Direct detection of tetrahydrobiopterin (BH4) and dopamine in rat brain using liquid chromatography coupled electrospray tandem mass spectrometry
Direct detection of tetrahydrobiopterin (BH4) and dopamine in rat brain using liquid chromatography coupled electrospray tandem mass spectrometry
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DOI：
10.1016/j.bbrc.2012.02.064
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发表时间：
2012-03-23
影响因子：
3.1
通讯作者：
Kim, Hyung-Gun
中科院分区：
文献类型：
--
作者：
Kim, Hak Rim;Kim, Tae-Hyun;Kim, Hyung-Gun
开发了一种简单快速的液相色谱串联质谱 (LC-MS/MS) 方法，使用 ε-乙酰胺己酸 (AACA) 作为内标，定量大鼠脑中的四氢生物蝶呤 (BH4) 和多巴胺。用乙腈沉淀样品中的蛋白质，然后使用 Sepax Polar-Imidazor（2.1 x 100 mm，内径，3 μ m）柱分离上清液，使用 10 mM 甲酸铵的乙腈/水（75:25，v/v）混合物作为流动相，流速为 300 μl/min。在三重四极杆质谱仪上进行定量，采用电喷雾电离，操作条件为多反应监测 (MRM)，正离子模式为 BH4 的 m/z 242.1 -> 166.0、多巴胺的 m/z 154.1 -> 90.0 和 AACA (IS) 的 m/z 174.1 -> 114.0。总色谱运行时间为 5.5 分钟。该方法经过样品分析验证：检测限为 10 ng/g。在大鼠大脑中，BH4 (r(2) = 0.995) 的校准曲线在 10-2000 ng/g 之间呈线性，多巴胺 (r(2) = 0.997) 的校准曲线在 10-5000 ng/g 之间呈线性。因此，获得了良好相关的 LC-ESI/MS/MS 结果，并发现它是定量分析大鼠脑中 BH4 和多巴胺的有力工具。 (C) 2012 Elsevier Inc. 保留所有权利。
A simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of tetrahydrobiopterin (BH4) and dopamine in rat brain using epsilon-acetamidocaproic acid (AACA) as an internal standard. Proteins in the samples were precipitated with acetonitrile and then the supernatants were separated by a Sepax Polar-Imidazole (2.1 x 100 mm, i.d., 3 mu m) column using a mixture of 10 mM ammonium formate in acetonitrile/water (75:25, v/v) as the mobile phase at a flow rate of 300 mu l/min. Quantification was performed on a triple quadrupole mass spectrometer employing electrospray ionization with the operating conditions as multiple reaction monitoring (MRM) and positive ion mode from m/z 242.1 -> 166.0 for BH4, m/z 154.1 -> 90.0 for dopamine and m/z 174.1 -> 114.0 for AACA (IS). The total chromatographic run time was for 5.5 min. The method was validated for the analysis of samples: the limit of detection was 10 ng/g. The calibration curve was linear between 10-2000 ng/g for BH4 (r(2) = 0.995) and 10-5000 ng/g for dopamine (r(2) = 0.997) in the rat brain. Thus, good correlated LC-ESI/MS/MS results were obtained and found to be a powerful tool for the quantitative analysis of BH4 and dopamine in the rat brain. (C) 2012 Elsevier Inc. All rights reserved.