用液相色谱-串联质谱法测定人血浆中血小板活化因子  

Quantitative determination of platelet activating factor in human plasma by LC-MS/MS method

作　　者：李宁[1] 李水军[2] 王文义[3] 王逸平[3] 顾水明[4] 

机构地区：[1]上海交通大学附属第六人民医院心内科,上海200233 [2]上海市徐汇区中心医院中心实验室,上海200031 [3]中国科学院上海药物研究所药物研究国家重点实验室,上海201203 [4]上海市徐汇区中心医院心内科,上海200031

出　　处：《药学服务与研究》2008年第2期112-115,共4页Pharmaceutical Care and Research

基　　金：上海市科委生物医药基金(No.044119657);上海市卫生局重点专科建设项目(No.05Ⅱ028)

摘　　要：目的：建立一种人血浆中血小板活化因子（PAF）的液相色谱－串联质谱测定方法（LC-MS／MS），从而反映临床冠心病独立风险预测因子脂蛋白相关磷脂酶A2（LP－PLA2）活性。方法：血浆添加底物PAF后，37℃孵育10min，加入甲醇沉淀蛋白质，离心取上清进样。血浆PAF经HPLC法分离，串联质谱法定量检测。以PAF的单位时间减少量来衡量LP—PLAa的活性。结果：在1～50μg／mL添加浓度范围内线性关系良好，线性方程y=0．284c＋0．214，r=0．9991（n=6）。低、中、高浓度（3、20、40μg／mL）质控品的批内、批间精密度分别为2．08％～4．75％（n=6）和5．47％～5．86％（n=18），准确度为93．50％～97．66％，PAF和内标非布索坦的回收率为97．36％～100．8％（n=6）。稳定性研究结果表明，PAF在样品预处理后12h和冻融3次后的准确度为99．96此～107．1％（，产6），说明稳定性良好。应用本方法测定10例冠心病患者和健康对照组的PAF反应量，测定结果分别为（22．77±1．26）mg／mL和（19．62±3．94）mg／mL，说明冠心病患者LP—PLA2活性明显高于健康对照组（P＜0．05）。结论：本方法专一灵敏、准确可靠、重复性好、检测通量高，可作为心血管疾病诊断指标之一，用于临床冠心病与缺血性卒中风险预测的研究。Objective： To establish a LC-MS/MS method for determining the concentration of platelet activating factor （PAF） in human plasma and to know the activity of lipoprotein-associated phospholipase A2 （LP-PLA2）, an independent risk factor for coronary artery disease （CAD） in clinical practice. Methods： After addition of PAF, the plasma sample was incubated at 37 ℃ for 10 min, then immediately pretreated with methanol for deproteinization. The supernatant was separated with liquid chromatography and detected with tandem mass spectrometry. The LP-PLA2activity was assessed from the velocity of the decrease in PAF in human plasma. Results： Good linear relation over the range of 1-50 μg/mL of PAF was obtained with regression equation y=0. 284 c＋0. 914. r=0.999 1（n=6）. At low,middle and high concentrations of quality control sample （3, 20, 40 μg/mL）,the intra- and inter-batch RSD were 2.08%-4.75%（n=6） and 5.47%-5.86%（n=18）, respectively. Accuracy was 93.50%-97.66% and recovery of PAF and febuxostat （internal standard） was 97.36%-100.8%（n=6）. Sample was stable for 12 halter preparation and for 3 freeze-thaw cycles with accuracy ranging from 99.96% to 107.1%（n=6）. Results of PAF determination by the developed method showed that PAF concentration of CAD group was significantly higher than that of healthy controls [（22.77 ±1. 26） mg/mL vs （19.62± 3.94） mg/mL, P〈0.05], which indicated a higher activity of LP-PLA2 in CAD patients than in healthy conlrols. Conclusion： The developed ducible, reliable and has the advantage of high throughput detection, used as a diagnostic marker for CAD and ischemic apoplexy. LC-MS/MS method, which is specific, accurate, repros suitable for determining LP-PLA2 activity which can be used as a diagnostic marker for CAD and ischemic apoplexy.

关 键 词：色谱法 液相 串联质谱法 血小板活化因子 脂蛋白相关磷脂酶A2 

分 类 号：R969.11[医药卫生—药理学]