Kim Tae-Hyun, Choi Juhee, Kim Hyung-Gun, Kim Hak Rim
Department of Pharmacology, College of Medicine, Dankook University, Cheonan 330-714, Republic of Korea ; Bioresources Regional Innovation Center, Soon Chun Hyang University, Asan 336-745, Republic of Korea.
Department of Pharmacology, College of Medicine, Dankook University, Cheonan 330-714, Republic of Korea.
J Anal Methods Chem. 2014;2014:506870. doi: 10.1155/2014/506870. Epub 2014 Sep 3.
A simple and rapid liquid chromatography tandem mass spectrometry method has been developed for the determination of BH4, DA, 5-HT, NE, EP, Glu, and GABA in mouse brain using epsilon-acetamidocaproic acid and isotopically labeled neurotransmitters as internal standards. Proteins in the samples were precipitated by adding acetonitrile, and then the supernatants were separated by a Sepax Polar-Imidazole (2.1 mm × 100 mm, i.d., 3 μm) column by adding a mixture of 10 mM ammonium formate in acetonitrile/water (75 : 25, v/v, 300 μl/min) for BH4 and DA. To assay 5-HT, NE, EP, Glu, and GABA; a Luna 3 μ C18 (3.0 mm × 150 mm, i.d., 3 μm) column was used by adding a mixture of 1% formic acid in acetonitrile/water (20 : 80, v/v, 350 μl/min). The total chromatographic run time was 5.5 min. The method was validated for the analysis of samples. The calibration curve was linear between 10 and 2000 ng/g for BH4 (r(2) = 0.995) , 10 and 5000 ng/g for DA (r(2) = 0.997) , 20 and 10000 ng/g for 5-HT (r(2) = 0.994) , NE (r(2) = 0.993) , and EP (r(2) = 0.993) , and 0.2 and 200 μg/g for Glu (r(2) = 0.996) and GABA (r(2) = 0.999) in the mouse brain tissues. As stated above, LC-MS/MS results were obtained and established to be a useful tool for the quantitative analysis of BH4, DA, 5-HT, NE, EP, Glu, and GABA in the experimental rodent brain.
已开发出一种简单快速的液相色谱串联质谱法，以ε-乙酰氨基己酸和同位素标记的神经递质作为内标，用于测定小鼠脑组织中的四氢生物蝶呤（BH4）、多巴胺（DA）、5-羟色胺（5-HT）、去甲肾上腺素（NE）、肾上腺素（EP）、谷氨酸（Glu）和γ-氨基丁酸（GABA）。通过加入乙腈使样品中的蛋白质沉淀，然后将上清液用Sepax Polar-Imidazole柱（2.1 mm×100 mm，内径，3μm）进行分离，用于测定BH4和DA时，流动相为含10 mM甲酸铵的乙腈/水混合液（75∶25，v/v，300μl/min）。用于测定5-HT、NE、EP、Glu和GABA时，使用Luna 3μ C18柱（3.0 mm×150 mm，内径，3μm），流动相为含1%甲酸的乙腈/水混合液（20∶80，v/v，350μl/min）。总色谱运行时间为5.5分钟。该方法经样品分析验证。校准曲线在小鼠脑组织中，BH4为10至2000 ng/g时呈线性（r(2)=0.995），DA为10至5000 ng/g时呈线性（r(2)=0.997），5-HT、NE和EP为20至10000 ng/g时呈线性（r(2)=0.994），Glu和GABA为0.2至200μg/g时呈线性（r(2)=0.996和r(2)=0.999）。如上所述，获得了液相色谱-串联质谱结果，并确定其为实验啮齿动物脑组织中BH4、DA、5-HT、NE、EP、Glu和GABA定量分析的有用工具。
