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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-甲酸铵缓冲液(15 mmol/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0.62 mol/L,线性范围均为0.62~400 mol/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3 h内完成测定或可在3 h内经乙腈处理后放置于4℃,24 h内测定完毕。
Objective To establish a method for the simultaneous determination of free choline and betaine in serum by high performance liquid chromatography tandem mass spectrometry (HPLC-MS / MS) and to observe the stability of two substances in serum. Methods acetonitrile as deproteinizing agent, d9-choline chloride, d9-betaine as internal standard, SiO2 column separation. The mobile phase consisted of acetonitrile-ammonium formate buffer (15 mmol / L, pH = 3.5), isocratic elution and mass spectrometry. Results The free choline and betaine in serum were well separated. The minimum detectable limit of quantification was 0.62 mol / L, the linear range was 0.62 ~ 400 mol / L, the intra-day coefficient of variation was 2.50% -7.23% 4.49% ~ 14.79%. The recoveries of free choline and betaine ranged from 88.64% to 98.23%. Stability studies, the serum temperature, time on free choline and betaine concentrations are affected, repeated freezing and thawing only had a significant impact on the betaine. Conclusion The simultaneous determination of free choline and betaine in serum by high performance liquid chromatography-tandem mass spectrometry has good accuracy and precision, and provides a simple and rapid detection method for clinical and scientific research. It is recommended that the assay be completed within 3 h of serum separation or be treated with acetonitrile at 3 h and then placed at 4 ° C. The assay is completed within 24 h.