高效液相色谱—电化学法检测蜜蜂脑部单胺类神经递质及其酸性代谢产物含量

建立同时测定蜜蜂脑部神经物质左旋多巴(l-DOPA)、去甲肾上腺素(NE)、3,4-二羟基苯乙酸(DOPAC)、多巴胺(DA)、5-羟吲哚乙酸(5-HIAA)、高香草酸(HVA)和5-羟色胺(5-HT)的高效液相色谱—电化学检测(HPLC-ECD)法.采用Acclaim C18色谱柱,以磷酸二氢钠、辛烷磺酸钠、柠檬酸、乙二胺四乙酸及乙腈的混合液为流动相,流速0.2 m L·min-1,进样量10μL,柱温38℃,检测电压300 m V.蜜蜂脑组织样品经高氯酸、焦亚硫酸钠和乙二胺四乙酸混合溶液去除蛋白,再由0.22μm滤膜过滤后上样检测.结果表明:脑组织中各待测神经物质线性回归方程相关系数均大于0.997,加样回收率平均值大于90%,日内、日间精密度指数RSD平均小于5.7%.经初步研究,意大利蜜蜂脑部l-DOPA、NE、DOPAC、DA和5-HIAA的含量分别为(0.232±0.107)、(1.288±0.630)、(2.555±1.201)、(0.574±0.233)、(0.393±0.123)ng·脑-1,中华蜜蜂脑部的对应物质含量分别为(0.331±0.162)、(0.894±0.395)、(2.819±0.974)、(0.636±0.255)、(0.353±0.163)ng·脑-1.

Applications of high performance liquid chromatography-electrochemical method in content determination of monoamine neurotransmitters and their metabolites in honeybee brains

A method based on high liquid chromatography-electrochemical ( HPLC-ECD) was established to simultaneously detect neuro substances in honeybee brains, which included levodopa ( l-DOPA) , norepinephrine ( NE) , 3,4-dihydroxyphenylacetic acid (DOPAC), dopamine (DA), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA) and serotonin (5-HT). Mono-amine transmitters and their metabolites were isolated by an Acclaim C18 column ( 2. 2 μm, 2. 1 × 100 mm) in 38 ℃ in mobile phase, which consisted of NaH2 PO4 , sodium 1-octanesulfonate, citric acid, EDTA and acetonitrile, at a flow rate of 0. 2 mL?min-1 . After proteins of honeybee brains were precipitated by mixture of perchloric acid, Na2 S2 O5 and EDTA, 10 μL supernatant was filtrated with 0.22 μm membrane, and followed by being detected at 300 mV. Results showed that the correlation coefficient of each standard curve was over 0.997, with average relative standard differences ( RSD) being less than 5.7% in inter-or intra-day as-say for all neuro substances. Average recovery rate was higher than 90%. Contents of l-DOPA, NE, DOPAC, DA and 5-HIAA in A-pis mellifera brains were (0.232±0.107), (1.288±0.630), (2.555±1.201), (0.574±0.233), (0.393±0.123) ng?brain-1, re-spectively. The corresponding results in Apis cerana brains were (0.331±0.162), (0.894±0.395), (2.819±0.974), (0.636± 0.255), (0.353±0.163) ng?brain-1, respectively.