Hitoshi Nohta

High-performance liquid chromatography of plasma catecholamines using 1,2-diphenylethylenediamine as precolumn fluorescence derivatization reagent

A simple, rapid and highly sensitive method for the determination of catecholamines (norepinephrine, epinephrine and dopamine) in human plasma is described which employs high-performance liquid chromatography with fluorescence detection. After cation-exchange chromatography on a Toyopak SP cartridge, the catecholamines and isoproterenol (internal standard) in 500 microliters of plasma are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine in aqueous acetonitrile. These compounds are separated within 8 min on a reversed-phase column, TSK-gel ODS-120T, with isocratic elution using a mixture of water, methanol and acetonitrile containing a Tris--hydrochloric acid buffer (pH 7.0). The detection limit for each catecholamine is ca. 2 fmol in a 100-microliters injection volume. N-Methyldopamine can also be used as internal standard.

Spectrofluorimetric determination of catecholamines with 1,2-diphenylethylenediamine

Abstract A sensitive spectrolfuorimetric method for the determination of catecholamines with 1,2-diphenylethylenediamine is described. The method is based on the reaction of catechol compounds in neutral medium with 1,2-diphenylethylenediamine in the presence of hexacyanoferrate(III) and glycine at ambient temperature. The fluorescence produced shows excitation and emission maxima around 345 and 480 nm, respectively. The method is simple, selective for catechol compounds and very sensitive. Catecholamines can be determined at concentrations as low as 15–20 pmol ml −1 .

Luminol-type chemiluminescence derivatization reagents for liquid chromatography and capillary electrophoresis.

The present paper provides the principles for chemiluminescence of luminol-type compounds and their wide and powerful application to the detection system in liquid chromatography and capillary electrophoresis as derivatization reagents. The reagents can be classified into two types, chemiluminescence labeling and chemiluminogenic reagents. The former reagents are highly chemiluminescent themselves and used for tagging their intense chemiluminophores to analytes, whereas the latter are weakly chemiluminescent but generate intense chemiluminescence by reaction with analytes. The liquid chromatographic methods utilizing chemiluminescence derivatizing reactions with luminol-type reagents allow the analytes to be detected at pmol-sub-fmol levels. Furthermore, the chemiluminogenic reactions show high selectivity owing to their selective reaction against the analytes permitting facile and reproducible detection.

Aromatic glycinonitriles and methylamines as pre-column fluorescence derivatization reagents for catecholamines

The reactivity of some catecholamines with aromatic glycinonitriles (AGN) (four species) and aromatic methylamines (AMA) (five species) was investigated in detail, to find pre-column fluorescence derivatization reagents for catecholamines. Of the nine reagents tested, 2-phenylglycinonitrile (PGN) and benzylamine (BA) were shown to be the best reagents in terms of selectivity and sensitivity. The reagents react selectively with catecholamines under mild conditions in the presence of ammonium molybdate and sodium periodate for PGN and potassium hexacyanoferrate(III) for BA to give fluorescent derivatives. The derivatives of four catecholamines (epinephrine (E), norepinephrine (NE), dopamine (DA) and isoproterenol (IP)) could be separated within 13 min by reversed-phase liquid chromatography with isocratic elution and measured fluorimetrically. The detection limits (signal-to-noise ratio = 3) are in the range 5.2–11.0 fmol for PGN and 1.6–100 fmol for BA in a 50 μl injection volume. The liquid chromatographie (LC) methods with PGN and BA were successfully applied to the determination of some catecholamines in human urine.

Increased serotonin release in mice frontal cortex and hippocampus induced by acute physiological stressors

The effects of acute physiological stressors (5 s tail pinch, handling and forced swimming at +25 and +5 degrees C for 3 min each) on serotonin (5-HT) release in the mouse brain were investigated using in vivo microdialysis. The extracellular 5-HT levels were determined by a newly developed highly-sensitive and selective high-performance liquid chromatography method based on derivatization with benzylamine and fluorescence detection. The basal levels of 5-HT in 3 min microdialysates from the ventral hippocampus and frontal cortex were 0.68+/-0.21 and 0.75+/-0.28 fmol/6 microl (n=24), respectively. All three stressors caused an immediate, significant and reversible increase (handling: 150%; swimming: 240%) of extracellular 5-HT levels in both brain structures, suggesting a more dynamic role played by the serotonergic system in response to acute stress.

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.

Fluorogenic reactions for biomedical chromatography

A number of fluorogenic reactions, which have been used for HPLC detection systems by means of pre- and/or postcolumn derivatization, are surveyed with respect to both sensitivity and selectivity for the determination of biomedically important substances. For the derivatization of the substances, two types of fluorogenic reactions, fluorescence-generating and fluorescence-tagging, have been studied. The former are usable in most instances for both pre- and postcolumn derivatization methods, and the latter only for precolumn derivatization methods. HPLC methods utilizing the fluorogenic reactions allow analytes to be detected at picomole-subfemtomole levels. In the fluorescence-generating reactions, several fluorogenic reagents possessing two or more reactive sites in the molecule, which show molecular recognition for a variety of analytes, permit facile and reproducible detection in HPLC because there are fewer interferences from biological matrices.

Simultaneous determination of 5-hydroxyindoles and catechols by high-performance liquid chromatography with fluorescence detection following derivatization with benzylamine and 1,2-diphenylethylenediamine.

A highly selective and sensitive method for the simultaneous determination of 5-hydroxyindoles and catechols (serotonin, norepinephrine, dopamine and related compounds) by high-performance liquid chromatography with fluorescence detection is described. The method is based on the two-step precolumn derivatization of 5-hydroxyindoles and catechols with benzylamine (BA) and 1,2-diphenylethylenediamine (DPE), respectively, resulting in highly fluorescent and stable benzoxazole derivatives. The first derivatization with BA proceeds at room temperature (ca. 23 degrees C) for 2 min in a mixture of 0.3 M 3-cyclohexylamino-1-propanesulfonic acid buffer (pH 10.0) and methanol in the presence of potassium hexacyanoferrate(III). The subsequent second derivatization with DPE is carried out at 50 degrees C for 20 min in the presence of glycine. The resulting fluorescent derivatives of five 5-hydroxyindoles and seven catechols are separated on a reversed-phase column (150 x 1.5 mm I.D., packed with C18 silica, 5 microm) with isocratic elution using a mixture of acetonitrile-15 mM acetate buffer (pH 4.5) (34:66, v/v) containing 1 mM octanesulfonic acid sodium salt, and are detected spectrofluorimetrically at 480 nm with excitation at 345 nm. The detection limits (signal-to-noise ratio of 3) of the related compounds are 80 amol to 86 fmol for a 20-microl injection.

High-sensitive liquid chromatographic method for determination of neuronal release of serotonin, noradrenaline and dopamine monitored by microdialysis in the rat prefrontal cortex

A high-sensitive liquid chromatographic method based on precolumn derivatization and fluorescence detection allowing simultaneous determination of serotonin (5-HT), noradrenaline (NA) and dopamine (DA) in brain microdialysis samples is described. 5-HT, NA and DA were derivatized with benzylamine and 1,2-diphenylethylenediamine in the presence of potassium hexacyanoferrate(III) and glycine, which yielded to highly fluorescent and stable benzoxazoles. The derivatized samples were separated on a microbore column (150 mm x 1.0mm i.d., packed with C18 silica, 5 microm) within 60 min. The mobile phase consisted of acetonitrile-Briton-Robinson buffer (pH 7.2) (32:68, v/v) containing 5 mM Na2EDTA and 5 mM octanesulfonic acid sodium salt. The detection limits (signal-to-noise ratio of 3) for 5-HT, NA and DA were 76, 42 and 95 amol/10 microl injected on-column, respectively. Microdialysis samples were collected at 10-min intervals from the probes implanted in the prefrontal cortex of awake rats. The basal levels of 5-HT, NA and DA were 7.3 +/- 0.7, 5.3 +/- 0.31 and 8.1 +/- 0.47 fmol/5 microl (mean +/- S.E.M., n = 5). Following 90-min perfusion with tetrodotoxin (1 microM) or calcium-free Ringer solution, the DA and NA levels were reduced to about 15 and 20%, respectively and the 5-HT levels to 45 and 60% of the basal levels, respectively. Reserpine, 12h after a dose of 5mg/kg i.p., reduced the extracellular 5-HT, NA and DA concentrations to about 34, 39 and 32% of the basal levels, respectively. In conclusion, the preset microdialysis/analytical method enables simultaneous monitoring of basal and pharmacologically reduced neuronal release of 5-HT, NA and DA in the rat brain.

Determination of histamine in microdialysis samples from rat brain by microbore column liquid chromatography following intramolecular excimer-forming derivatization with pyrene-labeling reagent.

This paper describes a sensitive and selective liquid chromatographic method with fluorescence detection for determination of histamine in brain microdialysis samples from awake rats. Samples containing histamine (10 microl) were derivatized with 20 microl of the reagent consisting of 3 mM 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), 3 mM potassium carbonate and acetonitrile (1:1:18, v/v), thereafter 20 microl volume was injected onto the microbore column packed with C18 silica gel. The histamine derivative contained two pyrene moieties, which generated intramolecular excimer fluorescence (450-540 nm) and allowed clear discrimination from the monomer fluorescence (360-420 nm) emitted by PSE itself. The separation of histamine-pyrene derivative was achieved within 25 min, the detection limit (S/N=3) was 0.3 fmol histamine in 20 microl injected. The basal extracellular levels of histamine collected in 10-min fractions (fmol per 10 microl, mean+/-S.D., not corrected for recovery, n=10 rats) were 35.45+/-4.56 (hypothalamus), 9.05+/-1.56 (prefrontal cortex), 7.83+/-0.86 (hippocampus) and 6.54+/-0.66 (striatum). The voltage-sensitive release of histamine was evaluated by perfusing the probes with high (100 mM) concentration of potassium ions or with sodium channel blocker tetrodotoxin (1 microM), and the calcium-dependent release was tested by perfusion with calcium-free Ringer solution. These data, together with physiologically induced increase of extracellular histamine in four examined brain regions during forced swimming demonstrate that this method is suitable for high-sensitive determination of neuronally released histamine under various pharmacological and physiological conditions.