UPLC-MS/MS同时测定大鼠脑微透析样品中11个神经递质含量  

Simultaneous determination of 11 neurotransmitters in brain microdialysis samples from rats by UPLC-MS/MS

作　　者：潘凌云[1] 王雨 刘新华[1] 贾益群 PAN Ling-yun;WANG Yu;LIU Xin-hua;JIA Yi-qun(Experiment Center for Science and Technology,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学科技实验中心,上海201203

出　　处：《中国中药杂志》2022年第12期3242-3250,共9页China Journal of Chinese Materia Medica

基　　金：上海中医药大学杏林青年学者人才项目(2017年)。

摘　　要：应用超高效液相色谱串联质谱(UPLC-MS/MS)建立一种测定大鼠脑透析液中乙酰胆碱、谷氨酸、甘氨酸、去甲肾上腺素等11个神经递质的检测方法。取大鼠脑透析液20μL,加入乙腈-水(4∶1)60μL稀释,离心10 min(10000 r·min^(-1)),取5μL进行UPLC-MS/MS分析。选用Waters ACQUITY BEH amide色谱柱(2.1 mm×100 mm,1.7μm),柱温35℃,流动相为乙腈-0.2%甲酸水溶液,采用梯度洗脱法,流速0.35 mL·min^(-1)。离子源为电喷雾离子源,扫描模式为正离子模式,分析时间(duration)为3.5 min,多反应监测模式(MRM)。并应用该方法考察了11个神经递质的体外探针回收率与灌流速度/神经递质浓度水平之间的关系,以及考察葛根素单用及合用冰片后对大鼠纹状体中11种神经递质含量的影响。结果表明该方法线性关系良好,线性相关系数均大于0.99,最低定量限均小于12.5 nmol·L^(-1),11个神经递质的批内及批间精密度RSD均小于9.3%,准确度RE在-8.4%~9.5%。稳定性、回收率和基质效应等均符合生物样品分析要求。实验表明,给于葛根素之后大鼠脑纹状体内的大部分神经递质水平相较空白组发生了明显的改变;而大鼠同时给予冰片及葛根素之后,除多巴胺、去甲肾上腺素外,其余神经递质的变化程度较单用葛根素的变化程度小。该研究建立的UPLC-MS/MS分析方法能够对透析样品中11个神经递质完成定量检测,为考察健康和疾病动物模型中神经递质的变化提供了高效的检测方法。This study established a method for simultaneous determination of 11 neurotransmitters,such as acetylcholine,glutamic acid,glycine,and norepinephrine from rat brain microdialysis samples using UPLC-MS/MS.A total of 20μL of rat brain dialysate was diluted with 60μL of acetonitrile-water(4∶1)and centrifuged for 10 min at 10000 r·min^(-1),and 5μL was injected into UPLC-MS/MS system for assay.Chromatographic separation was performed on a Waters ACQUITY BEH amide column(2.1 mm×100 mm,1.7μm)with gradient elution using acetonitrile/0.2%formic acid-water as mobile phases with a flow rate of 0.35 mL·min^(-1)and column temperature of 35℃.The eluate was detected by multiple-reaction monitoring(MRM)scanning with an electrospray ionization source operating in the positive ionization mode with an analysis duration of 3.5 min.The relationship between the recovery rate of 11 neurotransmitters and the perfusion rate or the concentration of neurotransmitters was investigated.Furthermore,the effects of puerarin alone or combined with borneol on the content of 11 neurotransmitters in the striatum of rats were investigated.The results showed the calibration curves displayed good linear regression with coefficients all greater than 0.99 and the lower limit of quantification(LLOQ)less than 12.5 nmol·L^(-1)for each analyte.The within-run and between-run precision(RSD)of the 11 neurotransmitters at low,medium,and high levels was less than 9.3%,and the relative error of the accuracy ranged from-8.4%to 9.5%.The stability,recovery,and matrix effects were in line with the biological sample analysis requirements.As revealed by experimental results,the levels of most neurotransmitters in the brain striatum changed significantly after rats were treated with puerarin as compared with the conditions in the blank group.Except for dopamine and norepinephrine,the degree of changes of other neurotransmitters in the combination group(borneol and puerarin)was less than that of the puerarin group.The established UPLC-MS/MS method could be ap

关 键 词：UPLC-MS/MS 微透析 探针回收率 神经递质 

分 类 号：R285.5[医药卫生—中药学]