LC-MS/MS方法定量测定人血浆中亮氨酸浓度及^(13)C-亮氨酸丰度  

Quantitative determination of leucine concentration and the abundance of ^(13)C-leucine in human plasma by LC-MS/MS

作　　者：杨芬[1] 王洪允[1] 胡蓓[1] 江骥[1] 

机构地区：[1]中国医学科学院北京协和医院,北京100730

出　　处：《药物分析杂志》2013年第8期1322-1325,1330,共5页Chinese Journal of Pharmaceutical Analysis

基　　金："重大新药创制"科技重大专项基金(编号:2008ZX09312-016)

摘　　要：目的:为了考察进食状态下不同时间点氨基酸的氧化分解状态,从而了解机体对氨基酸的需要量,建立了一种灵敏、特异、高效的同时检测血浆中亮氨酸浓度及13C亮氨酸丰度的LC-MS/MS定量分析方法。方法:用乙腈沉淀蛋白法处理血浆样本。采用Atlantics色谱柱(4.6 mm×100 mm,3.5μm);流动相为乙腈-水(0.1%甲酸)(5∶95),流速为1 mL.min-1,进样量为20μL。采用串联四极杆质谱在ESI正离子电离模式下,应用多反应监测(MRM)扫描模式测定亮氨酸(m/z 132.1→86.0)和13C-亮氨酸(m/z 133.1→86.0)。样品分析时间为3 min。结果:血浆中亮氨酸的浓度在50～1000 nmol.mL-1范围内,13C-亮氨酸/亮氨酸的摩尔浓度比值在0.01～0.5范围内线性关系良好(r>0.99),日内、日间精密度和准确度符合要求。稳定性考察项目的结果均符合要求。结论:本文建立了同时测定血浆中亮氨酸浓度和13C-亮氨酸丰度的LC-MS/MS方法。该方法灵敏度高,特异性好,各项方法学考核的结果均符合要求,可用于考察氨基酸氧化分解状态的临床研究。Objective： To evaluate the catabolism level of amino acid and investigate the amino acid requirement of the body,a specific,sensitive and efficient LC-MS / MS method will be developed for the simultaneous determination of leucine concentration and 13C-leucine enrichment in human plasma.Method： The plasma samples were extracted with acetonitrile.The HPLC separation was performed on an Atlantics（4.6 mm × 100 mm,3.5 μm） column using acetonitrile-water（0.1% formic acid） 5∶ 95 as mobile phase with the isocratic flow rate at 1 mL.min-1.Detection was performed on a tandem quadrapole mass spectrometer using positive electrospray ionization;multiple reactions monitoring（MRM） scan mode was applied to detect mass transitions of leucine（m / z： 132.1→86.0） and 13C-leucine（m / z： 133.1→86.0）.Each analytical run was 3 min.Results： The calibration curves were linear over the concentration range of 50-1000 nmol.mL-1 and the abundance ratio range of 0.01-0.5（r 0.99）.The intra and inter batch precision（RSD,%） and accuracy（RE,%） met the acceptance criteria.Conclusion： A sensitive and specific LC-MS / MS method for the determination of leucine concentration and 13C-leucine enrichment in human plasma is developed.The method is validated with respect to specificity,accuracy,precision and stability.Taken together,the results demonstrate the established method is suitable to evaluate the catabolism level of protein and amino acid in clinical research.

关 键 词：亮氨酸 13C-亮氨酸 氨基酸氧化分解 血浆样品 同位素丰度 多反应监测 液相质谱联用 

分 类 号：R917[医药卫生—药物分析学]