柱前衍生化UPLC-MS/MS测定12种单糖含量的方法学研究及其应用
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摘要
建立了1-苯基-3-甲基-5-吡唑啉酮(PMP)柱前衍生化UPLC-MS/MS检测12种单糖的方法,同时对蜜环菌中多糖的单糖组成成分进行了分析。安捷伦ZORBAX RRHD Eclipse Plus C18分析柱(2. 1 mm×100 mm,1. 8μm),95%乙腈(A)和乙酸铵-5%乙腈-水(B)为流动相梯度洗脱,质谱为电喷雾离子源(ESI)接口,采用负离子多反应监测模式(MRM)进行检测。结果表明,基于UPLC-MS/MS所建立的单糖检测方法,12种单糖成分在各自线性范围内线性关系良好(R2> 0. 990),加样回收率为92. 30%～105. 6%。在蜜环菌样品中,可检测出除阿洛糖外的11种单糖组分。该试验所建立的方法稳健,重复性和准确度高,适用于蜜环菌等的单糖成分的含量测定。
This study is to establish a pre-column derivatization procedure with 1-phenyl-3-methyl-5-pyrazolone(PMP) UPLC-MS/MS method for the determination of the monosaccharide composition of 12 polysaccharides. At the same time,the monosaccharide components of polysaccharides in Armillaria gallica were analyzed. The separation was performed on a ACQUITY ZORBAX RRHD Eclipse Plus C18 column(2. 1 mm × 100 mm,1. 8 μm),using 95% acetonitrile(A) and ammonium acetate-5% acetonitrile-water(B) as mobile phase with gradient elution. The target components were detected in multiple-reaction monitoring(MRM) mode by mass spectrometry with electrospray ionization(ESI) source operated in ionization mode. The results showed that based on the monosaccharides detection method established by UPLC-MS/MS,the linearity of the 12 monosaccharides components were linear in their linear range(R2> 0. 990),and the recovery rate were 92. 30%-105. 6%. 11 monosaccharides such as fructose,mannose,and glucose were detected in A. gallica samples. The method established in this experiment is robust,highly reproducible and accurate,and is suitable for the determination of monosaccharide components such as A. gallica.
This study is to establish a pre-column derivatization procedure with 1-phenyl-3-methyl-5-pyrazolone(PMP) UPLC-MS/MS method for the determination of the monosaccharide composition of 12 polysaccharides. At the same time,the monosaccharide components of polysaccharides in Armillaria gallica were analyzed. The separation was performed on a ACQUITY ZORBAX RRHD Eclipse Plus C18 column(2. 1 mm × 100 mm,1. 8 μm),using 95% acetonitrile(A) and ammonium acetate-5% acetonitrile-water(B) as mobile phase with gradient elution. The target components were detected in multiple-reaction monitoring(MRM) mode by mass spectrometry with electrospray ionization(ESI) source operated in ionization mode. The results showed that based on the monosaccharides detection method established by UPLC-MS/MS,the linearity of the 12 monosaccharides components were linear in their linear range(R2> 0. 990),and the recovery rate were 92. 30%-105. 6%. 11 monosaccharides such as fructose,mannose,and glucose were detected in A. gallica samples. The method established in this experiment is robust,highly reproducible and accurate,and is suitable for the determination of monosaccharide components such as A. gallica.
引文
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[2] Barile D,Rastall R A. Human milk and related oligosaccharides as prebiotics[J]. Curr Opin Biotechnol,2013,24(2):214.
[3] Musilova S,Rada V,Vlkova E,et al. Beneficial effects of human milk oligosaccharides on gut microbiota[J]. Benefic Microbes,2014,5(3):273.
[4] Smilowitz J T,Lebrilla C B,Mills D A,et al. Breast milk oligosaccharides:structure-function relationships in the neonate[J].Annu Rev Nutr,2014,34(34):143.
[5] Qian Z G. Cellulase-assisted extraction of polysaccharides from Cucurbita moschata and their antibacterial activity[J]. Carbohyd Poly,2014,101:432.
[6] Ji Y B,Ji C F,Yue L. Human gastric cancer cell line SGC-7901apoptosis induced by SFPS-B2 via a mitochondrial-mediated pathway[J]. Bio Med Mater Eng,2014,24(1):1141.
[7] Li C,Tian Z N,Cai J P. et al. Panax ginseng polysaccharide induces apoptosis by targeting Twist/AKR1C2/NF-1 pathway inhuman gastric cancer[J]. Carbohyd Poly,2014,102:103.
[8]徐灿,刁嘉茵,王淑美,等.银耳多糖的药理研究进展[J].今日药学,2018,28(3):207.
[9]邓来明,肖正华,陈定宇.黄芪多糖对糖尿病足溃疡成纤维细胞AGEs及RAGEmRNA表达的影响[J].今日药学,2014,24(5):313.
[10]柴银,王虹,刘雅萌,等.离子色谱测定多糖的单糖组成的方法学研究及其应用[J].药物生物技术,2016(4):332.
[11] Ma J,Adler L,Srzednicki G,et al. Quantitative determination of non-starch polysaccharides in foods using gas chromatography with flame ionization detection[J]. Food Chem,2017,220:100.
[12] Medeiros P M,Simoneit B R. Analysis of sugars in environmental samples by gas chromatography-mass spectrometry[J]. J Chromatogr A,2007,1141(2):271.
[13] Zhang Z,Khan N M,Nunez K M,et al. Complete monosaccharide analysis by high-performance anion-exchange chromatography with pulsed amperometric detection[J]. Anal Chem,2012,84(9):4104.
[14] Li H,Long C,Zhou J,et al. Rapid analysis of mono-saccharides and oligo-saccharides in hydrolysates of lignocellulosic biomass by HPLC[J]. Biotechnol Lett,2013,35(9):1405.
[15] Honda S,Suzuki S,Taga A. Analysis of carbohydrates as 1-phenyl-3-methyl-5-pyrazolone derivatives by capillary/microchip electrophoresis and capillary electro chromatography[J]. J Pharm Biomed Anal,2003,30(6):1689.
[16]徐锦堂.中国天麻栽培学[M].北京:北京医科大学,中国协和医科大学联合出版社,1993.
[17]顾雅君,张明艳,唐宏亮,等.蜜环菌和天麻、猪苓的关系及尚未大量人工驯化原因[J].中国食用菌,2008,27(6):20.
[18]陈明义,李福后,边银丙.蜜环菌不同菌株对天麻产量的影响[J].食用菌学报,2004,11(1):46.
[19]董燕婧,程访,许小珍,等.白及内生真菌的分离鉴定及其胞外多糖的抗氧化活性分析[J].中国实验方剂学杂志,2018,24(14):24.
[20]胡蓉蓉,姚鑫. UPLC-MS/MS测定银杏叶提取物中10个黄酮类成分的含量[J].中国实验方剂学杂志,2017,23(24):90.
[21]王笑笑,周勇,徐国群,等.黄酮类成分在蜂蜜抗菌性中的效能研究[J].中国现代应用药学,2017,34(3):363.