LC-MS/MS方法检测血中甲基丙二酸含量及应用分析

目的 建立一种LC-MS/MS方法 测定血清MMA,用于MMA血症的诊断以及治疗效果的监测.方法 收集2009年4-12月205份健康体检者和146份患者血清,用液-液萃取方法 萃取样本,用自制饱和盐酸-正丁醇进行衍生,采用氮气吹干,色谱柱为Discovery C18(50 mm×2.1 mm,5 μm),流动相为甲醇和水(含0.1%甲酸,V/V),梯度洗脱,液相分离后进入串联质谱进行分析测定,选择性反应监测模式,以标准品制作标准曲线,同位素内标法定量,以血清样本测定2、25、80μg/L3个浓度的加样回收率,以质控样本测定准确度、精密度和稳定性.检测205份健康人血清,用于临床验证.数字表法随机抽取13份血清,测定结果 与德国MDI实验室进行比对,用配对t检验分析测定数据.结果 方法 线性范围2～100μg/L,标准曲线相关系数R2＞0.995.MMA衍生物保留时间为10.5 min,在此实验条件下丁二酸与MMA不互相干扰,批内RSD≤6.4%,批间RSD≤5.0%,回收率为96.42%～103.33%,检出限为1 μg/L,分析准确度94.2%～108.2%.样本常温放置至少6 h稳定、-20℃下至少可以存放70 d稳定、冻融10次稳定,衍生物在4 ℃下至少存放5 d稳定.溶血样本组与未溶血样本组测定值中位数(四分位数)分别为102.53(13.84～302.33)μg/L和39.52(11.94～203.08)μg/L,差异有统计学意义(T=8,P＜0.05).本实验室与德国MDI实验室测定结果 中位数(四分位数)分别为32.82(24.50～100.42)μg/L和32.20(26.65～93.30)μg/L,差异无统计学意义(T=7,P＞0.05).健康成年人(18～58岁)158名,血清MMA测定值(18.46±10.49)μg/L,健康未成年人(1～17岁)47名,血清MMA测定值(22.38±11.45)μg/L.结论 成功建立了LC-MS/MS方法 准确测定血清MMA浓度,样本前处理简单,方法 灵敏度高,特异性强,可重复性好,可以用于MMA血症的筛查、诊断和治疗效果的监测.

Analysis of blood methylmalonic acid with a liquid chromatography-tandem mass spectrometry method and its application

Objective To establish a LC-MS/MS method for the determination of MMA in serum,and provide a assay for the diagnosis and screening of methylmalonic academia in the clinic. Methods MMA was extracted from 205 serum samples from healthy controls and 146 serum samples from patients with liquidliquid extraction method with MTBE as the extraction solvent. The supernatant was transferred to a tube and dried with nitrogen gas. Then the residual was derived with HCI-BuOH mixed agent to give a product, which was analyzed directly by LC-MS-MS system with a gradient elution, selective reaction monitor, a Discovery C18 column (50 mm × 2. 1 mm ,5 μm) as the isolation column and a mobile phase consisting of methanol and water (0. 1% formic acid, V/V), respectively. The concentration of MMA was detected with the isotope internal standard method. The stand curve was employed with a series of calibrators. The recovery was estimated with the 3 serum samples with the concentrations of 2, 25, 80 μg/L respectively. The accuracy,precision and stability were also tested with quality control samples. Moreover, the range of concentrations in healthy people were detected to investigate the influence of hemolysis on the detection results. Thirteen samples were randomly tested according to the digital chart. The testing results were compared with the results provided by Medical Diagnositic institution (MDI) in Germany. The paired t-test was applied to statistical analysis. Results The linear range of this method was 2-100 μg/L, and the correlation coefficient (R2 ) was more than 0. 995. The retention time of MMA derivative was 10. 5 min. Succinic acid and MMA were not found to interfere with each other. The within-run RSD was less than 6. 4%, and the between-run RSD was less than 5.0%. The recovery rates were from 96. 42% to 103. 33%. The limit of quantification was 1 μg/L.The accuracies of the method were form 94. 2% to 108. 2%. The samples were stable for 6 h at room temperature and stable for 70 d even keep at - 20 ℃. The samples were stable after 10 freeze-thaw cycles. The derivatives of MMA were found to be stable at least for 5 d at 4 ℃. The medians of the hemolysis group and the normal group were 102.53 (13.84-302.33) μg/L and 39.52 (11.94-203.08) μg/L,respectively. There was significant difference between the 2 groups ( T = 8, P ＜ 0. 05 ). The medians of comparison test in our laboratory and the MDI were 32. 82(24. 50-100. 42) μg/L and 32. 20(26. 65-93. 30)μg/L There was no significant difference between the 2 groups( T=7 ,P ＞0. 05 ). The mean value (-x± s)of 158 healthy adults( 18-58 years old) and 47 healthy teenages( 1-17 years old)were ( 18.46 ± 10.49 )μg/L and (22. 38 ± 11.45) μg/L, respectively. Conclusions A LC-MS/MS method for analysis of MMA in serum is established successfully. The quantitative method is simple and accurate with good sensitivity,specificity and repeatability. The method can be applied for diagnosis, screening and monitoring of methylmalonic acidemia.