UPLC-MS/MS法测定何首乌及其混伪品中多糖的单糖组成

Determination of monosaccharide composition of polysaccharides in Polygonum officinale and its adulterants by UPLC⁃MS/MS

目的:建立测定何首乌多糖的单糖组成超高效液相色谱串联四极杆质谱(UPLC-MS/MS)分析方法,并用以区分何首乌生品、制品、夜交藤(同一植物的不同部位)及市场常见混伪品(黄药子、红药子、白首乌)。方法:采用水提醇沉法获得样品中多糖成分,以三氟乙酸水解多糖,水解产物加入1-苯基-3-甲基-5-吡唑啉酮(PMP)进行衍生化。以Waters ACQUITY UPLC BEH C18柱(100 mm×2.1 mm,1.7μm)为分析柱;乙腈和缓冲盐溶液(1 mmol·L^(-1)乙酸铵-0.175%乙酸)为流动相进行梯度洗脱分离;流速0.2 mL·min^(-1);柱温35℃;进样量2μL。电喷雾正离子多反应监测(MRM)模式检测,采用内标法进行定量。结果:何首乌生品中的多糖成分由甘露糖、核糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖、木糖、岩藻糖等10种单糖组成,其含量百分比分别为0.38%,0.08%,0.99%,0.70%,8.56%,84.55%,2.11%,2.45%,0.11%,0.08%。制何首乌多糖的单糖组成与生品一致,但单糖含量存在一定差异,主要体现在葡萄糖和半乳糖醛酸上。夜交藤中多糖成分的单糖组成种类与生品不同。常见3种混伪品与生品的多糖成分也存在较大差异,主要表现在单糖组成含量不同,生品多糖中的葡萄糖含量高于3种混伪品,半乳糖含量低于3种混伪品。结论:建立了PMP柱前衍生-UPLC-MS/MS法对何首乌多糖成分的单糖组成进行分析,并用以何首乌生品与炮制品、夜交藤、常见混伪品的鉴别分析,该方法灵敏度高、分析时间短,可为何首乌质控方法的完善及常见混伪品的区分提供参考。

Objective:To establish an ultra⁃performance liquid chromatography⁃tandem quadrupole mass spectrometry(UPLC⁃MS/MS)method for the determination of the monosaccharide composition of polysaccharides from Polygonum multiflorum Thunb.,which can be used to reveal the differences between its raw herb,processing herb,tuber fleeceflower stem(different parts)and mixed forgeries in the market,i.e.,Airpotato Yam Rhizome,Ciliatenerve Knotweed Root,and Bunge Auriculate Root.Methods:The polysaccharides were obtained by hydrolysis using trifluoroacetic acid and the hydrolysis product was derivatized by adding 1⁃phenyl⁃3⁃methyl⁃5⁃pyrazolinone(PMP).The separation was carried out on a Waters ACQUITY UPLC BEH C18 analytical column(100 mm×2.1 mm,1.7μm)with acetonitrile and buffered salt solution(1 mmol·L^(-1) ammonium acetate⁃0.175%acetic acid)as the mobile phase using a gradient elution.The separation was carried out at a flow rate of 0.2 mL·min^(-1).The column temperature was set at 35℃and the injection volume was 2μL.The determination was carried out in multiple reaction monitoring(MRM)in electrospray positive ionization mode and quantified by internal standard method.Results:The polysaccharide composition of raw herb consisted of 10 monosaccharides:mannose,ribose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,arabinose,xylose and fucose,with the content percentages of 0.38%,0.08%,0.99%,0.70%,8.56%,84.55%,2.11%,2.45%,0.11%and 0.08%.The monosaccharide composition of the processing herb is the same as that of the raw herb,but differences exist in the monosaccharide content,mainly in glucose and galacturonic acid.The types of monosaccharide composition in tuber fleeceflower stem are different from those of the raw herb.There are also major differences between the three mixes and the raw herb,mainly in the composition of the monosaccharides.The glucose content of the raw herb was higher than that of the three mixes,while the lower level of galactose.Conclusion:A PMP pre⁃column derivatization⁃UPLC⁃MS/MS method was developed for the analysis of the monosaccharide composition of polysaccharides from Polygonum multiflorum Thunb..The method was also used for the identification and analysis of its raw herb and processing herb,tuber fleeceflower stem,and mixed forgeries.The method is of high sensitivity and short analysis time,which can provide a reference for the improvement of its quality control and the differentiation of common mixed forgeries.