柱前衍生高效液相色谱-质谱法测定血浆中的氨基葡萄糖浓度  

Determination of Glucosamine in Human Plasma by HPLC-ESI-MS with Pre-Column Derivatization

作　　者：陈继涛[1] 刘国柱[1] 陈波[1] 

机构地区：[1]化学生物学及中药分析教育部重点实验室湖南师范大学化学化工学院,中国长沙410081

出　　处：《湖南师范大学自然科学学报》2010年第2期77-82,共6页Journal of Natural Science of Hunan Normal University

基　　金：国家自然科学基金资助项目(20875028);国家"973"资助项目(2006CB504701);湖南省科技计划资助项目(2008FJ3063)

摘　　要：采用柱前衍生高效液相色谱-电喷雾质谱法（HPLC-ESI-MS）测定了人血浆中的氨基葡萄糖浓度.该方法以氨基半乳糖为内标,用丙酮沉淀蛋白后,加入5μL三乙胺和10μL异硫氰酸苯脂后,在60℃的恒温水浴中反应1 h,用氮气吹干、流动相溶解后于离心机上以12 000 r/min离心5 min后进样20μL分析;以甲醇与水作流动相,经过Ultimate-XB C18柱（4.6 mm×250 mm,5μm,Welch Materials）在梯度模式下分离后1∶4分流,以0.2 mL/min的流速进质谱分析.实验结果表明：氨基葡萄糖的回归方程为Y=6.70×10-4X＋1.11×10-2（r2=0.996）,在0.10-5.00μg/mL范围内线性关系良好;最低定量限为0.10 mg/L;氨基葡萄糖和内标的萃取回收率分别为88.3%-92.1%和85.2%;日内、日间精密度的RSD值分别为〈6.0%、〈5.0%,稳定性的RSD〈7.5%.所建立的方法准确度好、灵敏度高、稳定性好,适合于血浆中的氨基葡萄糖的含量测定.A high performance liquid chromatography-electrospray ionization mass spectrometry（HPLC-ESI-MS） method with pre-column derivatization was established and validated for the determination of glucosamine in human plasma.In this paper D（＋）-Galactosamine was selected as internal standard（I.S.） for the quantification of target compound.Plasma sample were deproteinized by acetone,and the supernatant was transferred into a clean polyethylene tube,then 5 μL triethylamine and 10 μL phenylisothiocyanate was added,and allow them to react for 1 h in the constant temperature water bath about 60 ℃.After derivatization,the sample solution was dried by nitrogen,dissolved with mobile phase and centrifuged at 12 000 r/min for 5 min.The supernatant was transferred into separate vials;sequentially 20 μL was injected onto the column for analysis.The chromatographic separation was performed on an Ultimate-XB C18 column（4.6 mm×250 mm,5 μm,Welch Materials） with a mobile phase containing methanol and water eluting on the gradient mode.The outlet of column was splited by the ratio of 1∶4 and only 0.2 mL/min was delivered into the MS.The results showed equation of linear regression was Y=6.70×10-4X＋1.11×10-2（r2=0.996）which showed a good linearity in the range of 0.10~5.00 mg/L.The limit of quantification（LOQ） was 0.10 μg/mL.Extraction recoveries of glucosamine and I.S in plasma were 88.3%~92.1% and 85.2%,respectively.The coefficient variations were less than 6.0%,5.0%,and 7.5% for the precision of intra-day,inter-day and stability,respectively.The method with good accuracy,high sensitivity and good stability was applied to the determination of glucosamine in human plasma.

关 键 词：高效液相色谱-电喷雾质谱法 氨基葡萄糖 血浆 柱前衍生 

分 类 号：O621[理学—有机化学]