Identification of novel urinary metabolites of the lipid peroxidation product 4-hydroxy-2-nonenal in rats

Following iv administration of 4-hydroxy-2-nonenal (HNE) and [4-3H]HNE to rats, 15 polar urinary metabolites accounting for about 50% of the urinary radioactivity were separated by HPLC. Among them, eight major compounds and tritiated water were quantified. The metabolites were unequivocally characterized using GC/MS and ESI/MS/MS/MS. Most of "HNE polar metabolites" originate from omega-oxidation of 4-hydroxy-2-nonenoic acid (HNA): 9-hydroxy-HNA, its mercapturic acid conjugate, and two diastereoisomers of the corresponding lactone. The oxidation of 9-hydroxy-HNA by alcohol and aldehyde dehydrogenases leads to the excretion of 9-carboxy-HNA and of the corresponding lactone mercapturic acid conjugate. 1, 4-Dihydroxy-2-nonene (DHN) originating from the reduction of HNE by alcohol dehydrogenase was to a lesser extent omega-hydroxylated, leading to 9-hydroxy-DHN which was excreted as a mercapturic acid conjugate (two diastereoisomers).     

Action of DNA-gyrase inhibiting derivatives of 4-oxo-1, 4-dihydro-3-pyridinecarboxylic acid against Trypanosoma brucei

Ten derivatives of 4-oxo-1,4-dihydro-3-pyridinecarboxylic acid as DNA-gyrase inhibitors were evaluated in vitro and in vivo against Trypanosoma brucei brucei IPP. Two compounds were were active at 100 microM in vitro after 1 h incubation time. After 24 h incubation, 8 compounds were active and the most interesting compound, 1-(4-hydroxy-2-methyl-phenyl)-6-[2-(4,5-dichloro-phenyl)-ethenyl] -4-oxo-1, 4-dihydropyridine-3-carboxylic acid (compound No. 8) was trypanocidal at 10 microM. The trypanocidal effects were not noted in vivo after subcutaneous treatment administered as a single dose of 50 mumols/kg. The in vitro trypanocidal effect is correlated with the DNA-gyrase inhibition.     

Synthesis and in vitro microsomal metabolism of 4-ethyl-5-(4-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione and its potential metabolites

Although most triazoline-3-thione derivatives (cyclic thiosemicarbazides) are important compounds possessing some biological and pharmacological activities, no literature was found showing their metabolic reactions with hepatic microsomal preparations. We, therefore, planned to study the in vitro microsomal metabolism of a prototype, 4-ethyl-5-(4-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (A). The substrate (A) and its potential metabolites i.e. the corresponding dealkylation (A1), desulphuration (A2) and S-oxidation (A3) products were synthesized and characterized by spectral methods. The substrate and its potential metabolites were separated by a reverse phase HPLC. A was incubated with rat microsomal preparations fortified with NADPH and extracted into DCM; concentrated under a stream of N2 at 20 degrees C and analyzed by HPLC. The results indicated that A was metabolically inert and failed to produce the corresponding desulphuration (triazole-3-one) and S-oxidation (sulphenic acid) metabolites which would lead to pharmacological and toxicological alterations compared to the parent molecule. However, a small amount of dealkylated product (A1) was observed as a metabolite together with two unidentified metabolites.     

Studies on 5-lipoxygenase inhibitors. Synthesis of and structure--activity relationship in p-hydroxyarylalkenylbenzoazoles

In a previous paper we reported that 2-(p-hydroxyarylbutadienyl)benzoxazoles are highly potent 5-lipoxygenase inhibitors. We synthesized their ethenyl homologues and benzothiazole derivatives, and evaluated their 5-lipoxygenase inhibitory activity in vitro with cell-free rat basophilic leukemia (RBL-1). In most cases the replacement of benzoxazolyl with benzothiazolyl resulted in an enhancement of the activity. All compounds with butadienyl spacers tested herein exhibited strong inhibitory activities. While most of the ethenyl homologues showed weaker activities than their corresponding butadienyl homologues, some ethenyl compounds in the benzothiazole derivatives were found to be as potent as their corresponding butadienyl homologues. The inhibitory activity was also affected by the variation in the p-hydroxyaryl functionality.     

Nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol in Fischer 344 rats: comparisons with 2- and 4-chloroaniline and 2- and 4-aminophenol

Nephrotoxicity occurs following intraperitoneal (i.p.) administration of 2-chloroaniline or 4-chloroaniline hydrochloride to Fischer 344 rats, but the nephrotoxicant chemical species and mechanism of nephrotoxicity are unknown. The purpose of this study was to evaluate the in vivo and in vitro nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol, metabolites of 4-chloroaniline and 2-chloroaniline. A comparison was also made between the nephrotoxic potential of the aminochlorophenols and the corresponding aminophenols to examine the effect of adding a chloride group on the nephrotoxic potential of the animophenols. Male Fischer 344 rats (4/group) were given an i.p. injection of a chloroaniline or aminochlorophenol hydrochloride (1.5 mmol/kg), and aminophenol (1.0 or 1.5 mmol/kg), or vehicle, and renal function monitored at 24 and 48 h. Both aminochlorophenols induced smaller and fewer renal effects that the parent chloroanilenes in vivo. Also, 4-aminophenol was markedly more potent as a nephrotoxicant that 4-amino-3-chlorophenol, while 2-aminophenol and 2-amino-5-chlorophenol induced only mild change in renal function. In vitro, the phenolic compounds reduce p-aminohippurate accumulation by renal cortical slices at bath concentrations of 0.01 mM, while a bath concentration of 0.50 mM or greater was required for the chloroanilines. However, all compounds reduced tetraethylammonium accumulation at bath concentrations of 0.1-0.5 mM or greater. These results indicate that extrarenally-produced aminochlorophenol metabolites do not contribute to the mechanism of chloroaniline nephrotoxicity. Also, the reduced nephrotoxic potential of 4-amino-3-chlorophenol compared to 4-aminophenol could result from an altered ability of the aminochlorophenol to redox cycle or form conjugates.     

Pathways of formation of 2-, 3- and 4-bromophenol from bromobenzene. Proposed mechanism for C-S lyase reactions of cysteine conjugates

Bromobenzene is metabolized by the rat and guinea pig to 2-, 3- and 4-bromophenol. 3-Bromophenol is formed through the sulfur-series pathway to phenols. This route involves the enterohepatic circulation; the key intermediate is the S-(2-hydroxy-4-bromocyclohexa-3,5-dienyl)-L-cysteine derived from the 4-S-glutathione conjugate of the 3,4-oxide. A sulfonium ion C-S lyase reaction is proposed in order to account for the pyridoxal phosphate-dependent cleavage/aromatization step, and a C-S beta-lyase reaction sequence is also proposed for the formation of bromodihydrobenzene thiolols. This route of phenol formation may prove to be a general one for aromatic hydrocarbons and closely related compounds that show arene oxide conjugation with glutathione. 2-Bromophenol is formed predominately by spontaneous isomerization of the 2,3-oxide. 4-Bromophenol is formed by the sulfur-series route from the S-(2-hydroxy-5-bromocyclohexa-3,5-dienyl)-L-cysteine. Additional in vivo routes to 3- and 4-bromophenol involve dehydration/aromatization of the 3,4-dihydro-3,4-diol, possibly by way of conjugates; these routes have transient ketonic intermediates. The pathways from bromobenzene to phenols and to sulfur-containing metabolites derived from premercapturic acids show species and dosage variation.     

Identification, inheritance, and linkage of B-G-like and MHC class I genes in cranes

Trans-4-hydroxy-2-nonenal (HNE) is a potent cytotoxic and genotoxic compound originating from the peroxidation of n-6 polyunsaturated fatty acids. Its metabolism has been previously studied in the rat (Alary et al. 1995. Chem. Res. Toxicol., 8: 35-39). In addition to major urinary mercapturic derivatives, some polar urinary metabolites were isolated and could correspond to hydroxylated compounds. 4-Hydroxynonenoic acid (HNA), resulting from the oxidation of the HNE carbonyl group, is a medium chain fatty acid and its omega-hydroxylation might be hypothesized. Therefore, the involvement of the CYP 4A family isoenzymes in the metabolism of [3H]HNE has been investigated in vivo using inducer treatments (fibrates) in wild-type or in peroxisome proliferator-activated receptor alpha (PPARalpha)-deficient mice. In wild-type mice, but not in PPARalpha (-/-) mice, fibrate treatments resulted in an increase of two urinary metabolites characterized, after HPLC purifications and mass spectrometry analyses, as the omega-hydroxylated metabolite of HNA, i.e., 4,9-dihydroxy-2-nonenoic acid, and its oxidized form, 4-hydroxy-2-nonene-1,9-dicarboxylic acid. The formation of the latter is correlated accurately to laurate hydroxylase activity studied concurrently in microsomes prepared from the liver of these animals. Basal levels of these two metabolites were measured in urine of normal and PPARalpha-deficient mice. These results are in accord with an implication of the P450 4A family in the extended oxidative metabolism of 4-HNE.     

(3R,4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl]chroman-4,7-diol: a conformationally restricted analogue of the NR2B subtype-selective NMDA antagonist (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)- 1-propanol

(1S,2S)-1-(4-Hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606, 1) is a recently described antagonist of N-methyl-D-aspartate (NMDA) receptors containing the NR2B subunit. In the present study, the optimal orientation of compounds of this structural type for their receptor was explored. Tethering of the pendent methyl group of 1 to the phenolic aromatic ring via an oxygen atom prevents rotation about the central portion of the molecule. Several of the new chromanol compounds have high affinity for the racemic [3H]CP-101,606 binding site on the NMDA receptor and protect against glutamate toxicity in cultured hippocampal neurons. The new ring caused a change in the stereochemical preference of the receptor-cis (erythro) compounds had better affinity for the receptor than the trans isomers. Computational studies suggest that steric interactions between the pendent methyl group and the phenol ring in the acyclic series determine which structures can best fit the receptor. The chromanol analogue, (3R,4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1- yl]chroman-4,7-diol (12a, CP-283,097), was found to possess potency and selectivity comparable to CP-101,606. Thus 12a is a new tool to explore the function of the NR2B-containing NMDA receptors.     

Noncanonical conformation of nucleic acids: structure with slipped-loops, occurring in DNA oligonucleotides

A HPLC method was developed and validated for the quantitation of 9-cis-retinoic acid (ALRT1057) and its major metabolite, 4-oxo-9-cis-retinoic acid (LG100182) in human plasma. Samples were buffered and extracted with methyl-tert-butyl-ether. The analytes and an I.S. were separated on a C18 HPLC column using a shallow gradient of 70-89% organic solvent. The analytes were quantitated by UV detection at 348 nm. Selectivity against endogenous compounds and potential metabolites (retinol, all trans-, 13-cis-, and 4-hydroxy-9-cis-retinoic acid) was demonstrated. The run time was 29 min. The standard curve was linear from 2.5 to 450 ng ml-1. Interassay precision for both analytes in quality control samples was less than 5.0% RSD. Accuracy was within 11.0% RE for both compounds. Analyte stability during sample storage, extraction processing, and chromatography was established. Method ruggedness was tested by two analysts and on two HPLC systems. This method has been applied to the quantitation of clinical samples.     

Formation of catechol estrogen glutathione conjugates and gamma-glutamyl transpeptidase-dependent nephrotoxicity of 17beta-estradiol in the golden Syrian hamster

In an animal model of hormone-mediated carcinogenesis, male golden Syrian hamsters develop renal carcinoma following prolonged exposure to 17beta-estradiol. The basis for the species and tissue specificity is unclear. Detailed information on the disposition of 17beta-estradiol in this model is lacking. Because catechol estrogens have been implicated in this model of carcinogenesis, we investigated the metabolism and nephrotoxicity of 17beta-estradiol in golden Syrian hamsters, with emphasis on the formation of catechol estrogen thioethers. 17beta-Estradiol (50 micromol/kg, i.p.) is a mild nephrotoxicant, causing significant elevations in the urinary excretion of gamma-glutamyl transpeptidase (gamma-GT), alkaline phosphatase, glutathione S-transferase (GST) and glucose. Increases in renal protein carbonyls and lipid hydroperoxides, which are markers of oxidative damage, also occur after administration of 17beta-estradiol (50 micromol/kg, i.p.). 17beta-Estradiol-mediated nephrotoxicity is reduced by treating animals with acivicin, an inhibitor of gamma-GT, implying that toxicity is mediated by metabolites requiring metabolism by this enzyme. Following administration of 17beta-[14C]estradiol (100 micromol/kg) to hamsters, 9.7% of the dose is recovered in bile after 5 h, the majority (7.9%) representing aqueous metabolites. Seven catechol estrogen GSH conjugates were identified, 2-hydroxy-1,4-bis-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-estrone, 2-hydroxy-1-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-17beta-estradiol, and 2-hydroxy-1-(glutathion-S-yl)-17beta-estradiol. At 5.4 micromol/kg of 17beta-estradiol, a dose-reflective of daily exposure levels in the hamster model of nephrocarcinogenicity, 12% of the dose is recovered within 5 h as a combination of GSH conjugates of 2- and 4-hydroxy-17beta-estradiol and 2- and 4-hydroxyestrone. In summary, oxidation of catechol estrogens, followed by GSH conjugation, occurs in vivo and 17beta-estradiol is a mild nephrotoxicant in a manner dependent on the activity of gamma-GT.     

Syntheses and evaluation of benzodiazaborine compounds against M. tuberculosis H37Rv in vitro

The synthesis of six benzo[e]diazaborine compounds and thier in vitro evaluation against M. tuberculosis H37Rv is described. The compounds 1,2-dihydro-1-hydroxy-2-phenyl-2,4,1-benzo[e]diazaborin-3(4H)-one, 4, and 1,2-dihydro-1-hydroxy-2-(3-pyridyl)-2,4,1-benzo[e]diazaborin-3(4H) - thione, (5), showed the greatest inhibitory activity.     

The metabolism of zingerone, a pungent principle of ginger

1. The metabolism of 4-(4-hydroxy-3-methoxyphenyl)butan-2-one (zingerone), a pungent principle of ginger, has been investigated in rats. 2. Oral or intraperitoneal dosage (100mg/kg) of zingerone resulted in the urinary excretion of most metabolites within 24 h, mainly as glucuronide and/or sulphate conjugates. While zingerone itself accounted for roughly 50-55% of the dose, reduction to the corresponding carbinol (11-13%) also occurred. Side chain oxidation took place at all three available sites and oxidation at the 3-position, giving rise to C6-C2 metabolites, predominated. About 95-97% of the dose was accounted for. 3. Appreciable (40% in 12 h) biliary excretion occurred. Biliary studies and studies in vitro using caecal micro-organisms indicated that several O-demethylated metabolites found in the urine are of bacterial origin.     

Analytical method development for the simultaneous quantitation of dexmedetomidine and three potential metabolites in plasma

Dexmedetomidine, a novel alpha 2-adrenergic receptor agonist, is being developed as an anesthetic adjunct for perioperative use. An assay method has been developed for the sensitive quantitation of dexmedetomidine and three metabolites in plasma: MPV-1305, MPV-1306 and MPV-1709. The method involves solid-phase extraction (C18 cartridge) of dexmedetomidine and metabolites followed by a two-step derivatization. The first step utilized BF3-MeOH to simultaneously mask a primary alcohol in MPV-1305 and a carboxylic acid in MPV-1306. The second step applied PFB-Cl to derivatize the imidazole ring for sensitive detection of these compounds by GC-negative chemical ionization MS at pg/ml concentrations. MPV-1709 was not derivatized in the process and was detected by GC-positive chemical ionization MS. Optimization of extraction and derivatization is discussed. The method is suitable for quantitation of dexmedetomidine, MPV-1305, MPV-1306 and MPV-1709 over concentration ranges of 0.1, 40, 0.5-100, 0.5-100 and 1.0-500 ng/ml, respectively. The method showed excellent specificity, linearity and sensitivity and is useful for profiling the pharmacokinetic disposition of these compounds.     

Severe meningoencephalitis in Borrelia burgdorferi infection]

A gas chromatographic-mass spectrometric method was used to separate quinine and its metabolites present in urine after oral dosing of 300 mg quinine in humans. The technique allowed the separation of quinine and ten metabolites. Four of these metabolites were definitely identified as 3-hydroxyquinine, 2'-quinone, O-desmethylquinine and 10,11-dihydroxydihydroquinine, by comparing their methane chemical ionization mass spectra with those of authentic standards prepared by organic synthesis. Six other metabolites are described for the first time in human urine. From their electron impact and chemical ionization mass spectra, we propose these compounds to be 3-hydroxy-2'-quinone, O-desmethyl-2'-quinone, O-desmethyl-3-hydroxyquinine, O-desmethyl-3-hydroxy-2'-quinone, 10,11-dihydroxydihydro-2'-quinone and 10,11-dihydroxydihydro-O-desmethylquinine. These secondary metabolites probably arose from further biotransformation of the four primary metabolites.     

Determination of four anabolic steroid metabolites by gas chromatography/mass spectrometry with negative ion chemical ionization and tandem mass spectrometry

A gas chromatography/mass spectrometry method is described which uses negative ion chemical ionization and tandem mass spectrometry for the determination of anabolic steroid metabolites. Four anabolic steroid metabolites to be derivatized by Pentafluoropropionic anhydride (PFPA) were determined using gas chromatography/mass spectrometry (GC/MS) with negative chemical ionization (NCI) and NCI/MS/MS. The repeatability and reproducibility of this procedure were in the range of 5.3-9.7% and 6.1-10.2%, respectively. This method of derivatization with PFPA for NCI and NCI/MS/MS was useful to determine four metabolites of nandrolone, dromostanolone, methenolone and boldenone. The derivatized metabolites of boldenone could be detected to 2 ppb and the other three steroids could be detected to 25 ppb in urine at a signal-to-noise ratio of S/N = 3.     

Metabolism of isbufylline in humans. Isolation, identification, and synthesis of plasma and urine metabolites

Isbufylline metabolism after oral administration to humans was studied. The main metabolites detected by the HPLC method, in plasma, were 1-methyl-7-(2-hydroxy-2-methyl-propyl) xanthine (I), 1,3-dimethyl-7-(2-hydroxy-2-methyl-propyl) xanthine (II), and 1-methyl-7-(2-methyl-propyl) xanthine (III). The main metabolites detected in urine were 1-methyl-7-(2-hydroxy-2-methyl-propyl) xanthine (I), 1,3-dimethyl-7-(2-carboxy-propyl) xanthine (IV), and 1,3-dimethyl-7-(2-hydroxymethyl-propyl) xanthine glucuronic acid (V)-Gluc. They were isolated by HPLC, identified by GC/MS, HPLC/MS, or HPLC/MS/MS, and finally synthesized. Recovery of these metabolites, along with the absence of unmetabolized isbufylline in the urine, indicated biotransformation and renal excretion as the main routes of isbufylline elimination in humans. HPLC quantitation of the characterized urine metabolites revealed that 49% of the drug was eliminated as (I), 9% as (V)-Gluc, and 5% as (IV).     

Representation of foveal visual fields in the ventral bank of the superior temporal sulcus in the posterior inferotemporal cortex of the macaque monkey

A fast and sensitive analytical method was developed to characterize artemether and its metabolites in small amounts in body fluids. The extracts were derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide, separated on an optimized capillary gas chromatographic system and identified by chemical ionization mass spectrometry by using ammonia as reagent gas. The analytical assay is demonstrated on samples extracted from bovine hemoglobin, rat blood and dog blood. Full mass spectra of artemether and three metabolites were obtained at a level of 1 x 10(-6) g/ml.     

Molecular basis of P450 inhibition and activation: implications for drug development and drug therapy

In previous studies many benzimidazole, imidazole and benzothiazole derivatives had been synthesized and their antimicrobial activities were tested in vitro conditions. Four of these compounds showed minimal inhibitory concentrations (MIC) of 5-25 micrograms/ml against standard strains and clinical isolates. In order to determine whether these four compounds can be used for therapeutic purpose, their serum MIC values and side effects on hepatic and renal functions were determined. Different concentrations of the compounds were tested on Wistar rats. Compound 1 was administered orally, intramuscularly and intravenously; compounds 2, 3 and 4 were given orally and intramuscularly. Blood samples were taken 4 and 24 h after administration of the compounds. Serum MIC values were investigated by bioassay and serum levels of biochemical parameters by autoanalyzer. None of the tested compounds showed antimicrobial activity at their serum concentrations. Although creatinine activity was found at normal levels in all experiments, compounds 1 and 2 caused a significant increase in blood urea nitrogen (BUN) level. The values of aspartate aminotransferase and/or alanine aminotransferase and/or alkaline phosphatase which are characteristic for liver function were generally found at high levels. According to these results, it can be concluded that the tested compounds caused damage in liver and biliary tracts without antimicrobial activity by their serum concentrations.     

Sensitive and selective detection of urinary 1-nitropyrene metabolites following administration of a single intragastric dose of diesel exhaust particles (SRM 2975) to rats

1-Nitropyrene (1-NP) has been proposed as a marker for exposure to diesel exhaust particles (DEP). Since the extent of the actual intake of 1-NP adsorbed on DEP will be relatively low, sensitive and selective methods are needed regarding human exposure assessment. Two analytical methods are presented for the assessment of 1-NP metabolites in urine of male Sprague-Dawley rats administered a single intragastric dose of native DEP (SRM 2975, 20 mg, 35.7 microgram of 1-NP/g). Enzymatically hydrolyzed urine was extracted using Blue Rayon. The extracts were analyzed directly, using HPLC with postcolumn on-line reduction and fluorescence detection (HPLC-Flu), or were processed further for GC/MS/MS analysis. Although sensitive to several metabolites, the HPLC-Flu method lacked selectivity for quantitation of some important metabolites in rat urinary extracts, and therefore seems suitable for screening purposes only. With regard to GC/MS/MS analysis, derivatization with heptafluorobutyrylimidazole (HFBI) yielded low limits of determination for hydroxy-1-aminopyrenes, hydroxy-N-acetyl-1-aminopyrenes (converted to derivatized hydroxy-1-aminopyrenes by the reagent), and 1-aminopyrene (1.8-9.2 fmol on the column). Derivatization of hydroxy-1-nitropyrenes yielded relatively high limits of determination, and therefore, hydroxy-1-nitropyrenes were reduced to hydroxy-1-aminopyrenes prior to derivatization with HFBI. Intragastric administration of DEP to rats resulted in urinary excretion of 6-hydroxy-N-acetyl-1-aminopyrene, 8-hydroxy-N-acetyl-1-aminopyrene, 6-hydroxy-1-nitropyrene, 8-hydroxy-1-nitropyrene, and 3-hydroxy-1-nitropyrene (7, 1.2, 1.6, 0.3, and 0.5% of the dose within 12 h, respectively). 1-Nitropyrene, N-acetyl-1-aminopyrene, and 3-, 6-, and 8-hydroxy-1-aminopyrene were not observed as urinary metabolites following administration of a single dose of DEP. The observed excretion pattern and urinary metabolite concentrations suggest that 1-NP present on unmodified DEP becomes bioavailable to a large extent and is metabolized in the same way as was previously observed following administration of pure 1-NP. The presented methods are promising for assessment of human exposure to 1-NP, e.g., following exposure to DEP, because of the possibility of analyzing large volumes of urine, the conversion of three types of metabolites to one (the amino metabolites), and the low detection limits that are achieved.     

Capillary zone electrophoresis with laser-induced fluorescence detection for the determination of nanomolar concentrations of noradrenaline and dopamine: application to brain microdialysate analysis

Determination of catecholamines by capillary zone electrophoresis with laser-induced fluorescence detection was performed on low-concentration samples, which were derivatized with naphthalene-2,3-dicarboxaldehyde to give highly fluorescent compounds. When the borate concentration in the derivatization medium was decreased from 130 to 13 mM, sensitivity for noradrenaline (NA) and dopamine (DA) was greatly enhanced while resolution between these two compounds decreased. A 50 mM borate concentration in derivatization medium was chosen since it provided maximal resolution between NA and DA, together with a high separation efficiency (3.1 million theoretical plates per meter for DA). The injection of 2.4 nL of a NA and DA solution derivatized at 10(-9) M produced peaks with signal-to-noise ratio of 8:1 and 3:1, respectively, corresponding to 1.8 amol of each catecholamine. The calibration curves were linear when NA and DA solutions were derivatized at concentrations ranging from 10(-6) to 10(-9) M. This method was used to determine NA in brain extracellular fluid: a peak corresponding to a basal level of 5 x 10(-9) M endogeneous NA was observed in microdialysates from the medial frontal cortex of the rat, and its nature was confirmed by both electrophoretic and pharmacological validations.