柱前衍生化高效液相色谱法用于生地黄和熟地黄中多糖的分析研究  

Study on polysaccharide in Rehmanniae Radix and Rehmanniae Radix Praeparata by high performance liquid chromatography with pre-column derivatization

作　　者：李明华[1] 孙红梅 程显隆[1] 魏锋[1] 马双成[1] LI Ming-hua;SUN Hong-mei;CHENG Xian-long;WEI Feng;MA Shuang-cheng(National Institutes for Food and Drug Control,Beijing 102629,China;Infinitus(China)Co.,Ltd.,Jiangmen 529100,China)

机构地区：[1]中国食品药品检定研究院,北京102629 [2]无限极(中国)有限公司,江门529100

出　　处：《药物分析杂志》2023年第4期602-611,共10页Chinese Journal of Pharmaceutical Analysis

摘　　要：目的:测定生地黄和熟地黄中多糖的单糖组成及含量,分析生地黄和熟地黄之间的差异。方法:采用三氟乙酸对提取的生地黄和熟地黄中的多糖进行酸水解,用1-苯基-3-甲基-5-吡唑啉酮(PMP)对酸解后的单糖进行衍生化。采用Waters Symmetry-C_(18)(250 mm×4.6 mm,5μm)色谱柱,以0.05 mol·L^(-1)磷酸盐缓冲液(pH 6.8)为流动相A,乙腈为流动相B,梯度洗脱,流速1.0 mL·min^(-1),检测波长250 nm,柱温30℃。采用主成分分析和方差分析,对地黄多糖水解后各单糖含量及其比值进行分析。结果:半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖进样质量依次在0.02~1.82、0.10~3.91、0.09~7.08和0.03~1.69μg范围内与峰面积呈良好的线性关系(r>0.9993),回收率为97.6%~103.7%,重复性RSD(n=6)为3.2%~4.7%,中间精密度均符合要求。生地黄和熟地黄多糖水解后均含有上述4个单糖,二者差异不明显,但生地黄中葡萄糖含量/半乳糖含量比值>0.4,熟地黄中此比值基本<0.4。结论:本方法重复性好,准确度高,可用于生地黄和熟地黄样品中多糖水解产物单糖的含量测定;生地黄和熟地黄之间葡萄糖含量/半乳糖含量比值存在一定程度差异。Objective:To determine the monosaccharide composition and contents of polysaccharides in Rehmanniae Radix and Rehmanniae Radix Praeparata,and analyze the differences between them.Methods:The extracted polysaccharide were hydrolyzed by trifluoroacetic acid,and the monosaccharides were derivatized by 1-phenyl-3-methyl-5-pyrazolone(PMP).Waters Symmetry-C_(18)column(250 mm×4.6 mm,5μm)was adopted,with gradient elution of 0.05 mol·L^(-1)phosphate buffer(pH 6.8,mobile phase A)and acetonitrile(mobile phase B).The flow rate was 1.0 mL·min^(-1),the detection wavelength was 250 nm,and the column temperature was 30℃.Principal component analysis and variance analysis were used to analyze the contents and ratios of monosaccharides after hydrolysis of Rehmanniae Radix and Rehmanniae Radix Praeparata.Results:Weights of galacturonic acid,glucose,galactose and arabinose showed a good linear relationship with the peak areas in the range of 0.02-1.82、0.10-3.91、0.09-7.08 and 0.03-1.69μg,respectively(r>0.9993).The recovery rates were from 97.6%to 103.7%.The RSDs(n=6)for repeatability tests were from 3.2%to 4.7%.The intermediate precision met the requirements.After hydrolysis,the polysaccharides in Rehmanniae Radix and Rehmanniae Radix Praeparata both contained the four monosaccharides above with no significant difference between them.But the ratio of glucose content to galactose content in Rehmanniae Radix was greater than 0.4,and that in Rehmanniae Radix Praeparata was basically less than 0.4.Conclusion:The method is reproducible and accurate,and suitable for the determination of monosaccharides which are the hydrolysates of polysaccharides in Rehmanniae Radix and Rehmanniae Radix Praeparata.There are some differences between Rehmanniae Radix and Rehmanniae Radix Praeparata on the ratio of glucose content to galactose content.

关 键 词：生地黄 熟地黄 半乳糖醛酸 葡萄糖 半乳糖 阿拉伯糖 柱前衍生化高效液相色谱法 

分 类 号：R917[医药卫生—药物分析学]