Antibodies to quinolinic acid and the determination of its cellular distribution within the rat immune system

Antibodies to quinolinic acid were produced in rabbits with protein-conjugated and gold particle-adsorbed quinolinic acid. Quinolinic acid immunoreactivity was below detection limits in carbodiimide-fixed rat brain. In contrast, strong quinolinic acid immunoreactivity was observed in spleen cells with variable, complex morphology located predominantly in the periarterial lymphocyte sheaths. In the thymus, quinolinic acid immunoreactivity was observed in cells with variable morphology, located almost exclusively in the medulla. Lymph nodes and gut-associated lymphoid tissue contained many, strongly stained cells of similar complex morphology in perifollicular areas. Immunoreactivity in liver and lung was restricted to widely scattered, perivascular cells and alveolar cells respectively. Additional stained cells with complex morphology were observed in bronchus-associated lymphoid tissue, in skin, and in the lamina propria of intestinal villi. Follicles in all secondary lymphoid organs were diffusely stained, ranging from mildly to moderately immunoreactive in spleen, to intensely immunoreactive in gut-associated lymphoid tissue. These results suggest that quinolinic acid is an immune system-specific molecule. Two hypothetical schemes are proposed to account for high levels of quinolinic acid in specific cells of the immune system.     

High-performance liquid chromatographic separation of microcystins derivatized with a highly fluorescent dienophile

Microcystins are potent hepatotoxins produced by cyanobacteria, and are also tumor promoters as well as potent inhibitors of the catalytic subunits of protein phosphatases 1 and 2A. In order to establish a physicochemical method for individual detection and determination of trace amounts of microcystins, we developed a derivatization method for fluorescence (FL) and chemiluminescence (CL) detection, in which a highly fluorescent dienophile, DMEQ-TAD (4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl) ethyl]-1,2,4-triazoline-3,5-dione), was used as the labeling reagent. DMEQ-TAD reacted smoothly with the conjugated diene of the Adda moiety to give 2 stereoisomers of the adducts. As a result of the extensive experiments, the following reaction conditions were optimized for the labeling: sample amount, 10 micrograms; reaction solvent, DMF:acetonitrile (1:1); reaction time, 15 minutes; reaction temperature, 70 degrees C; amount of DMEQ-TAD used relative to that of microcystin, 80 equivalent. The resulting 6 adducts from microcystins-LR, -YR, and -RR can be separated from one another using the following reversed phase HPLC conditions in combination with a clean-up using ODS silica gel: column, Cosmosil 5C18-AR (150 x 4.6 I.D. mm); mobile phase, methanol:0.05M phosphate buffer (pH 3) (1:1); flow rate, 1.0 ml/min; detection, FL lambda ex 370 nm, lambda em 440 nm. The detection limits of the DMEQ-TAD derivatives were estimated to be 100 and 500 pg for LR, and 65 and 2,500 pg for RR using FL and CL detections, respectively; and the detection behavior was different from that of the Dns-Cys derivatives, which were more sensitive to CL than FL.     

The use of dominant-negative mutations to elucidate signal transduction pathways in lymphocytes

A simple procedure for the determination of amphetamine in urine with minimal sample preparation is described. This method involves direct addition of human urine to an acetone-dansyl chloride solution for simultaneous deproteinization and fluorescence derivatization. The derivatized amphetamine is then measured by HPLC with fluorescence detection. It eliminates the extraction procedures often required by other HPLC or GC methods. The effects of pH, temperature and reaction time on the derivatization reaction were investigated. The stability of amphetamine-dansyl chloride in different storage conditions was examined. The detection limit and linearity associated with this assay are discussed.     

Isolation and expression of a mouse CB1 cannabinoid receptor gene. Comparison of binding properties with those of native CB1 receptors in mouse brain and N18TG2 neuroblastoma cells

The chiral separation of enantiomeric forms of derivatized amino acids have been achieved based on a metalchelate chiral capillary electrophoretic method and a cyclodextrin mediated host-guest interaction approach in micellar electrokinetic chromatography (MEKC) mode with laser-induced fluorescence detection. This approach has been applied to the determination of enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics, BMY-45012 and its analogs. Amino acids were analyzed by complete hydrolysis and the hydrolysate was derivatized with either dansyl chloride for UV absorbance detection or fluorescein isothiocyanate for laser based fluorescence detection. The presence of several amino acids, serine and beta-hydroxyl-N-methy-valine in the proposed structure have been confirmed as D-serine and L-beta-hydroxyl-N-methy-valine enantiomeric forms by both chiral capillary electrophoresis (chiral CE) and MEKC approaches. A non-chiral amino acid, sarcosine, was also confirmed. These methodologies provide a quick and sensitive approach for the determination of amino acids racemization of pharmaceutical natural products and have proven to be useful for structural elucidation refinement.     

Synthesis, Characterization and Fluorescence Properties of Europium, Terbium Complexes with Biphenyl-4-Carboxylic Acid and o-Phenanthroline

Two series of solid complexes of europium and terbium with biphenyl 4-carboxylic acid and phen were synthesized and characterized in this report. Their elemental analysis, molar conductivities and TG-DTA studies indicate that the complexes have the composition of Eu(phen)L3·1/2H2O, Eu0.5RE0.5(phen)L3·1/2H2O; Tb(phen)L3·H2O and Tb0.5RE0.5(Phen)L3·1/2H2O. (RE=Y3 , La3 and Gd3 ; L=biphenyl 4-carboxylic acid; phen=o-Phenanthroline). The studies of their IR, UV 1H NMR and molar conductivities demonstrate that biphenyl 4-carboylic acid is bounded with RE(Ⅲ) ion. Rare earth ions coordinate with two nitrogen atoms of phen molecules directly in these rare earth complexes. The fluorescence spectra and fluorescence lifetimes of the rare earth complexes show that the fluorescent intensity and lifetime of a series of europium complexes are longer than those of the series of terbium complexes as having the some ligands. There are better fluorescent intensity and lifetime of hetero-nuclear rare earth complexes than homo-nuclear rare earth complexes for europium complexes. The fluorescence emission intensity of Eu3 is raised by inert fluorescent rare earth ions (Y3 , Gd3 and La3 ), but in Tb3 hetero-nuclear rare earth complexes the intensity of Tb3 ions are quenched by the inert fluorescent rare earth ions.     

Sensitive determination of a new antiarrhythmic agent, trecetilide, in plasma by high-performance liquid chromatography with fluorescence detection

Two high-performance liquid chromatography (HPLC) methods were developed for the determination of trecetilide in plasma samples. Differing only in the addition of a derivatization step and different detection wavelengths, the two methods encompassed a wide concentration range. In both methods, plasma samples (0.1 ml) with added internal standard were applied to solid-phase extraction discs containing a non-polar/strong cation mixed-phase, washed and eluted with an acetone-acetonitrile triethylamine mixture. The eluate was evaporated to dryness, and either reconstituted and directly injected onto an HPLC column or first derivatized with 1-naphthyl isocyanate before HPLC analysis. In both methods, the separation was performed isocratically on a cyano analytical column utilizing a mobile phase composed of acetonitrile-pH 7.9 phosphate buffer (70:30, v/v). The column effluent was monitored by fluorescence detection at 290/345 nm (with derivatization) or 235/320 nm (without derivatization). The limits of detection and quantitation of the assay were 0.57 and 1.9 ng/ml, respectively, when derivatization was used, or 4.3 and 14 ng/ml, respectively, without derivatization.     

Quantification of carnitine, acetylcarnitine, and total carnitine in tissues by high-performance liquid chromatography: the effect of exercise on carnitine homeostasis in man

A method for the quantitative determination of carnitine, acetylcarnitine, and total carnitine in tissue was developed for application to clinical research and diagnosis. Human skeletal muscle and heart specimens (10-20 mg) were homogenized in 1 ml of water. Aliquots of the resulting homogenates (50 microliters) were extracted with 1.0 ml of acetonitrile:methanol (3:1) and the carnitine-related compounds were isolated using columns containing 300 mg of silica gel. Samples were then derivatized with 4'-bromophenacyl trifluoromethanesulfonate for spectrophotometric detection or 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate for fluorescence detection and quantified by high-performance liquid chromatography. Fluorometric detection of 2-(2,3-naphthalimino)ethyl ester derivatives afforded a 500-fold increase in sensitivity when compared to derivatization with 4'-bromophenacyl trifluoromethanesulfonate. This methodology permitted detection of acetylcarnitine in dilute human muscle homogenates at quantities of 790 fmol of acetylcarnitine injected. The method was applied to a series of human skeletal muscle biopsy samples obtained from subjects performing exercise at high work loads. The method permitted quantification of carnitine, acetylcarnitine, and total carnitine (sum of carnitine and all acylcarnitines) and demonstrated the specific redistribution of the carnitine pool from carnitine to acetylcarnitine with exercise above the lactate threshold. This HPLC method is facile, and provides a sensitive and specific approach for use in human biopsy specimens.     

Modification of fluorescent properties of norfloxacin in the presence of certain antacids

Norfloxacin (NFLX), a broad spectrum antibacterial quinolone, is a very thermostable but photosensitive drug, especially in solution leading to the formation of an ethylenediamine degradate. The modification of the fluorescent properties of NFLX in acid solution after exposure to fluorescent light and the degradation mechanisms were studied. Two analytical methods were previously developed and validated for NFLX, ultraviolet spectrophotometry (UV) and spectrofluorimetry (FL). Data obtained using both methods in the analysis of remaining NFLX in terms of percent recoveries revealed that there was no statistically significant modifications of the UV signal and of the recoveries obtained by the method. However, an important increase of the fluorescent signal after light exposure of NFLX solutions appeared, which led to an increase of the average recovery up to 270% over 15 months. Using a previously validated HPLC method for the photostability studies of NFLX, a loss of 5% with respect to the initial drug amount was observed. The study of UV and fluorescence spectra evidenced the formation of the degradation product, which induced significant modification of the fluorescent properties of NFLX samples. These results clearly indicated that FL analysis definitively is the method of choice and can be used to study the photodegradation of NFLX.     

Capillary zone electrophoresis separation of tryptophan and its metabolites, including quinolinic acid

Capillary zone electrophoresis with UV absorbance detection was used to separate tryptophan and ten of its metabolites. Run buffers of pH 4.0-10.0 were evaluated for their effect on resolution; a pH 9.6 buffer was found to give optimum separation of all components. Ethylenediaminetetraacetic acid (EDTA), which prevents complexation of some analytes with polyvalent cations, was included in the run buffer to insure good peak shape and reproducible mobilities. The resulting method was used to detect the presence of quinolinic acid in a urine sample.     

Pharmacokinetics of a discontinuous absorption process of oxolinic acid in turbot, Scophthalmus maximus, after a single oral administration

1. The pharmacokinetics of oxolinic acid have been studied in 500 g turbot (Scophthalmus maximus). The fish were kept in seawater at 16 degrees C with a 15 h/9 h photoperiod. Oxolinic acid was administered orally via a stomach tube at a single dose of 10 mg/kg of body weight. Serum concentrations of oxolinic acid were determined by a (HPLC) using liquid phase extraction with an internal standard and a fluorescence detection. 2. The pharmacokinetic process was not significantly sex-influenced. The short elimination phase of the oxolinic acid in turbot after oral administration was similar to the elimination after intravascular administration. The serum concentration profile of oxolinic acid was better described by a discontinuous absorption model than by compartment models using continuous absorption processes. The absorption of oxolinic acid in turbot was characterized by two distinct phases after a lag time of about 2 h. A time (Tmax) of 12 h was necessary to reach the peak serum concentration (Cmax) of 1.41 microg/ml. The oral bioavailability was 27.9%. 3. Based on the minimum inhibitory concentration for susceptible strains, and especially Vibrio anguillarum, the oxolinic acid could be effective in turbot after an oral treatment of 10 mg/kg/day.     

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.     

An automated method for the determination of subnanogram concentrations of eprinomectin in bovine plasma

Eprinomectin is a potent anthelmintic compound that kills certain parasitic nematodes and arthropods of cattle. A sensitive and automated bioanalytical assay was developed for quantitation of eprinomectin in bovine plasma in support of clinical development of eprinomectin for use in all classes of cattle. This assay determined the concentration of eprinomectin in plasma by reversed-phase high performance liquid chromatography (HPLC) with fluorometric detection. Plasma sample preparation included liquid extraction performed by the Packard MultiPROBE robotics workstation, followed by solid phase extraction performed by the Gilson ASPEC XL automated workstation. The HPLC assay included automated pre-column derivatization with a fluorogenic reagent system which included trifluoroacetic anhydride and N-methylimidazole as the catalyst. This reversed-phase chromatographic analysis was based on the fluorescence detection of derivatized eprinomectin and an internal standard, L-648 548, which was similarly derivatized by the fluorogenic reagents. The assay was automated and validated for two concentration ranges of 0.05-10 and 0.5-200 ng ml-1. The lower limit of quantitation of eprinomectin in plasma was 0.05 ng ml-1. The %RSD of the assay was 10% or better at all concentrations. This automated analysis of eprinomectin was used for high-throughput clinical assays with acceptable accuracy and precision.     

Highly effective separation and highly sensitive detection for clinical chemistry and biochemistry

Highly effective separation and highly sensitive detection reagents for clinical chemistry and biochemistry were developed and their applications were investigated. The sensitive detection of carboxylic acids was accomplished using 9-anthryldiazomethane (ADAM) which gave intensely fluorescent derivatives from carboxylic acids without catalysts or heating. 1-Pyrenyldiazomethane was then synthesized and proved to react also readily with carboxylic acid and more sensitive than ADAM. The optical resolution of amino acid enantiomers was achieved using 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC). GITC derivatized enantiomeric amino acids under mild conditions to give highly hydrophobic diastereomers which could be resolved on conventional reversed phase columns and easily detected by the absorption based on the thiourea structure. Then we devised an o-phthalaldehyde-N-acetylcystein reagent (OPA/NAcCys) giving diastereomers which were also resolved on a reversed phase column and detected fluorometrically with excellent sensitivity. OPA/NAcCys was useful for the assay of D-amino acids in the blood of uremic patients. The hypochlorite-thiamine method for the assay of proteins and peptides was established providing sensitive fluorometry, which well reflected the number of peptide groups in the molecule. This principle was applied to the assay using N-chlorodansylamide, designed for the fluorometry of peptides. The alkaline ninhydrin method was applied to the detection of guanidino compounds in the blood of uremic patients. Several fluorometric methods for the simultaneous detection of reducing and non-reducing carbohydrates, and guanidines were found to be useful reagents for this purpose because these compounds were resistant to the oxidation with periodate. Then protamine-bound columns were prepared for the separation of carbohydrates on HPLC, which showed excellent recovery of reducing carbohydrates in comparison with conventional alkylamino columns.     

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.     

High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine cysteine conjugate beta-lyase activity: application to S-1,2-dichlorovinyl-L-cysteine and S-2-benzothiazolyl-L-cysteine

An HPLC-fluorescence assay has been developed for the determination of the activity of rat renal cytosolic cysteine conjugate beta-lyase. The method is based on isocratic HPLC separation and fluorescence detection of pyruvic acid, derivatized with o-phenylenediamine (OPD), and is shown to be rapid, specific, and very sensitive. The assay has been evaluated with two model substrates for rat renal cytosolic beta-lyase, notably S-1,2-dichorovinyl-L-cysteine (DCVC) and S-2-benzothiazolyl-L-cysteine (BTC). Equimolar formation of pyruvic acid and 2-mercaptobenzothiazole, a chromophoric thiol, indicated that pyruvic acid formation actually reflects the beta-elimination activity of beta-lyase during the beta-elimination of BTC. From this it follows that the pyruvic acid assay can be applied to the measurement of the beta-elimination activity of this enzyme, independent of the presence of chromophoric groups or radiolabels in substrates. Due to the large linear range and the very high sensitivity of the present HPLC-fluorescence assay (detection limit, 7.5 pmol of pyruvic acid), both good and poor substrates of beta-lyase can be measured. Enzyme kinetic data are presented for the model substrates BTC and DCVC and for four structurally related S-2,2-difluoroethyl-L-cysteine conjugates.     

Analysis of aliphatic dicarboxylic acids in pharmaceuticals and cosmetics by liquid chromatography (HPLC) with fluorescence detection

2-Bromoacetyl-6-methoxynaphthalene has been found to be a useful prechromatographic fluorescent labelling reagent for the analysis of dicarboxylic acids. The derivatization reaction of azelaic acid and meglutol with this reagent yieded stable and highly fluorescent diesters which could be analysed by reversed-phase HPLC with fluorescence detection. According to the nature of the sample, the derivatization reaction could be carried out in acetonitrile or in an aqueous micellar system. The proposed methods proved to be suitable for the quality control of various complex pharmaceutical and cosmetic formulations of the azelaic acid and meglutol.     

Methamphetamine-stimulated striatal dopamine release declines rapidly over time following microdialysis probe insertion

A sensitive and selective HPLC method for the determination of ethambutol in human plasma and urine was developed. Ethambutol was extracted from basified plasma samples (0.2 ml) with diethyl ether, back-extracted into 0.01 M phosphoric acid and derivatized with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole. After 30 min at 80 degrees C and elimination of the reactive excess, the compound was determined by reversed-phase liquid chromatography. urine was analysed for ethambutol after dilution 1:200 with distilled water and derivatization as described for plasma. Quantification in plasma and urine was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human plasma and urine was observed. The limit of quantification was 10 ng/ml in plasma and 10 micrograms/ml in urine. The suitability of the method for in vivo samples was checked by analysis of plasma and urine samples drawn from healthy volunteers who had received a 1200-mg oral dose of the test compound.     

Potentiation of paclitaxel cytotoxicity by inostamycin in human small cell lung carcinoma, Ms-1 cells

A robust new analytical method has been developed for the determination of 5-fluorouracil (5-FU) in human plasma samples using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The method is based on a liquid-liquid extraction procedure, precolumn derivatization, reversed-phase HPLC separation, and detection using atmospheric pressure chemical ionization and selected reaction monitoring. The derivatization agent used was 4-bromomethyl-7-methoxycoumarin. The internal standard for the assay procedure was a stable isotope labeled analog of 5-FU. The lower limit of quantitation was 1.0 ng/mL using 500 microL aliquots of plasma. Sample throughput on the mass spectrometer was approximately 17 samples/h (3.5 min/sample). The method was fully validated. The recovery of 5-FU averaged 76.1%. The accuracy of the assay, assessed from quality control samples, ranged from 99.1% to 104.3% (% theoretical). The overall interassay precision (% RSD) was 2.7%, and the intraassay precision (% RSD) ranged from 1.5% to 3.9%. The derivatized samples were found to be stable under sample analysis conditions and during refrigerator storage. The method was specific for the determination of 5-FU.     

Functional dyspepsia in liver cirrhosis]

Precolumn fluorescence derivatization for the determination of the antagonist [Arg6,D-Trp7,9,MePhe8]-Substance P?6-11? (antagonist G) using benzoin in HPLC was studied. Under the conditions chosen (0.067 M NaOH, heating at 100 degrees C for 10 s), a good yield of fluorescent derivatives was obtained and no methodology-related degradation occurred. The detection limit of antagonist G was 0.21 nmol/ml. The method has been applied to the selective and sensitive detection of arginine-containing fragments in degradation products of antagonist G.     

General protease assay method coupling solid-phase substrate extraction and capillary electrophoresis

Capillary electrophoresis with laser-induced fluorescence detection was used to develop a universal, highly specific protease assay. In this method, a peptide, biotinylated at the N-terminus, is labeled with fluorescein at a lysine residue near the C-terminus. Impurities are removed from the fluorescence labeling mixture by solid-phase extraction of the substrate on immobilized streptavidin, followed by extensive washing. The purified fluorescent substrate is dissociated from the streptavidin and incubated with the protease. The peptide sequence between the biotin and fluorescent label contains the cleavage sequence of the protease of interest. After cleavage, the fluorescent product does not contain a biotin group. A second solid-phase extraction is used to remove unreacted substrate to dramatically lower the background signal. The product is detected by capillary electrophoresis, which provides powerful discrimination against products generated by nonspecific proteases. With chymotrypsin as a test protease, product was detected with as little as 10 pg/mL (4.6 x 10(-13) M) chymotrypsin, or 5 amol of enzyme in the 10-microL sample volume.