CN111337609B - 一种基于lc-ms同时测定多种神经递质的人血清测定方法 - Google Patents
一种基于lc-ms同时测定多种神经递质的人血清测定方法 Download PDFInfo
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Abstract
本发明提供一种基于LC‑MS同时测定多种神经递质的人血清测定方法,属于生物检测技术领域,通过采用甲酸乙腈溶液进行液‑液萃取,提取样品混合物中的神经递质,再使用HILIC色谱将神经递质分离后,采用标准曲线法进行串联质谱分析,具有选择性好、灵敏度高、分析时间短等优点。
Description
技术领域
本发明涉及生物检测技术领域,具体涉及一种基于LC-MS同时测定15种神经递质的人血清测定方法。
背景技术
神经递质是突触间进行信号传递的一种化学“信使”分子,通过与效应器或者神经元细胞上的受体特异性结合而发挥其生理作用。它广泛参与运动、认知学习、记忆、情绪,睡眠和觉醒、应激反应等多种生理活动。神经递质的异常往往与抑郁症、阿尔兹海默症、帕金森病、精神分裂症、癫痫等多种精神类疾病和神经类疾病的形成有关。神经递质的测定已成为疾病诊断和监测以及治疗干预的重要手段,有效的神经递质检测对于疾病诊疗乃至新药研发都至关重要。但由于神经递质类化合物具有分子量小、极性大、稳定性差、内源干扰物质较多等特点,直接检测的难度较大。
发明内容
本发明的目的是提供一种基于LC-MS同时测定多种神经递质的人血清测定方法,通过采用甲酸乙腈溶液进行液-液萃取,提取样品混合物中的神经递质,再使用HILIC色谱将神经递质分离后,采用标准曲线法进行串联质谱分析,具有选择性好、灵敏度高、分析时间短等优点。
为了实现上述目的,本发明采用的技术方案如下:
一种基于LC-MS同时测定多种神经递质的人血清测定方法,包括如下步骤:
步骤S1样本处理,将待测血清样本加入含甲酸的乙腈溶液中,再进行冰浴超声波处理,低温孵育,低温高速离心,取上清进行冷冻干燥,再加入含甲酸的乙腈-水溶液复溶,再次低温高速离心,取上清液上机备用;
步骤S2色谱分离,取步骤S1获得的上清液进行色谱分离,A相为含25mM甲酸铵的0.1%甲酸水溶液,B相为含0.1%甲酸的乙腈溶液,液相梯度如下,0-1min,B相维持在95%;1-14min,B相从95%线性变化至65%;14-16min,B相从65%线性变化至40%;16-18min,B相维持在40%;18-18.1min,B相从40%线性变化至95%;18.1-23min,B相维持在95%;
步骤S3质谱分析,步骤S2分离后的样品进行正离子扫描模式的质谱分析,离子源(ESI源)的条件如下,离子源温度(source temperature):450℃,辅助气压1(ion SourceGas1):60,辅助气压2(Ion Source Gas2):60,气帘气压(Curtain gas):30,离子喷雾电压(ionSapary Voltage Floating):5000V。
根据以上方案,所述的步骤S1的待测血清样本与含甲酸的乙腈溶液体积比例为血清样本:含甲酸的乙腈溶液=1:4,甲酸含量为1%;所述的甲酸乙腈溶液和甲酸乙腈-水溶液中均含有1%的甲酸,其中甲酸乙腈-水溶液中乙腈与水的体积比例=4:1。
根据以上方案,所述的冰浴超声波处理中的超声波功率为180W,处理时间为20-40min;所述的低温孵育是-20℃孵育1h;所述的低温高速离心是使用14000rcf 4℃离心20min。
根据以上方案,所述的步骤S2中的色谱分离的色谱柱温度为35℃,流速为300μL/min。
根据以上方案,步骤S3中的质谱分析的各离子对及其对应的去簇电压、碰撞电压和碰撞池出口电压参数如下:
Compound | Q1/m/z | Q3/m/z | DP/V | CE/V | CXP/V |
r-Amino-butyric acid | 104 | 87.1 | 40 | 15 | 20 |
DOPA | 198.1 | 151.8 | 40 | 25 | 15 |
Dopamine | 154.1 | 137 | 30 | 16 | 15 |
Epinephrine | 184.1 | 107.1 | 35 | 29 | 15 |
Norepinephrine | 170.3 | 152 | 45 | 17 | 15 |
5-HIAA | 192 | 146.1 | 50 | 19 | 15 |
Serotonin | 177 | 160.1 | 50 | 18 | 20 |
5-Hydroxy-L-tryptophan | 221.2 | 134.1 | 40 | 33 | 15 |
3-methoxytyramine | 168.2 | 151 | 40 | 15 | 15 |
Acetylcholine | 146.1 | 60.1 | 50 | 16 | 20 |
Histamine | 112.1 | 95.1 | 60 | 22 | 20 |
Normetanephrine | 184.1 | 106 | 30 | 24 | 20 |
Tyramine | 138 | 76.9 | 30 | 36 | 15 |
glutamate | 148.1 | 84.1 | 40 | 21 | 15 |
Glutamine | 147 | 84.1 | 50 | 24 | 15 |
所述的步骤S3中的质谱分析采用多反应监测模式(MRM模式)检测待测离子对。
本发明的有益效果是:
通过对色谱和质谱的实验参数进行优化,利用液相色谱进行分离,再配合质谱的分析,实现同时测定15种神经递质,包括r-Amino-butyric acid、DOPA、Dopamine、Epinephrine、Norepinephrine、5-HIAA、Serotonin、5-Hydroxy-L-tryptophan、3-methoxytyramine、Acetylcholine、Histamine、Normetanephrine、Tyramine、glutamate和Glutamine,具有分辨率高、通量大、速度快等的优势,同时具有可预期的临床应用前景。
具体实施方式
下面结合实施例对本发明的技术方案进行说明。
一种基于LC-MS同时测定多种神经递质的人血清测定方法,包括如下步骤:
步骤S1样本处理,将待测血清样本按体积比例,血清样本:甲酸乙腈溶液=1:4加入到含甲酸1%的乙腈溶液中,冰浴并使用180W的超声波处理30min,再用-20℃孵育1h,使用14000rcf 4℃离心20min取上清,再进行冷冻干燥,再加入含1%甲酸的乙腈-水溶液复溶,甲酸乙腈-水溶液中乙腈与水的体积比例=4:1,再使用14000rcf 4℃离心20min,取上清液备用;
步骤S2色谱分离,取步骤S1获得的上清液采用Agilent 1290Infinity LC超高效液相色谱系统,色谱柱温度为35℃,流速为300μL/min,液相梯度如下,0-1min,B相维持在95%;1-14min,B相从95%线性变化至65%;14-16min,B相从65%线性变化至40%;16-18min,B相维持在40%;18-18.1min,B相从40%线性变化至95%;18.1-23min,B相维持在95%;
步骤S3质谱分析,步骤S2分离后的样品采用5500QTRAP质谱仪(AB SCIEX)在正离子扫描模式的质谱分析,离子源的条件如下,source temperature:450℃,Ion SourceGas1(Gas1):60,Ion Source Gas2(Gas2):60,Curtain gas(CUR):30,ionSapary VoltageFloating(ISVF):5000V。
质谱分析的各离子对及其对应的去簇电压、碰撞电压和碰撞池出口电压参数如下:
所述的步骤S3中的质谱分析采用多反应监测模式检测待测离子对。
本实施例中用15种神经递质的标准品采用上述方法制得标准曲线,15种神经递质的标准曲线如下:
神经递质种类 | 质量信息 | 保持时间 | 标准曲线 | R2 |
r-Amino-butyric acid | 104.0/87.1 | 9.28739311 | y=420.23248x-215245 | 0.99817 |
DOPA | 198.1/151.8 | 10.34674953 | y=118.90085x-496181 | 0.99833 |
Dopamine | 154.1/137.0 | 7.432457009 | y=8989.87326x-76952.2 | 0.99817 |
Epinephrine | 184.1/107.1 | 7.611214982 | y=3444.79744x+161807 | 0.997 |
Norepinephrine | 170.3/152.0 | 8.553900642 | y=112.50354x-134374 | 0.99725 |
5-HIAA | 192.0/146.1 | 1.378657632 | y=184.33853x+102808 | 0.99713 |
Serotonin | 177.0/160.1 | 6.44926457 | y=9599.13413x-39503.6 | 0.9997 |
5-Hydroxy-L-tryptophan | 221.2/134.1 | 9.030580354 | y=110.40864x-109911.1 | 0.99792 |
3-methoxytyramine | 168.2/151.0 | 6.062569477 | y=21042.99952x+799211 | 0.99777 |
Acetylcholine | 146.1/60.1 | 4.61499318 | y=43369x-401678 | 0.99367 |
Histamine | 112.1/95.1 | 13.2691355 | y=45598.2x+978545 | 0.99807 |
Normetanephrine | 184.1/106.0 | 7.056986166 | y=373.40994x+115491 | 0.99877 |
Tyramine | 138.0/76.9 | 6.098622047 | y=6420.39016x-1057.54323 | 0.99824 |
glutamate | 148.1/84.1 | 11.80192524 | y=266.63448x+66783 | 0.99924 |
Glutamine | 147.0/84.1 | 11.8687508 | y=289.81324x-271673 | 0.99959 |
本实施例中的样本检测结果如下(nM/L):
以上实施例仅用以说明而非限制本发明的技术方案,尽管上述实施例对本发明进行了详细说明,本领域的相关技术人员应当理解:可以对本发明进行修改或者同等替换,但不脱离本发明精神和范围的任何修改和局部替换均应涵盖在本发明的权利要求范围内。
Claims (4)
1.一种基于LC-MS同时测定多种神经递质的人血清测定方法,其特征在于,包括如下步骤:
步骤S1样本处理,将待测血清样本加入含甲酸的乙腈溶液中,待测血清样本与含甲酸的乙腈溶液体积比例为血清样本:含甲酸的乙腈溶液=1:4,甲酸含量为1%,再进行冰浴超声波处理,所述的冰浴超声波处理中的超声波功率为180W,处理时间为20-40min,低温孵育,低温高速离心,取上清液体进行冷冻干燥,再加入含甲酸的乙腈-水溶液复溶,再次低温高速离心,取上清液上机备用;
步骤S2色谱分离,取步骤S1获得的上清液进行色谱分离,A相为含25mM甲酸铵的0.1%甲酸水溶液,B相为含0.1%甲酸的乙腈溶液,液相梯度如下,0-1min,B相维持在95%;1-14min,B相从95%线性变化至65%;14-16min,B相从65%线性变化至40%;16-18min,B相维持在40%;18-18.1min,B相从40%线性变化至95%;18.1-23min,B相维持在95%;
步骤S3质谱分析,步骤S2分离后的样品进行正离子扫描模式的质谱分析,离子源的条件如下,离子源温度:450℃,辅助气压1:60,辅助气压2:60,气帘气压:30,离子喷雾电压:5000V;
质谱分析的各离子对及其对应的去簇电压、碰撞电压和碰撞池出口电压参数如下:
所述的步骤S3中的质谱分析采用多反应监测模式检测待测离子对。
2.根据权利要求1所述的基于LC-MS同时测定多种神经递质的人血清测定方法,其特征在于,所述含甲酸的乙腈-水溶液中含有1%的甲酸,其中乙腈与水的体积比例=4:1。
3.根据权利要求1所述的基于LC-MS同时测定多种神经递质的人血清测定方法,其特征在于,所述的低温孵育是-20℃孵育1h;所述的低温高速离心是使用14000rcf 4℃离心20min。
4.根据权利要求1所述的基于LC-MS同时测定多种神经递质的人血清测定方法,其特征在于,所述的步骤S2中的色谱分离的色谱柱温度为35℃,流速为300μL/min。
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