液相色谱-质谱联用技术检测大鼠血浆、肾上腺及下丘脑中神经递质含量

建立了一种液相色谱-质谱联用技术同时检测γ-氨基丁酸(GABA)、5-羟色胺(5-HT)、酪氨酸(L-tyr)、多巴胺(DA)、去甲肾上腺素(NE)和肾上腺素(E)6种神经递质的方法,并将本方法用于大鼠肾上腺、下丘脑及血浆中神经递质的检测。以丹磺酰氯为衍生化试剂,以5-羟基色氨酸和咖啡酸为内标,采用Ulti Mate 3000 RSLC色谱系统、Thermo C_(18)色谱柱(50 mm×3 mm,2.7μm),流动相为0.1%甲酸和甲醇溶液梯度洗脱,流速0.2 mL/min,进样量2 μL,多反应检测(MRM),正离子模式。GABA、5-HT、L-Tyr、DA、NE、E线性检测范围分别为0.26~620.80 μmol/L、0.034~11.20 μmol/L、1.20~88.00 μmol/L、0.03~41.02 μmol/L、0.01~47.20 μmol/L、0.01~90.24 μmol/L,加标回收率为91.16%~116.20%。本方法可快速准确检测大鼠肾上腺、血浆及下丘脑中神经递质含量,为药理实验的中的指标分析提供了检测技术支持。     

高压液相色谱磷钼杂多酸修饰电极安培检测儿茶酚胺类神经递质的研究

研究了磷钼杂多酸修饰电极对去甲肾上腺素(NE)、肾上腺素(E)和多巴胺(DA)等儿茶酚胺类神经递质的催化氧化作用,探讨了催化机理,并采用高压液相色谱电化学方法对其进行了分离检测,NE、DA和E的线性范围(mol)依次为8.0×10^-11～2.0×10^-8、8.0×10^-11～2.0×10^-8、4.0×10^-11～2.0×10^-8;检测限(mol))依次为4.0×10^-11、4.0×10^-11、2.0×10^-11.7次平行测定的相对标准偏差(％)依次为1.6、2.0和4.5.将此法用于鼠脑组织中神经递质的测定,获得满意的结果.     

杯芳烃涂层毛细管分离单胺类神经递质

本文设计制作了一种对烯丙基杯 [4 ]芳烃涂层毛细管。用于毛细管电泳分离的结果表明 ,此管能分离 5 羟色胺 ( 5 HT)、多巴胺 (DA)、去甲肾上腺素 (NE)、肾上腺素 (E)等结构相近的单胺类神经递质 ,而无涂层管则不能。本涂层管在pH4～ 9范围内的电渗较无涂层管下降约 75% ,且当pH <8时 ,电渗随pH的变化接近于线性关系 ,有利于高重现分离。杯芳烃涂层毛细管不降低紫外检测灵敏度。其主要问题是酚羟基对胺类仍有吸附 (或静电 )作用     

大鼠脑组织中不同部位单胺类神经递质含量的HPLC测定

单胺类神经递质,多巴胺(DA)、去甲肾上腺素(NA)和肾上腺素(A)是哺乳动物和人类中枢重要的信息传递物质。了解脑组织中各部位单胺类神经递质的含量对于研究其生理功能、有关疾病的诊断和药物及手术疗效的评价均有重要的意义。     

掺铂／聚3-甲基噻吩修饰电极研究氨酪酸对大鼠脑纹状体中单胺类神经递质影响

氨酪酸(GABA)是哺乳动物中枢神经系统中重要的抑制性神经递质.本研究探讨了外源性GABA对单胺类神经递质及其代谢产物的影响.研制了一种新型掺铂/聚3-甲基噻吩玻碳修饰电极,利用原子力显微镜(AFM)对该电极进行了表征,通过循环伏安法(CV)和微分脉冲伏安法(DPV)研究了单胺类神经递质及其代谢产物在该修饰电极上的电化学行为,结果表明:该修饰电极对单胺类神经递质及其代谢产物有显著的催化作用,电极的灵敏度高,稳定性和重现性好.在液相色谱电化学检测实验中,去甲肾上腺素(NE)、多巴胺(DA)、3,4-二羟基苯乙酸(DOPAC)、5-羟色胺(5-HT)和5-羟吲哚乙酸(5-HIAA)在修饰电极上的电流响应灵敏.5种被测物的检测线性范围宽达3个数量级以上,检出限分别为2.0×10-10 mol/L(NE)、1.0×10-10 mol/L(DA)、3.0×10-10 mol/L(DOPAC)、4.0×10-10 mol/L(5-HT)和7.0×10-10 mol/L(5-HIAA).将该方法与微渗析活体取样技术联用,研究了GABA对大鼠脑纹状体中单胺类神经递质及其代谢产物的影响.     

双柱双泵高效液相色谱-电化学检测法测定尿中儿茶酚胺

儿茶酚胺类(CAs)化合物是人体内重要的生理活性物质,主要包括肾上腺素(E)、去甲肾上腺素(NE)及多巴胺(DA)。尿中CAs的水平不仅可作为嗜铬细胞瘤等疾病的诊断标准,而且与肾功能关系密切,因此具有重要的临床诊断和研究价值。 CAs在体液中含量低,以往用萤光分光光度法只能测E和NE的总量,无法测DA。采用HPLC-UV法虽能分别测定上述三种物     

雄黄对大鼠脑组织中单胺类神经递质及其代谢产物的影响

将32只Wistar大鼠随机分为对照组(口服0.5%羧甲基纤维素钠溶液)以及低剂量(0.3g/kg)、中剂量(0.9g/kg)和高剂量(2.7g/kg)雄黄混悬液处理组,通过4周连续灌胃给服雄黄混悬液;采用高效液相色谱-电化学法测定了大鼠脑组织中单胺类神经递质及其代谢产物的含量,研究了雄黄对大鼠脑组织单胺类神经递质及其代谢产物的影响.结果表明,与对照组比较,雄黄染毒组大鼠脑组织中NE、DOPAC和DA含量均呈增加趋势,而5-HIAA呈降低趋势.雄黄可对大鼠脑组织单胺类神经递质及其代谢产物产生影响,单胺类神经能系统可能是雄黄毒性作用的靶点之一.     

维生素E的交流示波极谱法测定

在 1mol/LKCl- 3mol/LHCl- 1mol/LH2 SO4 -无水乙醇 ( 0 .0 5∶1.5∶0 .1∶15V/V )混合溶剂中 ,以Ce(Ⅳ )与维生素E之间的氧化还原反应为基础 ,用Ce(Ⅳ )直接滴定维生素E ,在交流示波极谱图上 ,以Ce(Ⅳ )切口的出现指示滴定终点 ,从而测定VE。此法简单易行 ,且灵敏准确 ,终点直观。     

壳聚糖-碳纳米管修饰电极的电催化作用研究

报道了一种新型的壳聚糖-碳纳米管修饰电极(MC/GCE)的电化学特性.此修饰电极对神经递质去甲肾上腺素(NE)表现出很强的电催化效应,在pH 5.5的磷酸盐缓冲溶液中,NE的峰电流与浓度在5.0×10-6～1.0×10-4 mol/L范围内呈线性关系.此外,其它生物分子如多巴胺(DA)、尿酸(UA)在MC/GCE上也有很好的电化学响应.修饰电极表现出很好的稳定性和选择性.     

高效液相色谱法测定尿液中儿茶酚胺类物质

本文报道用高效液相色谱法(HPLC)分离,荧光检测器检测儿茶酚胺含量的新方法。该法可同时分离测定去甲肾上腺素(NE)、肾上腺素(E)和多巴胺(DA)三种组分,三种组分的浓度与其峰面积均呈良好的线性关系。方法灵敏度高,选择性好,测定结果令人满意,可在临床推广应用。     

径向电场调制毛细管电泳法用于神经递质分离

利用双向电场控制毛细管电泳系统，考察了神经递质的分离。在pH2.5的0.01mol/L磷酸盐缓冲体系中，通过加入20％（V／V）正丙醇，改善了多巴胺和5－羟色胺的分离效果，但仍不太理想。通过施加径向电场，可进一步提高分离度。本研究不仅拓宽了径向电场调控的样品分离范围，而且为生物活性物质的痕量分析提供了参考依据。     

高效液相色谱-荧光检测流速梯度法测定去卵巢小鼠脑内单胺类神经递质

 建立了同时检测去卵巢(OVX)小鼠脑内的去甲肾上腺素(NE)、多巴胺(DA)、 5-羟色胺(5-HT)3种神经递质的高效液相色谱方法。采用的色谱条件为：C18柱分离,荧光检测器检测,流动相为0.1　mol/L的 KH2PO4缓冲液-甲醇(体积比为9∶1),设置单泵流速梯度洗脱。方法的检测限(S/N=3)均在10 nmol/L以下,绝对回收率均在80％以上,样品日内测定峰面积的相对标准偏差在3.03%以下。该方法具有检测灵敏度高、样品制备简便等优点,对小鼠脑内的3种单胺类递质的检测具有实用价值。     

柱前衍生-高效液相色谱法同时测定血清中氨基酸类及单胺类神经递质

以邻苯二甲醛（o-phthalaldehyde，OPA）为衍生试剂，建立了柱前衍生-高效液相色谱（HPLC）同时测定血清中氨基酸类神经递质牛磺酸（Tau）、谷氨酸（Glu）、甘氨酸（Gly）、γ-氨基丁酸（γ-GABA）和单胺类神经递质多巴胺（DA）含量的分析方法。血清与乙醇以1：2的体积比混合，进行蛋白质沉淀后离心，取其上清液，氮吹至近干。前处理后的样品与OPA进行柱前衍生，衍生化产物采用Luna 5u C18色谱柱（250 mm×4.6 mm，5 μm）分离，以柠檬酸-乙酸钠缓冲溶液（pH 3.73）为流动相A、乙腈为流动相B进行梯度洗脱，流速为1.0 mL/min，柱温为30℃，检测波长为338 nm。5种神经递质在各自范围内线性关系良好（r²≥0.9866），检出限为0.10~0.40 μmol/L，不同加标水平下目标物的加标回收率为87.57%~115.31%，相对标准偏差均低于7.80%。方法操作简单，灵敏度高，精密度、线性关系和回收率等方法学指标较好，可实现血清中氨基酸类及单胺类神经递质的同时检测。     

高效液相色谱同时检测生物样本中8种单胺类神经递质

建立一种快速、准确测定生物样品中左旋多巴(L-DOPA)、去甲肾上腺素(NE)、肾上腺素(E)、多巴柯(DOPAC)、多巴胺(DA)、5-羟吲哚乙酸(5-HIAA)、高香草酸(HVA)及5-羟色胺(5-HT)8种递质含量的高效液相色谱- 电化学检测方法.使8种物质在25 min于单一流动相、单流速、单通道检测器情况下达到良好的分离效果.采用ESA MD-150色谱柱 (150 mm×3.2 mm, 3 μm),流动相为50 mmol/L柠檬酸、50 mmol/L无水乙酸钠、0.5 mmol/L 1-庚烷磺酸钠、0.5 mmol/L乙二胺四乙酸二钠、5 mmol/L三乙胺,pH 3.5,在甲醇浓度为5%～10%,流速0.3～0.5 mL/min, 柱温为30 ℃时,都能使8种物质很好分离,其中在甲醇浓度8%,流速0.4 mL/min,检测到前5种物质线性范围为0.005～10 nmol/L; 后3种0.001～10 nmol/L,8种物质相关系数在0.994～0.999之间,检出限在pmol/L水平;回收率在80.3%～102.1%之间,相对偏差在1.4%～4.8%之间.且对样本处理和保存方法进行了探讨.     

In vivo monitoring of the monoamine neurotransmitters in the rat brain by microdialysis coupled with the liquid chromatography dual electrochemical detector

The carbon film based ring-disk dual electrodes in the thin-layer radial flow cell are used as the dual electrochemical detector (DECD) for liquid chromatography (LC) to determine the monoamine neurotransmitters. Cyclic voltammetric experiments show there has great difference in the reversibility of the oxidative reactions of dopamine and ascorbate. Therefore the ring-disk dual electrode arrangement in the radial flow cell can effectively remove the interference of ascorbate and determine dopamine in the LC-DECD. In order to obtain the better collection efficiency (CE) and better peak current of analytes in the LC-DECD, several operational parameters have been investigated: flow rate, pH and the working potentials. Under the optimum conditions, the method shows a good stability and reproducibility to determine dopamine (DA), norepinephrine (NE), 5-hydroxytryptamine (5-HT), epinephrine (E) and 3,4-dihydroxyphenylacetic acid (DOPAC). The limits of detection are 0.1 pmol for DA, 0.1 pmol for NE, 0.1 pmol for 5-HT, 1.0 pmol for E and 0.1 pmol for DOPAC. The application of this method, coupled with microdialysis sampling, for the in vivo determination of the monoamine neurotransmitters in the striatum of the rat brain is satisfactory.     

Silica stationary phase-based on-line sample enrichment coupled with LC-MS/MS for the quantification of dopamine,serotonin and their metabolites in rat brain microdialysates

Accurate measurement of trace levels of endogenous compounds remains challenging despite advancements in analytical technologies. In particular, monoamine neurotransmitters such as dopamine (DA) and serotonin (5-HT) are polar compounds with low molecular weights, which complicates the optimization of retention and detection on liquid chromatography-mass spectrometry (LC-MS). Microdialysis is an important sampling technique to collect extracellular fluid from the brain of living animals. However, the very low basal concentrations of the neurotransmitters, small sample volume (maximum 30 μL) and the absence of matrix-matching calibrators are limitations of a microdialysate as an analytical sample. In the present study, an LC-MS/MS method was developed and fully validated for the quantification of DA, 5-HT and their main metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), in microdialysates from the rat nucleus accumbens shell. To improve the method sensitivity and accuracy, on-line sample enrichment using silica stationary phase was employed, before which any other sample pretreatment was not performed. The validation results proved the method to be selective, sensitive, accurate and precise, with acceptable linearity within calibration ranges. The lower limits of quantification were 0.025, 0.1, 0.5, 25 and 2.5 ng/mL for 5-HT, DA, 5-HIAA, HVA and DOPAC, respectively. This is a powerful analytical method to determine endogenous concentrations of those compounds in microdialysates from the rat nucleus accumbens and will be very useful to further study on the pathophysiological functions of monoamine neurotramsmitters in vivo.     

Derivatization chemistries for determination of serotonin, norepinephrine and dopamine in brain microdialysis samples by liquid chromatography with fluorescence detection

The present paper provides an overview on currently developed derivatization chemistries and techniques for determination of monoamine neurotransmitters serotonin (5-HT), norepinephrine (NE) and dopamine (DA) in microdialysis samples by microbore liquid chromatography with fluorescence detection. In mild alkaline conditions, 5-hydroxyindoles and catecholamines react with benzylamine (BA), forming highly fluorescent 2-phenyl-4,5-pyrrolobenzoxazoles and 2-phenyl(4,5-dihydropyrrolo) [2,3-f]benzoxazoles, respectively. However, for derivatization of DA a higher fluorescence intensity was achieved for reaction with 1,2-diphenylethylenediamine (DPE) rather than with BA, therefore for simultaneous determination of 5-HT, NE and DA in brain microdialysates, a two-step derivatization with BA followed by DPE was developed. The detection limits for 5-HT, NE and DA were 0.2, 0.08 and 0.13 fmol, respectively, in an injection volume of 20 microL, which corresponds to concentrations of 30, 12 and 19.5 pm, respectively in standard solution prior to derivatization. The experimental data presented demonstrate the ability of the technique to simultaneously monitor neuronally releasable pools of monoamine neurotransmitters in the rat and mouse brains at basal conditions and following pharmacological treatments or physiological stimuli. These techniques play an important role in drug discovery and clinical investigation of psychiatric and neurological diseases such as depression, schizophrenia and Parkinson's disease.     

蒙脱土修饰碳纤维电极的制备及其应用于活体测定脑神经递质

制备了蒙脱土修饰碳纤维电极,研究了其对神经递质多巴胺(DA)及5羟色胺(5HT)的富集作用,以及对负电性的代谢产物3,4二羟基苯乙酸(DOPAC)、5羟吲哚乙酸(5HIAA)及脑内大量存在的抗坏血酸(AA)排斥性能.该电极具有很高的灵敏度、分辨率和抗干扰性,对DA的检测下限达16×10^-8mol/L,优于传统的Nafion修饰电极,对5HT的检测下限为1×10^-7mol/L.在动物活体分析中,使用该电极成功地检测了大鼠双侧颈总动脉结扎再灌损伤时,脑纹状体中神经递质DA浓度的变化.     

Determination and Application of Nineteen Monoamines in the Gut Microbiota Targeting Phenylalanine,Tryptophan, and Glutamic Acid Metabolic Pathways

It has been reported that monoamine neurotransmitters can be produced by gut microbiota, and that several related metabolites of amino acids in these pathways are associated with nervous system (NVS) diseases. Herein, we focused on three pathways, namely, phenylalanine (Phe), tryptophan (Trp), and glutamic acid (Glu), and established an underivatized liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantification of nineteen monoamine neurotransmitters and related metabolites in the gut microbiota. The neurotransmitters and related metabolites included Phe, tyrosine (Tyr), l-dopa (Dopa), dopamine (DA), 3-methoxytyramine, Trp, hydroxytryptophan, 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), kynurenine (KN), kynurenic acid (KYNA), melatonin, tryptamine (TA), indole-3-lactic acid (ILA), indole-3-acetic acid (IAA), indolyl-3-propionic acid (IPA), Glu, gamma-aminobutyric acid (GABA), and acetylcholine (Ach). A fluoro-phenyl bonded column was used for separation, and the mobile phase consisted of methanol:acetonitrile (1:1) and water, with 0.2% formic acid in both phases. The compounds exhibited symmetric peak shapes and sufficient sensitivity under a total analysis time of 8.5 min. The method was fully validated with acceptable linearity, accuracy, precision, matrix effect, extraction recovery, and stability. The results showed that neurotransmitters, such as Dopa, DA, 5-HT, GABA, and Ach, were present in the gut microbiota. The metabolic pathway of Trp was disordered under depression, with lower levels of 5-HT, 5-HIAA, KN, KYNA, TA, ILA, IAA, IPA, and Glu, and a higher ratio of KYNA/KN. In addition, some first-line NVS drugs, such as sertraline, imipramine, and chlorpromazine, showed regulatory potential on these pathways in the gut microbiota.     

Simultaneous determination of monoamine and amino acid neurotransmitters in rat endbrain tissues by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification

A sensitive and efficient method for simultaneous determination of glutamic acid (Glu), gamma-amino-butyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat endbrains was developed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line mass spectrometric identification following derivatization with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). Different parameters which influenced derivatization and separation were optimized. The complete separation of five neurotransmitter (NT) derivatives was performed on a reversed-phase Hypersil BDS-C(18) column with a gradient elution. The rapid structure identification of five neurotransmitter derivatives was carried out by on-line mass spectrometry with electrospray ionization (ESI) source in positive ion mode, and the BCEOC-labeled derivatives were characterized by easy-to-interpret mass spectra. Stability of derivatives, repeatability, precision and accuracy were evaluated and the results were excellent for efficient HPLC analysis. The quantitative linear range of five neurotransmitters were 2.441-2x10(4) nM, and limits of detection were in the range of 0.398-1.258 nM (S/N=3:1). The changes of their concentrations in endbrains of three rat groups were also studied using this HPLC fluorescence detection method. The results indicated that exhausting exercise could obviously influence the concentrations of neurotransmitters in rat endbrains. The established method exhibited excellent validity, high sensitivity and convenience, and provided a new technique for simultaneous analysis of monoamine and amino acid neurotransmitters in rat brain.