高效液相色谱-程序波长紫外检测法同时测定血浆中的色氨酸及其代谢物

建立了一种高效液相色谱-程序波长紫外检测法同时测定血浆中色氨酸(Trp)及其主要代谢产物犬尿氨酸(Kyn)和5-羟色胺(5-HT)。以茶碱为内标(IS),采用BDS-Hypersil-C8柱(150 mm×4.6 mm, 5 μm)分离。流动相为10 mmol/L醋酸钠缓冲液(pH 4.5)-乙腈(94:6, v/v),流速为0.6 mL/min;柱温为25 ℃;紫外检测波长设定: Kyn和IS为360 nm, 5-HT为220 nm, Trp为302 nm。3种物质的平均回收率为87%～113%;线性范围分别为3.97～400 μmol/L(Trp), 0.421～20.2 μmol/L(Kyn), 4.36～980 nmol/L(5-HT);检出限分别为0.134 μmol/L(Trp), 0.0160 μmol/L(Kyn), 2.03 nmol/L(5-HT)。利用该方法对15例抑郁症患者和15例健康志愿者的血浆进行测定,结果表明两组间Trp的代谢存在显著的差异。     

在线衍生-高效液相色谱法同时测定血浆中的色氨酸及其代谢物

建立了醋酸锌在线衍生高效液相色谱法同时测定血浆中色氨酸(Trp)、犬尿氨酸(Kyn)、5-羟吲哚乙酸(5-Hiaa)和犬尿喹啉酸(Kyna)的方法。以3-硝基酪氨酸为内标(IS),采用Hypersil C-18柱(250 mm×4.0 mm, 5 μ m),以250 mmol/L醋酸锌溶液(pH 5.5)-乙腈(95:5, v/v)为流动相,流速为0.8 mL/min,柱温30℃。荧光检测波长设定:5-Hiaa为278 nm(λ_ex)/343 nm(λ_em), Kyna为244 nm(λ_ex)/400 nm(λ_em);紫外检测波长设定:Kyn和IS为360 nm, Trp为302 nm。4种物质的回收率在91.62%~114.17%之间;线性范围分别为2.50~320.00 μ mol/L(Trp), 0.32~15.36 μ mol/L(Kyn), 3.27~104.60 nmol/L(5-Hiaa), 14.00~464.80 nmol/L(Kyna);检出限分别为0.078 μ mol/L(Trp), 0.056 μ mol/L(Kyn), 0.690 nmol/L(5-Hiaa), 1.290 nmol/L(Kyna)。利用该方法对30例正常孕妇和28例女性健康志愿者的血浆进行测定,结果表明两组间Trp, Kyn和Kyna含量有显著性差异。该方法操作简便,重复性好,灵敏度高,适合于临床检测。     

高效液相色谱法同时测定血清中的犬尿氨酸和色氨酸

建立了一种能同时检测血清中的犬尿氨酸(kynurenine,Kyn)和色氨酸(tryptophan,Trp)的高效液相色谱-紫外检测法。采用的色谱柱为Symmetry Shield RP-C18柱(150 mm×3.9 mm i.d.,5 μm),流动相为15 mmol/L乙酸钠-乙酸溶液(含2.7%乙腈,pH 3.6),流速为1.0 mL/min,紫外检测波长为225 nm。血清标本经5.0%(体积分数)高氯酸溶液去除蛋白质后取上清液直接进样分析测定。研究结果表明,Kyn保留时间为3.5 min,线性范围为0.098~49 μmol/L,最低检出浓度为0.02 μmol/L,回收率为90.82%~93.45%;Trp保留时间为8.1 min,线性范围为4.9~490 μmol/L,最低检出浓度为0.20 μmol/L,回收率为95.51%~98.67%。Kyn和Trp日内、日间测定的相对标准偏差均小于4%,苯丙氨酸、酪氨酸、5-羟色胺和犬尿喹啉酸等物质对该法均无干扰。该方法简便、快速、稳定、可行,可应用于临床和科研工作。     

超高效液相色谱-四极杆-飞行时间质谱检测和鉴定猪尿中氯丙那林的主要代谢产物

采用超高效液相色谱-四极杆-飞行时间质谱(UPLC/Q-TOF MS)检测和鉴定了猪尿中氯丙那林的主要代谢产物,并讨论了氯丙那林在猪体内的主要代谢途径。按10 mg/kg(b. w.)的剂量口服灌食氯丙那林,分别采集给药前及给药后的猪尿液样品。采用UPLC/Q-TOF MS对样品进行分析,并应用质量亏损过滤和离子色谱峰提取等数据处理技术,在给药后24 h内的猪尿中检测和鉴定了9种氯丙那林的代谢产物,其中,Ⅰ相代谢产物2种,Ⅱ相代谢产物7种。然后,根据氯丙那林原形和代谢产物的碎片离子特征,对代谢产物的结构进行鉴定。最后,根据所鉴定的代谢产物,推测氯丙那林在猪体内的代谢途径包括苯环羟基化、β -羟基和仲氨基的葡萄糖醛酸轭合、羟基化后的葡萄糖醛酸和硫酸轭合等。研究结果表明,羟基化氯丙那林及其轭合产物的相对含量大于60%,明显高于氯丙那林原形及其轭合产物,是尿液中的主要代谢产物。本研究将为确定氯丙那林在动物体内的残留标示物及加强对氯丙那林非法使用的监控提供科学依据。     

超高效液相色谱-四极杆-飞行时间质谱检测和鉴定猪尿中氯丙那林的主要代谢产物北大核心CSCD

采用超高效液相色谱-四极杆-飞行时间质谱(UPLC/Q-TOF MS)检测和鉴定了猪尿中氯丙那林的主要代谢产物,并讨论了氯丙那林在猪体内的主要代谢途径。按10 mg/kg(b.w.)的剂量口服灌食氯丙那林,分别采集给药前及给药后的猪尿液样品。采用UPLC/Q-TOF MS对样品进行分析,并应用质量亏损过滤和离子色谱峰提取等数据处理技术,在给药后24 h内的猪尿中检测和鉴定了9种氯丙那林的代谢产物,其中,Ⅰ相代谢产物2种,Ⅱ相代谢产物7种。然后,根据氯丙那林原形和代谢产物的碎片离子特征,对代谢产物的结构进行鉴定。最后,根据所鉴定的代谢产物,推测氯丙那林在猪体内的代谢途径包括苯环羟基化、β-羟基和仲氨基的葡萄糖醛酸轭合、羟基化后的葡萄糖醛酸和硫酸轭合等。研究结果表明,羟基化氯丙那林及其轭合产物的相对含量大于60%,明显高于氯丙那林原形及其轭合产物,是尿液中的主要代谢产物。本研究将为确定氯丙那林在动物体内的残留标示物及加强对氯丙那林非法使用的监控提供科学依据。     

高效液相色谱-荧光检测法同时测定血清中的犬尿氨酸和犬尿喹啉酸

建立了在线衍生、双波长高效液相色谱-荧光检测器同时检测血清中犬尿氨酸(kynurenine, Kyn)和犬尿喹啉酸(kynurenic acid, KYNA)含量的方法。血清标本经5%高氯酸溶液去除蛋白质后,上清液直接进样分析测定。采用的色谱柱为Hypersil C8柱;流动相为0.25 mol/L醋酸锌-50 mmol/L醋酸溶液(含3%乙腈),流速为1.5 mL/min。在0～10 min时间段,在激发波长和发射波长分别为365 nm和480 nm时检测Kyn;10 min后,在激发波长和发射波长分别变换为344 nm和404 nm时检测KYNA。Kyn的保留时间约为8.1 min,线性范围为98～19600 nmol/L,最低检出浓度为50 nmol/L,平均回收率为94.88%,日内、日间测定值的相对标准偏差(RSD)均低于4%。KYNA的保留时间约为13.0 min,线性范围为2.62～1047 nmol/L,最低检出浓度为0.11 nmol/L,平均回收率为102.72%,日内、日间测定的RSD均低于4%。苯丙氨酸、酪氨酸、色氨酸和5-羟色胺等物质对目标物的检测无干扰。71例健康成人血清中,Kyn和KYNA含量分别为(1.40±0.34) μmol/L和(24.22±8.67) nmol/L。该方法简便、快速、灵敏、特异,适于临床和科研应用。     

同位素稀释超高效液相色谱串联质谱测定尿液中苯系物和三氯乙烯代谢产物

建立同位素内标稀释,超高效液相色谱串联质谱(Uhra performance liquid chromatography-tandem mass sepctrometry,UPLC-MS/MS)同时测定尿中苯系物和三氯乙烯代谢产物的方法,并用于孕妇尿样的分析.尿样过经含同位素内标的醋酸铵缓冲液稀释10倍后,过滤,取滤液进样分析.采用C18色谱柱,梯度洗脱进行UPLC分离,电喷雾负离子扫描多反应监测(Multiple reaction monitoring,MRM)模式检测.方法检出限在0.0350～ 1.75 μg/L之间,方法的日内和日间精密度分别为1.2％～9.1％和2.0％～9.7％;用所建立的方法测定了650个孕妇尿液样品,并统计分析了其地区差异,加标回收率为85.0％～104％.所建立的方法快速灵敏,适合尿液样品的批量分析.     

人尿中诺龙和炔诺酮代谢物的气相色谱-质谱分析

本文对合成的19-去甲雄烷醇酮的四个差向异构体混合物进行气相色谱分离,对诺龙和炔诺酮用药后,留取的人尿样通过化学预处理,MSTFA/TMSI衍生化反应,GC/MS分析,确证了尿样中诺龙的两个主要代谢产物为19-去甲雄酮和19-去甲原胆烷醇酮,炔诺酮的两个主要代谢物是四氢炔诺酮(THINE)的两个形式,可能是3α-OH-5α-THINE和3α-OH-5β-THINE,在炔诺酮代谢前期,还发现有诺龙的上述两个代谢物存在。     

氯丙嗪在大鼠体内的代谢研究

采用气相色谱-质谱法(GC-MS)检测大鼠尿液中的氯丙嗪及其代谢产物,并对其进行质谱解析,推测氯丙嗪在大鼠体内的代谢途径.通过对Wistar大鼠进行灌胃服用药物后收集24 h内尿样,经固相萃取(SPE)净化提取后,采用GC-MS(EI,PCI)检测.对比空白尿液与阳性尿液萃取液的质谱图,并根据质谱裂解规律鉴定其结构.实...     

大鼠尿中硝基安定与尼美西泮及其代谢物的GC-MS检测

采用GC-MS比较了硝基安定和尼美西泮在大鼠体内产生的代谢产物,确定了尼美西泮在大鼠体内的代谢途径.给大鼠急性喂食硝基安定和尼美西泮,收集24 h内尿液,尿样用β葡萄糖醛酸酶水解,Oasis(R)HLB柱固相提取,以DB-35 MS柱为分离柱,气相色谱-质谱联用检测.从大鼠尿液中检出3种硝基安定代谢物:7-乙酰氨基硝基安定、7-氨基硝基安定和2-氨基-5-硝基苯基苯甲酮;4种尼美西泮代谢物:7-乙酰氨基尼美西泮、7-乙酰氨基硝基安定、7-氨基尼美西泮和2-氨基-5-硝基苯基苯甲酮,检出少量尼美西泮原体.并推断出2种药物在大鼠体内的代谢途径.     

Separation and determination of L-tryptophan and its metabolites by capillary micellar electrokinetic chromatography with amperometric detection

A high-performance method of capillary micellar electrokinetic chromatography (CMEKC) with amperometric detection (AD), using a newly designed pre-aligned electrochemical cell, has been developed for the separation and determination of L-tryptophan (Trp) and its eight metabolites including 3-hydroxy-L-kynurenine (3-HK), 5-hydroxy-L-tryptophan (5-HTP), L-kynurenine (KN), 5-hydroxyindole-3-acetic acid (5-HIAA), xanthurenic acid (XA), indole-3-pyruvic acid (IPA), 5-hydroxytryptamine (5-HT), and tryptamine (Tryp). A carbon disk electrode was used as the working electrode and the optimal detection potential was 0.85 V (versus Ag/AgCl). At 24 kV of applied voltage, the nine compounds were completely separated, within 23 min, in a 10 mol/L Na(2)HPO(4)-NaOH buffer (pH 11.0) containing 40 mmol/L sodium dodecyl sulfate (SDS) and 3% methanol (v/v). A good linear relationship was obtained for all analytes in this paper and the detection limits of 3-HK, 5-HTP, KN, Trp, 5-HIAA, XA, IPA, 5-HT, and Tryp were 7.42, 5.18, 34.6, 3.99, 15.1, 12.7, 260, 6.72, and 8.01 nmol/L, respectively. This method has been applied to analyze the metabolism of Trp in rabbit urine.     

Simultaneous determination of tryptophan,kynurenine, kynurenic acid,xanthurenic acid and 5‐hydroxytryptamine in human plasma by LC‐MS/MS and its application to acute myocardial infarction monitoring

Reliable methods for the determination of tryptophan and its metabolites are vital to the monitoring of biochemical states during the initiation and progression of cardiovascular disease. In the present study, a single‐run liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous determination of tryptophan (Trp) and its metabolites, including kynurenine (Kyn), kynurenic acid (KA), xanthurenic acid (XA) and 5‐hydroxytryptamine (5‐HT), in human plasma. The plasma samples were prepared using a protein precipitation approach, and the chromatographic separation was performed by gradient elution on a C₁₈ column within a total analysis time of 3.5 min. The calibration ranges were 40–20,000 ng/mL for Trp, 4–2000 ng/mL for Kyn, 0.2–100 ng/mL for KA, 0.4–200 ng/mL for XA and 1–500 ng/mL for 5‐HT, and the precision and accuracy were acceptable. The evaluation of recovery and internal standard‐normalized matrix effect proved that the sample preparation approach was effective and the matrix effect could be negligible. The newly developed method was successfully applied to the analysis of plasma samples from healthy individuals and myocardial infarction patients. The findings suggested that the plasma concentrations of Trp, Kyn, 5‐HT as well as the concentration ratios of Kyn/Trp and Trp/5‐HT might serve as biomarkers for the monitoring of acute myocardial infarction.     

色氨酸及其主要代谢产物的分离和在生物样品中的测定

建立以乙酸缓冲系统和甲醇作流动相、电化学和紫外检测器联用的高效液相色谱法,分离和测定了色氨酸经5-羟色胺和犬尿酸原两条主要代谢途径的8种代谢物。使用三氯乙酸作离子对试剂以延长3-羟犬尿酸原的保留时间,分析了流动相pH值和三氯乙酸浓度对各物质分离的影响及检测条件。结果表明,pH值及三氯乙酸浓度对各物质保留时间有明显影响,可作为控制分离的主要因素。此外,对生物样品中各物质分离和检测条件进行了讨论。     

Ultrasensitive detection of indoleamines by combination of nanoparticle-based extraction with capillary electrophoresis/laser-induced native fluorescence

For the first time, citrate-capped gold nanoparticles (citrate-AuNPs) have been used for the selective extraction of indoleamines – 5-hydroxytryptophan (5-HTP), tryptophan (Trp), tryptamine (TA), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) – prior to their analysis by capillary electrophoresis/laser-induced native fluorescence (CE/LINF). The extinction spectra obtained for the citrate-AuNPs in the presence of indoleamines revealed that 5-HTP, 5-HT, and 5-HIAA were extracted mainly because of van der Waals interactions between the indole ring and the citrate-AuNPs (hydrophobic surface), while 5-HT and TA were extracted by electrostatic attractions between the amine group of the indoleamines and the citrate ligands adsorbed on the AuNP surface. The extracted indoleamines could be liberated from the AuNP surface by the addition of high concentrations of 2-mercaptoethanol (2-ME), which binds strongly to the AuNPs. The sensitivity of this method to indoleamines could be significantly enhanced by increasing the AuNP concentration, incubation time, and sample volume. Under optimal extraction and separation conditions, the combination of NP-based extraction and CE-LINF provided 48-, 4077-, 985-, 920-, and 4030-fold improvements in the limits of detection (signal-to-noise ratio of 3) for 5-HTP, Trp, TA, 5-HT, and 5-HIAA as compared to the analysis of five indoleamines by CE-LINF. In addition, this proposed method was successfully used for the determination of TA and 5-HT in urine.     

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.     

高效液相色谱-串联质谱法测定兔尿中溴氰菊酯及其毒性生物标志物

建立了一种应用高效液相色谱-串联质谱（HPLC-MS/MS）测定兔尿中与溴氰菊酯毒性相关的多种生物标志物的检测方法。分析物包括溴氰菊酯及其代谢产物1R,3R-二溴菊酸、3-苯氧基苯甲酸,以及5种生物标志物5-羟色胺、5-羟基吲哚乙酸、3-硝基丙酸、8-羟基脱氧鸟苷和6-甲氧基鸟嘌呤。样品经硅藻土基质固相分散萃取、三氯乙酸沉淀蛋白质和HLB固相萃取小柱净化,使用电喷雾离子源,在多反应监测模式下正负切换采集测定,其中溴氰菊酯、5-羟色胺、5-羟基吲哚乙酸、8-羟基脱氧鸟苷和6-甲氧基鸟嘌呤采用正离子模式,1R,3R-二溴菊酸、3-苯氧基苯甲酸和3-硝基丙酸采用负离子模式。基质校准曲线外标法定量。结果表明,7种生物标志物在各自的浓度范围内线性关系良好（R²不小于0.9914）,5-羟基吲哚乙酸的检出限和定量限分别为20 μg/L和50 μg/L,其余化合物的检出限和定量限分别为0.2~5.0 μg/L和0.5~10 μg/L;在兔尿中3个不同添加水平的平均回收率为74.2%~98.7%,相对标准偏差（RSD）不大于12%,方法简单、快速、准确、灵敏,可作为溴氰菊酯暴露评估的检测方法。     

Development of a 6-hydroxychroman-based derivatization reagent: application to the analysis of 5-hydroxytryptamine and catecholamines by using high-performance liquid chromatography with electrochemical detection

We developed a novel derivatization reagent, (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (NPCA), for electrochemical (EC) detection in HPLC. NPCA was synthesized from (R)-(+)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (alpha-CA), which exhibits intense EC response. NPCA successfully yielded alpha-CA derivatives of primary amines by a two-step derivatization procedure. Following pre-column derivatization with NPCA, a simultaneous determination of alpha-CA derivatives of neuroactive monoamines [dopamine (DA), epinephrine, and 5-hydroxytryptamine (5-HT)], their monoamine oxidase metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindole-3-acetic acid) and their catechol-O-methyltransferase metabolites [3-methoxytyramine (3-MT) and normetanephrine (NMN)] was completely achieved using our HPLC-EC method. Using an HPLC equipped with coulometric electrode-array detection system, the resultant alpha-CA derivatives of NMN, 5-HT, DA and 3-MT showed intense EC responses, that were approximately 1.3, 1.4, 1.1 and 1.4-fold higher than the corresponding native forms, respectively. The detection limits were in the range of approximately 16-60 fmol on column (signal-to-noise ratio 3). The proposed HPLC method was applied to determine 5-HIAA, HVA, alpha-CA-5-HT and alpha-CA-DA in rat urine. As a consequence, these analytes were successfully determined with satisfactory precisions.     

Simultaneous determination of serotonin and 5-hydroxyindole-3-acetic acid in human urine by automated precolumn derivatization and semi-microbore column liquid chromatography with fluorescence detection.

An improved high-performance liquid-chromatographic (HPLC) assay for the simultaneous determination of serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in human urine is reported. Following the automated precolumn derivatization of urinary 5-HT and 5-HIAA with benzylamine, the derivatives are separated by isocratic elution on a reversed-phase C18 semi-microbore column, and are fluorometrically detected at an excitation wavelength of 345 nm and an emission wavelength of 480 nm. The detection limits of 5-HT and 5-HIAA are 7 and 5 nmol/l in urine (0.7 and 0.5 fmol/20-microl injection). The proposed automatic method permits a highly selective and sensitive determination of 5-HT and 5-HIAA in human urine without any sample purification.