Quantitative determination of 5-hydroxytryptophan in dissected brain regions by on-line trace enrichment HPLC with electrochemical detection

A sensitive, specific and rapid quantitative HPLC assay for 5-hydroxytryptophan (5-HTP) in samples of brain regions of widely differing size is described. The method utilizes off-line prepurification of tissue supernatants on gravity-fed strong cation exchange columns, on-line enrichment of the entire cation exchange column eluate on short reverse phase enrichment precolumns, further separation by reverse phase chromatography on an analytical column and electrochemical detection. On-line trace enrichment permits the efficient incorporation of off-line column chromatography to maximize assay specificity without compromising assay sensitivity. A reliable, working limit of detection of 200 pg 5-HTP/sample permits the estimation of in vivo tryptophan hydroxylase activity by determining the rate of 5-HTP accumulation following L-aromatic amino acid decarboxylase inhibition in small discrete brain regions or larger tissue samples only poorly innervated by 5-HT terminals.     

III. simultaneous determination of catecholamines in rat brain by reversed-phase liquid chromatography with electrochemical detection

Recently, an assay method for the determination of norepinephrine and dopamine in biological specimens by application of high performance liquid chromatography with electrochemical detection has been developed. Application of a microparticle reversed-phase column has made clean separation and detection of trace amounts of each amine possible. This paper presents an assay method for the simultaneous measurement of norepinephrine, dopamine and epinephrine with N-methyldopamine as the internal standard. Incorporation of an effective sensitivity-shift technique resulted in a remarkable increase in sensitivity with this method. The assay limit for quantitation was approximately 50 pg for all amines. Applicability was also studied following administration of reserpine or fusaric acid.     

High-performance liquid chromatographic determination of indoleamines, dopamine, and norepinephrine in rat brain with fluorometric detection

A high-performance liquid chromatography-fluorescence procedure for the determination of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tryptophan, dopamine, and norepinephrine has been developed. The method uses an ion-pairing system on an Ultrasphere ODS (5-microns) column with detector wavelength settings of excitation at 290 nm and emission at 330 nm. The procedure has been used to quantitate these indoleamines and catecholamines in rat brain tissue after homogenization in a perchloric acid solution; an aliquot of this solution is injected directly onto the HPLC column. Column sensitivities range from 6.1 pmol for tryptophan to 1.1 pmol for 5-hydroxytryptamine.     

In vivo comparison of norepinephrine and dopamine release in rat brain by simultaneous measurements with fast-scan cyclic voltammetry

Brain norepinephrine and dopamine regulate a variety of critical behaviors such as stress, learning, memory, and drug addiction. In this study, we demonstrate differences in the regulation of in vivo neurotransmission for dopamine in the anterior nucleus accumbens (NAc) and norepinephrine in the ventral bed nucleus of the stria terminalis (vBNST) of the anesthetized rat. Release of the two catecholamines was measured simultaneously using fast-scan cyclic voltammetry at two different carbon-fiber microelectrodes, each implanted in the brain region of interest. Simultaneous dopamine and norepinephrine release was evoked by electrical stimulation of a region where the ventral noradrenergic bundle, the pathway of noradrenergic neurons, courses through the ventral tegmental area/substantia nigra, the origin of dopaminergic cell bodies. The release and uptake of norepinephrine in the vBNST were both significantly slower than for dopamine in the NAc. Pharmacological manipulations in the same animal demonstrated that the two catecholamines are differently regulated. The combination of a dopamine autoreceptor antagonist and amphetamine significantly increased basal extracellular dopamine whereas a norepinephrine autoreceptor antagonist and amphetamine did not change basal norepinephrine concentration. α-Methyl-p-tyrosine, a tyrosine hydroxylase inhibitor, decreased electrically evoked dopamine release faster than norepinephrine. The dual-microelectrode fast-scan cyclic voltammetry technique along with anatomical and pharmacological evidence confirms that dopamine in the NAc and norepinephrine in the vBNST can be monitored selectively and simultaneously in the same animal. The high temporal and spatial resolution of the technique enabled us to examine differences in the dynamics of extracellular norepinephrine and dopamine concurrently in two different limbic structures.     

Syntheses of the sulfoconjugated isomers of norepinephrine and dopamine, controlled by HPLC with ultraviolet detection

The physiological significance of sulfoconjugated catecholamines and their involvement in clinical disorders, e.g. hypertension and Parkinsonism, is poorly investigated. For this reason, the sulfoconjugated isomers of dopamine as well as of norepinephrine were synthesized by modified methods. All isomers and their intermediates could be detected by a reversed-phase high-performance liquid chromatography with ultraviolet detection (HPLC-UV) with short retention times and a good reproducibility. Ion-exchange chromatography with an extended column length improved the separation of the reaction products, and the immediate control by HPLC-UV enabled precise cutting of the fractions. The selection of the fractions with the optimum ratios of product/by-product resulted in improved yields and highest purity. All by-products, e.g. dopamine sulfonic acids, were less than 0.04%, as detected by HPLC-UV and, in addition, the contamination by free catecholamines was only 41 x 10(-4)-87 x 10(-4)%, as measured by HPLC with electrochemical detection (HPLC-ED). The purity was further demonstrated in two highly sensitive biological assays: cAMP production in human mononuclear leukocytes and aggregation of human platelets. The sulfoconjugated catecholamines were characterized by melting point, thin-layer chromatography, infrared spectrum, HPLC-UV, elemental analysis, and unequivocally identified by 1H-NMR.     

Measurement of acetylcholine and choline in brain by HPLC with electrochemical detection

A simple and rapid method for measuring acetylcholine and choline using high performance liquid chromatography (HPLC) with electrochemical detection is presented. Acetylcholine and choline were first separated using reverse-phase chromatography; acetylcholine was then hydrolyzed post-column to choline by acetylcholinesterase. Choline was oxidized enzymatically by choline oxidase to betaine and hydrogen peroxide, and the peroxide was detected electrochemically. Changes in methodology from previous procedures include a different mobile phase, controlled heating of chromatography column and post-column reaction coil, and a different extraction method for quaternary amines. The changes resulted in less inhibition of derivatizing enzymes by mobile phase, narrow and consistent elution of peaks, and a rapid and efficient extraction of quaternary amines. Measurement of acetylcholine and choline in brain tissue was found to be replicable, and the levels agreed with literature 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.     

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.     

A new on-line dual enzymatic method for simultaneous quantification of cholesterol and triglycerides in lipoproteins by HPLC

We describe an on-line dual detection method using HPLC for lipoprotein analysis that allows simultaneous determination of cholesterol and triglyceride profiles from a single injection of sample. Two different gel permeation columns, TSKgel LipopropakXL and Superose 6HR, were applied to the dual detection system, evaluating analytical performance of the proposed method and the columns by analyzing serum samples from human and nonhuman subjects. Both TSK and Superose columns produced good within-day imprecision values less than 4.7% for cholesterol and 4.2% for triglyceride determination. Linear regression analysis showed the results from the Superose column (y) correlated well with those from the TSK column (x): y = 0.969x + 5.44 (r = 0.990) for total cholesterol (mg/dl), y = 1.08x - 11.14 (r = 0.985) for total triglycerides (mg/dl), and y = 1.093x - 0.06 (r = 0.978) for the ratios of triglycerides to cholesterol (mg/mg). Furthermore, the cholesterol and triglyceride profiles elucidated the differences in the resolution ability of the columns, which have not been apparent from a single lipid profile. We conclude that the dual detection concept with proper choice of column and enzymic reagents specific to the objectives of the particular study can facilitate studies of lipoprotein metabolism.     

Determination of the dopamine agonist rotigotine in microdialysates from the rat brain by microbore column liquid chromatography with electrochemical detection

Rotigotine, an investigational dopamine agonist formulated as a patch, is being studied in Parkinson's disease. A microdialysis technique, in combination with microbore column liquid chromatography and electrochemical detection, was developed to monitor rotigotine levels in the brain. Microdialysis probes were inserted into the striata of anesthetized rats, and samples were collected during perfusion with Ringer's solution. Rotigotine was separated using a C18 reversed-phase column. The mobile phase consisted of 50mM Na(2)HPO(4) x 2H(2)O, 2.5 mM sodium octyl sulfonate, and pH 4.5; 35% volume to volume acetonitrile. The flow rate was 30 microl/min, and the potential of the glassy carbon electrode was set to +850 mV. The method allowed monitoring of the time course of brain extracellular rotigotine levels with a detection limit of 1 nM following either intravenous (0.5 mg/kg) or subcutaneous (5.0 mg/kg) rotigotine injection.     

Determination of DOPA,dopamine, DOPAC,epinephrine, norepinephrine, α-monofluoromethyldopa and α-difluoromethyldopa in various tissues of mice and rats using reversed-phase ion-pair liquid chromatography with electrochemical detection

A method for the determination of catecholic amino acids and amines by reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection has been developed. By using octanesulfonic acid for ion pairing and by optimising ionic strength, pH and methanol concentration of the mobile phase, separation was achieved of 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxypehnylacetic acid (DOPAC), norepinephrine (NE), epinephrine (EPI), and dopamine (DA). α-Difluoromethyldopa (DFMD) and α-monofluoromethyldopa (MFMD), two potent enzyme-activated irreversible inhibitors of aromatic amino acid decarboxylase were also separated from the natural catechols. Concentrations of catechols and inhibitors were measured in brains, hearts and kidneys of mice treated with small repeated doses of MFMD. The method has also been applied to the determination of catechols in other organs such as prostates and seminal vesicles of rats and in smaller tissues like mesenteric arteries. A semi-automated procedure making use of an automatic sample processor and a digital integrator permitted the analysis of as many as sixty samples per day.     

Improved method for determination of catecholamines in rat brain by isolation on boric acid gel and high-performance liquid chromatography with electrochemical detection

An improved sensitive, simple and time-saving method for determining catecholamine (CA) in rat brain is described. The method involves isolation on boric acid gel and high-performance liquid chromatography with electrochemical detection. Boric acid gel effectively adsorbs CA at weakly alkaline pH and the over-all recoveries of 5 ng and 10 ng samples of authentic norepinephrine (NE) and dopamine (DA) added to a homogenate of rat brain were 98.9 ± 9.2% and 103.4 ± 9.3% for NE and 96.2 ± 4.6% and 99.4 ± 4.8 for DA, respectively. Intra-assay variation was 5.3% (5 ng) and 3.0% (10 ng) for NE and 4.4% (5 ng) and 3.8% (10 ng) for DA. Inter-assay variation was 7.7% (1 ng) for NE and 5.0% (1 ng) for DA. With this analytical system, the lowest amount of NE or DA detectable was 40 pg. Application of this method to determination of the DA and NE contents of rat hypothalamus during estrous cycle revealed significant increases in the turnovers of both in the proestrus stage. This method should be useful for routine determination of plasma NE and DA because it is sensitive and inexpensive.     

Determination of the primary metabolite of central nervous system norepinephrine, 3-methoxy-4-hydroxy-phenethyleneglycol, in mouse brain and brain perfusate by high-performance liquid chromatography with electrochemical detection

Assays are described for the determination of picomole levels of 3-methoxy-4-hydroxy-phenethyleneglycol (MHPG) in mouse brain and in the perfusate of an intact mouse brain. High-performance liquid chromatography with electrochemical detection yielded a MHPG detection limit of 0.37 pmol. This technique offers a sensitive and inexpensive alternative to gas chromatography with mass spectrometry.     

Depletion of epinephrine in rat hypothalamus by a dopamine agonist,pergolide

The i.p. injection of pergolide mesylate, a dopamine agonist, at doses of 0.3–0.6 mg/kg led to a decrease in epinephrine concentration in rat hypothalamus. After a 0.6 mg/kg dose of pergolide mesylate, epinephrine concentration in hypothalamus decreased within 2 hr, reached a minimum concentration at about 8 hrs, and then returned toward control values. Norepinephrine N-methyltransferase activity was not decreased after pergolide injection in vivo nor was it inhibited by pergolide added in vitro at concentrations as high as 10^–3 M. Higher i.p. doses of less potent dopamine agonists, apomorphine (10 mg/kg) and lergotrile (3 mg/kg), also decreased epinephrine concentration in hypothalamus. The pergolideinduced decrease in hypothalamic epinephrine concentration was prevented by pretreatment with haloperidol or spiperone., antagonists of dopamine receptors. Activation of dopamine receptors appears to result in a decrease in epinephrine concentration in rat brain, possibly due to, enhanced release of epinephrine.     

Determination of 5-hydroxytryptophan, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid in 20 rat brain nuclei using liquid chromatography with electrochemical detection

High-performance liquid chromatography with electrochemical detection is utilized for the simultaneous determination of serotonin, its precursor 5-hydroxytryptophan, and its major metabolite 5-hydroxyindoleacetic acid in nervous tissue samples. Tissue preparation required only homogenization in acidic solution and centrifugation prior to application to the chromatograph. Detection limits in the low picogram range were obtained for those indoles separated. This assay was used in combination with a micropunch dissection technique of 20 discrete rat brain nuclei to measure serotonin, its precursor, and major metabolite. The specificity of the assay was checked with pharmacological experiments aimed to increase or decrease serotonin levels. Pargyline, a monoamine oxidase inhibitor, led to a marked increase in serotonin and a decrease of 5-hydroxyindoleacetic acid while p-chlorophenylalanine, by blocking the conversion of tryptophan to 5-hydroxytryptophan, selectively depleted 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid.     

Determination of dopamine, norepinephrine, and related trace amines by prechromatographic derivatization with naphthalene-2,3-dicarboxaldehyde

Dopamine, norepinephrine, octopamine, tyramine, and 3,4-dihydroxybenzylamine react readily with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide ion under mild conditions to give highly fluorescent cyanobenz[f]isoindole (CBI) products. The CBI products exhibit good solution chemical stability. The high-fluorescence quantum efficiency of the CBI fluorophore and the ability to excite these adducts in the visible region (420-450 nm) enhance the sensitivity and selectivity of this derivatization detection technique. The CBI products of catecholamines and "trace" amines are readily separated by reverse-phase HPLC giving detection limits in the 20 to 60 fmol range (S/N = 3). A prechromatographic derivatization HPLC assay for the trace analysis of dopamine and norepinephrine in urine is described.     

Determination of reduced glutathione in guinea pig and rat tissue by HPLC with electrochemical detection

A method using HPLC with electrochemical detection has been developed for the determination of GSH in tissue. The method is based upon the separation of GSH from other components by cation exchange chromatography coupled with the electrochemical oxidation of GSH to the corresponding disulfide. Detection limits of ca. 5 × 10^?12 moles GSH were established and the method was used to measure GSH content of rat and guinea pig brain, liver and synaptosome preparations.     

The simultaneous electrochemical detection of ascorbic acid, dopamine, and uric acid using graphene/size-selected Pt nanocomposites

In this study, a graphene/Pt-modified glassy carbon (GC) electrode was created to simultaneously characterize ascorbic acid (AA), dopamine (DA), and uric acid (UA) levels via cyclic voltammetry (CV) and differential pulse voltammetry (DPV). During the preparation of the nanocomposite, size-selected Pt nanoparticles with a mean diameter of 1.7 nm were self-assembled onto the graphene surface. In the simultaneous detection of the three aforementioned analytes using CV, the electrochemical potential differences among the three detected peaks were 185 mV (AA to DA), 144 mV (DA to UA), and 329 mV (AA and UA), respectively. In comparison to the CV results of bare GC and graphene-modified GC electrodes, the large electrochemical potential difference that is achieved via the use of the graphene/Pt nanocomposites is essential to the distinguishing of these three analytes. An optimized adsorption of size-selected Pt colloidal nanoparticles onto the graphene surface results in a graphene/Pt nanocomposite that can provide a good platform for the routine analysis of AA, DA, and UA.