A Rapid Sample Preparation Procedure Using MonoSpin CBA and Amide Columns for Tetrodotoxin Detection in Serum and Urine Using LC-MS/MS Analysis
A Rapid Sample Preparation Procedure Using MonoSpin CBA and Amide Columns for Tetrodotoxin Detection in Serum and Urine Using LC-MS/MS Analysis
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DOI：
10.1007/s10337-014-2663-3
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发表时间：
2014-05-01
期刊：
影响因子：
1.7
通讯作者：
Inokuchi, Sadaki
中科院分区：
文献类型：
--
作者：
Saito, Takeshi;Miura, Naoya;Inokuchi, Sadaki
河豚毒素（TTX）中毒的临床诊断可能是困难的，因为缺乏特征性的形态学表现和筛查试验，如免疫测定。在这里，我们提出了一个充分验证的方法分析TTX在血清和尿液中。在该方法中，使用MonoSpin CBA或酰胺柱提取血清和尿液样品，然后进行LC-MS/MS分析。用0.1 mL 10%乙酸溶液从柱上洗脱TTX，并直接注入LC-MS/MS。（Zorbax HILIC Plus 2.1 × 100 mm，3.5 μ m）用于等度洗脱，用含甲酸的10 mM甲酸铵的移动的流动相（95：5，v/v），沿着用5 mM三氟乙酸和2%乙腈。使用Agilent 6410质谱仪，利用正电喷雾电离和多反应监测检测TTX。血清和尿液的定量限分别确定为1和0.5 ng/mL（-1）。血清和尿液的检测限分别为0.5和0.25 ng/mL（-1）。日内和日间精密度范围为1.5%至8.5%。两种基质的回收率均> 86.5%。在该方法中，进样到LC-MS/MS之前的样品制备过程大约需要10-15分钟，这将提取时间减少到以前方法的十分之一。通过对河豚鱼中毒患者体内生物物质的分析，进一步验证了该方法的应用。
Clinical diagnosis of tetrodotoxin (TTX) poisoning can be difficult because of the lack of characteristic morphological findings and a screening test, such as an immunoassay. Here, we present a fully validated method for the analysis of TTX in serum and urine. In this method, serum and urine samples were extracted using MonoSpin CBA or amide columns, followed by LC-MS/MS analysis. The TTX was eluted from the column by 0.1 mL of 10 % acetic acid solution, and was directly injected into LC-MS/MS. An Agilent 1200 HPLC system equipped with a HILIC separation column (Zorbax HILIC Plus 2.1 x 100 mm, 3.5 mu m) was used for isocratic elution, with a mobile phase of 10 mM ammonium formate with formic acid (95:5, v/v), along with 5 mM trifluoroacetic acid and 2 % acetonitrile. TTX was detected with an Agilent 6410 mass spectrometer utilizing positive electrospray ionization and multiple reaction monitoring. Limits of quantification for serum and urine were established to be 1 and 0.5 ng mL(-1), respectively. Limits of detection for serum and urine were 0.5 and 0.25 ng mL(-1), respectively. Intra-day and inter-day precision varied from 1.5 to 8.5 %. The recovery was > 86.5 % for both matrices. In this method, the sample preparation process prior to injection into the LC-MS/MS takes approximately 10-15 min, which reduces the extraction time to one-tenth of that of previous methods. The application of this method was further verified by analysis of biological materials from a patient suffering from puffer fish poisoning.