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.     

Study of catecholamines in patient urine samples by capillary electrophoresis.

Capillary zone electrophoresis with photodiode array detection at 220 nm was used for analysis of catechol compounds in human urine. The method was optimized with reference compounds 3,4-dihydroxybenzylamine, adrenaline, noradrenaline, normetanephrine, dopamine, dopac (homogensitic acid), methanephrine, vanillyl-mandelic acid, 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid and 3-methoxytyramic acid at pH 4.0 and 8.0 for their electrophoretic separation. The UV spectra of the catechols were detected at a concentration of 20 microM. Repeatability of the method calculated using the absolute migration times of the catechols was below 1.5% and using the peak areas below 5%. The patient samples were hydrolyzed by 0.5 M acid or base solutions. In the studies, a few patient samples were analyzed using 3,4-dihydroxybenzylamine as an internal standard. In the hydrolysis steps needed for their detection in urine, all the other catecholamines, except 5-HIAA, did not decompose to detectable species at 220 or 254 nm. The concentrations of the catecholamines observed in real samples were at nM levels.     

Alcohol biomarker analysis: simultaneous determination of 5-hydroxytryptophol glucuronide and 5-hydroxyindoleacetic acid by direct injection of urine using ultra-performance liquid chromatography-tandem mass spectrometry

A direct ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) for simultaneous measurement of urinary 5-hydroxytryptophol glucuronide (GTOL) and 5-hydroxyindoleacetic acid (5-HIAA) was developed. The GTOL/5-HIAA ratio is used as an alcohol biomarker with clinical and forensic applications. The method involved dilution of the urine sample with deuterated analogues (internal standards), reversed-phase chromatography with gradient elution, electrospray ionisation and monitoring of two product ions per analyte in selected reaction monitoring mode. The measuring ranges were 6.7-10 000 nmol/l for GTOL and 0.07-100 micromol/l for 5-HIAA. The intra- and inter-assay imprecision, expressed as the coefficient of variation, was below 7%. Influence from ion suppression was noted for both compounds but was compensated for by the use of co-eluting internal standards. The accuracy in analytical recovery of added substance to urine samples was 96 and 98%, respectively, for GTOL and 5-HIAA. Method comparison with GC-MS for GTOL in 25 authentic patient samples confirmed the accuracy of the method with a median ratio between methods (GC-MS to UPLC-MS/MS) of 1.14 (r(2) = 0.975). The difference is explained by the fact that the GC-MS method also measures unconjugated 5-hydroxytryptophol naturally present in urine. The comparison with data for 5-HIAA obtained by an HPLC method demonstrated a median ratio of 1.05 between the methods. The UPLC-MS/MS method was capable of measuring endogenous GTOL and 5-HIAA levels in urine, which agreed with the literature data. In conclusion, a fully validated and robust direct method for the routine measurement of urinary GTOL and 5-HIAA was developed.     

Voltammetric determination of 5-hydroxydole-3-acetic acid in human gastric juice

A method for the determination of the major serotonin metabolite — 5-hydroxyindole-3-acetic acid (5-HIAA) in human gastric juice by cyclic voltammetry was described. The measurement conditions were investigated. The potential window was chosen from +0.1 to +0.9 V, the supporting electrolyte was 0.025 M PBS solution (pH 2.0). The method allowed determination in the concentration range from 2.0 ×10⁻⁷ to 2.0 ×10 ⁻⁵ M and a detection limit of 80 nM. When samples of gastric juice were analyzed with the method, we obtained the mean content of 5-HIAA in the gastric juice. Meanwhile, interference from other ions and substances were examined. The experimental results indicate that the method for the determination of gastric juice samples is successful.     

Simultaneous determination of the major acidic metabolites of catecholamines and serotonin in urine by liquid chromatography with electrochemical detection after a one-step sample clean-up on Sephadex G-10; influence of vanilla and banana ingestion

A simple method is described for the simultaneous determination of vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HIAA) and homovanillic acid (HVA) in urine. The compounds are isolated by a one-step sample clean-up on Sephadex G-10, separated by ion-pair reversed-phase liquid chromatography and detected electrochemically. A single analysis is completed within 65 min. Sample clean-up did not cause losses of the compounds of interest. The detection limits in urine were 0.4, 0.8, 1.0 and 1.6 mumol/l for VMA, DOPAC, 5-HIAA and HVA, respectively. 3,4-Dihydroxymandelic acid and vanillic acid (VA) were also detectable, but, under the chromatographic conditions used, they were not resolved from interfering components. VA and 5-HIAA could be analysed separately in the Sephadex G-10 eluate if more restrictive sampling conditions were used. Ingestion of bananas caused an increase of VMA, DOPAC, 5-HIAA and HVA in 24-h urine. After ingestion of vanilla an increased excretion of VA was observed, while the excretion of VMA, DOPAC and HVA was unaffected.     

鼠脑组织中6-羟基-1-甲基-1,2,3,4-四氢-β-咔啉及其相关生物胺的高效液相色谱-电化学检测

建立了大鼠脑组织中6-羟基-1-甲基-1,2,3,4-四氢-β-咔啉(6-OH-MTHβC)、5-羟色胺(5-HT)和5-羟吲哚乙酸(5-HIAA)含量的高效液相色谱-库仑阵列电化学检测(HPLC-ECD)方法。采用的色谱柱为DiscoveryHS F5柱(250 mm×4.6 mm,5 μm),流动相为缓冲液（40 mmol/L柠檬酸+20 mmol/L磷酸氢二钠+0.3 mmol/L乙二胺四乙酸二钠,pH 4.0）-甲醇（体积比为78∶22）混合液,流速为1 mL/min。6-OH-MTHβC、5-HT、5-HIAA在1.0～500.0 μg/L范围内线性关系良好(r>0.9992),检出限分别为0.56,0.26,0.53 μg/L,日内和日间精密度(以相对标准偏差表示)均低于6.1%,回收率分别为87.1%～98.2%,87.0%～95.3%,90.1%～97.7%。用该方法检测新生7 d的SD胎鼠脑内6-OH-MTHβC及5-HT、5-HIAA的含量,发现SD胎鼠在急性酒精中毒8 h后6-OH-MTHβC显著上升(P<0.05);而5-HT和5-HIAA的含量有所下降,但无显著性差异。该法简便、稳定、灵敏度高,适用于测定鼠脑组织中6-OH-MTHβC和5-HT,5-HIAA含量的相关研究。     

Rapid determination of 5-hydroxytryptamine in whole blood by liquid chromatography with fluorimetric detection.

A rapid and simple reversed-phase (using muBondapak C18 as the stationary phase) liquid chromatographic method with fluorimetric detection is described for the quantitation of 5-hydroxytryptamine in whole blood. The rapidity and simplicity of the method are explained by the absence of a pretreatment. 5-Fluoro-dl-tryptophan was used as internal standard. The mobile phase was 0.01 M phosphate buffer (pH 4.5) with 0.0025 M 1-heptanesulfonic acid and 20% methanol. The detection wavelength were 302 nm for excitation and 340 nm for emission. Analysis time was 10 min with retention times for 5-hydroxytryptamine of 9 min and for 5-fluoro-dl-tryptophan of 7 min. This method is proposed for biological exploration of psychiatric disorders involving 5-hydroxytryptamine and would be useful for tryptophan.     

Impact of halides on the simultaneous separation of aromatic amines and their acidic metabolites by capillary electrophoresis with laser-induced native fluorescence detection under acidic conditions

This paper describes a simple, sensitive, efficient, and rapid method for simultaneous analysis of biologically active amines and acids by capillary electrophoresis in conjunction with laser-induced native fluorescence detection (CE-LINF) using a diode pumped solid state nanolaser at 266 nm. In order to optimize resolution of the amines that were prepared in 10.0 mM formate-Tris (FT) solutions, 10.0 mM FT solutions with and without containing halides were used to fill the capillary and reservoirs, respectively. The electrophoretic mobilities of tryptamine (TA) and serotonin (5-HT) at pH 4.0 decrease with the increase in halide concentration (0-10.0 mM). Taken together with a great effect of iodide than other halides, we suggest that the formation of ion pairs is a main contributor for altering the migration of the amines. In order to simultaneously analyze the amines and their metabolites (acids) at low pH, a high bulk EOF is required. The analysis of 10 anlytes including amines and acids was completed within 12 min by CE-LINF using a capillary treated with 0.5M NaOH and then filled with 10.0 mM FT solutions (pH 4.0) containing 10.0 mM KCl prior to analysis. The limits of detection for TA and 5-hydroxyindole-3-acetic acid (5-HIAA) are 0.12 and 6.0 nM, respectively. The present method has been further validated by analyzing urine samples, with an RSD less than 3.1% (migration times) and 3.9% (concentration).     

Mass fragmentographic determination of 5-hydroxytryptamine and 5-hydroxyindole-3-acetic acid in brain tissue using deuterated internal standards.

A mass fragmentographic method for the determination of 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in the same extract of rat brain tissue is decribed. Deuterium-labelled analogues were used as internal standards. 5-HT and 5-HIAA were separated by solvent extraction and pentafluoropropionyl derivatives were prepared for the mass fragmentographic analysis. Multiple ion analysis confirmed the identity of 5-HT and 5-HIAA in the rat brain. At the mass numbers used routinely for the determination of 5-HT and 5-HIAA in the rat brain. At the mass numbers used routinely for the determination of 5-HT and 5-HIAA, the experimental error was below 3% (calculated from mean values of 0.05 and 0.24 nmole, respectively). The recovery of the authentic compounds added to brain extracts was more than 95%. The levels of 5-HT and 5-HIAA in the rat brain were 2.95 +/- 0.16 and 0.64 +/- 0.18 nmole/g, respectively. More than 100 samples could be analyzed within 3 days. The presence of 5-hydroxytryptophol in rat brain was also investigated, but none could be detected either as a conjugate or as the free alcohol.     

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.     

Highly sensitive analysis of cholesterol and sitosterol in foods and human biosamples by liquid chromatography with fluorescence detection

A simple and sensitive method is described for the quantitative analysis of important animal and plant sterols (cholesterol and sitosterol) by liquid chromatography with fluorimetric detection. The method is based on the derivatization of cholesterol and sitosterol with a fluorescent reagent (naproxen acyl chloride) in toluene. The resulting derivatives were isocratically separated on a C(8) column with a mixed solvent of methanol-isopropanol-water (90:5:5, v/v) as a mobile phase and monitored with a fluorimetric detector (excitation 231 nm and emission 352 nm). The linear range for the quantitation of cholesterol or sitosterol was 0.1-2.0 microM with a detection limit (S/N=3 with 10 microl injected) of about 25 nM. Recoveries of cholesterol spiked in milk (n=5) ranged over 99-104% with relative standard deviations (RSD) less than 6.0%. Application of the method to the analysis of cholesterol or sitosterol in milk, saliva and urine proved simple and feasible.     

Determination of residues of azamethiphos in salmon tissue by liquid chromatography with fluorescence detection.

A liquid chromatographic (LC) method with fluorescence detection (FLD) is described for determining residues of the pesticide azamethiphos (AZA) in salmon tissue. The sample is extracted with ethyl acetate, centrifuged, dehydrated with anhydrous sodium sulfate, evaporated, reconstituted in water, and defatted with hexane. The aqueous phase is passed through a C18 solid-phase extraction (SPE) column. The SPE column is eluted with methanol, and the eluate is evaporated to dryness and then taken up in 10% acetonitrile (ACN) in water. The analyte is determined by LC using a C18 column, ACN-H2O (32 + 68) mobile phase, and FLD with excitation at 230 nm and emission at 345 nm. Composited salmon tissues were fortified with AZA at 5, 10, 21, 42, and 83 ng/g or ppb (target level, X = 10 ng/g). Overall recoveries were 86%, with between-day variability of 5.3%. The method detection limit was calculated as 1.2 ppb AZA based on a 5 g sample. The limit of quantitation as determined empirically by this method is the lower limit of the standard curve, approximately 5 ppb.     

四川凉山杜鹃及其中成药药效成分的研究Ⅰ.槲皮素和山萘酚的快速直接测定

 用反相 HPLC法测定了凉山杜鹃叶子、浸膏和口服液中的两种黄酮类物质——槲皮素和山萘酚的含量。分析方法简便、快速、灵敏。所用分析柱为 Zorbax SB-C1 8,流动相为体积分数为 60 %的甲醇水溶液 ,检测波长为3 60 nm。     

高效液相色谱法测定柳树皮提取物中的水杨甙

 建立了一种测定柳树皮提取物中水杨甙的反相高效液相色谱法。该法采用KromasilC18(4 6mmi.d .× 2 5 0mm ,5 μm)色谱柱 ,以甲醇 0 0 1mol/LKH2 PO4 缓冲液 (pH 4 0 1) (体积比为 15∶85 )的混合溶液作流动相 ,流速为1 0mL/min ,紫外检测波长为 2 6 5nm ,灵敏度为 0 0 4AUFS。在 8 89mg/L～ 2 84 40mg/L的范围内 ,水杨甙的峰面积Y与其质量浓度X的线性关系良好 ,回归方程为Y =- 2 91 5 2 6 0 +5 0 0 0 0 34X(r =0 9998)。水杨甙的平均回收率为 96 1%～ 10 1 2 % (n =5 ) ,相对标准偏差 (RSD)为 1 43%。该法操作简便、快速、准确。     

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。     

Magnetic nanoparticles grafted with β-cyclodextrin for solid-phase extraction of 5-hydroxy-3-indole acetic acid

We describe the synthesis of ß-cyclodextrin modified magnetic nanoparticles (CD-mNPs) as a material for solid-phase extraction of the cancer biomarker 5-hydroxy-indole-3-acetic acid (5-HIAA) from urine. The CD-mNPs were characterized by TEM, FTIR, and XRD, and the kinetics and adsorption isotherms were studied. The strong interaction between the CD-mNPs and 5-HIAA is the main driving force for recognition and extraction, while the magnetic core of the NPs allows their separation from the sample matrix. Recovery of 5-HIAA from the adsorbent using an adequate solvent regenerated the adsorbent for further use. 5-HIAA was then quantified by fluorometry of its complex with ß-CD. The method works in the 1?×?10^?7 to 1?×?10^?5 mol L^?1 (R² 0.9982–0.9996) concentration range, and the limits of detection (3σ) and quantification (10 σ) of the method are 1.2?×?10^?8 mol L^?1 and 4.01?×?10^?8 mol L^?1 5-HIAA, respectively. The recovery of 5-HIAA from urine samples spiked with 5-HIAA in three concentrations (1.4?×?10^?6, 4.50?×?10^?6 and 1.0?×?10^?5 mol L^?1) are within 63?±?3 %.

反相高效液相色谱法测定盐酸索他洛尔

 建立了用于盐酸索他洛尔的含量测定、有关物质的检查和稳定性考察的 RP-HPL C法。采用 ODS柱、体积分数为 0 .1 %的乙酸水溶液 -乙腈 (体积比为 80∶ 2 0 )为流动相的色谱条件 ,以磺胺二甲基嘧啶为内标物 ,测定的线性范围为 5～ 45 mg/ L(r=0 .9991 ) ,日内精密度为 0 .2 0 % ,日间精密度为 0 .93 %。     

Determination of estriol and creatinine in urine by high performance liquid chromatography

A quantitative method for the determination of estriol (E3) and creatinine (C) in random urine by high performance liquid chromatography is described. The mobile phase was a mixed solution of methanol and phosphate buffer (0.025 M, pH 6.5) and the detection wavelength was at 205 nm. The method was simple, rapid and accurate. The OCV for E3 and C using this method were 1.7-3.4% and 2.2-2.5%, respectively. The RCV for E3 and C were 6.2-7.0% and 4.5-6.9%, respectively. The recoveries were 87-104% for E3 and 98-103% for C, respectively. The method has been used for clinical determinations.     

High Pressure Liquid Chromatographic Analysis and Dissolution of Famotidine in Tablet Formulation

Abstract

A reversed phase high pressure liquid chromatographic method was developed for the quantitation of famotidine in tablet formulation using a mobile phase consisting of 0.1M phosphate buffer (84%), acetonitrile (11%) and methanol (5%) at a pH of 6.5. The detection wavelength was set at 285 nm. The method is precise and adaptable for quality control purposes. The use of the analytical method hi studying tablet dissolution is described.     

Determination of flumequine in channel catfish by liquid chromatography with fluorescence detection.

Rapid methods are described for determination of flumequine (FLU) residues in muscle and plasma of farm-raised channel catfish (Ictalurus punctatus). FLU residues were extracted from tissues with an acidified methanol solution, and extracts were cleaned up on C18 solid-phase extraction cartridges. FLU concentrations were determined by liquid chromatography (LC) using a C18 analytical column and fluorescence detection (excitation, 325 nm; emission, 360 nm). Mean recoveries of FLU from fortified muscle were 87-94% at 5 levels ranging from 10 to 160 ppb (5 replicates per level). FLU recoveries from fortified plasma were 92-97% at 5 levels ranging from 20 to 320 ppb. Limits of detection (signal-to-noise ratio, 3:1) for the method as described were 3 and 6 ppb for muscle and plasma, respectively. Relative standard deviations (RSDs) for recoveries were < or = 12%. Live catfish were dosed with 14C-labeled or unlabeled FLU to generate incurred residues. Recoveries of 14C residues throughout extraction and cleanup were 90 and 94% for muscle and plasma, respectively. RSDs for incurred FLU at 2 levels in muscle and plasma ranged from 2 to 6%. The identity of FLU in incurred tissues was confirmed by LC/mass spectrometry.