New antioxidant mixture for long term stability of serotonin, dopamine and their metabolites in automated microbore liquid chromatography with dual electrochemical detection

An automated microbore liquid chromatographic assay with dual electrochemical detection is described for the determination of serotonin, dopamine and their metabolites, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Due to the chemical instability of the compounds, the addition of an antioxidant is required for automated analysis over a long period of time (e.g., 20 h). Therefore, the time stability of these substances was tested with different antioxidants. The stability for serotonin and 5-hydroxyindoleacetic acid was poor in acidic medium containing Na₂EDTA but could greatly be improved by the addition of

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-cysteine and ascorbic acid. Using this assay, the neurotransmitters and their metabolites could easily be determined in microdialysates obtained from different rat brain areas.     

New dual electrochemical detector for microbore liquid chromatography determination of dopamine and serotonin in rat striatum dialysates

A new type of liquid chromatographic (LC) dual thin-layer amperometric detector for the simultaneous measurement of trace levels of dopamine and serotonin in microdialysates is described. The concentrations of these analytes in rat dialysates are usually in the sub-nanomolar concentration range (typically, 0.10–5.00 pg in 5-μl dialysates). With this dual electrode, a glass-lined microbore column provides excellent sensitivity, selectivity, and separation. In addition, a three- to five-fold improvement in anodic current or cathodic responses over conventional dual electrodes in microbore LC can be achieved. Due to the irreversible electrochemical properties of some interference peaks, this dual electrode provides reliable measurement of dopamine based on the cathodic signal. The detection limit (signal-to-noise RATIO=3) of this assay is 0.02 pg per injection for dopamine or serotonin. This new dual electrode allows the simultaneous measurements of basal dopamine and serotonin in rat striatum dialysates without the use of re-uptake inhibitors in perfusion medium.     

Determination of serotonin, catecholamines and their metabolites by direct injection of supernatants from chicken brain tissue homogenate using liquid chromatography with electrochemical detection

An isocratic liquid chromatographic method with electrochemical detection for the determination of

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-3,4-dihydroxyphenylalanine, dopamine, norepinephrine, epinephrine, serotonin, and their major metabolites, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylacetic acid and 5-hydroxyindole-3-acetic acid in chicken brain tissue is described. Chickens were killed at different ages, the brains were quickly frozen and 300-μm cryostat sections were made. From these sections, two to six tissue micropunches (1 mm in diameter) were punched out from 20 different areas of the hypothalamus and homogenated in 100 μl 0.1 M perchloric acid which included 0.01% cysteine as antioxidant. Fifty-μl supernatants were injected directly onto the LC system, separated on a 3-μm Phase II ODS column (100×3.2 mm I.D.) and detected by an electrochemical detector at a potential of +0.75 V. Standard curves, recoveries, analytical precision and detection limits were investigated for each monoamine neurotransmitter and its metabolites. The method was applied to study the influence of food restriction on the concentration of monoamine neurotransmitters in different brain areas, known to be involved in feeding and reproductive behaviour of female broiler chickens. Over 1000 micropunched tissue samples from ad libitum fed and food-restricted female broiler chickens were analyzed. Our results provide a possible role for catecholamines and indolamines in the altered feeding and reproductive behaviour of the broiler chicken.     

On-line coupling of automated solid-phase extraction with high-performance liquid chromatography and electrochemical detection: Quantitation of oxidizable drugs of abuse and their metabolites in plasma and urine

The concentration effect of automated on-line solid-phase extraction (SPE) in combination with HPLC and very sensitive electrochemical detection was employed for the determination of N-ethyl-4-hydroxy-3-methoxy-amphetamine (HMEA, the main metabolite of the ecstasy analogue MDE), Δ⁹-tetrahydrocannabinol (THC) and 11-nor-Δ⁹-tetrahydrocannabinol-carboxylic acid (THC-COOH) in plasma and urine in comparison to a previously published psilocin assay. For the SPE either CBA (functional group: carboxypropyl)- or CH (functional group: cyclohexyl)-sorbent was used. The following separation was carried out on a reversed-phase column (LiChroCart, Superspher 60 RP select B from Merck). Depending on the hydrodynamic voltammogram of the analyzed substance the oxidation potential varied from 920 mV up to 1.2 V. In spite of using high potentials, precision and accuracy were always within the accepted statistical requirements. The limits of quantitation were between 5 ng/ml (THC, THC-COOH in plasma) and 20 ng/ml (HMEA in plasma). Advantages of on-line SPE in comparison with off-line methods were less manual effort, evidently smaller volumes (≤400 μl) of plasma or urine and almost always higher recovery rates (>93%). The assays have been successfully proven with real biological samples and found suitable for use in routine analysis.     

Reverse-phase high-performance liquid chromatographic separation and electrochemical detection of norepinephrine,dopamine, serotonin,and related major metabolites

A convenient method for the determination of biogenic amines and their metabolites in small samples of brain tissue weighing from 0.5 to 5 mg, based on reverse-phase chromatography, is described. The biogenic amines, norepinephrine, dopamine, and serotonin, and the metabolites normetanephrine and 3-methoxytyramine were separated by ion-pair chromatography on a μBondapak phenyl column with an aqueous eluant, while the metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3-methoxy-4-hydroxyphenylglycol, and 5-hydroxyindoleacetic acid were separated on a μBondapak C18 column with a methanol aqueous mobile phase. The sensitivity of the method is in the picogram range, from 20 to 100 pg, depending on the substances analyzed.     

Analysis of monoamines and their metabolites by high performance liquid chromatography with coulometric electrochemical detection

High performance liquid chromatography with coulometric electrochemical detection has been used to achieve simultaneous determination of norepinephrine, epinephrine, 5-hydroxytryptophan, normetanephrine, dopamine, metanephrine, 3,4-dihydroxyphenylacetic acid, N-acetyldopamine, tyramine, tryptophan, 5-hydroxyindoleacetic acid, 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, homovanillic acid, tyrosine, p-octopamine, N-acetyl-p-octopamine, and p-synephrine. The procedure has been applied to study monoamine degradation in the insect brain and to demonstrate that N-acetylation rather than oxidative deamination is the primary route of monoamine catabolism in insects.     

Determination of uric acid in human saliva by high-performance liquid chromatography with amperometric electrochemical detection

The aim of the present study is to establish a highly sensitive method for the determination of uric acid (UA) in human saliva. The monitoring of UA levels in less invasive biological samples such as saliva is suggested for the diagnosis and therapy of gout, hyperuricemia, and the Lesch-Nyhan syndrome, and for detecting such conditions as alcohol dependence, obesity, diabetes, high cholesterol, high blood pressure, kidney disease, and heart disease. Reversed-phase high-performance liquid chromatography with electrochemical detection (HPLC-ED) was employed for the determination of UA obtained by solid-phase extraction from saliva. To quantify UA, we compared the ED efficiencies of an amperometric ED (Ampero-ED) with a single electrode and a coulometric ED (Coulo-ED) with a multiple electrode array. The results showed that the detection limits (S/N=3) were 3 nM for Ampero-ED and 6 nM for Coulo-ED, and the linearity of the calibration curves of 60-6000 nM had correlation coefficients exceeding 0.999. In addition, the total analytical time was 10 min. In the sample preparation of UA in saliva, an Oasis MAX solid-phase cartridge was used. The recoveries of UA spiked at 0.6 and 3 microM in saliva were above 95% with a relative standard deviation (RSD) of less than 15%. Therefore, the present method may be used in the routine and diagnostic determination of UA in human saliva.     

Simultaneous measurement of dopamine and its metabolites, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and tryptophan in brain tissue using liquid chromatography and electrochemical detection

An assay has been developed for brain tryptophan using reverse-phase liquid chromatography with electrochemical detection. The method simultaneously assays dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). The method does not require elution from ion exchange resins. After deproteinization and centrifugation samples are injected directly onto the chromatographic column. It was found that small changes in mobile phase pH markedly influenced the retention time of tryptophan while elution of the indoleamines and catecholamines did not change. The assay of these endogenous compounds in a single injection proved not expedient but inexpensive. Values obtained using alumina and ion exchange resins yielded comparable values.     

Determination of dopamine, homovanillic acid and 3,4-dihydroxyphenylacetic acid in rat brain striatum by high-performance liquid chromatography with electrochemical detection

Two procedures using liquid chromatography with electrochemical detection are described for the determination of dopamine (DA) and its two acidic metabolites, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), in subregions of rat striatum and nucleus accumbens. A strong cation-exchange column was used for DA analysis and a C₁ reversed-phase column was used for the analysis of the metabolites. Effects of pH, temperature and percentage of methanol on the retention time of HVA and DOPAC were studied. Levels of these compounds in the subregions of rat striatum and nucleus accumbens are reported.     

Simultaneous determination of norepinephrine,serotonin, and 5-hydroxyindole-3-acetic acid in microdialysis samples from rat brain by microbore column liquid chromatography with fluorescence detection following derivatization with benzylamine

A microbore column liquid chromatographic method for the simultaneous determination of norepinephrine (NE), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5HIAA) in microdialysis samples from rat brain is described. The method is based on precolumn derivatization of NE, 5HT, and 5HIAA with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in the corresponding highly fluorescent and stable benzoxazole derivatives. A 15-microl sample was mixed with 15 microl derivatization reagent solution containing 0.3M 3-cyclohexylaminopropanesulfonic acid buffer (pH 12.0), 0.5M benzylamine, 10mM potassium hexacyanoferrate(III), and methanol (1/1/1/12, v/v/v/v). The derivatization was carried out at 50 degrees C for 20 min. The benzylamine derivatives of NE, 5HT, and 5HIAA were separated on a reversed-phase column (100 x 1.0mm i.d., packed with C18 silica, 5 microm) within 30 min. The mobile phase consisted of 15 mM acetate buffer (pH 5.0) and acetonitrile (31%, v/v); the flow rate was 50 microl/min. The detection limits (signal-to-noise ratio of 3) for NE, 5HT, and 5HIAA in the injection volume of 20 microl were 90, 210, and 260 amol, respectively. Microdialysis samples were collected in 7.5-min intervals from the probes implanted in the hippocampus and prefrontal cortex of awake rats. The basal levels of NE, 5HT, and 5HIAA in the dialysates from the hippocampus were 4.2+/-0.5, 4.9+/-0.6, and 934.1 +/- 63.4 fmol/20 microl, and those from the prefrontal cortex were 6.0+/-1.2,5.51.3, and 669.1 +/- 96.0 fmol/20 microl (mean +/- SE, n=25), respectively. The NE and 5HT levels were altered by perfusion of high-potassium or low-calcium solution and following antidepressant drugs imipramine and desipramine. It is concluded that the new fluorescence derivatization method in combination with microbore column liquid chromatography allows the simultaneous determination of NE, 5HT, and 5HIAA in the microdialysis samples at higher sensitivity, providing easier maintenance in routine use than that achieved by high-performance liquid chromatographic methods with electrochemical detection.     

Determination of 5-aminolevulinic acid in blood plasma, tissues and cell cultures by high-performance liquid chromatography with electrochemical detection

A simple and sensitive method for determining 5-aminolevulinic acid (ALA) in biological samples is described. ALA is derivatized with o-phthaldehyde to give a compound with favorable properties for high-performance liquid chromatography with electrochemical detection. The method does not require extensive pretreatment of the samples and its detection limit is in the range of 1 pmol/20 μl injection. This method was applied to the determination of plasma ALA from normal and lead-exposed subjects, where 0.26±0.08 μM (n=30) and 2.6±0.75 μM (n=30), respectively were found. We also determined ALA in rat tissues, namely liver and brain, and the uptake of ALA by cultured fibroblasts and hepatocytes to illustrate the diversified applicability of the method.     

High-performance liquid chromatography with electrochemical detection for the determination of levodopa, catecholamines and their metabolites in rat brain dialysates

A high-performance liquid chromatographic assay with electrochemical detection is described for the simultaneous determination of levodopa, 3-O-methyldopa, dopamine, dihydroxyphenylacetic acid, homovanillic acid, 3-methoxytyramine, noradrenaline, adrenaline, 3-methoxy-4-hydroxyphenylethylene glycol and 5-hydroxyindoleacetic acid in rat brain dialysates. Samples are obtained in vivo using the microdialysis technique. Microdialysis probes are placed in the brain area to be studied and neurochemicals are collected by perfusion of the probe with modified Ringer's solution. Direct injection of the dialysates allows rapid and reliable results to be obtained.     

Clinical chemistry of serotonin and metabolites

Analyses of serotonin and other 5-hydroxyindoles, such as its precursor 5-hydroxytryptophan and major metabolite 5-hydroxyindoleacetic acid (5-HIAA), are indispensable for the elucidation of their (patho)physiological roles. In clinical chemistry attention is mainly focused on the diagnosis and follow-up of carcinoid tumours. For this most laboratories routinely measure urinary 5-HIAA. More recently, measurements of serotonin in platelets and urine have been advocated. Platelet serotonin may be the most sensitive indole marker for the detection of carcinoid tumours that secrete only small amounts of serotonin and/or its precursor 5-hydroxytryptophan. Although several chromatographic techniques have emerged for the analysis of tryptophan-related indoles, HPLC with either electrochemical or fluorometric detection have become the methods of choice for their quantification. HPLC-based methods combine selectivity, sensitivity and high precision, and enable the simultaneous investigation of several metabolically related indoles. This review aims to place the analysis of indoles in biological matrices in a biochemical, physiological and clinical perspective and highlights several important steps in their chromatographic analysis and quantification.     

Whole blood serotonin and plasma tryptophan using high-pressure liquid chromatography with electrochemical detection

Methods are detailed for whole blood serotonin (5-HT), equivalent to platelet 5-HT, and plasma tryptophan. These assays may be carried out on the same blood sample which need be no more than 1 ml, are rapid, and avoid the difficulties, such as protein precipitation and fluorophor formation, encountered in other methods. The linearity of the methods is extensive and allows for accurate measurement of low concentrations. Data are given for normal humans and for patients receiving psychiatric treatment for depression.     

Quantitation of daunorubicin and its metabolites by high-performance liquid chromatography with electrochemical detection

A selective and sensitive high-performance liquid chromatographic method was developed for the separation and quantitation of daunorubicin and its metabolites in serum, plasma, and other biological fluids. Daunorubicin and metabolites in human plasma were injected directly into the high-performance liquid chromatography system via a loop-column to pre-extract the drugs from the plasma, and quantitated against a multilevel calibration curve with adriamycin as the internal standard. The column effluent was monitored with an electrochemical detector at an applied oxidative potential of 0.65 V and by fluorescence. Daunorubicin and four metabolites were separted and characterized by this method. In a blinded evaluation of accuracy and precision, the mean coefficients of variation were 3.8, 3.6 and 9.8% at concentrations of 150, 75 and 15 ng/ml, respectively, and blank samples gave negligible readings. The amperometric sensitivity was greater than achieved by fluorescence detection, and offers an alternative method for quantitation of these compounds. The new method has a limit of detection of less than 2 ng on column, allowing quantitation of < 10 ng/ml in plasma samples without organic extraction prior to chromatographic analysis.     

Optimized isocratic conditions for the simultaneous determination of serotonin precursors and metabolites by reversed-phase high-pressure liquid chromatography with electrochemical detection

We describe an analytical procedure for the simultaneous determination of 5-hydroxyindole derivatives using high-pressure liquid chromatography with electrochemical detection. The procedure clearly resolves 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxytryptophol, and 5-hydroxyindole-3-acetic acid. The C-18 extraction column methodology and high-pressure liquid chromatography-electrochemical detection parameters have been developed to provide a rapid, sensitive, and reproducible quantitative determination of these 5-hydroxyindoles with picogram sensitivity. Chromatograms obtained from the analysis of whole normal mouse brain by the present technique clearly resolve the 5-hydroxyindoles and appear to be uncomplicated by interfering substances.     

Simultaneous determination of tricarboxylic acid cycle metabolites by high-performance liquid chromatography with ultraviolet detection

Here we describe a simple high-performance liquid chromatography (HPLC) procedure for the simultaneous detection and quantitation in standard solutions of 13 important metabolites of cellular energy metabolism, including 9 tricarboxylic acid (TCA) cycle components and 4 additional metabolites. The metabolites are detected by their absorbance at 210 nm. The procedure does not require prior derivatization, and an analysis can be carried out at ambient temperature within 15 min. The significance of the current work is that the current HPLC procedure should motivate the development of simplified TCA cycle enzyme assays, isotopomer analysis, and determination of selected TCA metabolite levels in plasma/tissues.     

Methods for the determination of seven selective serotonin reuptake inhibitors and three active metabolites in human serum using high-performance liquid chromatography and gas chromatography

This paper describes a set of simple and sensitive multiresidue methods for the determination of the specific serotonin reuptake inhibitors (SSRIs) used as antidepressant drugs, and some of their respective active metabolites in human serum. It involves liquid–liquid extraction procedures followed by gas chromatography coupled to nitrogen phosphorus detection or isocratic reversed-phase high-performance liquid chromatography combined with fluorescence detection (HPLC–FL), depending on the analytes. Extraction recoveries were between 71 and 96% for the eight SSRIs and their metabolites analysed by GC and between 41 and 77% for the two of them analysed by HPLC. Limits of detection (LODs) and limits of quantitation (LOQs) ranged, respectively, from 2.5 to 5 μg/l and from 10 to 20 μg/l. Intra-assay and inter-assay precision was studied at three and four concentration levels, respectively, and was less than 19% for all compounds. Accuracy was also satisfactory for all. An excellent linearity was observed from the LOQs up to 1000 μg/l for milnacipram and paroxetine and from each LOQ up to 400 mg/l for the other compounds. The performance of the methods described thus allows the therapeutic drug monitoring of the currently commercialised SSRIs.     

Simultaneous determination of dopamine, ascorbic acid, and uric acid using carbon ionic liquid electrode

A recently constructed carbon composite electrode using room temperature ionic liquid as pasting binder was employed as a novel electrode for sensitive, simultaneous determination of dopamine (DA), ascorbic acid (AA), and uric acid (UA). The apparent reversibility and kinetics of the electrochemical reaction for DA, AA, and UA found were improved significantly compared to those obtained using a conventional carbon paste electrode. The results show that carbon ionic liquid electrode (CILE) reduces the overpotential of DA, AA, and UA oxidation, without showing any fouling effect due to the deposition of their oxidized products. In the case of DA, the oxidation and reduction peak potentials appear at 210 and 135mV (vs Ag/AgCl, KCl, 3.0M), respectively, and the CILE shows a significantly better reversibility for dopamine. The oxidation peak due to the oxidation of AA occurs at about 60mV. For UA, a sharp oxidation peak at 340mV and a small reduction peak at 250mV are obtained at CILE. Differential pulse voltammetry was used for the simultaneous determination of ternary mixtures of DA, AA, and UA. Relative standard deviation for DA, AA, and UA determinations were less than 3.0% and DA, AA, and UA can be determined in the ranges of 2.0x10(-6)-1.5x10(-3), 5.0x10(-5)-7.4x10(-3), and 2.0x10(-6)-2.2x10(-4)M, respectively. The method was applied to the determination of DA, AA, and UA in human blood serum and urine samples.     

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection has been developed for the simultaneous determination of epirubicin, 13-S-dihydroepirubicin, doxorubicin and 13-S-dihydrodoxorubicin in human plasma. An aliquot of 200 μl plasma, spiked with internal standard, was extracted by solid-phase extraction using polymeric adsorbent columns. Chromatography was performed using a C₁₈ reversed-phase column with a mobile phase consisting of water–acetonitrile (71:29, v/v) containing 0.05 M Na₂HPO₄ and 0.05% v/v triethylamine adjusted to pH 4.6 with citric acid. Linearity of the method was obtained in the concentration range of 1–500 ng/ml for all the analytes. Analytical recoveries of the analytes ranged from 89 to 93%. The assay can be used for the simultaneous determination of the four analytes, or for epirubicin and its metabolite or doxorubicin and its metabolite, using the other parent drug as an internal standard. The method was applied to analyze human plasma samples from patients treated with epirubicin using doxorubicin as an internal standard.