LC-MS/MS法检测猪内脏和肌肉中N-乙酰神经氨酸、N-羟乙酰氨酸和脱氨神经氨酸浓度的研究  

Determination for the Contents of Neu5Ac, Neu5Gc and KDN in Organ Meats and Muscle of Pigs by LC-MS/MS

作　　者：吉素娜 王芳[1] 张盼望 陈越[1] 杨长伟[1] 王冰[1] 

机构地区：[1]厦门大学医学院基础医学系神经营养研究室,福建厦门361102

出　　处：《现代生物医学进展》2017年第4期610-614,623,共6页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(31271038)

摘　　要：目的:建立液相色谱-三重四级杆质谱联用法(LC-MS/MS)测定猪内脏和肌肉组织中唾液酸N-乙酰神经氨酸(Neu5Ac)、N-羟乙酰神经氨酸(Neu5Gc)和脱氨神经氨酸(KDN)浓度的分析方法。方法:将内脏肉组织匀浆后,利用三氟乙酸(TFA)将结合态的唾液酸从糖脂或糖蛋白链上解离下来后进样分析。色谱条件:色谱柱为ZORBAX Eclipse Plus C18(5μm,4.6×250 mm,Waters),流动相为0.1%醋酸铵-100%乙腈(9:1),柱温:25℃,流速800μL·min^(-1),进样量20μL。质谱条件:离子源为电喷雾化离子源(ESI),扫描方式为多重反应监测(MRM),监测离子对:Neu5Ac:307.7→87.0,Neu5Gc:323.3→116.0,KDN:266.7→87.0,13C3Neu5Ac内标品:310.9→90.0。结果:Neu5Ac、Neu5Gc和KDN分别在0.1~20.0、0.05~10.00和0.005~1.000μmol·L^(-1)的浓度范围内和对照品与内标峰面积比值线性关系良好,重复性平均RSD为1.2%,稳定性平均RSD为1.9%,日间和日内精密度试验RSD均小于6.7%,平均回收率为92.9~106.4%。结论:本方法简便、快速、灵敏度高,可广泛运用于组织和体液中唾液酸的测定。Objective： To establish an LC-MS/MS method for the detection of N-acetylneuraminic acid（Neu5Ac）,N-glycolylneuraminic acid（Neu5Gc） and 2-keto-3-deoxy-D-glycero-D-galactonononic acid（KDN） in organ meats and muscle of pigs.Methods： Tissues were acid hydrolyzed with trifluoroacetic acid（TFA） to get ganglioside-bound and glycoprotein-bound sialic acid,followed by injected and analyzed. The analysis was performed on an ZORBAX Eclipse Plus C18（5 μm, 4.6 ×250 mm, Waters）column, and the mobile phase consisted of 0.1 % ammonium^-100 % acetonitrile（9：1）, the flow rate was 800 μL/min, the temperature was maintained at 25 ℃, injection volume was set up 20 μL. Negative electrospray ionization（ESI） and multiple reaction monitoring（MRM） mode was used, MRM： Neu5Ac： 307.7→87.0, Neu5Gc： 323.3 →116.0, KDN： 266.7→87.0, 13C3Neu5Ac（IS）： 310.9→90.0.Results： The ratio of internal with standard sample peak area and content of Neu5 Ac, Neu5 Gc and KDN showed good linearity in the range of 0.1~20.0, 0.05~10.00, and 0.005~1.000 μmol/L, RSDs of the repeatability and stability were 1.2 % and 1.9 %, Intra-and inter-day RSDs were all lower than 6.7%, the recoveries of sialic acid were 92.9~106.4%. Conclusion： The established method is simple,accurate and rapid, higher sensitivity and can be applied to determination of Neu5 Gc, Neu5 Ac and KDN in tissues and fluid of animals and human.

关 键 词：液相色谱-三重四级杆质谱联用 N-羟乙酰神经氨酸 N-乙酰神经氨酸 脱氨神经氨酸 猪内脏组织 

分 类 号：R-33[医药卫生] R446