Investigation of the interaction between serotonin-related compounds and phenylboronate moieties in monolithic silica disk-packed spin columns.

Solid-phase extraction technologies are widely used for sample pretreatment in bioanalysis. Monolithic silica disk-packed spin columns modified with phenylboronate moieties have been developed for the selective extraction of cis-diol compounds such as catecholamines. However, in our preliminary studies, serotonin was found to also be extracted in this treatment, along with catecholamines. In this study, the interaction between serotonin-related compounds (serotonin, tryptophan, 5-hydroxy-tryptophan and 5-hydroxyindoleacetic acid) and phenylboronate moieties was investigated. We found that only serotonin was extracted with phenylboronate-modified monolithic silica, whereas tryptophan, 5-hydroxy-tryptophan and 5-hydroxyindoleacetic acid were not. Hydrophobic interactions rather than ionic interactions were the primary factor for the adsorption of serotonin to phenylboronate. Finally, the selective pretreatment procedure for catecholamines was improved: thus, the method could be applied for the pretreatment of bio-samples.

Determination of urinary 5-hydroxyindoleacetic acid by combining Dµ-SPE using carbon coated TiO2 nanotubes and LC-MS/MS.

BACKGROUND: In this article, carbon coated titanium dioxide nanotubes (TiO2-NT@C) are employed for the determination of 5-hydroxyindole-3-acetic acid in urine by LC-MS/MS. RESULTS: All the variables involved in the extraction have been studied and optimized in depth. The method has been analytically characterized on the basis of its linearity, accuracy, sensitivity and precision. The LOD is 155.8 µg/l while the repeatability and the reproducibility, expressed as RSD, are better than 5.42 and 5.25%, respectively. The obtained relative recovery is 115%. CONCLUSION: TiO2-NT@C permit the efficient extraction of 5-hydroxyindole-3-acetic acid from complex biological samples such as urine allowing its sensitive determination by LC-MS/MS.

Polypyrrole hollow fiber for solid phase extraction.

We have developed a solid-phase extraction method based on conductive polypyrrole (PPy) hollow fibers which were fabricated by electrospinning and in situ polymerization. The electrospun poly (e-caprolactone) (PCL) fibers were employed as templates for the in situ surface polymerization of PPy under mechanical stirring or ultrasonication to obtain burr-shaped or smooth fiber shells, respectively. Hollow PPy fibers, achieved by removing the PCL templates, were the ideal sorbents for solid phase extraction of polar compounds due to their inherent multi-functionalities. By using the hollow PPy fibers, two important neuroendocrine markers of behavioural disorders, 5-hydroxyindole-3-acetic acid and homovanillic acid, were successfully extracted. Under the optimized conditions, the absolute recoveries of the above two neuroendocrine markers were 90.7% and 92.4%, respectively, in human plasma. Due to its simplicity, selectivity and sensitivity, the method may be applied to quantitatively analyse the concentrations of polar species in complex matrix samples.

Filmy channel microchip with amperometric detection.

In this article, a new type of microchip with filmy channels and a sample-injection fracture is introduced. Unlike commercial microchip, new microchip possessed filmy channel with width 2-3 mm. The effective cooling ability made filmy channel microchip restrain the generation of Joule heat even under electric field of 588 V/cm. Moreover, wider channel could be more easily modified to prevent the absorption of samples, load more samples and result in a higher sensitivity. Sample-injection fracture was first applied to match the filmy channel in microchip. Equipped with an amperometric detector, the characteristics of the newly designed filmy channel microchip had been studied and the results showed that it had good reproducibility, higher sensitivity and excellent separation ability. The microchip was also applied to separate L-tryptophan's metabolites, namely 5-hydroxy-L-tryptophan, 5-hydroxytryptamine and 5-hydroxy-indole-3-acetic acid.

Online extraction of 5-hydroxyindole acetic acid from urine for analysis by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Urinary 5-hydroxyindole acetic acid (5-HIAA) is a useful marker for the turnover of tryptophan metabolites in the diagnosis and monitoring of carcinoid tumours and the carcinoid syndrome. We have developed a simple and cost-effective assay for urinary 5-HIAA using liquid chromatography-tandem mass spectrometry (LC-MS/MS) incorporating an online sample clean-up process to replace a liquid chromatography electrochemical (LC-EC) technique. METHODS: Acidified urine was serially diluted in ammonium acetate buffer followed by ammonium acetate buffer enriched with 5-hydroxyindole-3-acetic-2,2-D2 acid internal standard. A 2.1 x 10 mm C18 column was used for primary online clean-up and eluted with 100% methanolic mobile phase onto a second dC18 Atlantis 2.1 x 20 mm column. Analytes were detected by mass spectrometry using transitions 192.1 > 146.3 and 194.1 > 148.0 for 5-HIAA and deuterated analyte, respectively. RESULTS: Run time was 3 min with 5-HIAA eluting at 1.37 min. The inter and intra-assay imprecision and accuracies of the three levels of inhouse quality control (QC) (30, 300 and 600 micromol/L) were acceptable with coefficient of variations (CVs) and deviation from target values <12% (n = 15). The average recovery of 5-HIAA spiked into urine was 93.7% with no ion suppression observed. The limit of detection was 2.8 and lower limit of quantification 4.0 micromol/L. Passing-Bablok regression of LC-EC with LC-MS/MS results showed good agreement between the methods, the relationship described as LC-MS/MS = 1.01(LC-EC)-1.22. No systematic or proportional biases were observed over the working range of the method. The assay was linear to at least 2000 micromol/L. CONCLUSIONS: We have developed a robust method offering a more than six-fold improvement in linearity compared to the existing LC-EC method.

High-performance liquid chromatography of monoamines on phenyl-bound sorbents using organic free mobile phases.

Tryptophan and its metabolites, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindolacetic acid, as well as dopamine, homovanilic acid and 2,3-dihydroxyphenylacetic acid, were separated on phenyl bound silica gel using isocratic elution with phosphate buffer. The method was successfully transferred to several other phenyl HPLC columns from different manufacturers simply by adjusting the pH of the buffer. The method has been validated by the determination of the level of monoamines in rat hypothalamus.

In vivo control of 5-hydroxytryptamine release by terminal autoreceptors in rat brain areas differentially innervated by the dorsal and median raphe nuclei.

Selective serotonin reuptake inhibitors (SSRIs) reduce the 5-HT release in vivo. This effect is due to the activation of somatodendritic 5-HT1A receptors and it displays a regional pattern comparable to that of selective 5-HT1A agonists, i.e., preferentially in forebrain areas innervated by the dorsal raphe nucleus (DRN). However, despite a comparatively lower 5-HT1A-mediated inhibition of 5-HT release and a greater density of serotonergic uptake sites in hippocampus, the net elevation produced by the systemic administration of SSRIs is similar in various forebrain areas, regardless of the origin of serotonergic fibres. As terminal autoreceptors may also limit the SSRI-induced elevations of 5-HT in the extracellular brain space, we reasoned that a differential control of 5-HT release by terminal autoreceptors in DRN- and median raphe-innervated areas might be accountable. To examine this possibility, we have conducted a regional microdialysis study in the DRN, MRN and four forebrain regions preferentially innervated either by the DRN (frontal cortex, striatum) or the median raphe nucleus (MRN; dorsal and ventral hippocampus) using freely moving rats. Dialysis probes were perfused with 1 microM of the SSRI citalopram to augment the endogenous tone on terminal 5-HT autoreceptors. The non-selective 5-HT1 antagonist methiothepin (10 and 100 microM, dissolved in the dialysis fluid) increased extracellular 5-HT in frontal cortex and dorsal hippocampus in a concentration-dependent manner. The 5-HT(1B/1D) antagonist GR 127935 was ineffective at 10 microM and tended to reduce 5-HT in dorsal hippocampus at 100 microM. The local infusion of 100 microM methiothepin significantly elevated the extracellular 5-HT concentration to 142-173% of baseline (mean values of 260 min post-administration) in the DRN, MRN, frontal cortex, striatum and hippocampus (dorsal and ventral). Comparable elevations were noted in the four forebrain regions examined. As observed in frontal cortex and dorsal hippocampus, the perfusion of 10 microM GR 127935 did not elevate 5-HT in DRN. MRN, striatum or ventral hippocampus. Because the stimulated 5-HT release in the DRN has been suggested to be under control of 5-HT(1B/1D) receptors, we examined the possible contribution of these receptor subtypes to the effects of methiothepin in the DRN. The perfusion of sumatriptan (0.01-10 microM) or GR 127935 (0.01-10 microM) did not significantly modify the 5-HT concentration in dialysates from the DRN. Thus, the present data suggest that the comparable effects of SSRIs in DRN- and MRN-innervated forebrain regions are not explained by a preferential attenuation of 5-HT release by terminal 5-HT1B autoreceptors in hippocampus, an area with a low inhibitory influence of somatodendritic 5-HT1A receptors. Methiothepin-sensitive autoreceptors (possibly 5-HT1B) appear to play an important role not only in the projection areas but also with respect to the control of 5-HT release in the DRN and MRN. In addition, our findings indicate that GR 127935 is not an effective antagonist of the actions of 5-HT at rat terminal autoreceptors.

The role of tryptophan, 5-hydroxy indole-3-acetic acid and their protein binding in uremic patients.

An on-line high performance liquid chromatography (HPLC) method of analysis with electrochemical detection was developed to quantitate catecholamines, tryptophan (Trp) and 5-hydroxy indoleacetic acid (5-HIAA) in muscle and plasma samples from healthy subjects and uraemic patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Equilibrium dialysis was performed on the plasma samples from 14 healthy and 14 uraemic subjects for the separation of free Trp and its metabolites from bound Trp. The results demonstrate abnormal Trp-albumin binding probably with accumulation of indolic metabolites and abnormally high muscle Trp concentration in uraemic patients.

Serotonin-induced stimulation of cortisol secretion from human adrenocortical tissue is mediated through activation of a serotonin4 receptor subtype.

The occurrence of serotonin in the human adrenal gland was demonstrated both by immuno-histochemical and biochemical approaches. Using specific polyclonal antibodies to serotonin, the presence of numerous immunoreactive cells was revealed by means of the peroxidase-antiperoxidase technique. These cells exhibited the morphological characteristics of mast cells. Combination of high performance liquid chromatography and electrochemical detection showed the presence of substantial amounts of both serotonin and its metabolite 5-hydroxyindolacetic acid in adrenocortical extracts. The role of serotonin in the regulation of steroidogenesis from human adrenocortical slices was studied in vitro using a perifusion system technique coupled to a specific radioimmunoassay for cortisol. Graded doses of serotonin (from 10(-8) M to 3 x 10(-7) M) increased cortisol production in a dose-dependent manner. Prolonged exposure of adrenal fragments to serotonin (10(-7) M) induced a biphasic response, i.e. a rapid and transient increase in cortisol secretion followed by a plateau phase, suggesting the existence of a desensitization phenomenon. The stimulatory effect of serotonin (10(-7) M) was not altered during infusion of the serotonin1 and/or serotonin2 receptor antagonists methysergide (10(-6) M) and ketanserin (10(-6) M), respectively. In contrast, ICS 205 930 (10(-6) M), a non-selective serotonin3/serotonin4 antagonist, totally abolished the response of adrenal slices to serotonin (10(-7) M). The benzamide derivative zacopride, considered as a serotonin4 agonist, induced a robust stimulation of cortisol secretion. In addition, the corticotropic effects of serotonin (10(-7) M) and zacopride (10(-6) M) were not additive. Incubation of adrenocortical fragments with zacopride (10(-6) M) or serotonin (10(-6) M) caused a significant increase in cAMP formation. Taken together, these data suggest that serotonin, locally released by intra-adrenal mast-like cells, may act as a paracrine factor to stimulate cortisol secretion in man. Our results also indicate that serotonin-induced corticosteroid production is mediated through activation of a serotonin4 receptor subtype positively coupled to adenylate cyclase.

Combined administration of a non-neurotoxic 3,4-methylenedioxymethamphetamine analogue with amphetamine produces serotonin neurotoxicity in rats.

In the present study, a central serotonin neurotoxicity was induced by combining a non-neurotoxic 3,4-methylenedioxymethamphetamine analogue, 5-methoxy-6-methyl-2-aminoindan (MMAI), with the non-vesicular dopamine (DA) releaser, S-(+)-amphetamine (Amp). With the multiple dosing regimen utilized neither drug alone resulted in any changes in serotonergic parameters, including 5-HT, 5-HIAA and the number of 5-HT uptake sites. However, MMAI (10 mg/kg) in combination with Amp (2 x 2.5 mg/kg) did result in a long-term 20% decrease in cortical serotonergic parameters. The same dose of Amp plus 20 mg/kg MMAI resulted in a 50 to 60% reduction. Effects in the hippocampus and caudate nucleus were similar. These data support the hypothesis that DA release plays a critical role in the serotonin neurotoxicity of substituted amphetamines.

Time-course variations induced by pargyline on the 5-hydroxyindole compounds measured in the nucleus raphe dorsalis and in blood: a voltammetric and HPLC approach in the rat.

Ninety min after pargyline (Pargy, 75 mg/kg) injection, the +300 mV voltammetric signal, measured in the nucleus raphe dorsalis (nRD) of freely moving rats, disappeared completely. This effect was also followed (2-7 h post-injection) by the reappearance of a peak (post-Pargy-peak) at the same +300 mV potential. The height of this post-Pargy signal was still decreased by Pargy (30 or 75 mg/kg). Endogenous 5-hydroxyindoleacetic acid (5-HIAA) contents, measured with HPLC in the nRD of Pargy-treated rats, exhibited analogous variations. Inhibition of monoamine oxidases by Pargy seems thus to be effective in blocking 5-HIAA production over only 2 h. The 15-fold increase of 5-HT endogenous content in the nRD (3 h after injection) was not reflected in the voltammetric extracellular measurements performed 90 min to 7 h after Pargy injection; this increase is suspected to be mainly intracellular.

Modification of reversed-phase separations of small molecules using non-ionic surfactants and mixed ionic-non-ionic surfactants.

The effects of non-ionic surfactants, Tween 20 and Tween 60, on reversed-phase separations of small molecules have been examined. Tween compounds were found to partition irreversibly into the ODS material used, markedly decreasing capacity factors for the compounds tested. Compounds which could hydrogen bond were less affected. Ion pairing using either anionic or cationic surfactants was possible in the presence of the non-ionic surfactants. While reversed-phase effects predominate under these conditions, secondary effects on retention order were observed and attributed to hydrogen bonding. Primary amines were retained longer than the corresponding secondary amine while catechols were retained longer than the corresponding methoxyphenols.

Simultaneous determination of homovanillic acid, 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in brain tissue, using a double-column procedure.

A double-column procedure for simultaneous determination of homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in brain tissue was described. These compounds extracted with perchloric acid were adsorbed on Sephadex G-10 in the column. HVA and DOPAC were desorbed from Sephadex G-10 with 0.01 N HCl and then retained on QAE-Sephadex A-25 (acetate form) placed beneath the Sephadex G-10 column. HVA and DOPAC were eluted with small volume of 0.2 mol/l NaCl. 5-HIAA remaining on the Sephadex G-10 was eluted with 0.05 mol/l phosphate buffer at pH 7.5. Each substance was determined fluorometrically. The recovery rates of HVA, DOPAC and 5-HIAA were more than 80, 80 and 85%, respectively. Effects of oxypertine on dopamine (DA) neurons in the rat brain were investigated using the method described here. Oxypertine caused marked increases in the levels of HVA and DOPAC in cortex and striatum of the rat brain, without any obvious change in the level of 5-HIAA after the intraperitoneal administration of 10 mg/kg oxypertine. These findings suggest that the favorable antipsychotic action of oxypertine may be due not only to a marked reduction in brain norepinephrine level, but to a blockade of DA receptors.

Liquid-chromatographic determination of indole-3-acetic acid and 5-hydroxyindole-3-acetic acid in human plasma.

We describe a "high-performance" reversed-phase liquid-chromatographic method for determination of indole-3-acetic acid (I) and 5-hydroxyindole-3-acetic acid (II) in human plasma. I is eluted at 1.0 mL/min with a mixture of 1-pentanesulfonic acid (pH 3.1), methanol, and water. It is detected by fluorometry. A mixture of citric acid/sodium phosphate solution (pH 4.8) and methanol, at 1.5 mL/min, is used to elute II, which is detected with an electrochemical cell. Platelet-poor plasma samples were pretreated with HCl, perchloric acid, and trichloroacetic acid for protein precipitation. Best results were obtained with the last (protein precipitation is incomplete with HCl, while recoveries of I are concentration dependent with perchloric acid). Analytical recoveries were 58% (SD 3.1%, CV 5.3%, n = 12) and 79% (SD 3.3%, CV 5.3%, n = 9) for I and II, respectively. Concentrations of I and II in plasma ranged from 0.61 to 3.32 (mean 1.54, SD 0.59, n = 15) mumol/L and from 33.0 to 102.6 (mean 51.8, SD 20.1, n = 16) nmol/L, respectively.

Urinary 4-hydroxy-3-methoxymandelic (vanillylmandelic) acid, 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid, and 5-hydroxy-3-indoleacetic acid determined by liquid chromatography with electrochemical detection.

We describe a simple liquid-chromatographic assay of urinary 4-hydroxy-3-methoxymandelic (vanillylmandelic) acid, 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid, and 5-hydroxy-3-indoleacetic acid with electrochemical detection, with direct injection of the sample. The first two analytes are measured simultaneously; 5-hydroxy-3-indoleacetic acid is measured separately. Chromatographic conditions for assay of the three were: column temperature, 65 and 60 degrees C; mobile phase, potassium phosphate buffer (0.2 mol/L, pH 3.0) for 6 min, then potassium phosphate buffer plus acetonitrile (9/1 by vol) for 20 min; flow rate, 0.7 mL/min; oxidation potential, 600 and 450 mV vs an Ag/AgCl reference electrode; and sensitivity, 40 and 160 nA at full scale. Values so obtained agreed well with those obtained for samples that were first solvent-extracted.

Isolation of tryptophan, 5-HT, and 5HIAA from single brain areas using disposable columns packed with sephadex G-10 resin.

Using disposable Bio-Rad Columns packed with Sephadex G-10 resin (7 x 40 mm resin column), tryptophan, 5HT, and 5HIAA can be isolated from single discrete brain areas. The preparation of Sephadex G-10 columns requires a minimum amount of time, and once prepared, the columns require very little maintenance. Since the procedures involve gel filtration and not affinity chromatography, equilibration of the resin to its appropriate ionic strength or pH is not required. Brain tissue weighing 25-200 mg is homogenized in 1 ml of 0.4 M HClO4. After centrifugation at 4800 g, the supernatant is decanted onto the columns. Washing and eluting involve twelve 1 ml applications of either a phosphate buffer or a carbonate-NH4OH solution. Tryptophan and 5HIAA are eluted with the third, fourth, and fifth washings while 5HT is eluted with the last five washings. Tryptophan, 5HT and 5HIAA are all assayed by fluorometric techniques. These methods provide recovery values, reproducibility, and sensitivity comparable with cation exchange and some solvent extract methods.

A simple determination of serotonin, 5-hydroxyindoleacetic acid and 5-hydroxytryptophan decarboxylase activity in rat brain areas and parallel correlation among the levels.

We devised a simple method for the separation of serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) using small P-cellulose and DEAE-Sephadex columns, respectively. Both substances were estimated fluorometrically by the reaction with o-phthalaldehyde (OPA). In this method, some known interfering substances in addition to many primary amines and amino acids were separated from 5HT and 5HIAA. The recoveries of 5HT and 5HIAA were 80 and 71%, respectively. The fluorescence intensities of 30 pmol of 5HT and 15 pmol of 5HIAA were about twice over the reagent blank. The complete separation of 5-hydroxytryptophan (5HTP) from 5HT made it possible to determine 5HTP decarboxylase activity in rat brain areas without using an isotope-labeled substrate. The method allows for quantitation of the enzyme activity in mg amounts of the brain tissues. The activities in discrete areas were in good parallel with the 5HT contents, except for the striatum. The levels of 5HIAA were also much in parallel with those of 5HT.

[Simultaneous extraction and fluorimetric determination of noradrenaline, dopamine, serotonin and 5-hydroxyindoleacetic acid in separate small brain segments]. / Odnovremennaia ékstraktsiia i fliuorimetricheskoe opredelenie noradrenalina, dofamina, serotonina i 5-oksiindoluksusnoi kisloty v otdel'nykh nebol'shikh uchastkakh mozga.

Highly-sensitive and reproducible method is developed for simultaneous assay of noradrenaline, dopamine, serotonin and 5-hydroxyindolylacetic acid in small samples of brain tissue. The procedure comprised extraction of the substances with acidified butanol, reextraction of the compounds with phosphate buffer in presence of isooctane, separation of the amines form precursors and metabolites using ion exchange resin Amberlite CG-50 and of 5-hydroxyindolylacetic acid--on Sephadex G-10.