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.     

Liquid chromatography with amperometric detection at a nano crystalline Ce-doped lead dioxide film modified electrode for determination of (R)-Salsolinol, (R)-N-methylsalsolinol and monoamine neurotransmitters in Parkinsonian patients' cerebrospinal fluid

The fabrication and application of a novel electrochemical detector (ED) with nano crystalline Ce-doped lead dioxide film chemically modified electrode (CME) for liquid chromatography (LC) were described. The Ce-doped PbO(2) film was characterized by X-ray diffractometer (XRD) and scanning tunnel microscope (STM). The electrochemical behaviors of (R)-Salsolinol ((R)-Sal) at the CME were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). It was found that the CME exhibited an efficiently electrocatalytic effect on the current responses of (R)-Sal, (R)-N-methylsalsolinol ((R)-NMSal) and monoamine neurotransmitters. In LC-ED, all (R)-Sal, (R)-NMSal, dopamine (DA), norepinephrine (NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) had good and stable current responses at the CME. The linear ranges of the nine analytes were over three orders of magnitude (R(2) > 0.995). The application of this method coupled with microdialysis sampling for the determination of (R)-Sal, (R)-NMSal and monoamine neurotransmitters in Parkinsonian patients' cerebrospinal fluid (CSF) was 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.     

高效液相-电化学检测法研究针刺对家兔血浆及脑组织中单胺类神经递质的影响

 通过高效液相 电化学检测法测定了电针刺激家兔肾旁穴前后其血浆及脑组织中单胺类神经递质去甲肾上腺素 (NE)、肾上腺素 (E)、多巴胺 (DA)和 5 羟色胺 (5 HT)含量的变化 ;研究了神经递质的变化与家兔繁殖能力的关系。该实验采用ODS柱 ,流动相为V(0 0 2mol/L柠檬酸三钠 0 0 5mol/L磷酸氢二钠 )∶V(甲醇 ) =95∶5的溶液 ,用电化学检测器检测。实验证明 ,血浆及脑组织中NE ,E ,DA和 5 HT的含量在针刺肾旁穴前后都有了显著的变化 ,说明针刺能激发家兔脑内神经元的活动 ,导致相应的递质含量的变化 ,同时使得血浆中相应含量也发生变化。     

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。     

Sensitive determination of norepinephrine,epinephrine, dopamine and 5‐hydroxytryptamine by coupling HPLC with [Ag(HIO6)2]5−–luminol chemiluminescence detection

Based on the enhancing effects of norepinephrine (NE), epinephrine (EP), dopamine (DA) and 5‐hydroxytryptamine (5‐HT) on the chemiluminescence (CL) reaction between [Ag(HIO₆)₂]^5? and luminol in alkaline solution, a high‐performance liquid chromatography (HPLC) method with CL detection was explored for the sensitive determination of monoamine neurotransmitters for the first time. The UV–visible absorption spectra were recorded to study the enhancement mechanism of monoamine neurotransmitters on the CL of [Ag(HIO₆)₂]^5? and luminol reaction. The HPLC separation of NE, EP, DA and 5‐HT was achieved with isocratic elution using a mixture of aqueous 0.2% phosphoric acid and methanol (5:95, v/v) within 11.0 min. Under the optimized conditions, the detection limits of NE, EP, DA, and 5‐HT were 4.8, 0.9, 1.9 and 2.3 ng/mL, respectively, corresponding to 17.6–96.0 pg for 20 μL sample injection. The recoveries of monoamine neurotransmitters in rat brain were >95.6% with the precisions expressed by RSD <5.0%. The validated HPLC‐CL method was successfully applied for the quantification of NE, EP, DA and 5‐HT in rat brain. This method has promising potential for some biological and clinical investigations focusing on the levels of monoamine neurotransmitters. Copyright © 2016 John Wiley & Sons, Ltd.     

高效液相色谱/电化学法测定大鼠血液和脑组织中单胺类物质的含量

建立了高效液相色谱／电化学检测法测定大鼠脑组织和血液中单胺递质及其代谢产物的方法。能同时测定去甲肾上腺素（NE）、肾上腺素（E）、3，4-二羟基苯乙酸（DOPAC）、多巴胺（DA）、高香草酸（HAV）、5-羟色胺（5-HT），并能和内标3，4-二羟卞胺（DHBA）分离良好。本方法快速、简便，回收率为92％-105％，线性范围2．8-680ng／mL，检出限为0．05ng（S／N=3）。本方法已应用在服用中药的大鼠下丘脑组织及血液的测定中，数据显示，本法能满足测定要求。     

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.     

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.     

Rapid procedure for chromatographic isolation of DOPA, DOPAC, epinephrine, norepinephrine and dopamine from a single urinary sample at endogenous levels

A three-step procedure has been investigated to extract 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), epinephrine (E), norepinephrine (NE) and dopamine (DA) from a single urinary sample with the object of obtaining extracts pure enough for specific fluorimetric assay. The procedure described in this paper results from the combination of urine purification on an aluminum oxide column, separation by ion-exchange chromatography of the DOPA-DOPAC fraction from catecholamines, and ether isolation of DOPAC from DOPA. The whole procedure is rapid and easily performed in one work day. Extraction recoveries were 72.4 +- 3.5%, 76 +- 2%, 85.7 +- 3.3%, 85.6 +- 1.4% and 92.4 +- 5.5% for DOPA, DOPAC, E, NE and DA respectively (n=6). The lowest amounts of the five catechols that could be detected in urinary samples by a combination of this extraction procedure and the methods of assay used in our laboratory were 15 ng for DOPA, 40 ng for NE, 20 ng for E, 152 ng for DA and 2.95 micrograms for DOPAC. Urinary volumes convenient for accurate estimation of each compound were 20 ml for healthy human subjects. For pathological or pharmacological purposes, 5 ml of human urine were sufficient. The daily excretion of DOPA, DOPAC, E, NE and DA found by this procedure agrees with data obtained by other authors in healthy subjects. In pathological samples, our three-step procedure led to lower amounts than methods using alumina purification only. The discrepancies between the two methods are discussed in terms of development of internal standards, relative specificity of fluorimetric assays, values of blank eluates, and the possibility of interference from unknown abnormal body metabolites or pharmacological drugs not eliminated by a single-step alumina purification.     

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     

Determination of 5-Hydroxytryptamine, Norepinephrine, Dopamine and Their Metabolites in Rat Brain Tissue by LC–ESI–MS–MS

A liquid chromatography–electrospray ionization tandem mass spectrometry method has been developed to perform the determination of 5-hydroxytryptamine (5-HT), norepinephrine (NE), dopamine (DA) and their metabolites, i.e., 5-hydroxyindole-3-acetic acid (5-HIAA), 4-hydroxy-3-methoxyphenylglycol (MHPG) sulfate, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat brain tissue. Analytes were separated on a Thermo C₁₈ column (4.6 mm × 250 mm, 5 μm, SN: 1245575T, Thermo electron corporation, USA) with a mobile phase of 0.05% formic acid/acetonitrile (92:8 for ESI⁺, 82:18 for ESI^?, v/v) at the flow-rate of 0.8 mL min^?1. The LC system was coupled to a Waters Micromass Quattro Premier XE tandem quadruple mass spectrometer. MS acquisition of 5-HT, NE and DA was performed in positive electrospray ionization multiple reaction monitoring (MRM) mode, while negative electrospray ionization MRM mode was used to monitor their metabolites. The calibration curves were linear within the concentration range of 4–4,450 ng mL^?1 for 5-HT, 4–4,110 ng mL^?1 for NE and 4–4,100 ng mL^?1 for DA (r ≥ 0.999). The limit of quantitation was 4 ng mL^?1. 5-HIAA, MHPG, DOPAC and HVA have good linearity within the range of 12–1,000 ng mL^?1(r ≥ 0.998) and the limit of quantitation was 12 ng mL^?1. The intra- and inter-day RSD were lower than 8.45%. The method is sensitive, fast, accurate and usable for quantity determination of monoamine neurotransmitters and their metabolites in neuropsychiatric diseases.     

Liquid chromatography-electrochemical detection for studying the effects of tetrahydrobiopterin on monoamine neurotransmitters in rat striatum

BH4 is the natural cofactor of two important amino acid hydroxylases: tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine from tyrosine, and tryptophan hydroxylase, the rate-limiting enzyme in the biosynthesis of serotonin from …     

Microdialysate analysis of monoamine neurotransmitters—A versatile and sensitive LC–MS/MS method

We have developed and validated a sensitive method for the simultaneous determination of some monoamine neurotransmitters like dopamine (DA), norepinephrine (NE) and serotonin (5-HT) in rat brain microdialysate using high-performance liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). Sensitivity enhancement has been achieved by amine derivatization with the reagent (5-N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) under mild conditions. The use of the selected reaction monitoring (SRM) mode has allowed detection of the analytes at a concentration of 30 pM (lower limit of quantification, LLOQ, signal-to-noise ratio higher than 5) with an accuracy of ≤3.80% and a precision of ±7.39 (%CV) for all neurotransmitters. Derivatization improves resolution and chromatographic retention times (3 min) by lipophilization. Linearity has been good (R > 0.99) over a large concentration range (30–50,000 pM). The intra and inter-batch accuracy and precision were not greater than 4.8% and 6.4%, respectively. Therefore, the method was successfully applied for monitoring the concentration changes of neurotransmitters in microdialysis samples deriving from striatum rat brain region after amphetamine administration (3 mg kg⁻¹, i.p.).     

The Electrochemical Behavior of Neurotransmitters at a Poly (Pyrrole‐ β‐Cyclodextrin) Modified Glassy Carbon Electrode

Abstract

A glassy carbon electrode surface was modified with an electropolymerized film made of pyrrole and β‐cyclodextrin 1∶1, in a 0.1 M LiClO₄ solution using cyclic voltammetry. The resulting modified electrode (Ppy/β‐CD) exhibits interesting electrocatalytic activity toward the electrochemical oxidation of neurotransmitters such as dopamine (DA) and norepinephrine (NE). Well‐resolved and reversible cyclic voltammograms (CVs) were obtained for these organic compounds in a 0.1 M H₂SO₄ solution. The effect of the pH on the voltammetric response of DA and NE was also investigated inside the range of pH 2.8–8.4. The oxidation current of norepinephrine increase linearly with the concentration inside the range of 4×10^?7 M–2×10^?6 M. The oxidation current of dopamine also followed the same trend range of 2×10^?6 M–10^?5 M. The detection limit was 6×10^?6 M for (DA) and 8×10^?7 M for (NE). As an example, the relative standard deviation for 1×10^?5 M of DA was 2.056%. The diffusion coefficients D for the molecules studied were determined by means of the measurement of the effect of the scan rate on the CVs of the neurotransmitters. All the results showed that the electron transfer was predominantly diffusion controlled at the conducting polymer/solution interface.

Interference phenomenon due to ascorbic acid (AA) toward the neurotransmitters was also investigated. The novel modified electrodes presented capability to resolve perfectly the AA and neurotransmitters oxidation peaks. This performance could be achieved even at a concentration 20 times higher than the neurotransmitters.

Finally, the newly fabricated Ppy/β‐CD film exhibits interesting analytical performances compared with other systems in the literature, such as higher sensitivity, rapid response, good mechanical stability and reproducibility.     

生物胺类神经递质的电化学检测研究进展

就1993-2005年生物胺类神经递质包括多巴胺、肾上腺素、去甲肾上腺素、5-羟色胺的各种电化学检测方法的应用研究和发展方向进行了评述。引用文献58篇。     

微柱液相色谱-双电极电化学法测定脑微透析液中单胺类递质的含量

用微柱液相色谱－双工作电极电化学检测法,分析了麻醉大白鼠纹状体微透析样品中的单胺类递质及其代谢产物的含量。无论在上游的氧化电位或下游的还原电位进行检测,至少在２～２００ｐｇ范围内,浓度与响应的线性关系良好,检测极限可达１ｐｇ以下。对于检测的优点和需要注意的问题作了扼要的讨论。     

High-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometric method for the analysis of catecholamines and metanephrines in human urine

An assay of norepinephrine (NE), epinephrine (E), dopamine (DA), normetanephrine (NE) and metanephrine (MN) based on high-performance liquid chromatography (HPLC) in combination with atmospheric pressure chemical ionization mass spectrometry (APcI-MS) is described. The catecholamines and metanephrines were extracted from urine and aqueous samples using Bio-Rex 70 cation-exchange resin and subjected to analysis by HPLC/APcI-MS. The separation was performed on a C18 column in 50 mM ammonium formate buffer, pH 3.0, using a flow rate of 0.8 mL/min. Acetonitrile was added post-column at a flow rate of 0.2 mL/min via a post-column addition tee. The total analysis time was 6.5 min. The quantitative analysis was performed using 3,4-dihydroxybenzylamine (DHBA) as the internal standard (I.S.). Selected ion monitoring detection was applied: m/z 170 (for NE), 184 (for E and NM), 154 (for DA), 198 (for MN) and 140 (for DHBA, I.S.). The limits of quantitation were 5 ng/mL for NE, E and DA and 2.5 ng/mL for NM and MN. The recovery ranged from 75 to 83% for each analyte. The method was found to be simple and highly selective for the determination of catecholamines and metanephrines in the urine of patients suspected of pheochromocytoma.     

Microchip capillary electrophoresis with a cellulose-DNA-modified screen-printed electrode for the analysis of neurotransmitters

A microfluidic chip based on capillary electrophoresis coupled with a cellulose-single-stranded DNA (cellulose-ssDNA) modified electrode was used for the simultaneous analysis of dopamine (DA), norepinephrine (NE), 3,4-dihydroxy-L-phenylalanine (L-DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), and ascorbic acid (AA). The modification of the electrode improved the electrophoretic analysis performance by lowering the detection potential and enhancing the signal-to-noise characteristic without surface poisoning of the electrode. The sensitivity of the modified electrode was about 12 times higher than those of the bare ones. The test compounds were separated using a 62 mm long separation channel at the separation field strength of +200 V/cm within 220 s in a 10 mM phosphate buffer (pH 7.4). The most favorable potential for the amperometric detection was 0.7 V (vs. Ag/AgCl). A reproducible response (relative standard deviation of 1.3, 1.3, 2.1, 3.1, 3.4% for DA, NE, L-DOPA, DOPAC, and AA, respectively, for n = 9) for repetitive sample injections reflected the negligible electrode fouling at the cellulose-ssDNA modified electrode. Square-wave voltammetric analyses reflected the sensitivities of the modified electrode for DA, NE, L-DOPA, DOPAC, and AA which were 1.78, 0.82, 0.69, 2.45, and 1.23 nC/microM with detection limits of 0.032, 0.93, 1.13, 0.31, and 0.62 microM, respectively. The applicability of this microsystem to real sample analysis was demonstrated.     

Determination of Catecholamine Concentration by Reverse-Phase Liquid Chromatography with Elecrtrochemical Detector in Plasma

The plasma catecholamine include mainly norepinephrine(NE),epinephrine(E) and dopamine (DA). The catecholamines(CA) are nervous mediator which are synthesized and release from sympathetic system. It is significant for diagnosing disease and researching surgical stress in clinic that determine the concentration of catecholamine in plasma.The analytic method had traditionally radioactive enzyme, fluorescent spectrometry and high performance liquid chromatography(HPLC). This paper describe an analytical technique that determine the concentration of NE, E and DA in plasma using HPLC-ED. The method is sensitive, fast and accuracy,and can be applied for the clinical diagnosis and laboratory research.