HPLC-MS/MS测定血浆中甜菜碱和游离胆碱及其稳定性研究  

Quantitative Measurement and Stability Study of Betaine and Free Choline in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry

作　　者：张珍真[1] 王佳佳[2] 谭炳炎[3] 王娴[1] 郑琳[3] 常颖[1] 朱惠莲[1] 

机构地区：[1]中山大学公共卫生学院营养学系,广东广州510080 [2]中山大学公共卫生学院卫生检验与检疫专业,广东广州510080 [3]中山大学公共卫生学院实验教学中心,广东广州510080

出　　处：《中山大学学报（医学科学版）》2011年第3期406-410,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(30872099);广东省科技计划项目(2008B030301100)

摘　　要：【目的】建立高效液相色谱串联质谱法(HPLC-MS/MS)测定血浆甜菜碱和游离胆碱水平,并研究血浆中两种物质的稳定性。【方法】以d9-氯化胆碱、d9-甜菜碱为内标,血浆经乙腈除蛋白后,采用SiO2柱(2.1mm×100mm,5μm)乙腈-甲酸铵缓冲液(15mmol/L,pH=3.5)等度洗脱,经电喷雾电离,选择多反应监测模式(MRM)同时测定血浆中甜菜碱和游离胆碱。【结果】血浆中甜菜碱和游离胆碱得到良好分离,最低定量限均为0.62μmol/L,线性范围均为0.62～400μmol/L。对游离胆碱的测定,日内相对标准差(RSD)为5.10%～5.85%,日间RSD为4.54%～13.26%,回收率94.15%～103.70%;对甜菜碱的测定,日内RSD为3.60%～7.30%,日间RSD为2.70%～8.10%,回收率90.03%～103.88%。稳定性研究中,游离胆碱和甜菜碱浓度随保存时间及温度增加,游离胆碱在血浆分离48h内变化小于15%,甜菜碱超过3h后变化即大于15%。3个反复冻融循环内游离胆碱浓度稳定;甜菜碱变化明显,第一个冻融循环后即增加至原始浓度3倍。【结论】高效液相色谱串联质谱法同时检测血浆中甜菜碱和游离胆碱是一种快速、准确且灵敏的方法。血浆保存时应注意保存温度、时间及反复冻融对血浆中甜菜碱和游离胆碱浓度的影响。[Objective]To establish a method for the quantitative measurement of betaine and free choline in plasma simultaneously using liquid chromatography tandem mass spectrometry（HPLC-MS/MS） and to study the stability of the two analytes in plasma.[Methods] Separation was achieved on a normal-phase silica column（2.1 mm × 100 mm,5 μm） with acetonitrile/15 mmol/L formic aicd（6：4v/v） under isocratic elution.A mass spectrometry was applied to measure the objective analytes.Acetonitrile,contained d9-choline and d9-betaine,as internal standards,was added into plasma to precipitate protein.[Results] For both betaine and choline,the lower limit of quantitation（LLOQ） was 0.62 μmol/L and the response was linerar over the range investigated（0.62 μmol/L-400.00 μmol/L,r2 〉0.999）.The within-day relative standard devition（RSD） were below 8% and between-day below 15%.The recoveries were over 90%.For the stability tests,the concentration of the two analytes increased with the storage temperature and time.Choline was stable throughout three freeze-thawn cycles while concentration of betaine had a significantly rise after the first cycle.[Conclusion] This is a technically simple and accurate method that can be carried out with HPLC-MS/MS.Attention should be paid to the critical effects of storage temperature and time and freeze-thawn cycle on the measurement.

关 键 词：血浆 甜菜碱 游离胆碱 稳定性 高效液相色谱串联质谱 

分 类 号：R151.2[医药卫生—营养与食品卫生学]