LC-MS／MS法测定大鼠脑组织中单胺类神经递质及代谢产物

LC-MS/MS determination of monoamine neurotransmitters and its metabolites in rat brain tissue

目的:建立 LC—MS/MS 法测定大鼠脑组织中5-羟色胺(5-HT)、去甲。肾上腺素(NE)、多巴胺(DA)、5-羟吲哚乙酸(5-HIAA)、3-甲氧基-4-羟基苯乙二醇(MHPG)、3,4-双羟苯乙酸(DOPAC)、高香草酸(HVA)的含量。方法:采用 Ther—mo C_(18)色谱柱(4.6 mm×250mm,5μm),柱温为室温;水(含0.05%的甲酸)-乙腈为流动相,流速为0.8 mL·min^(-1)。ESI^+多个反应监测(MRM)模式监测反应质荷比(m/z):177.06→59.92(5-HT),170.00→152.00(NE),154.00→137.00(DA);ESI^-多个反应监测(MRM)模式监测反应质荷比(m/z):190.00→145.90(5-HIAA),263.00→165.00和263.00→150.00(MHPG),166.90→122.80(DOPAC),180.70→136.60(HVA)。结果:5-HT、NE 和 DA 分别在4.45～4450 ng·mL^(-1)、4.11～4110 ng·mL^(-1)和4.10～4100 ng·mL^(-1)浓度范围内线性良好(r≥0.999),最低检测浓度分别为4.45,4.1l,4.10 ng·mL^(-1);5-HIAA、MHPG、DOPAC 和 HVA 在12.5～1000 ng·mL^(-1)浓度范围内线性良好(r≥0.998),最低检测浓度为12.5 ng·mL^(-1)。日内和日间 RSD 均小于8.5%,重复性好。结论:本方法灵敏、快捷、准确、专一性强,可用于神经精神疾病神经递质的研究与分析。

Objectives：To establish an LC - MS/MS method for the determination of 5 - hydroxytryptamine （5 - HT）, norepinephrine （ NE）, dopamine （ DA）, 5 - hydroxyindole - 3 - acetic acid （ 5 - HIAA ） ,4 - hrdroxy - 3 - methoxyphenylglycol（MHPG） ,3,4 -dihydroxyphenylacetic acid （DOPAC） and homovanillic acid（HVA） in rat brain tissue. Methods：A Thermo C18 column （4.6 mm × 250 mm,5 μm） was selected as separation column at room temperature, and the mobile phase consisted of distilled water （ containing 0. 05 % formic acid） and acetonitrile at the flow - rate of 0. 8 mL·min^- 1. MS acquisition of 5 - HT, NE and DA was performed in electrospray positive ionization multiple reaction monitoring （MRM） mode, by monitoring the reaction m/z 177.06→159. 92,170. 00→ 152. 00,154.00→137.00,respectively;Electrospray negative ionization MRM mode was used to monitor the reaction m/z 190. 00 → 145.90 （ 5 - HIAA ）, 263.00 →165.00,263.00 → 150. 00 （ MHPG ）, 166. 90 → 122.80 （DOPAC） and 180. 70→136.60 （HVA）. Results： The calibration curves were linear within the concentration range of 4.45 -4450 ng · mL^-1 for 5 -HT,4. 11 -4110 ng·mL^-1 for NE and 4. 10 -4100 ng ·mL^-1 for DA（ r≥ 0. 999）. The limit of quantitation was 4.45,4. 11,4. 10 ng·mL^-1 , respectively. 5 - HIAA, MHPG, DOPAC and HVA have good linearity within the range of 12. 5 - 1000 ng·mL^-1 （r≥0. 998） and the limit of quantitation was 12.5 ng·mL^-1. The intra - and inter - day RSD were 〈 8.5%. Conclusions：The method is sensitive,fast,accu rate and usable for quantity determination of monoamine neurotransmitters and their metabolites in neuropsychiatric diseases.