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.     

Effects of Transient Forebrain Ischemia and Pargyline on Extracellular Concentrations of Dopamine, Serotonin, and Their Metabolites in the Rat Striatum as Determined by In Vivo Microdialysis

Striatal microdialysis was performed in rats subjected to 20 min of transient forebrain ischemia produced by occlusion of the carotid arteries during hemorrhagic hypotension. Extracellular changes of dopamine, serotonin, and their metabolites were monitored before, during, and after the ischemic insult at 10-min intervals by on-line HPLC analysis. During ischemia, extracellular dopamine increased dramatically (156 times baseline), as did 3-methoxytyramine (3-MT), whereas 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) decreased (15-25% of baseline). Upon reperfusion, dopamine was cleared from the extracellular fluid within 40 min and reached a stable level (70% of baseline). DOPAC and HVA increased (250-330%) transiently and reached their maximum 1 h following reperfusion, whereas 3-MT decreased to undetectable levels within 20 min. Although baseline levels of serotonin were not detectable, serotonin and 5-hydroxyindoleacetic acid showed a qualitatively similar temporal pattern to dopamine and its acid metabolites. Killing rats by cervical dislocation produced changes in extracellular dopamine, serotonin, and their metabolites that were almost identical to those seen during ischemia. Pargyline pretreatment 2 h before ischemia had marginal effects on the postischemic clearing of dopamine. The pargyline pretreatment, however, did increase the survival rate of rats subjected to ischemia, and this protective effect might be due to the pargyline-induced blockade of the post-ischemic monoamine oxidase-mediated increase in dopamine metabolism and the concurrent production of the potentially neurotoxic molecule, hydrogen peroxide.     

Method for measuring endogenous 3-O-methyldopa in urine and plasma

The present report describes a method using column liquid chromatography with electrochemical detection for assaying concentrations of 3-O-methyldopa in urine and plasma. The technique combines a one-step sample preparation scheme with post-column flow-through electrodes in series, allowing adequate chromatographic separation of 3-O-methyldopa from other endogenous substances in urine. The validity of the method was confirmed by markedly decreased urinary 3-O-methyldopa levels after administration of an inhibitor of catechol-O-methyltransferase to rats, radioactivity in chromatographic fractions corresponding to 3-O-methyldopa in urine of rats undergoing infusion of [³H]-l-DOPA, and correlations between excretion rates of 3-O-methyldopa and catechols in humans. In healthy humans, urinary excretion of 3-O-methyldopa averaged 974 ± 707 (S.D.) nmol per day, and plasma levels of 3-O-methyldopa averaged 89 ± 32 nmol/l. The method should be useful in studies about the metabolism of endogenous and exogenous DOPA.     

A rapid and sensitive method for the analysis of brain monoamine neurotransmitters using ultra-fast liquid chromatography coupled to electrochemical detection

Electrochemical detection is often used to detect catecholamines and indolamines in brain samples that have been separated by conventional reverse-phase high performance liquid chromatography (HPLC). This paper presents the transfer of an existing chromatographic method for the determination of monoamines in brain tissues using 5 μm granulometry HPLC columns to columns with a particle diameter less than 3 μm. Several parameters (repeatability, linearity, accuracy, limit of detection, and stability of samples) for this new ultrafast high performance liquid chromatography (UHPLC) method were examined after optimization of the analytical conditions. The separation of seven compounds, noradrenaline, dopamine and three of its metabolites, dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine, and serotonin and its metabolite, 5-hydroxyindole-3-acetic acid was analyzed using this UHPLC-electrochemical detection method. The final method, which was applied to brain tissue extracts from mice, rats, and cats, decreased analysis time by a factor of 4 compared to HPLC, while guaranteeing good analytical performance.     

Simultaneous automatic determination of catecholamines and their 3-O-methyl metabolites in rat plasma by high-performance liquid chromatography using peroxyoxalate chemiluminescence reaction

A highly specific and sensitive automated high-performance liquid chromatographic method for the simultaneous determination of catecholamines (CAs; norepinephrine, epinephrine, and dopamine) and their 3-O-methyl metabolites (normetanephrine, metanephrine, and 3-methoxytyramine) is described. Automated precolumn ion-exchange extraction of diluted plasma is coupled with HPLC separation of CAs and their 3-O-methyl metabolites on an ODS column, postcolumn coulometric oxidation, fluorescence derivatization with ethylenediamine, and finally peroxyoxalate chemiluminescence reaction detection. The detection limits were about 3 fmol for norepinephrine, epinephrine, and dopamine, 5 fmol for normetanephrine, and 10 fmol for metanephrine and 3-methoxytyramine (signal-to-noise ratio of 3). Fifty microliters of rat plasma was used and 4-methoxytyramine was employed as an internal standard. The relative standard deviations for the method (n = 5) were 2.5-7.6% for the intraday assay and 6.3-9.1% for the interday assay. The method was applicable to the determination of normetanephrine and metanephrine in 50 microl of rat plasma.     

Simultaneous determination of 1-chloro-2,4-dinitrobenzene, 2,4-dinitrophenyl-S-glutathione and its metabolites for human placental disposition studies by high-performance liquid chromatography

We developed and validated an HPLC method for determination of 1-chloro-2,4-dinitrobenzene (CDNB) and its glutathione conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG) to study the kinetics and mechanisms involved in DNP-SG formation and efflux, as a probe for human placental metabolism and transport. This method combines use of 3 microm solid phase, rapid mobile phase gradient with dual wavelength ultraviolet detection to permit determination of a lipophilic parent compound and its hydrophilic metabolites in a single short run. The selectivity, linearity, accuracy, precision, relative recovery and stability of the assay are sufficient for determining CDNB, DNP-SG and its metabolites from buffer and tissue samples to support placental drug metabolism and transport studies.     

Retention mechanism of analytes in the solid-phase extraction process using molecularly imprinted polymers. Application to the extraction of triazines from complex matrices

The influence of sampling variables on the concentration of the dopamine metabolites 3-methoxytyramine (3MT), dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) was examined in equine urine. A logarithmic transformation of the data for all horses gave distribution which approximated the normal distributions for each metabolite. The mean urinary concentration of 3 MT in horses was 214 ng/mL and the application of a threshold with a probability of 1 in 10,000 gave an actionable level of 4 microg/mL. Environmental variables were not forensically significant in determining the population distribution. HVA was not found to be a reliable indicator of dopamine or levodopa administration.     

Application of the high-performance liquid chromatographic method for separation, purification and characterisation of p-bromophenylacetylurea and its metabolites

This study presents a HPLC method for the separation and purification of p-bromophenylacetylurea (BPAU) and its metabolites. The method effectively separated and purified BPAU and its metabolites. Three metabolites of BPAU, M1, M2 and M3 were characterised by mass spectroscopy and nuclear magnetic resonance. They are named as N′-hydroxy-p-bromophenylacetylurea, 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione and N′-methyl-p-bromophenylacetylurea, respectively. The major metabolic pathways of BPAU were proposed. The establishment of the HPLC method and characterisation of BPAU metabolites make it possible for further pharmacokinetic studies to explore the mechanism of BPAU-induced delayed neuropathy.     

Simultaneous determination of levodopa and 3-O-methyldopa in human plasma by liquid chromatography with electrochemical detection

A simple and rapid assay is described for the simultaneous analysis of levodopa (l-DOPA) and 3-O-methyldopa (3-OMD) in human plasma samples, applying an ion-pair reversed-phase liquid chromatographic method with electrochemical detection, designed for clinical trials performed to study the effect of peripheral catechol-O-methyltransferase inhibitors on the metabolism of l-DOPA. After protein precipitation of 100 microl plasma sample aliquots with perchloric acid, the analytes are directly injected, separated within 10 min and simultaneously quantified down to 20 ng/ml by an electrochemical detector equipped with a dual-electrode system operating in redox mode eliminating effectively potential endogenous and exogenous interferences. The intra-assay precision for l-DOPA and 3-OMD was 1.34-6.54 and 3.90-5.50%, whereas the inter-assay precision was 2.09-7.69 and 4.16-9.90%, respectively. The recoveries were close to 90% for l-DOPA and almost 100% for 3-OMD. Satisfactory storage stability was achieved for up to 16 weeks at -70 degrees C by stabilizing plasma samples with antioxidants.     

Simultaneous determination of the histamine H1-receptor antagonist ebastine and its two metabolites,carebastine and hydroxyebastine,in human plasma using high-performance liquid chromatography

Ebastine (CAS 90729-43-4) is an antiallergic agent which selectively and potently blocks histamine H₁-receptors in vivo. A simple and sensitive high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of ebastine and its two oxidized metabolites, carebastine (CAS 90729-42-3) and hydroxyebastine (M–OH), in human plasma. After a pretreatment of plasma sample by solid-phase extraction, ebastine and its metabolites were analyzed on an HPLC system with ultraviolet detection at 254 nm. Chromatography was performed on a cyano column (250×4.0 mm I.D.) at 40 °C with the mobile phase of acetonitrile–methanol–0.012 M ammonium acetate buffer (20:30:48, v/v/v) at a flow rate of 1.2 ml/min. Accurate determinations were possible over the concentration range of 3–1000 ng/ml for the three compounds using 1 ml plasma samples. The intra- and inter-day assay accuracy of this method were within 100±15% of nominal values and the precision did not exceed 12.4% of relative standard deviation. The lower limits of quantitation were 3 ng/ml for ebastine and its metabolites in human plasma. This method was satisfactorily applied to the determination of ebastine and its two oxidized metabolites in human plasma after oral administration of ebastine.     

Clinical pharmacology of the new COMT inhibitor CGP 28 014

CGP 28 014 is a specific inhibitor of catechol-O-methyltransferase (COMT) in vivo. In humans, the inhibition was assessed by measuring urinary excretion of isoquinolines and with the levodopa test. Following administration of CGP 28 014, urinary excretion of isoquinolines was significantly increased. In rats, CGP 28 014 reduced plasma and striatal concentrations of 3-O-methyldopa (30MD) in a dose-dependent manner. Acute and subchronic administration of CGP 28 014 alone or in combination with the peripherally acting decarboxylase inhibitor benserazide decreased plasma 30MD as an index of COMT inhibition by about 50%. There seems to be a close relationship between the time-course of plasma concentrations of CGP 28 014 and the extent of COMT inhibition assessed by the 30MD/DOPA ratio in plasma.     

Different action on dopamine catabolic pathways of two endogenous 1,2,3,4-tetrahydroisoquinolines with similar antidopaminergic properties.

The effect of single and multiple 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) and 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ) administration on concentrations of dopamine and its metabolites: homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxytyramine (3MT) in three brain areas was studied HPLC with electrochemical detection in Wistar rats. The rate of dopamine catabolism in the striatum along the N-oxidative and O-methylation pathways was assessed by calculation of the ratio of appropriate metabolites to dopamine concentration. In addition, the spontaneous and apomorphine-stimulated locomotor activity, and muscle rigidity was studied after acute administration of 1MeTIQ and 1BnTIQ. We have found that 1MeTIQ did not change the level of dopamine and HVA in all investigated structures both after a single and chronic administration. However, the levels of intermediary dopamine metabolites, DOPAC and 3MT, were distinctly affected. The level of DOPAC was strongly depressed (by 60-70%) while the level of extraneuronal matabolite 3MT was significantly elevated (by 170-200%). In contrast to 1MeTIQ, 1BnTIQ depressed the level of dopamine (by approximately 60%) and increased the level of total metabolite, HVA, (by 40%) especially in the striatum, but the levels of DOPAC and 3MT remained unchanged. The paper has shown that 1MeTIQ and 1BnTIQ produced different effects on dopamine catabolism. Potential neuroprotective compound 1MeTIQ did not change the rate of total dopamine catabolism, it strongly inhibited the monoamine oxidase (MAO)-dependent catabolic pathway and significantly activated the catechol-O-methyltransferase (COMT)-dependent O-methylation. In contrast 1BnTIQ, a compound with potential neurotoxic activity, produced the significant increase of the rate of dopamine metabolism with strong activation of the oxidative MAO-dependent catabolic pathway. Interestingly, both compounds produced similar antidopaminergic functional effects: antagonism of apomorphine hyperactivity and induction of muscle rigidity. The results may explain the biochemical basis of the neuroprotective and of the neurotoxic properties endogenous brain tetrahydroisoquinoline derivatives.     

Simultaneous quantitative determination of cilostazol and its metabolites in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cilostazol, a quinolinone derivative, and its known metabolites OPC-13015, OPC-13213, OPC-13217, OPC-13366, OPC-13269, OPC-13326 and OPC-13388 in human plasma was developed and validated. Cilostazol, its metabolites and two internal standards, OPC-3930 and OPC-13112, were extracted from human plasma by a combination of liquid–liquid and liquid–solid phase extractions, with combined organic solvents of n-butanol, methanol, chloroform, methyl-tert.-butyl ether, and a Sep-Pak silica column. The combined extract was then evaporated and the residue was reconstituted in ammonium acetate buffer (pH 6.5). The reconstituted solution was injected onto a HPLC system and was subjected to reversed-phase HPLC on a 5 μm ODS-80TM column to obtain quality chromatograph and good peak resolution. A gradient mobile phase with different percentages of acetonitrile in acetate buffer (pH 6.5) was used for the resolution of analytes. Cilostazol, its metabolites and the two internal standards were well separated at baseline from each other with resolution factor being 74 and 138. This HPLC method was demonstrated to be specific for all analytes of interest with no significant interference from the endogenous substances of human plasma. The lower limit of quantitation was 20 ng/ml for cilostazol and all metabolites. The method was validated initially for an extended linear range of 20–600 ng/ml for all metabolites and cilostazol, and has been revised later for a linear range of 20–1200 ng/ml for cilostazol and two major and active metabolites OPC-13015 and OPC-13213. The overall accuracy (relative recovery) of this method was established to be 98.5% to 104.9% for analytes with overall precision (CV) being 1.5% to 9.0%. The long-term stability of clinical plasma samples was established for at least one year at −20°C. Two internal standards of OPC-3930 and OPC-13112 were evaluated and validated. However, the data indicated that there was no significant difference for all accuracy and precision obtained by using either OPC-3930 or OPC-13112. OPC-3930 was chosen as the internal standard for the analysis of plasma samples from clinical studies due to its shorter retention time. During the validation standard curves had correlation coefficients greater than or equal to 0.998 for cilostazol and the seven metabolites. These data clearly demonstrate the reliability and reproducibility of the method.     

Comparative study of sulpiride and haloperidol on dopamine turnover in the rat brain

The effects of the neuroleptics, sulpiride and haloperidol, on dopamine (DA) turnover were compared following the acute and chronic administration of these drugs alone or in combination with levodopa or apomorphine. In the acute treatment, the increase in DA metabolites in the striatum and nucleus accumbens was more marked in the haloperidol-treated rats than in the sulpiridetreated rats. Following the additional administration of levodopa, however, the potency of the neuroleptics in elevating DA metabolites was reversed. A low dose of apomorphine induced a marked reduction in the striatal DA metabolite levels by approximately 50%. When rats were pretreated with the neuroleptics, haloperidol was more effective in preventing an apomorphine-induced reduction in DA metabolites. On repeated administration of the neuroleptics, a tolerance occurred in the striatum and nucleus accumbens, but not in the prefrontal cortex. This differential development of tolerance was observed in the different brain regions and with the different drugs administered. These results suggests that the pharmacological mechanism of sulpiride on DA turnover differs from that of haloperidol.     

HPLC-assay with electrochemical detection for the neurotoxin MPTP, its metabolite MPP+ and MPTP-analogues in biological samples after purification over Sephadex G10

A sensitive and selective method for assaying the neurotoxin MPTP and some MPTP-analogues in mouse brain and serum is described. The method is based on isolation of the compounds from biological samples on small Sephadex G10 columns followed by reverse phase HPLC with amperometric detection. HPLC separation was performed at pH 3, after which the pH was increased to 6.8 by mixing the column effluent with 0.5 M phosphate pH 9, to provide the conditions required for electrochemical detection. A metabolite of MPTP, MPP+, was determined as MPTP after reduction with NaBH4. This assay allows the determination of brain and serum concentrations in the pmol/g range of administered MPTP and MPTP-analogues and the effects of these substances on dopamine and its metabolites in the same tissue sample.     

Rapid analysis of metabolic stability of dopamine receptor antagonists and detection of their metabolites by liquid chromatography/tandem mass spectrometry

In vitro metabolic stability of dopamine D(3)/D(4) receptor antagonists and identification of their metabolites by high-performance liquid chromatography (HPLC) coupled with ion-trap mass spectrometry (ITMS) were assessed in rat liver microsomes. The compounds were divided into three cassette groups for rapid quantitative analysis of multiple drugs and simultaneous detection of their metabolites. The samples from incubation with rat liver microsomes were pooled into designed cassette groups and analyzed by HPLC/electrospray ITMS in full-scan mode. The metabolic stability of the drugs was determined by comparing their signals after incubation for 0 and for 30min. The metabolic stability of the examined dopamine receptor antagonists was in the range of 9.9-84.4%. In addition, the present cassette analysis allowed the simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent multistage tandem mass spectrometry on the ion-trap instrument allowed characterization of the structure of the detected metabolites. N,O-dealkylation and ring hydroxylation reactions were identified as major metabolic reactions in piperazinylalkylisoxazole derivatives. These results suggested that the present approach is useful for the rapid evaluation of metabolic stability and structural characterization of metabolites within a short period in new drug discovery.     

Simultaneous estimation of levodopa and carbidopa by RP‐HPLC using a fluorescence detector: its application to a pharmaceutical dosage form

A reverse‐phase high‐performance liquid chromatographic (RP‐HPLC) method was developed and validated for the simultaneous estimation of levodopa and carbidopa in bulk and pharmaceutical formulations. Chromatographic separation was achieved by using a C₁₈ reverse‐phase column and a mixture of an aqueous phase (10 mM potassium dihydrogen phosphate buffer, pH 4.0) and methanol (90:10 v/v) as the mobile phase. Quantitative analysis of levodopa and carbidopa was performed using a fluorescence detector at an excitation wavelength of 280 nm and an emission wavelength of 310 nm. The method was linear between 5 and 500 ng/mL for both levodopa and carbidopa. The detection limits for levodopa and carbidopa were 0.30 and 0.60 ng/mL, respectively, whereas the quantitation limit was 0.80 ng/mL for levodopa and 1.2 ng/mL for carbidopa. The method demonstrated good and consistent recoveries (99.63–100.80% for levodopa and 98.97–100.94% for carbidopa) with low interday and intraday relative standard deviation. The validated method was successfully applied to quantify levodopa and carbidopa simultaneously in a pharmaceutical formulation. The method was found to be precise, sensitive and accurate for the simultaneous determination levodopa and carbidopa in bulk and pharmaceutical formulations. Copyright © 2014 John Wiley & Sons, Ltd.     

Changes in Neostriatal Dopamine Concentrations in Response to Levodopa Infusions

Abstract: Levodopa was infused under various circumstances of pretreatment into the ear veins of unanesthetized rabbits. Concentrations of neostriatal dopamine formed in response to levodopa administration were determined. The aim was to characterize the temporal relationship between the concentrations of levodopa in plasma and dopamine in the neostriatum. When plasma levodopa was maintained constant by i.v. infusion, the concentration of neostriatal dopamine reached a plateau by 1 h. Increases in dopamine were proportional to the amount of precursor in plasma. The tissue half-life of this dopamine in normal rabbits was not more than 15 min. Half-lives of comparable duration for striatal dopamine were calculated from rabbits treated chronically with levodopa, and from rabbits with monoamine-depleting lesions. The results show that the concentration of dopamine in rabbit neostriatum correlates closely with the concentration of levodopa in plasma. Concurrent analyses of neocortical tissues indicate that the neostriatum may not be different from other brain regions with regard to dopamine storage mechanisms. Interpretation of the results in terms of the clinical use of levodopa suggests that the durations of short-term effects (measured in h) of the drugs are paralleled by changes in concentration of brain dopamine.     

Neurotoxic action of 6-hydroxydopamine on the nigrostriatal dopaminergic pathway in rats sensitized with D-amphetamine.

To determine whether behavioral sensitization produced by prolonged D-amphetamine administration affects susceptibility of nigrostriatal dopaminergic neurons to the neurotoxic actions of 6-hydroxydopamine (6-OHDA), rats were treated daily from the 23 rd day after birth for 11 consecutive days with D-amphetamine (1.0 mg/kg s.c.) or saline. On the last day of treatment, one group primed with D-amphetamine and one control group of rats were tested to confirm behavioral sensitization development. The remaining animals were additionally treated on the 34 th day (one day after the last D-amphetamine injection) with 6-OHDA HBr (300 microg in 10 microl i.c.v., salt form, half in each lateral ventricle) or its vehicle. Four weeks later the levels of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-metoxytyramine (3-MT), as well as 5-hydroxytrypatmine (5-HT) and its metabolite 5-hydroxyindoleacteic acid (5-HIAA) were assayed in the striatum, by HPLC/ED. In rats with behavioral sensitization, 6-OHDA reduced endogenous dopamine and its metabolites content to a comparable degree in comparison to controls. This finding indicates that presumed up-regulation of the dopamine transporter in the behaviorially sensitized rats did not increase the neurotoxicity of a high dose of 6-OHDA.     

左旋多巴甲酯在PLGA微球的稳定性研究

目的：考察左旋多巴甲酯在PLGA微球中的稳定性并探讨其稳定方法。方法：利用HPLC的方法考察了左旋多巴甲酯在不同pH值和光照的环境中和微球里的稳定性。结果：左旋多巴甲酯在pH3中稳定,在微球中也可以稳定一周。结论：包封左旋多巴甲酯在PLGA微球中,是一种有效地保护了左旋多巴甲酯在微球中的活性,可以实现长效缓释,是一种可行的方案。