怀牛膝多糖的柱前衍生化-HPLC指纹图谱建立及单糖成分含量测定

目的:建立怀牛膝多糖的柱前衍生化-高效液相色谱(HPLC)指纹图谱,测定其主要单糖成分的含量,为怀牛膝饮片的质量评价提供参考.方法:以10批不同生产厂家的怀牛膝饮片为样品,采用水提醇沉法提取其多糖后,以三氟乙酸进行水解,再采用1-苯基-3-甲基-5-吡唑啉酮衍生化处理后,进行HPLC分析.色谱柱为Hanbon Hede...

Establishment of Pre-column Derivatization-HPLC Fingerprint and Content Determination of Monosaccharide Composition from Achyranthes bidentata Polysaccharides

OBJECTIVE:To establish pre-column derivatization-HPLC fingerprint of polysaccharide from Achyranthes bidentata,and to determine the contents of monosaccharide components,so as to provide reference for quality evaluation of A.bidentata decoction pieces.METHODS:Taking 10 batches of A.bidentata decoction pieces from different manufacturers as samples,the polysaccharides were extracted by water extraction and alcohol precipitation,hydrolyzed by trifluoroacetic acid,and then derivatized by 1-phenyl-3-methyl-5-pyrazolone for HPLC analysis.The determination was performed on Hanbon Hedera C18 column with column temperature of 30℃at the flow rate of 1.2 mL/min.The mobile phase consisted of acetonitrile-0.02 mol/L ammonium acetate solution(gradient elution).The detection wavelength was set at 250 nm,and sample size was 20μL.HPLC fingerprint was established and similarity evaluation was performed for 10 batches of A.bidentata polysaccharide by using TCM Chromatogramic Fingerprint Similarity Evaluation System(2012A edition).The common peak was identified by comparing with the reference substance,and cluster analysis was performed by using SPSS 25.0 software.The contents of identified monosaccharides were determined by HPLC.RESULTS:The similarity of 10 batches of A.bidentata polysaccharide were all higher than 0.95.Nine common peaks were fixed and a total of 5 common peaks were identified,namely anhydrous glucose(peak 1),mannose(peak 2),rhamnose(peak 4),galacturonic acid(peak 5)and arabinose(peak 7).Results of cluster analysis showed that S1 sample was classified into one category;S2,S5,S8 and S9 samples were clustered into one category;S3,S4,S6,S7 and S10 samples were clustered into one category.Results of content determination showed that the contents of anhydrous glucose in 10 batches of samples ranged from 6.17 to 17.55 mg/g;those of mannose ranged from 3.31 to 7.66 mg/g;those of rhamnose ranged from 38.80 to 73.97 mg/g;those of galacturonic acid ranged from 2.49 to 8.95 mg/g;those of arabinose ranged from 11.30 to 28.58 mg/g.CONCLUSIONS:Established pre-column derivatization HPLC fingerprints and content determination method can provide reference for quality evaluation of A.bidentata decoction pieces.