Zhou Jianke，Han Kang，Zhang Li，Shi Zhihong
(College of Chemistry and Environmental Science, Hebei University; Key Laboratory of Analytical Science and Technology of Hebei Province, Baoding, 071002, China)
AbstractIn this paper, a method for the determination of monosaccharide composition in coking malt by HPLC was established. The derivatization of monosaccharide with 1-phenyl-3-methyl-5-pyrazolone (PMP) was simplified. Four monosaccharide derivatives have been well separated by using C18 column. The mobile phase consisted of acetonitrile-ammonium acetate buffer (pH5.5) (71.5:28.5, v/v) and the detection wavelength was set at 250nm. The linear ranges of mannose, rhamnose, glucose and xylose were 0.10 mmol/L-0.001 mmol/L and correlation coefficients ranged from 0.9995-0.9999. The RSD values were all below 3.5% and the average recoveries ranged from 84.2%-108.8%.
As early as in 1994, acrylamide was listed as a “potential human carcinogen” by the International Agency for Research on Cancer (IARC). From then on, acrylamide attracted the extensive concerns from the scientists all over the world. So far, the formation mechanism of acrylamide in food is not very clear. But it is generally believed that acrylamide is produced primarily at high-temperature through the Maillard reaction in food which is rich in starch . Coking malt, a kind of commonly used Chinese herb, is rich in starch and is processed from malt at high temperature. Trace acrylamide has been detected in it by our research group. The relationship between acrylamide and monosaccharide composition need to be further studied. This paper described the determination of the monosaccharide composition in processed Chinese herb for the first time.
A considerable amount of researches have already been carried out in recent years for the determination of . monosaccharides. Since monosaccharides encompass a number of homologues having very similar structures and many of them exist concurrently in the samples, monosaccharide analysis inevitably requires high-resolution separation techniques. However, these compounds generally have low intrinsic UV spectral activity. Therefore the derivatization of monosaccharide is indispensable to obtain highly sensitive detection . The reagent 1-phenyl-3-methyl-5-pyrazolone (PMP) is one of the popularderivatising agent that can react with reducingmonosaccharide under mild conditions, requiring no acid catalyst and causing no desialylation and isomerization. PMP yields strong UV absorbance at 245 nm. This derivatization method was first developed for the analysis of monosaccharide by Honda . PMP derivatization increases the hydrophobicity of monosaccharide, therefore HPLC is quite suitable for analyzing PMP derivatives . In this paper, on the basis of previous research work, the RP-HPLC analysis method is established to detect monosaccharide composition in coking maltbyimplified sample processing and derivative method.
2.1. Apparatus and reagents
The HPLC system consisted of LC-10AT solvent delivery pump, 7725i injector and SPD-10A UV-VIS detector from Shimadzu Corporation (Japan).The N-2000 Double-Channel Chromatograph Data Operation Station and Software were purchased from Intelligent Information Engineering Research Institute of Zhejiang University. The UV-265 spectrophotometer was from Shimadzu Corporation (Japan). XW-80A Vortex was from Instrumental Factory of Shanghai Medical University.
PMP was purchased fromBeijing Purchasing and Supplying Station of Chinese Pharmaceutical Companies (China). Trifluoroacetic acid (TFA) was obtained from Kermel (Tianjin, China). HPLC grade solvents such as methanol and acetonitrile were used to prepare the mobile phase. Trichloromethane, Ammonia water ammonium acetate were analytical grade reagent. D-xylose (Xyl) and L-rhamnose (Rham) were purchased from Beijing Chemical Reagent Company (Beijing, China), D-glucose (Glu) from Kermel (Tianjin, China), D-mannose (Man) from Shanghai Chemical Reagent Company (Shanghai, China). These monosaccharide reagents were all of analytical grade.
2.2 Chromatographic conditions
Separation was performed using Diamonsil C18 (250*4.6mm i.d., 5m) column. The temperature of the column was kept at room temperature. The optimized mobile phase was acetonitrile-100mmol/L ammonium acetate buffer (pH 5.5) (21.5:78.5, v/v). The detection was carried out at the wavelength of 250 nm. The flow rate was 1.0ml/min and the injection volume was 5.0l.
2.3 Hydrolysis of polysaccharide
Coking malt was grinded and 2.0 g of the sample was accurately weighed and placed in a small beaker, then 20mL of twice-distilled water was added. After boiling for 5 min, the solution was ultrasonic extracted for 10 min, and then stood for 10 min. Supernatant was filtered through filter paper. The leftover was washed for three times. Then supernatant was merged and diluted to 20 ml with water. The solution was finally filtered through 0.45m membrane. 100L sample solution was mixed with 4mol/L trifluoroacetic acid (100L) in frosted cone pipe with stopper (2ml). It was sealed under a nitrogen atmosphere and kept at 110 ºC for 2h in a temperature controlled aluminum heater .
2.4 Preparation of PMP derivatives
2.4.1 Derivatives of standard monosaccharide mixture
Man (0.0180g), Glu (0.0180g), Rham (0.0182g) and Xyl (0.0150g) were all dissolved in ammonia water(5ml) . A 100L of this solution was mixed with a 0.5mol/L methanol solution of PMP (100μl), and the mixture was allowed to react at 70 ºCfor 30 min. After cooling to room temperature, the reaction mixture was dried by a nitrogen stream at 100 ºCfor discarding ammonia. The residue was dissolved in distilled water and trichloromethane (1ml each). The organic phase was discarded after vigorous shake and centrifugation (3000 r/min) to remove the excess reagents. This extraction process was repeated three times, and then the aqueous layer was diluted with 20 times water before HPLC analysis.
2.4.2 Derivatives of hydrolysate of polysaccharide and analysis
The product of hydrolytic reaction mentioned in 2.3 was dried by a nitrogen stream at 100 ºC. The residue was then dissolved with 100μL ammonia liquor and 0.5mol/L methanol solution of PMP (100l). Then the mixture was treated using the method mentioned in 2.4.1. Finally, aqueous layer was diluted 20 times for HPLC analysis.
3. RESULTS AND DISCUSSION
3.1 Hydrolysis and derivatization of polysaccharide
Hydrolysis reaction was often performed in an ampoule in other references . In our method, the frosted cone tube was sealed with sealing membrane under a nitrogen atmosphere, so the troublesome step of sealing with alcohol spray torch was omitted. Meanwhile, experimental cost was lowered. In the literature , aqueous HCl was needed to neutralize aqueous NaOH after derivative reactions. While in our experiment, using ammonia liquor instead, the excess ammonia was simply removed under nitrogen stream which avoided the neutralization procedure and the drying step under vacuum 
3.2 Selection of mobile phase
Derivatives of monosaccharides could not be well separated using the mixture of acetonitrile-water as mobile phase. The optimized mobile phase was acetonitrile-100mmol/L ammonium acetate buffer. The volume of acetonitrile influenced the separation greatly when the concentration of acetonitrile increased from 20% to 22%. Considering both the separation and retention time, the volume of acetonitrile was chosen to be 21.5% for further study.
3.3 Linear equations, correlation coefficients and detection limits
A series of working solutions were prepared by diluting the mixed standard solution containing four kinds of monosaccharide (0.02mmol/mL each). They were treated using the method mentioned in 2.4.1 and the final solution was detected under the optimized chromatographic conditions. The calibration graphs of the standard compounds were found to be linear over the concentration range studied. The linear equations and the detection limits of the derivatives are listed in Table 1.
Table 1 Linearity and the detection limits of PMP derivatives of four kinds of monosaccharide
Y-peak area; X-the concentration of the detected compound
3.4Recoveries and precision
The recoveries of the method were studied. The standard solutions of different concentrations were added into coking malt sample solutions. Then the spiked sample solutions were treated with the same method as described in 2.3 and 2.4. The concentrations of Man, Rham and Xyl were examined under the optimized chromatographic conditions and the recoveries could be calculated according to the linear equation. The precision of the method was determined by preparing the sample with the same method as described in 2.3 and 2.4 for each analysis, and RSD values were determined by 5 repeated analyses. As for Glu, the coking malt sample solution was diluted 100 times to carry on the similar operation again. The results are given in Table 2.
Table 2Recoveries and precision
3.5 Analysis of coking malt samples
Coking malt samples were analyzed using the established method, and the chromatograms are shown in Figure 1.The use of merely monosaccharide standard compounds which the laboratory have had, four monosaccharide were identified by the retained value qualitative method, they were Man, Rha, Glu and Xyl. The molar ratio of Man, Rha, Glu and Xyl in coking malt was 1.82:1.00:209.44:2.67 by HPLC analysis and molar ratio calculation. The RSD values of four kinds of monosaccharide were all lower than 3.5%. The repeatability of detected results was satisfactory.
Figure 1Chromatograms of spiked coking malt sample (A) and coking malt sample (B)
1-Mannose; 2-Rhamnose; 3-Glucose; 4-Xylose
A simple, rapid and reproducible method for the derivatization of monosaccharide by PMP was established, which avoided the procedures of the drying of hydrolysates and derivative solution. The experimental results demonstrated that the developed method was suitable for the analysis of monosaccharides by HPLC.
ACKNOWLEDGEMENTThis work was supported by the National Natural Science Foundation of China (No. 20575016).