruction of M and G, and the chromatographic separation conditions
Accepted 5 January 2014
Available online 10 January 2014
were also optimized. A satisfactory resolution of M and G was achieved with a KP-C18 column using
0.1 mol/L phosphate buffer (pH 7.0)-acetonitrile (83/17, v/v) as a mobile phase, after PSS was hydrolyzed
with 0.1 mol/L sulfuric acid and labeled with 1-phenyl-3-methyl -5-pyrazolone. The M/G ratio of PSS
Keywords:
Propylene glycol alginate sodium sulfate determined by this method was in good accordance with that obtained by the 1 H NMR method with a
Pre-column derivatization desulfurization strategy. Our method is rapid, sensitive, accurate and reproducible. The limit of detection
Hydrolysis was found to be 0.25 ␮g/mL for M and 0.40 ␮g/mL for G.
M/G ratio © 2014 Elsevier Ltd. All rights reserved.
1-Phenyl-3-methyl-5-pyrazolone.

1. Introduction Nuclear magnetic resonance (NMR) as a simple and non-

destructive method has been widely used for determining the M/G
Alginate is a linear polysaccharide composed of (1→4) linked ratio in alginate (Heyraud et al., 1996; Burana-osot et al., 2009;
␤-D-mannuronic acid (M) and its C-5 epimer ␣-L-guluronic acid Aida, Yamagata, Watanabe, Richard, & Smith, 2010). However, it is
(G) (Rahelivao, Andriamanantoanina, Heyraud, & Rinaudo, 2013). not suitable for the analysis of sulfated alginate derivatives because
Alginate and its sulfated derivatives have various biological activ- the presence of sulfate groups affect the resolution of anomeric
ities, such as anti-inflammatory and anticoagulant actions (Zhao proton signals. The commonly used method for M/G ratio deter-
et al., 2007; Fan et al., 2011), and they have been extensively mination of alginate by anion exchange liquid chromatography is
used as wound dressing materials (Pawar & Edgar, 2012). Pro- rapid and simple (Gacesa, Squire, & Winterburn, 1983). However,
pylene glycol alginate sodium sulfate (PSS), a heparinoid-active the lactone forms of uronic acids interfere with the quantification
sulfated polysaccharide (Fig. 1) prepared by chemical sulfation of of M and G. It is also not suitable for the microanalysis of alginate
low-molecular-weight alginate, has been commonly used as an sulfated derivatives in biological samples.
anti-cardiovascular disease drug in China for nearly 30 years (Li, Su, Here, we report a reversed-phase high performance liquid
& Guan, 2012). However, the activities of PSS are influenced by the chromatographic (HPLC) method using 1-phenyl-3-methyl-5-
uronic acid composition (M/G ratio), degree of sulfate substitution pyrazolone (PMP) as a pre-column derivatization reagent for the
and weight-averaged molecular weight (Fan et al., 2011; Lin et al., separation and analysis of M and G. This method is rapid, accurate
2007). The M/G ratio in PSS plays an important role in its anticoag- and sensitive for determination of the M/G ratio in PSS, and it is
ulation, blood lipid regulation activities and side effects. There is a suitable for quality control and microanalysis of PSS.
need to develop a reliable and sensitive method for determination
of the M/G ratio of PSS for quality control and clinical purposes.
2. Experimental

2.1. Chemicals
∗ Corresponding author at: Key Laboratory of Marine Drugs, Ministry of Educa-
tion, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road,
Qingdao, Shandong 266003, China. Tel.: +86 532 82 03 15 60;
M and G monosaccharide standards were provided by the School
fax: +86 532 82 03 15 60. of Medicine and Pharmacy at the Ocean University of China (Qing-
E-mail addresses: [email protected], [email protected] (X. Zhao). dao, China). PMP was purchased from Sigma–Aldrich (Shanghai,

0144-8617/$ – see front matter © 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2014.01.011
24 J. Wu et al. / Carbohydrate Polymers 104 (2014) 23–28

2.5. Determination of the M/G ratio of PSS

PSS was hydrolyzed and derivatizated with PMP, and then sep-
arated at the optimal chromatographic conditions as mentioned
above. The M/G ratio of PSS was calculated by comparing the peak
area values of M or G to each calibrated standard curve. Calibra-
tion curves of M and G were constructed by plotting the peak areas
against the concentrations of their monosaccharide standards on
each analysis day using freshly prepared calibration standards.

Fig. 1. Chemical structure of propylene glycol alginate sodium sulfate (PSS). R1 = Na, 2.6. Desulfation of PSS
CH2 CH(OH)CH3 ; R2 = H, SO3 Na.

Desulfation of PSS was performed according to the method

China). HPLC-grade acetonitrile was purchased from Merck KGaA described by Falshaw and Furneaux (1998). Briefly, PSS (30 mg)
(Germany). Propylene glycol alginate sodium sulfate (PSS) was pro- was dissolved in water and passed through an ion-exchange col-
vided by Qingdao Chiatai Haier Pharmaceutical Co., Ltd (Qingdao, umn (732 resin, H+ form), and was then neutralized with pyridine
China). All other chemicals and solvents were of analytical grade to pH 7.0 and lyophilized to give a pyridinium salt. The salt was dis-
unless otherwise specified. solved in 10 mL of dimethyl sulfoxide (DMSO) containing 10% (v/v)
of anhydrous methanol and 1% pyridine. After stirring at 100 ◦ C
2.2. Hydrolysis of PSS for 4 h, the reaction mixture was adjusted to pH 9.0 and dialyzed.
The purified desulfated product was recovered by alcohol precipita-
The hydrolysis of PSS was carried out by four commonly used tion and desulfation was confirmed by the disappearance of sulfate
different degradation methods for polysaccharides as follows: (1) peaks in its infrared spectrum.
PSS was dissolved in dilute sulfuric acid (0.1 mol/L) at a concentra-
tion of 5 mg/mL, hydrolyzed in a sealed glass ampoule at 120 ◦ C for 2.7. 1H NMR analysis
4 h, and then neutralized to pH 7.0 with NaOH after it was cooled
to room temperature; (2) PSS was suspended in 2 mol/L trifluo- The M/G ratio of PSS, the desulfurization product of PSS and
roacetic acid (TFA) at a concentration of 5 mg/mL, hydrolyzed in a its intermediate (low-molecular-weight alginate) were compared
sealed glass ampoule at 110 ◦ C for 6 h. The TFA was removed by with a nuclear magnetic resonance (NMR) method. Briefly, the sam-
rotary evaporation and washed out by water (Wang, Zhao, Yu, Li, ple (20 mg) was freeze-dried three times with 1 mL of D2 O (99.96%)
& Hao, 2009; Dai et al., 2010); (3) PSS was dissolved in 0.1 mol/L to remove all exchangeable protons. The 1 H NMR spectra were
HCl at a concentration of 20 mg/mL, hydrolyzed under microwave recorded at 20 ◦ C on a Bruker Avance 500 MHz spectrometer using
irradiation (1600 W) at 130 ◦ C for 15 min (Hu et al., 2013), and then deuterated acetone as an internal standard. The M/G ratio was cal-
neutralized with NaOH; and (4) PSS was hydrolyzed by the con- culated as previously reported (Rahelivao et al., 2013) and three
ventional acid hydrolysis method (with 80% H2 SO4 at 20 ◦ C for 18 h peak areas (P1 , signal at 5.1 ppm, H1 -G; P2 , signal at 4.7 ppm, H1 -M
and then 2 mol/L H2 SO4 at 100 ◦ C for 6 h) as described by Haug and H5 -GM; P3 , signal at 4.5 ppm, H5 -GG) defined the M/G ratio
and Larsen (1962). All experiments were operated in parallel three (M/G ratio = [P2 – P1 + P3 ]/P1 ).
times, and all hydrolysates were diluted to the same concentration
before derivatization. 3. Results and discussion

2.3. Derivatization procedure 3.1. Optimization of separation conditions

The derivatization reactions of PSS hydrolysate, M and G The separation conditions of M and G derivatives were inves-
monosaccharide standards with PMP were carried out essentially tigated by changing the pH value of the phosphate buffer, the
as previously described (Honda et al., 1989; Wang et al., 2009). proportion of acetonitrile, the flow rate and loading quantity of the
Briefly, 100 ␮L of 0.3 mol/L NaOH and 120 ␮L methanolic solution sample based on previous trials (Wang et al., 2009). Results showed
of PMP (0.5 mol/L) were added to 100 ␮L of the sample solution, and that the retention behavior and resolution of M and G standard
the mixture was heated to 70 ◦ C and incubated for 1 h. The reaction PMP derivatives were significantly affected by the pH value of the
mixture was neutralized with 100 ␮L of 0.3 mol/L HCl after it was phosphate buffer and the proportion of acetonitrile. The separation
cooled to room temperature and was then extracted with chlo- degree of the two PMP derivatives was improved and the reten-
roform (500 ␮L each) three times. The aqueous layer was filtered tion time was reduced with pH values increasing from 6.5 to 7.4,
through a 0.22 ␮m micron membrane filter before HPLC analysis. when the acetonitrile proportion remained at 17% (Fig. 2A). How-
ever, the column pressure increased rapidly at high pH values. The
2.4. HPLC analysis elution of M and G derivatives was earlier but the degree of separa-
tion was lower when the proportion of acetonitrile increased from
The Agilent 1260 HPLC system consisted of a binary pump 15% to 20% (Fig. 2B). The peaks of the two PMP derivatives were
(G1311C), an autosampler (G1329B), a variable wavelength detec- broad and even tailed at low proportions of acetonitrile, and 17%
tor (VWD, G1314F) and a system controller. The data were (v/v) of acetonitrile in the mobile phase provided the best sepa-
collected using an Open LAB CDS Chemstation Edition (version ration degree. The separation degree of two PMP derivatives was
C.01.05) provided by the Agilent Company (USA). A KP-C18 column improved slightly when the flow rate decreased from 1.0 mL/min
(150 × 4.6 mm, 5 ␮m), purchased from Zhongran Technology Co., to 0.5 mL/min (Fig. 2D), while the loading quantity of the sam-
Ltd. (Huaian, China), was used for HPLC analysis. Chromatographic ple had no significant effect on the separation degree of M and G
separation of PMP derivatives was carried out using 0.1 mol/L phos- derivatives (Fig. 2C). Considering the separation degree, peak shape,
phate buffer (pH 7.0) and acetonitrile at a ratio of 83:17 (v/v, %) as column pressure and analysis efficiency altogether, the optimal
a mobile phase at a flow rate of 0.8 mL/min. The temperature of the chromatographic conditions were chosen as follows (with a sep-
column was maintained at 30 ◦ C and detected by VWD at 245 nm. aration degree above 2.0): the mobile phase should be 0.1 mol/L
 J. Wu et al. / Carbohydrate Polymers 104 (2014) 23–28 25

Fig. 2. The influences of chromatographic separation conditions on the retention behavior and resolution of M and G standard PMP derivatives. (A) Influence of the pH value
of phosphate buffer (acetonitrile proportion 17%). (B) Influence of the acetonitrile proportion (phosphate buffer pH 7.0). (C) Influence of the loading quantity of sample
(phosphate buffer pH 7.0 and acetonitrile proportion 17%). (D) Influence of the flow rate (phosphate buffer pH 7.0 and acetonitrile proportion 17%).

phosphate buffer (pH 7.0) and acetonitrile in a ratio of 83:17 (v/v, To further explore the optimal conditions for dilute sulfuric acid
%) at a flow rate of 0.8 mL/min. hydrolysis of PSS, an orthogonal scheme of three factors and three
levels, namely, the degradation temperature (110 ◦ C, 120 ◦ C, 130 ◦ C;
factor A), degradation time (3 h, 4 h, 5 h; factor B) and concentration
3.2. The choice of degradation methods
of sulfuric acid (1.0 mol/L, 0.1 mol/L, 0.01 mol/L; factor C), was car-
ried out as shown in Table 1. Results analyzed by orthogonal design
Uronic acids are liable to destruct during acid hydrolysis and the
assistant II V3.1 software showed that the concentration of sulfuric
losses of M and G are different using different degradation meth-
acid and degradation temperature were the main factors that influ-
ods, which cause a significant deviation in the M/G ratio determined
enced the peak areas of M and G derivatives, and the degradation
even in the same sample. To choose a suitable hydrolysis method
time had the least influence among three factors (RC > RA > RB ). The
for PSS, four commonly used degradation methods for polysaccha-
optimal conditions for hydrolysis of PSS were determined to be the
rides were evaluated in our experiments. Comparing the peak areas
following: the concentration of sulfuric acid should be 0.1 mol/L,
of M and G derivatives degraded by four different methods, which
the degradation time should be 4 h and the degradation tempera-
were determined at the same chromatographic separation condi-
ture should be 120 ◦ C.
tions (as shown in Fig. 3), the destruction degrees of M and G by
the microwave degradation method were the lowest (peak area,
M: 260.07; G: 146.04, mAu × min, Fig. 3D) and were approximately 3.3. Method validation
equal to those of the dilute sulfuric acid (0.1 mol/L) hydrolysis
method (peak area, M: 235.40; G: 133.69, mAu × min, Fig. 3C). The The proposed HPLC method was validated in terms of specificity,
losses of M and G were the highest using the TFA hydrolysis method linearity, detection limit, precision, repeatability and stability.
(peak area, M: 122.34; G: 18.11, mAu × min, Fig. 3B) and losses were As shown in Fig. 4, both M and G monosaccharide standards
next highest using the traditional acid degradation method (peak gave a single chromatographic peak because of the alkaline envi-
area, M: 173.46; G: 54.57, mAu × min, Fig. 3A). The M/G ratios of ronment in the PMP derivatization reaction, indicating that the
PSS degraded by the microwave degradation method or dilute sul- lactone forms of uronic acids did not interfere with the sep-
furic acid hydrolysis method were approximate to the M/G ratio aration and analysis of M and G. The retention times of two
determined by the 1 H NMR method, which suggested that the two peaks in PSS were consistent with M and G monosaccharide
methods had less uronic acid loss. Although the microwave degra- standards. The linear ranges of M and G were evaluated using
dation method was rapid and there was a low loss of uronic acid, their monosaccharide standards at nine different concentrations
it showed the poorest repeatability (RSD = 3.78%, n = 3). Therefore, (0.125 mg/mL, 0.25 mg/mL, 0.50 mg/mL, 0.75 mg/mL, 1.0 mg/mL,
compared with the other three methods, dilute sulfuric acid hydrol- 1.25 mg/mL, 1.50 mg/mL, 2.0 mg/mL and 2.5 mg/mL). The lin-
ysis was the best method for the hydrolysis of PSS, with low uronic ear correlations between the peak area (Y, mAu × min) and the
acid loss and good repeatability (RSD = 0.57%, n = 3). concentration (X, mg/mL) of M or G were excellent, and the
26 J. Wu et al. / Carbohydrate Polymers 104 (2014) 23–28

1000 1000
(A) (B)
2
800 800
2
600 600

400 400

200 1 200
1
0 0

0 2 4 6 8 10 12 14 min 0 2 4 6 8 10 12 14 min

1000 1000
(C) (D)
2
800 2 800

600 600
1
1
400 400

200 200

0 0

0 2 4 6 8 10 12 14 min 0 2 4 6 8 10 12 14 min

Fig. 3. Chromatograms of PSS degraded by four different methods: (A) conventional acid hydrolysis method with 80% H2 SO4 at 20 ◦ C for 18 h and then 2 mol/L H2 SO4 at
100 ◦ C for 6 h; (B) 2 mol/L TFA hydrolysis hydrolyzed at 110 ◦ C for 6 h; (C) 0.1 mol/L sulfuric acid hydrolyzed at 120 ◦ C for 4 h; (D) microwave irradiation (1600 W) at 130 ◦ C
for 15 min (1-guluronic acid, G; 2-mannuronic acid, M).

calibration curves were Y = 248.94X−9.2524 (R2 = 0.9955, M) and The intra-day relative standard deviation (RSD) for three PSS
Y = 131.75X + 4.1182 (R2 = 0.9954, G). The detector responses were concentrations (4 mg/mL, 5 mg/mL and 6 mg/mL) were 0.79%, 0.49%
different for the two uronic acids, and the relative response of and 0.23% (n = 6), respectively, and the inter-day RSD for the three
G was 52.9% when M was 100%. The limit of detection (LOD) at PSS concentrations were 2.23%, 1.64% and 1.20% (n = 5), respec-
the signal–noise ratio of 3 was determined to be 0.25 ␮g/mL for tively, indicating that the method precision and reproducibility
M and 0.40 ␮g/mL for G by successive dilutions of PMP-labeled were satisfactory. The stability of the PSS derivative stored at room
monosaccharide standards. The limit of quantification (LOQ) at temperature was determined at 0 h, 1 h, 3 h, 5 h, 8 h and 24 h, and
the signal–noise ratio of 10 was found to be 1.0 ␮g/mL for M and the RSD was 1.75% (n = 6), indicating that the PMP derivative was
1.5 ␮g/mL for G. stable within 24 h.

Table 1
Results of L9 (34 ) orthogonal test of PSS hydrolyzed by dilute sulfuric acid method.

No. Temperature Reaction time Acid concentration Peak area of M and G

(factor A, ◦ C) (factor B, h) (factor C, mol/L) (mAu × min)

1 130 5 1 32.82
2 130 4 0.1 225.43
3 130 3 0.01 125.57
4 120 5 0.1 329.16
5 120 4 0.01 113.69
6 120 3 1 279.47
7 110 5 0.01 45.95
8 110 4 1 343.76
9 110 3 0.1 116.51
Ka 1 127.94 135.98 87.67
K2 240.77 227.63 223.70
K3 168.74 173.85 95.07
Rb 112.83 91.65 128.63
a
K: mean value.
b
R: range analysis.
 J. Wu et al. / Carbohydrate Polymers 104 (2014) 23–28 27

Table 2
Comparison of M/G ratios of PSS, PMS, PGS and their desulfurization products, with the intermediates determined by the 1 H NMR method and HPLC methods, respectivelya .
1
HPLC method H NMR method

Sample M/G ratio Intermediate M/G ratio Desulfurization sample M/G ratio

PSS 0.94 LMWA 0.98 DPSS 0.88

PMS 4.04 PM 4.01 DPMS 3.97
PGS 0.12 PG 0.11 DPGS 0.13
a
(LMWA, low-molecular-weight alginate; PM, polymannuronate; PG, polyguluronate; DPSS, desulfurization product of PSS; DPMS, desulfurization product of PMS; DPGS,
desulfurization product of PGS).

mAU M Table 3
1400 M/G ratios of PSS samples that were collected from different manufacturers in China.

1200 G Sample Peak area Peak area M/G ratio RSD (%)
(average, ratio (n = 3)
1000 mAu × min)

800 G M

1 76.49 181.91 2.38 1.26 1.62

600
2 68.90 164.98 2.39 1.26 0.83
3 72.71 165.44 2.28 1.21 1.60
400 (C) 4 128.18 224.39 1.75 0.93 1.81
200 (B) 5 127.01 240.08 1.89 1.00 1.34
(A) 6 132.81 238.29 1.79 0.95 1.10
0 7 112.84 233.91 2.07 1.10 0.74
8 124.35 204.55 1.65 0.87 1.54
0 5 10 15 20 25 min
9 106.58 227.59 2.14 1.13 0.63
10 89.94 174.61 1.94 1.03 0.53
Fig. 4. Chromatograms of ␣-L-guluronic acid (A), ␤-D-mannuronic acid (B) and
PSS(C).

ratios of two other alginate sulfated derivatives; i.e., polyman-

3.4. M/G ratios of PSS from different manufacturers nuronate sulfate (PMS) and polyguluronate sulfate (PGS), and their
desulfurization product, intermediates (i.e., polymannuronate and
To further demonstrate that the proposed HPLC method is opti- polyguluronate, respectively) were also detected by NMR and HPLC
mal, we employed a desulfurization strategy to overcome the methods, respectively, to demonstrate the feasibility of the desul-
influence of the sulfate group on the resolution of anomeric proton furization tactics. Results showed that the M/G ratios of PSS, PMS
signals. We determined the M/G ratio of PSS by the NMR tech- and PGS determined by the HPLC method were in good accordance
nique using its intermediate (low-molecular-weight alginate) and with those obtained by the traditional non-destructive 1 H NMR
desulfurization product (as shown in Fig. 5). In addition, the M/G method (as shown in Table 2) using their desulfurization products.

Fig. 5. 1 H NMR spectra of PSS (A), the intermediate (low-molecular-weight alginate) of PSS (B) and the desulfurization product of PSS (C) recorded at 20 ◦ C on a Bruker
Avance 500 MHz spectrometer (P1 , signal at 5.1 ppm, H1 -G; P2 , signal at 4.7 ppm, H1 -M and H5 -GM; P3 , signal at 4.5 ppm, H5 -GG).
28 J. Wu et al. / Carbohydrate Polymers 104 (2014) 23–28

This showed that the HPLC method was accurate in determining Burana-osot, J., Hosoyama, S., Nagamoto, Y., Suzuki, S., Linhardt, R. J., & Toida, T.
the M/G ratio of PSS. The M/G ratio results for PSS samples that (2009). Photolytic depolymerization of alginate. Carbohydrate Research, 344,
2023–2027.
were collected from different manufacturers in China are shown in Dai, J., Wu, Y., Chen, S. W., Zhu, S., Yin, H. P., Wang, M., et al. (2010). Sugar
Table 3. There were obvious differences in the M/G ratios for the ten compositional determination of polysaccharides from Dunaliella salina by mod-
PSS samples, which likely result from the differences in raw mate- ified RP-HPLC method of precolumn derivatization with 1-phenyl-3-methyl-5
-pyrazolone. Carbohydrate Polymers, 82, 629–635.
rial alginates. This means it is necessary to strengthen the quality Falshaw, R., & Furneaux, R. H. (1998). Structural analysis of carrageenans
control of PSS. from the tetrasporic stages of the red algae, Gigartina lanceata and
Gigartina chapmanii (Gigartinaceae, Rhodophyta). Carbohydrate Research, 307,
325–338.
4. Conclusion Fan, L. H., Jiang, L., Xu, Y. M., Zhou, Y., Shen, Y., Xie, W. G., et al. (2011). Synthesis
and anticoagulant activity of sodium alginate sulfates. Carbohydrate Polymers,
A rapid and accurate HPLC with pre-column derivatization 83, 1797–1803.
Gacesa, P., Squire, A., & Winterburn, P. J. (1983). The determination of the uronic
method was developed for determination of the M/G ratio of PSS.
acid composition of alginates by anion-exchange liquid chromatography. Car-
This method was validated by comparison with the non-destructive bohydrate Research, 118, 1–8.
1 H NMR method. The HPLC separation conditions of M and G deriva- Haug, A., & Larsen, B. (1962). Quantitative determination of the uronic acid compo-
sition of alginates. Acta Chemica Scandinavica, 16, 1908–1918.
tives were optimized and the pH value of the phosphate buffer and
Heyraud, A., Gey, C., Leonard, C., Rochas, C. L., Girond, S., & Kloareg, B. (1996). NMR
the proportion of acetonitriles obviously affected the resolution of spectroscopy analysis of oligoguluronates and oligomannuronates prepared by
the two derivatives. The dilute sulfuric acid (0.1 mol/L) degradation acid or enzymatic hydrolysis of homopolymeric blocks of alginic acid application
method is suitable for the hydrolysis of PSS with low uronic acid to the determination of the substratespecificity of Haliotis tuberculata alginate
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loss, has good repeatability and is simple to operate, while high Honda, S., Akao, E., Suzuki, S., Okuda, M., Kakehi, K., & Nakamura, J.
losses of M and G are found in commonly used 2 mol/L TFA hydrol- (1989). High-performance liquid chromatography of reducing carbohy-
ysis and traditional sulfuric acid degradation methods. There were drates as strongly ultraviolet-absorbing and electrochemically sensitive
1-phenyl-3methyl-5-pyrazolone derivatives. Analytical Biochemistry, 180,
significant differences in the M/G ratios of PSS samples that were 351–357.
collected from different manufacturers in China. It is necessary to Hu, T., Li, C. X., Zhao, X., Li, G. S., Yu, G. L., & Guan, H. S. (2013). Preparation and char-
strengthen the quality control of PSS to ensure its clinical efficacy acterization of guluronic acid oligosaccharides degraded by a rapid microwave
irradiation method. Carbohydrate Research, 373, 53–58.
and reduce the incidence of adverse drug reactions. Li, C. X., Su, Y., & Guan, H. S. (2012). Progress of marine drug propylene glycol algi-
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This research was supported by National Science & Technol- alginate on anticoagulant activities. European Polymer Journal, 43, 3009–3015.
ogy Support Program of China (2013BAB01B02),), the Special Fund Pawar, S. N., & Edgar, K. J. (2012). Alginate derivatization: a review of chemistry,
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for Marine Scientific Research in the Public Interest (201005024),
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Qingdao Science & Technology Project (11-2-2-1-hy) and Shandong Structure and properties of three alginates from Madagascar seacoast. Food
Science & Technology Project (2011GSF11815). Hydrocolloids, 32, 143–146.
Wang, J. X., Zhao, X., Yu, G. L., Li, G. S., & Hao, C. (2009). Analysis of uronic acid
compositions in marine brown alga polysaccharides by precolumn derivati-
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