Zhao Yanyan, Liu Liyan, Han Yuanyuan, Bai Jie, Du Guangling, Gao Qian
Experimental Center of Medicine, Hebei University, Baoding 071000, China.
Se Pu. 2011 Feb;29(2):146-51. doi: 10.3724/sp.j.1123.2011.00146.
A method for the simultaneous detection of L-dihydroxyphenylalanine, norepinephrine, epinephrine, dopamine, 3,4-dihydroxyphenylacetic acid, serotonin hydrochloride, 5-hydroxyindole-3-acetic acid and homovanillic acid in the different sections of mouse brains was established by using high performance liquid chromatography (HPLC) with fluorescence detection and isocratic elution. Before analysis, the sample was deproteinized by 0.60 mol/L perchloric acid, followed by adjusting pH value of the sample with 1.20 mol/L K2HPO4, addition of 0.1 g/L L-cysteine as antioxidant and 0.50 mmol/L Na2EDTA as complexing agent. The separation column was a Shim-pack C18 column (250 mm x 4.6 mm, 5 microm) and the mobile phase (pH 3.8) was 13% methanol containing 50 mmol/L citric acid, 50 mmol/L sodium acetate, 0.5 mmol/L 1-heptanesulfonic acid sodium salt, 5 mmol/L triethylamine and 0.5 mmol/L Na2EDTA. The flow rate was 1.0 mL/min. The injection volume was 10 microL. The emission and excitation wavelengths were 330 nm and 280 nm, respectively. Under the optimized separation conditions, the calibration curves showed good linearity within the concentrations of 1.25 - 5000 microg/L (r > 0.9999). The limits of detection were between 0.20 - 5.00 microg/L, the average recoveries were between 94.83% and 99.19%, and the relative standard deviations (RSDs) were between 0.08% and 2.51%. The advantages of the method include easy and prompt operation, high recovery, low detection limit, good separation effect, high accuracy and precision. The method has practical value for detecting 8 monoamine neurotransmitters in biological samples.
采用高效液相色谱（HPLC）荧光检测和等度洗脱法，建立了同时检测小鼠脑不同区域中L-二羟基苯丙氨酸、去甲肾上腺素、肾上腺素、多巴胺、3,4-二羟基苯乙酸、盐酸5-羟色胺、5-羟基吲哚-3-乙酸和高香草酸的方法。分析前，样品用0.60 mol/L高氯酸脱蛋白，然后用1.20 mol/L K2HPO4调节样品pH值，加入0.1 g/L L-半胱氨酸作为抗氧化剂和0.50 mmol/L Na2EDTA作为络合剂。分离柱为Shim-pack C18柱（250 mm×4.6 mm，5 µm），流动相（pH 3.8）为含50 mmol/L柠檬酸、50 mmol/L醋酸钠、0.5 mmol/L 1-庚烷磺酸钠、5 mmol/L三乙胺和0.5 mmol/L Na2EDTA的13%甲醇。流速为1.0 mL/min。进样量为10 µL。发射波长和激发波长分别为330 nm和280 nm。在优化的分离条件下，校准曲线在1.25 - 5000 μg/L浓度范围内呈良好线性（r > 0.9999）。检测限在0.20 - 5.00 μg/L之间，平均回收率在94.83%至99.19%之间，相对标准偏差（RSD）在0.08%至2.51%之间。该方法具有操作简便快速、回收率高、检测限低、分离效果好、准确度和精密度高等优点。该方法对于检测生物样品中的8种单胺类神经递质具有实际应用价值。
