血清中游离胆碱和甜菜碱的高效液相色谱串联质谱法测定及稳定性研究  

DETERMINATION AND STABILITY STUDY OF FREE CHOLINE AND BETAINE IN SERUM BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY

作　　者：王佳佳[1] 张伟 谭炳炎[3] 王娴[4] 郑琳[3] 朱惠莲[4] 

机构地区：[1]中山大学公共卫生学院卫生检验与检疫专业 [2]广州市越秀区疾控中心 [3]中山大学公共卫生学院实验教学中心 [4]中山大学公共卫生学院营养学系,广州510080

出　　处：《营养学报》2011年第6期607-611,共5页Acta Nutrimenta Sinica

基　　金：国家自然科学基金(No.30872099)

摘　　要：目的建立一种高效液相色谱串联质谱法（HPLC—MS／MS）同时测定血清中游离胆碱和甜菜碱的方法，并观察血清中两种物质的稳定性。方法以乙腈为除蛋白剂，d9-氯化胆碱、d9-甜菜碱为内标，SiO2柱分离。流动相为乙腈一甲酸铵缓冲液（15mmol／L，pH=3．5），等度洗脱，质谱检测。结果血清中游离胆碱和甜菜碱得到良好分离，最低定量检测限均为0．62μmol／L，线性范围均为0．62-400μmol／L，日内变异系数为2．50％～7．23％，日间变异系数为4．49％-14．79％。游离胆碱和甜菜碱的回收率均在88．64％-98．23％。稳定性研究中，血清保存温度、时间对游离胆碱和甜菜碱浓度均有影响，反复冻融只对甜菜碱的影响明显。结论高效液相色谱串联质谱法同时检测血清中的游离胆碱和甜菜碱具有较好的准确度和精密性，为临床和科研提供了一种简单快速的检测方法。建议在血清分离后3h内完成测定或可在3h内经乙腈处理后放置于4℃，24h内测定完毕。Objective To develop a method for determination of free choline and betaine in serum simultaneously by liquid chromatography tandem mass spectrometry and to observe its stability. Method Acetonitrile was added into serum to precipitate protein and rig-choline and d9-betaine as internal standards. A normal-phase silica column was used to separate choline and betaine with acetonitrile / deionized water （60：40v/v, containing 15 mmol/L formic aicd in the water phase） under isocratic elution. Mass spectrometry was used to measure choline and betaine. Results For choline and betaine, the lower limit of quantitation was 0.62 μmol/L and the linearity was observed up to 400 μmol/L. Within- and between-day coefficient of variation were 2.50% -7.23% and 4.49%-14.79% respectively. The recovery rate was 88.64%- 98.23% respectively. For stability test, the stored temperature and time could affect the Concentration of the two analytes. Choline was stable throughout three freeze-thawn cycles. The stability of the extracted sample was observed at 4℃ for 24 h. Conclusion This rapid, sensitive, accurate and stable method is suitable in research practice and clinical settings. Choline and betaine should be determined in 3 h after serum separation or extracted as soon as possible then stored at 4℃ and analysed within 24 h.

关 键 词：胆碱 甜菜碱 稳定性 高效液相色谱串联质谱 

分 类 号：O657.63[理学—分析化学] R151[理学—化学]