Assessing the potential toxicity of MK-801 and remacemide: chronic exposure in juvenile rhesus monkeys.

The present experiment examined the effects of chronic exposure to either 0.1 or 1.0 mg/kg MK-801 [a selective N-methyl-D-aspartate (NMDA) receptor antagonist] or 20.0 or 50.0 mg/kg remacemide (an NMDA receptor antagonist which also blocks fast sodium channels) in juvenile rhesus monkeys. Endpoints were monitored to provide a general index of subjects' health and included measures of clinical chemistry, hematology, ophthalmology, spontaneous home-cage behavior, and peak drug plasma levels. In general, both drugs were well tolerated and produced no treatment-related effects during 2 years of dosing and assessment. Periodic plasma drug level determinations provided limited evidence that both compounds may induce their own metabolism. The present results contrast sharply with previously reported effects of long-lasting impairments in the acquisition of incremental learning and in the development of color and position discrimination in these same subjects. These observations highlight the importance of collecting a broad range of toxicology data, including tests of cognitive function, to make comprehensive assessments of new drug safety. In the present case, the less obvious effects of these drugs on cognition defined the toxicologic response.

Persistence of gentian violet and leucogentian violet in channel catfish (ictalurus punctatus) muscle after water-borne exposure.

Gentian violet is a triphenylmethane dye that is an antifungal/antiparastic agent. GV is similar to malachite green that has been used in the aquaculture industry for treatment or prevention of external fungal and parasitic infections in fish and fish eggs although it (MG) is not approved for this use. For these reasons, GV's potential for misuse by the aquaculture industry is high. The uptake and depletion of gentian violet (GV) were determined in channel catfish (Ictalurus punctatus) after water-borne exposure (100 ng ml(-1), 1 h) under simulated aquaculture farming conditions. Leucogentian violet (LGV) was rapidly formed, concentrated in the muscle tissue, and very slowly eliminated from muscle tissue. An isocratic (60% acetonitrile-40% water; 0.05 M ammonium acetate buffer, pH 4.5) HPLC system consisting of a 5 microm LC-CN 250x4.6 mm I.D. column, a 20x2.0 mm I.D. PbO2 oxidative post-column, and a UV-VIS detector set at 588 nm were used to determine uptake and depletion of tissue residues of GV and LGV with time. GV was rapidly depleted and converted to its major metabolite, LGV, which was detected out to 79 days. Therefore, LGV is the appropriate target analyte for monitoring exposure of channel catfish to GV.

Confirmation of malachite green, gentian violet and their leuco analogs in catfish and trout tissue by high-performance liquid chromatography utilizing electrochemistry with ultraviolet-visible diode array detection and fluorescence detection.

A sensitive analytical procedure for the confirmation of residues of malachite green (MG), gentian violet (GV) and their leuco analogs (LMG and LGV) in catfish and trout tissue at 10 ng/g is described. Frozen (-20 degrees C) fish fillets were cut into small pieces and homogenized in Waring blendors. The compounds of interest were extracted from 20-g amounts of homogenized fish tissue with acetonitrile-buffer, partitioned against methylene chloride, and isolated with tandem neutral alumina and propylsulfonic acid cation-exchange solid-phase extraction cartridges. Samples of 100 microl (0.8 g equiv.) were chromatographed isocratically in 10 min using an acetonitrile-buffer mobile phase on a short-chain deactivated (SCD) reversed-phase column (150x4.6 mm I.D.) in-line with a post-column oxidation coulometric electrochemical cell (EC), a UV-Vis diode array detector and a fluorescence detector.

Determination of sulfonamides in edible salmon tissue by liquid chromatography with postcolumn derivatization and fluorescence detection.

Fourteen sulfonamides-sulfanilamide, sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxypyridazine, sulfachloropyridazine, sulfamonomethoxine, sulfadoxine, sulfamethoxazole, sulfadimethoxine, and sulfaquinoxoline-residues of which could be found in aquacultured species, were separated in < 25 min by reversed-phase (C18) liquid chromatography (LC) with gradient elution. Analytes were extracted from edible salmon tissue (muscle and adhering skin) with acetonitrile-2% aqueous acetic acid, isolated with 2 liquid-liquid partitionings, and derivatized with fluorescamine after eluting from the column. The derivatives were detected by fluorescence. Recoveries (n = 4) from coho salmon fortified with sulfonamides at 5, 10, and 20 ng/g tissue averaged 79.7 +/- 7.3, 84.6 +/- 7.7, and 88.2 +/- 7.1%, respectively. Limits of quantitation were 5 ng/g tissue, for sulfanilamide, sulfamethoxypyridazine, and sulfaquinoxoline and 1 ng/g tissue for the remaining sulfonamides.

Determination of D-fenfluramine, D-norfenfluramine and fluoxetine in plasma, brain tissue and brain microdialysate using high-performance liquid chromatography after precolumn derivatization with dansyl chloride.

A HPLC method is described for the simultaneous determination of D-fenfluramine (FEN), D-norfenfluramine (NF) and fluoxetine (FLX) using fluorometric detection after precolumn derivatization with dansyl-chloride. The method has limits of quantitation of 200 fmol for FEN and NF, 500 fmol for FLX in brain microdialysate, and 1 pmol for NF and FEN, and 2 pmol for FLX in plasma. Brain tissue standards were linear between 5 and 200 pmol/mg for all three compounds. The inter-assay variability (relative standard deviation) was 6.6%, 6.9% and 9.3% for FEN, 4.6%, 3.7% and 7.9% for NF and 10.4%, 4.9% and 12.2% for FLX, for brain microdialysate (2 pmol/microl), plasma (2 pmol/ microl) and brain tissue (50 pmol/mg), respectively. Intra-assay variability was always lower, typically several times lower than inter-assay variability. Extraction recovery was 108% and 48% for FEN, 105% and 78% for NF and 94% and 45% for FLX, in plasma (2 pmol/microl) and brain tissue (5 pmol/mg), respectively. Due to the stability of the dansyl-chloride derivatives this method is well suited for an autoinjector after manual derivatization with dansyl chloride at room temperature for 4 h.

Simultaneous determination of malachite green, gentian violet and their leuco metabolites in catfish or trout tissue by high-performance liquid chromatography with visible detection.

A sensitive analytical procedure for the determination of residues of leucomalachite green (LMG)-malachite green (MG) and leucogentian violet (LGV)-gentian violet (GV) in catfish or trout tissue is presented. Frozen (-20 degrees C) fish fillets were cut into small pieces and blended in a Waring blender. A 20-g amount of homogenized fish tissue was extracted with acetonitrile-buffer, partitioned against methylene chloride, and cleaned up on tandem neutral alumina and propylsulfonic acid cation-exchange solid-phase extraction cartridges. Samples of 100 microliters (0.8 g equiv.) were chromatographed isocratically in 10 min using an acetonitrile-buffer mobile phase on a short-chain deactivated (SCD) reversed-phase column (250 x 4.6 mm I.D.) in-line with a post-column PbO2 oxidation reactor. The PbO2 post-column reactor efficiently oxidized LMG to the chromatic MG, and LGV to the chromatic GV permitting visible detection at 588 nm for all four compounds. Linearity was demonstrated with standards over the range of 0.5-50 ng per injection. Recoveries of LMG, MG, LGV and GV from catfish tissues fortified at 10 ng/g were 75.4 +/- 3.0, 61.3 +/- 4.1, 72.6 +/- 3.7 and 87.9 +/- 2.5, respectively, while trout tissues fortified at 10 ng/g yielded recoveries of 82.6 +/- 2.3, 48.6 +/- 1.8, 72.1 +/- 2.1 and 83.8 +/- 4.6 (mean +/- S.D., n = 4), respectively.

Confirmation of gentian violet and its metabolite leucogentian violet in catfish muscle using liquid chromatography combined with atmospheric pressure ionization mass spectrometry.

Gentian violet (GV) is a triphenylmethane dye antiseptic with potential for illegal use in livestock production, especially aquaculture where the related malachite green has been widely used. This potential misuse has regulatory importance because of the observed rodent carcinogenicity of GV. This report describes the use of online LC-APCI/MS for confirmation of incurred GV residues, and those of its principal metabolite, LGV, in catfish muscle following treatment of live catfish with GV under putative use conditions. LC with APCI/MS detection provided sensitive analysis of GV and LGV with estimated detection limits of < 1 pg observed for both compounds. Fragmentation of GV and LGV via in-source CID was effected by varying the sampling cone-skimmer voltage. Ion intensity data were collected using a rapid cone voltage switching procedure that permits selected ion acquisition under optimal conditions for the parent molecule and several selected fragment ions. For GV, four ions including the ionized molecule were used and for LGV, six ions including the protonated molecule were used. The levels of GV and LGV in muscle from fish dosed with 10 micrograms/l in aquarium water were determined by LC/VIS to be 0.5 and 44 ppb, respectively. Analysis of these samples yielded ion intensity ratios that agreed precisely between injections (< 5%) and accurately with those generated by a comparable amount of authentic GV and LGV (< 10% deviation). These results show the utility of on-line LC-APCI/MS to do both sensitive confirmatory analyses of incurred drug residues for use in monitoring the food supply.

Determination of leucogentian violet and gentian violet in catfish tissue by high-performance liquid chromatography with visible detection.

A sensitive analytical procedure for the determination of residues of leucogentian violet (LGV) and gentian violet (GV) in catfish tissue is presented. Frozen (-20 degrees C) catfish fillets were cut into chunks and then blended in a Waring blender. A 10-g amount of catfish muscle tissue was homogenized and extracted with acetonitrile-buffer, partitioned against methylene chloride, and cleaned up on tandem neutral alumina and propylsulfonic acid cation-exchange solid-phase extraction cartridges. Samples of 100 microliters (0.5 g equiv.) were chromatographed isocratically in 15 min using an acetonitrile-buffer mobile phase on a cyano phase column in-line with a post-column PbO2 oxidation reactor. The PbO2 post-column reactor efficiently oxidized the LGV to the chromatic GV permitting visible detection at 588 nm for both LGV and GV. Linearity was demonstrated with standards over the range 0.5-50 ng per injection. Recoveries of LGV and GV from catfish tissues fortified at 20, 10, and 1 ng/g were 83.1 +/- 1.2, 78.4 +/- 4.0, 84 +/- 8 and 92.7 +/- 1.8, 95.0 +/- 2.2, 93 +/- 2 (mean +/- S.D., n = 4), respectively.

Liquid chromatographic determination of sulfadiazine in salmon by postcolumn derivatization and fluorescence detection.

A reversed-phase (ODS-2) liquid chromatographic method was developed to determine low nanogram-per-gram levels of sulfadiazine (SDZ) in salmon muscle tissue. SDZ was extracted with acetonitrile-aqueous 2% acetic acid (pH 3.0), partitioned into methylene chloride, and cleaned up by using a strong-cation-exchange, solid-phase extraction cartridge. SDZ was derivatized postcolumn with fluorescamine and detected by fluorescence. The limit of detection was 0.2 ng SDZ/g tissue. Recoveries from coho salmon tissue fortified with 1, 5, 10, and 20 ng SDZ/g tissue averaged 84.5, 85.0, 83.6, and 83.9%, respectively; recoveries from Atlantic salmon tissue fortified with 10 ng SDZ/g tissue averaged 82.6%.

Formation of artifactual metabolites of doxylamine following acid hydrolysis.

This study describes the use of gas chromatographic-mass spectrometric, high-performance liquid chromatographic and capillary column gas chromatographic separation techniques in demonstrating the production of several artifactual compounds reported in the literature as metabolites of doxylamine. Rhesus monkey urinary extracts which contained doxylamine and doxylamine metabolites were examined with and without acid hydrolysis. The production of 1-phenyl-1-(2-pyridinyl)ethanol and 1-phenyl-1-(2-pyridinyl)ethylene under acid hydrolysis conditions was demonstrated. These artifactual products were shown to originate from the acid hydrolysis of 2-[1-phenyl-1-(2-pyridinyl)ethoxy] acetic acid and not from doxylamine.

Characterization, purification, and analysis of solvent yellow 33 and solvent green 3 dyes.

Thin-layer chromatography and high-performance liquid chromatography (HPLC) with linear photodiode array detection (LPDA) were used to separate impurities from two commercial dyes. Gravity flow liquid chromatography was used to purify reference standards of the dyes. Normal phase HPLC with LPDA detection was used to determine purities of submitted samples by comparing responses to those of the reference standards. Electron impact mass spectrometry and nuclear magnetic resonance were utilized to confirm structures of the dyes and their impurities.

Determination of triethylenetetramine dihydrochloride in aqueous solution by reversed-phase ion-pairing high performance liquid chromatography and conductivity detection.

Triethylenetetramine dihydrochloride (TETA) has been used for the treatment of Wilson's disease which is a metabolic disorder that prevents its victims from eliminating excess copper. TETA was scheduled for toxicological evaluation because of a deficiency of such information. Analytical chemical procedures to determine the purity of the drug as well as the proper concentration and stability of the drug in dosed water were prerequisites for the toxicological tests. A high performance liquid chromatography (HPLC) procedure employing ion-pairing and conductivity detection has been developed for the analysis of TETA in dosed water at levels as low as 10 micrograms/mL and for the determination of drug purity. The conductivity detector response was linear over the concentration range of 10 to 100 micrograms/mL. Data are presented concerning the stability of the drug in water during ambient storage and after autoclaving. An ancillary colorimetric procedure for the analysis of aqueous TETA solutions is also presented which is based on measuring the absorbance of the colored TETA copper chelate at 599 nm. The HPLC procedure is applicable to the analysis of TETA and the chemically similar polyamines spermidine and spermine in admixture.

Analysis of D-penicillamine by gas chromatography utilizing nitrogen--phosphorus detection.

A method is presented for the analysis of the "orphan" drug D-penicillamine (D-Pa), which is used for the treatment of the inherited rare copper metabolism dysfunction known as Wilson's disease, by assaying a derivative of the compound by gas chromatography employing a rubidium sensitized nitrogen--phosphorus detector. Analytical procedures are described for the analyses of residues of D-Pa X HCl salt in animal feed and for the analyses of the salt or free base from aqueous solutions by utilizing a single-step double derivatization with diazomethane--acetone. Stability data for D-Pa X HCl in animal feed and for the free base in water are presented. An ancillary fluorescence derivatization procedure for the analysis of D-Pa in water is also reported.

Simultaneous determination of trimellitic anhydride and its trimellitic acid impurity by GC/FID.

Trimellitic anhydride has widespread usage in a variety of industrial processes. Inhalation of the airborne dust and fumes generated by these processes is the primary route of occupational exposure leading to a variety of respiratory maladies. Because of its extensive industrial usage and limited toxicological data, trimellitic anhydride was scheduled for toxicological evaluation. Analytical purity certification and chemical characterization of the chemical were required as prerequisites for such toxicological tests. Therefore, a sensitive and specific procedure for the analysis of trimellitic anhydride and its trimellitic acid impurity in admixture was developed. After methylation with diazomethane, the compounds were assayed by gas chromatography using flame ionization detection. Chromatography on 5% SP-2100 yielded two well separated peaks, which were used for quantitation of the parent compounds. Confirmational identity of trimellitic anhydride was determined by direct insertion probe mass spectrometry, while that of the anhydride methyl derivative and trimellitic acid trimethyl derivative was determined by gas chromatography-mass spectrometry.

A "marker" technique to monitor treated industrial wastewater effluents.

In toxicological research with hazardous substances (e.g. carcinogens), wastewater effluent from the test facility must be free of such substances before discharge into the environment. An industrial wastewater processing employing adsorbers of carbon and XAD-2 resin is described; however, chemical assays of each batch of treated effluent must certify the absence of all test agents. Elution profiles and adsorption isotherm tests with the test agents vs. the two adsorbents provided the basis for a "marker" technique which should eliminate the necessity to assay for all test agents in each batch of processed effluent. A radionale is presented for periodic introduction of a "marker" (gentian violet) into the primary adsorbers. If detected in the effluent, the "marker", which elutes from the adsorbers before most of the test agents would signal impending depletion of the adsorbent which could then be replaced. Recommendations to modify the industrial wastewater treatment plant and to implement the "marker" technique are presented as cost-effective alternatives to extensive and laborious chemical assays.

Determination of gentian violet in animal feed, human urine, and wastewater by high pressure liquid chromatography.

Sensitive and specific procedures are described for the analysis of residues of gentian violet in animal feed, human urine, and wastewater at levels of 1000 ppm down to 10 ppb, 1 ppb, and 10 ppb, respectively. The cleaned-up extracts were analyzed by reverse-phase high pressure liquid chromatography by using an absorption detector at 588 nm. Recoveries of the compound from animal feed, human urine, and wastewater at the 10 ppb level were 79%, 58%, and 60%, respectively. Information concerning the stability of the compound in animal feed and the efficiency of extracting the residues is presented. Ancillary information is also reported concerning the separation and analysis of six related triphenylmethane dyes.

Determination of sodium phenobarbital in animal chow by high-pressure liquid chromatography.

An analytical procedure is described for determining residues of sodium phenobarbital in animal chow at levels as low as 0.14 ppm. The methanol extract is subjected to a liquid-liquid cleanup at pH 13 and 1, further cleaned up on a silica gel column and assayed by high-pressure liquid chromatography by using an ultraviolet absorption detector at 210 nm. Data concerning extraction efficiency, partition values and stability of the chemical in animal chow are also presented.