十种一碳代谢关键产物的HPLC-MS/MS 定量检测方法

目的:利用HPLC-MS/MS方法对十种一碳代谢相关产物进行定量分析。方法:采用Aglient ZORBAX SB-AQ C18柱(2.1mm×100 mm,3.5 m)、电喷雾离子源(ESI),以多离子反应监测方式(MRM)进行正离子检测。对游离叶酸(FA)、5-甲酰四氢叶酸(5-FT)、5-甲基四氢叶酸(5-MT)、S-腺苷蛋氨酸(SAM)、S-腺苷同型半胱氨酸(SAH)、胱硫醚(CYSTA)、组氨酸(HIS)、丝氨酸(SER)、蛋氨酸(MET)、同型半胱氨酸(HCY)进行定量分析。结果:FA、5-FT、5-MT、SAM、SAH、CYSTA、HIS、SER、MET、HCY的检测限分别为0.1 ng.L-1、0.25 ng.L-1、0.1 ng.L-1、0.1 ng.L-1、0.25 ng.L-1、0.25 ng.L-1、0.1 ng.L-1、0.025 ng.L-1、0.1 ng.L-1、0.1 ng.L-1。FA、5-FT、5-MT、SAH、CYSTA浓度测定方法线性范围为2~50 ng.L-1,SER、SAM浓度测定方法线性范围20~500 ng.L-1,MET、HCY浓度测定方法线性范围200~5000 ng.L-1,HIS浓度测定方法线性范围为400~10000 ng.L-1,r均在0.993以上,全部涵盖了已报道的血清中指标的含量范围。结论:建立了HPLC-MS/MS方法,可同时分析十种一碳代谢通路的关键产物,所需样品量少,检测速度快,同时实现分项检测,可为多种代谢性疾病系统性地检测一碳代谢中间产物体液分析方法建立实验条件基础。

Quantitative Method of 10 Pivotal Metabolites in One-Marbon Metabolismby High Performance Liquid Chromatography Tandem Mass Spectrometry

Objective: To quantify 10 pivotal compounds in one-carbon metabolism by using high-performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS).Methods: The separation was carried out on a Aglient ZORBAX SB-AQ C18 column(2.1 mm ×100 mm,3.5 m) at 25 ℃.ESI was performed in the positive-ion mode using the MRM mode.Using target ions at m/z 442.1-295.3 for folate acid,m/z 474.1-299.3 for 5-FT,m/z 460.0-313.0 for 5-MT,m/z 399.0-298.3 for SAM,m/z 385.0-136.0 for SAH,m/z 106.0-60.0 for CYSTA,m/z 156.0-110.0 for HIS,m/z 106.0-60.0 for SER,m/z 150.0-55.9 for MET,m/z 136.0-90.0 for HCY.Results: The LLOD of the compounds was 0.1 ng·L-1 for FA,0.25 ng·L-1 for 5-FT,0.1 ng·L-1 for 5-MT,0.1 ng·L-1 for SAM,0.25 ng·L-1 for SAH,0.25 ng·L-1 for CYSTA,0.1·ng L-1 for HIS,0.025 ng·L-1 for SER,0.1 ng·L-1 for MET,0.1 ng·L-1 for HCY.The calibration curve was obtained with good correlation(r ≥0.993),using the concentration range of 2~50 ng·L-1 for FA,5-FT,5-MT,SAH,and CYSTA;20~500 ng·L-1 for SER,and SAM;200~5 000 ng·L-1 for MET,and HCY;400~10 000ng·L-1 for HIS,coverd ranges of most compounds in serum that have published previously.Conclusion: A sensitive method for 10 pivotal compounds in one-carbon metabolism was developed.It should be a reliable method for quantifying many compounds simultaneously with less sample and short time.And this result can provide a quantitive detection method for the diagnosis and prevention of the diseases associated with one carbon metabolism.