Reactions of deoxyribonucleotide bases with sulfooxymethyl or halomethyl polycyclic aromatic hydrocarbons induce unwinding of DNA supercoils.

Torsional stress in double-stranded DNA enables and regulates facets of chromosomal metabolism, replication, and transcription and requires regulatory enzymatic systems including topoisomerases and histone methyltransferases. As such, this machinery may be subject to deleterious effects from reactive mutagens, including ones from carcinogenic polycyclic aromatic hydrocarbon (PAH) adduct formation with DNA. Supercoiled plasmid DNA was investigated for its torsional responses to adducts formed in vitro from PAH benzylic carbocation reactive intermediates created spontaneously by release of leaving groups. PAH sulfate esters were found to (1) unwind DNA in a concentration dependent manner, and (2) provide maximum unwinding in a pattern consistent with known carcinogenicities of the parent PAHs, that is, 6-methylbenzo[a]pyrene > 7,12-methylbenz[a]anthracene > 3-methylcholanthrene > 9-methylanthracene > 7-methylbenz[a]anthracene > 1-methylpyrene. Supercoil unwinding was demonstrated to be dependent on the presence of sulfate or chloride leaving groups such that reactive carbocations were generated in situ by hydrolysis. In silico modeling of intercalative complex topology showed PAH benzylic carbocation reactive functional groups in alignment with target nucleophiles on guanine bases in a 5'-dCdG-3' pocket in agreement with known formation of nucleotide adducts. Inhibitory or modulatory effects on PAH-induced supercoil unwinding were seen with ascorbic acid and an experimental antineoplastic agent Antineoplaston A10 in agreement with their known anticarcinogenic properties. In summary, the reactive PAH intermediates studied here undoubtedly participate in well-known mutational mechanisms such as frameshifts and apurinic site generation. However, they are also capable of random disruption of chromosomal supercoiling in a manner consistent with the known carcinogenicities of the parent compounds, and this mechanism may represent an additional detrimental motif worthy of further study for a more complete understanding of chemical carcinogenicity.

Mass spectrometric methods for evaluation of voriconazole avian pharmacokinetics and the inhibition of its cytochrome P450-induced metabolism.

Invasive fungal aspergillosis is a leading cause of morbidity and mortality in many species including avian species such as common ravens (Corvus corax). Methods were developed for mass spectral determination of voriconazole in raven plasma as a means of determining pharmacokinetics of this antifungal agent. Without further development, GC/MS/MS (gas chromatography-tandem quadrupole mass spectrometry) proved to be inferior to LC/MS/MS (liquid chromatography-tandem quadrupole mass spectrometry) for measurement of voriconazole levels in treated raven plasma owing to numerous heat-induced breakdown products despite protection of voriconazole functional groups with trimethylsilyl moieties. LC/MS/MS measurement revealed in multi-dosing experiments that the ravens were capable of rapid or ultrarapid metabolism of voriconazole. This accounted for the animals' inability to raise the drug into the therapeutic range regardless of dosing regimen unless cytochrome P450 (CYP) inhibitors were included. Strategic selection of CYP inhibitors showed that of four selected compounds including cimetidine, enrofloxacin and omeprazole, only ciprofloxacin (Cipro) was able to maintain voriconazole levels in the therapeutic range until the end of the dosing period. The optimal method of administration involved maintenance doses of voriconazole at 6 mg/kg and ciprofloxacin at 20 mg/kg. Higher doses of voriconazole such as 18 mg/kg were also tenable without apparent induction of toxicity. Although most species employ CYP2C19 to metabolize voriconazole, it was necessary to speculate that voriconazole might be subject to metabolism by CYP1A2 in the ravens to explain the utility of ciprofloxacin, a previously unknown enzymatic route. Finally, despite its widespread catalog of CYP inhibitions including CYP1A2 and CYP2C19, cimetidine may be inadequate at enhancing voriconazole levels owing to its known effects on raising gastric pH, a result that may limit voriconazole solubility.

MEASUREMENT OF PERSISTENT ORGANIC POLLUTANTS, PERFLUORINATED COMPOUNDS, AND TOXIC METALS IN THE BLOOD OF HUMBOLDT PENGUINS (SPHENISCUS HUMBOLDTI) AT PUNTA SAN JUAN, PERU USING DRIED BLOOD SPOTS.

The Humboldt penguin (Spheniscus humboldti) population at the Punta San Juan Marine Protected Area in Peru is considered critical to the long-term sustainability of this endangered species in Peru. Exposure of the rookery to environmental toxicants is a mounting concern because of regional growth of industries and human populations. Whole blood samples were collected from 30 free-ranging penguins in 2011 as part of a broader population health monitoring program. Dried blood spots (DBS) containing 50 µl of blood were prepared and analyzed to assess exposure to five groups of environmental contaminants. Concentrations of elements arsenic, cadmium, iron, lead, mercury, selenium, and thallium were analyzed using inductively coupled plasma mass spectrometry. Persistent organic pollutant concentrations were measured using gas chromatography-tandem mass spectrometry to analyze organochlorine pesticides (OCP; p,p'-DDT, p,p'-DDE, ß-hexachlorocyclohexane, t-nonachlor, and oxychlordane), polychlorinated biphenyls (congeners 138 and 153), and polybrominated flame retardants (polybrominated biphenyl-153 and polybrominated diphenyl ether congeners 47 and 99). Per- and polyfluoroalkyl substances, including perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid were measured using liquid chromatography-tandem mass spectrometry. Results revealed low levels of exposure to these selected contaminants, at levels not considered to be of concern for wildlife health. DBS methodology was considered effective in a field-based setting for quantification of whole blood concentrations of environmental contaminants in penguins.

Pharmacokinetics of Trazodone in Hispaniolan Parrots (Amazona ventralis).

The objective of this study was to establish the pharmacokinetics of a single oral dose of trazodone in the Hispaniolan Amazon parrot (Amazona ventralis). Trazodone is a selective serotonin antagonist and reuptake inhibitor used commonly in both human and veterinary medicine as an antidepressant behavioral modification medicine. A single oral dose of compounded trazodone hydrochloride solution (20 mg/mL) at 50 mg/kg was administered to a total of 7 healthy adult Hispaniolan Amazon parrots. The 7 healthy adult parrots ranged in age from 10 to 15 years and weighed 228 to 323g. Blood was collected at baseline (2 weeks before study) and at 1, 2, 4, 6, 10, and 14 hours post-drug administration. Plasma concentrations of both trazodone and its active metabolite m-chlorophenylpiperazine (mCPP) were measured via liquid chromatography tandem mass spectrometry. Noncompartmental pharmacokinetic analysis was completed. The half-life (t1/2) ± SD of trazodone for the Hispaniolan parrots was 1.89 ± 0.49 hours, and the t1/2 ± SD of mCPP metabolite was 1.9 ± 0.55 hours. Maximum serum drug concentrations, or Cmax (ng/mL), were 738.3 ± 285.3 for trazodone. Times to achieve Cmax (hours) for trazadone and the mCPP metabolite were 1 hour and 2 hours postdosing, respectively. While this study did not establish the behavioral effects of trazodone, no adverse side effects were observed throughout the 48-hour period following drug administration and blood collection. Our results indicate that the oral administration of a 50-mg/kg single dose of trazodone to Hispaniolan parrots may be considered a safe dose. Plasma concentrations are comparable to previously published values in humans, dogs, horses, and pigeons (Columba livia domestica) for up to 14 hours following dosing. This study indicates that further studies are needed to establish the pharmacodynamics and the efficacy of trazodone in the medical management of behavioral problems in psittacine species.

A review of current chemistry, pharmacology, and regulation of endogenous anabolic steroids testosterone, boldenone, and nandrolone in horses.

Anabolic androgenic steroids are synthetic substances related to the male sex hormones (androgens). These agents promote the growth of skeletal muscle (anabolic effects) and the development of male sexual characteristics (androgenic effects). Anabolic steroids have been illegally used for many years as performance-enhancing drugs in human, equine, and canine sports and as growth promoters in livestock reared to provide meat for human consumption. The analytical challenge to developing effective means of control within these fields has been exacerbated by the reported endogenous nature of some of these steroids. Anabolic steroids have been employed extensively in equine practice over the past 50 years. Their usefulness is largely dependent on subjective opinions, as only minimal studies investigating pharmacodynamics have been carried out in horses. Therefore, their use will vary markedly between practitioners depending on their personal experiences and pressures by trainers to use them. They form part of rational therapy in a variety of conditions. In addition to their use for increasing muscle mass, they are used to varying extents in the raising of yearlings and in the training and racing of horses with the view of improving performance. The use of these agents is prohibited in the horseracing industry by the Association of Racing Commissioners International (ARCI), International Federation of Horseracing Authorities (IFHA), and Fédération Equestre Internationale (FEI).

Gas chromatography-tandem mass spectrometric analysis of metaldehyde and its metabolite acetaldehyde in initial assessment of hemodialysis abatement of toxicity in live animals.

Metaldehyde consumption by pets and other mammals constitute medical emergencies ideally requiring rapid poison removal. The purpose of this study was three-fold: 1) development of a sensitive method for metaldehyde quantitation in patient serum samples by gas chromatography combined with tandem quadrupole mass spectrometry (GC/MS/MS); 2) development of a sensitive method for quantitation of the volatile metaldehyde metabolite acetaldehyde by headspace analysis combined with GC/MS/MS; and 3) an initial assessment of the efficacy of combined dialysis and hemoperfusion treatments in diminishing toxin loads in canine victims of metaldehyde poisoning. Both mass spectrometric approaches relied on Multiple Reaction Monitoring (MRM) methodologies. Metaldehyde extracted via liquid-liquid partitioning from serum was detected with a limit of quantitation (LOQ) of 7.3 ± 1.4 ng/mL with linearity in the range 1-250 ng/mL with accuracy improved by inclusion of a deuterated metaldehyde internal standard. Acetaldehyde was determined to have an LOQ of 0.39 µg/mL with linearity in the range 1-1000 µg/mL. The developed methodologies were applied to canine samples taken over various time points during dialysis treatment. Two of three canine patients showed significant abatement of metaldehyde levels by over 50-fold from initial concentrations while a third was shown to be negative with no measureable metaldehyde. The toxic metabolite acetaldehyde was found in one of the metaldehyde-poisoned patients and the detected acetaldehyde was also reduced by roughly 200-fold during the course of treatment. The designed mass spectrometric techniques were thus successful in demonstrating the efficacy of the applied dialysis-hemoperfusion methods which may find wider applicability against other potentially lethal toxins in poisoned patients in future studies.

Pentafluorobenzylation and detection of sodium monofluoroacetate (compound 1080) in veterinary samples using gas chromatography/tandem quadrupole mass spectrometry with multiple reaction monitoring.

RATIONALE: The analytical detection of chemical residues from sodium monofluoroacetate (MFA) ingestion in targeted predatory wildlife and in pesticide misuse incidents perpetrated against nuisance companion animals remains a concern in veterinary forensic toxicology. There is a current need for chemically stable sample extracts with reliable and specific diagnostic methods for trace quantities in diverse diagnostic matrices. METHODS: Biphasic pentafluorobenzylation provided a simple combined extraction and derivatization procedure for removing MFA in a chemically stable form from a complex matrix such as stomach contents. Analysis of the derivatized extract using gas chromatography/tandem quadrupole mass spectrometry (GC/MS/MS) with multiple reaction monitoring (MRM) approaches specific to MFA provided greater specificity than simple scan or selected ion monitoring approaches. RESULTS: Collision-induced dissociation in GC/MS/MS showed that pentafluorobenzyl (PFB)-derivatized MFA (M+ m/z 258) generated m/z 258➔130, 149, 161, 177, 178, 180.1, and 181.1 transitions. Of these, the transition m/z 258➔181 provided a peak for quantitation, whereas m/z 258➔161 and 258➔178 provided specificity for qualifying MFA. Similarly, PFB-derivatized 2-chloropropionic acid (M+ m/z 288) was used as an internal standard, which generated m/z 288➔181 and 161. Of these, the transition m/z 288➔181 provided a peak for quantitation, whereas m/z 288➔161 and 181➔161 served to qualify the internal standard. CONCLUSIONS: The method was validated with a calculated limit of detection of 0.35 ppm and limit of quantitation of 1.09 ppm MFA. The method should have adequate sensitivity and reliability for veterinary toxicology labs analyzing specimens from animals poisoned by this predacide.

Haloxyfop determination by gas chromatography/tandem mass spectrometry in eggs.

RATIONALE: Haloxyfop is a pre/post-emergence herbicide with known organ toxicities and teratogenic effects in mammals. The European Union Commission on Food Safety has an established maximum residue limit of 10 µg/kg in all agricultural products including eggs. A sensitive highly specific method would be of value in determination of haloxyfop residues in foodstuffs such as eggs. METHODS: The Michigan State University Veterinary Diagnostic Lab (MSU VDL) developed a method for the extraction of haloxyfop from eggs based on popular QuEChERS (quick, easy, cheap, effective, rugged, and safe) methodologies, essentially providing acetonitrile extracts following treatment with high ionic strength additives. Extracts derivatized with trimethylsilyl (TMS) groups were examined by gas chromatography/tandem mass spectrometry using developed multiple reaction monitoring (MRM) methodology. RESULTS: The MSU VDL received eggs from chickens exposed to 760 µg/kg haloxyfop in flaxseed. Haloxyfop-TMS m/z 374→73 MRM setting enabled quantitation across the 1-50 ppb range in comparison with an ibuprofen MRM transition as internal standard. CONCLUSIONS: The determined limit of quantitation was 2.5 ng/g, and the method successfully identified haloxyfop residues in five of six batches of the chicken eggs, with nonzero values ranging from 2.7 to 14.5 ng/g. These values were consistent with flaxseed incorporation into the diet at 4-7% and known excretion into eggs at 2-3% of daily haloxyfop exposure, and establish the utility of the method in identifying regulatory noncompliance and adulteration of food sources.

Phosphine detection in veterinary samples using headspace gas chromatography/tandem mass spectrometry with multiple reaction monitoring.

RATIONALE: Determination of phosphine exposure from zinc or aluminum phosphide fumigants continues to be a routine analytical requirement in veterinary forensic toxicology. There is a need for a more reliable and specific method than simple gas chromatography/mass spectrometry (GC/MS) analysis of sample solvent extracts, as GC/MS of extracts on capillary columns used for general screens involves significant interference from air peaks. METHODS: GC/MS/MS headspace analysis of acid-generated phosphine gas enabled study of the feasibility of devising multiple reaction monitoring (MRM) approaches to the determination of phosphine with greater specificity. RESULTS: Collision-induced dissociation in GC/MS/MS showed that phosphine generated m/z 34 → 31, 32 and 33 ion transitions by sequential proton release as well as minor transitions m/z 34 → 47, 34 → 63 and 63 → 31.5 by intermolecular collisions and double charging. Study of the formation of these product ions enabled development of MRM settings for a highly useful headspace method for phosphine detection. CONCLUSIONS: The method was validated over a working range of 5-100 ppm of phosphide generating phosphine gas which enabled retention of regular screen capillary columns without necessitating separation from air components. The method should have adequate sensitivity and reliability for veterinary toxicology laboratories confronting specimens from animals poisoned by crop fumigants.

Veterinary utility of dried blood spots for detailed analysis of chlorinated pesticides and polychlorinated biphenyls by gas chromatography tandem mass spectrometry.

Persistent organic pollutants (POPs) are organic compounds of anthropogenic origin that resist atmospheric and microbial degradation and thus persist in the environment and in food chains for exceptionally long periods of time. Veterinarians and wildlife researchers need simple methodologies for monitoring and measuring such compounds including two large and diverse categories, organochlorine pesticides (OCs) and polychlorinated biphenyls (PCBs), compounds that have been largely banned from production and use except for specific exceptions. We present development of methodologies for detection and quantitation of 22 OCs and 10 PCB congeners by tandem quadrupole gas chromatography-mass spectrometric analysis of Dried Blood Spots (DBS). Development was enabled by (1) optimization of suspension and extraction methodologies for DBS; (2) strategic streamlining and condensation of Multiple Reaction Monitoring (MRM) settings on GC/MS/MS; and (3) improvement of GC settings to accommodate all 32 compounds in a single chromatographic run per sample. The method was validated for parameters of linearity, limits of detection and quantitation, recovery and precision, and results from blood were shown to correlate well with those from DBS despite both being only 50 uL in volume. The method was applied successfully to blood samples from nine avian specimens submitted to the MSU Veterinary Diagnostic Lab, and all were shown to bear the burden of varying levels of OCs and/or PCB compounds.

Liquid chromatography/tandem mass spectrometric analysis of penicillamine for its pharmacokinetic evaluation in dogs.

Copper storage disease occurs in multiple dog breeds and is one of the most common causes of chronic hepatitis in this species. The disease is caused by hereditary defects in copper metabolism in conjunction with high dietary copper levels. The progressive copper accumulation leads to hepatitis, cirrhosis, and eventually death if left untreated. Copper chelators are critical in modulating the effects of this disease. It is therefore of significant practicality to understand the pharmacokinetic (PK) parameters of chelating agents, particularly since they are oftentimes quite expensive. A liquid chromatography-tandem mass spectrometric (LC/MS/MS) method was developed to measure plasma levels of one of the most common chelators, d-penicillamine. The compound was discovered to exist in two forms, monomeric and dimeric, and various chemical derivatizations were tried to force the compound into one form or the other. Eventually, the simplest approach was individual determination of penicillamine and its dimer, with summation of the two quantities. This enabled determination of canine PK parameters for penicillamine based on comparison of oral and intravenous administration of the drug, including time to maximum drug level (Tmax), concentration at maximum (Cmax), clearance (Cls) and volume of distribution (Vdss). The drug was found to exist predominantly in the dimeric form in plasma, which is incapable of chelating copper owing to lack of free sulfhydryl groups and must therefore provide a storage form of the drug in equilibrium with its monomeric form in vivo. Mechanisms are discussed for the electrospray-induced fragmentation of penicillamine as well as of its dimer.

Qualitative identification of imidacloprid in postmortem animal tissue by gas chromatography-tandem mass spectrometry.

During an avian mass mortality event investigation at the National Fish and Wildlife Forensic Laboratory in Ashland, OR, imidacloprid became an insecticide of concern. A qualitative analytical toxicology screen of seeds, plucks (tongue, esophagus, and trachea), and ventricular contents was requested. A method for the extraction and qualitative analysis of the insecticide in animal tissues was therefore developed. The procedure relies on a combined Food Emergency Response Network (FERN) and QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) approach to sample extraction followed by qualitative analysis by gas chromatography-tandem mass spectrometry. Since imidacloprid is not amenable to the conditions of gas chromatography, a trimethylsilyl derivative was created and characterized. Proposed mechanisms for the creation of this derivative and its mass spectrum are described. The imidacloprid-trimethylsilyl (TMS) derivative was detected in all samples submitted.

Veterinary utility of dried blood spots for analysis of toxic chlorinated hydrocarbons.

Dried blood spots (DBS) on filter paper provide a simple and convenient means of collecting, storing and shipping samples for veterinary diagnostics related to toxin exposures. This paper presents validation data on analysis of DBS for chlorinated persistent organic pollutants, specifically 4,4'-dichloro-diphenyl-trichloroethane (4,4'-DDT) and its breakdown product 4,4'-dichlorodiphenyl-dichloroethylene (4,4'-DDE), lindane and a representative polychlorinated biphenyl (PCB) congener PCB-153. Analysis was by gas chromatography with electron capture detection (GC-ECD). The method required one 12.5 mm diameter spot representing application of 50 µL of blood, and working limits of detection (LOD) for each of the compounds was 5 ppb. Data are presented on development and description of the method, assay precision, LOD and quantitation, linearity, accuracy, specificity, effects of long-term storage and ruggedness. The method was also applied to 27 avian DBS, and 4,4'-DDE was detected in the majority of samples.

Structure, function and carcinogenicity of metabolites of methylated and non-methylated polycyclic aromatic hydrocarbons: a comprehensive review.

The Unified Theory of PAH Carcinogenicity accommodates the activities of methylated and non-methylated polycyclic aromatic hydrocarbons (PAHs) and states that substitution of methyl groups on meso-methyl substituted PAHs with hydroxy, acetoxy, chloride, bromide or sulfuric acid ester groups imparts potent cancer producing properties. It incorporates specific predictions from past researchers on the mechanism of carcinogenesis by methyl-substituted hydrocarbons, including (1) requirement for metabolism to an ArCH2X type structure where X is a good leaving group and (2) biological substitution of a meso-methyl group at the most reactive center in non-methylated hydrocarbons. The Theory incorporates strong inferences of Fieser: (1) The mechanism of carcinogenesis involves a specific metabolic substitution of a hydrocarbon at its most reactive center and (2) Metabolic elimination of a carcinogen is a detoxifying process competitive with that of carcinogenesis and occurring by a different mechanism. According to this outlook, chemical or biochemical substitution of a methyl group at the reactive meso-position of non-methylated hydrocarbons is the first step in the mechanism of carcinogenesis for most, if not all, PAHs and the most potent metabolites of PAHs are to be found among the meso methyl-substituted hydrocarbons. Some PAHs and their known or potential metabolites and closely related compounds have been tested in rats for production of sarcomas at the site of subcutaneous injection and the results strongly support the specific predictions of the Unified Theory.

A Cytochrome P450-Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes.

Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.

Atypical bromethalin intoxication in a dog: pathologic features and identification of an isomeric breakdown product.

BACKGROUND: Definitive post mortem confirmation of intoxication by the neurotoxic rodenticide bromethalin can be challenging. Brain lesions are not specific and detection of bromethalin and its metabolites are unpredictable due to rapid photodegradation and inconsistent behavior in tissues. CASE PRESENTATION: A 2-year-old dog presented with rapid onset of severe muscle tremors and death within hours after a known ingestion of a reportedly low dosage of bromethalin and subsequent decontamination using activated charcoal. Marked meningeal hemorrhages and multifocal myelin sheath vacuolation were observed in the brain. A marked reactive astrocytosis and neuronal hypoxia/necrosis were identified using immunohistochemistry (IHC) for glial fibrillary acidic protein (GFAP) and for neuron specific protein (NeuN). Bromethalin exposure and tissue absorption was confirmed by identification of one of two isomeric 543.7 molecular weight (MW) breakdown products in the patient's adipose and kidney samples using gas chromatography (GC) combined with tandem quadrupole mass spectrometry (MS/MS). CONCLUSIONS: The severity of clinical signs and subsequent death of this dog was not expected with the low dosage of bromethalin reportedly ingested, and the use of activated charcoal possibly precipitated a hypernatremic status. Meningeal hemorrhages are atypical of bromethalin intoxication, and might have been caused by hyperthermia, secondary to tremors or hypernatremia. Identification of one of two isomeric breakdown products in the adipose tissue and kidney provides an additional molecule to the toxicologic testing regime for bromethalin intoxication.

Fatal diphenhydramine poisoning in a dog.

We report a fatal diphenhydramine poisoning of a 10-year-old, male poodle-cross dog with pre-existing conditions and suspected co-ingestion of ethanol. This case illustrates that diphenhydramine overdose can be fatal in certain circumstances and that analytical toxicology may play an important role in animal death investigations.

Empoisonnement mortel à la diphenhydramine chez un chien. Nous signalons un empoisonnement mortel à la diphenhydramine chez un caniche croisé mâle âgé de 10 ans ayant des conditions préexistantes et une co-ingestion soupçonnée d'éthanol. Ce cas illustre qu'une surdose de diphenhydramine peut être mortelle dans certaines circonstances et qu'une toxicologie analytique peut jouer un rôle important dans les enquêtes sur la mort d'animaux.(Traduit par Isabelle Vallières).

Dried blood spot analysis for elements of nutritional concern as demonstrated in studies of Galápagos land iguanas (Conolophus species).

BACKGROUND: Dried blood spot (DBS) technology is valuable in providing simple means of storing blood samples from wildlife with small blood volumes. Methods designed for heavy metal analysis on DBS become more useful if extended to elements of nutritional significance. PURPOSE: (1) Development of procedures for measuring Mn, Fe, Co, Cu, Zn, Se and Mo in DBS; (2) use the designed methods in health assessments of Galápagos land iguanas (Conolophus species). PROCEDURES: Elements were measured by inductively coupled plasma/mass spectrometry (ICP-MS) following acid digestion of whole blood or DBS from the same animal for direct comparison. Study animals comprised free-ranging iguanas from separate islands in the Galápagos archipelago. MAIN FINDINGS: DBS spikes (Mn, Fe, Co, Cu, Zn, Se and Mo) demonstrated accuracy to ∼100 ppb; reporting limits were set there except for Fe and Zn which were set at 1000 ppb. Plasma samples - generally preferable for nutritional element diagnostics - were submitted from Galápagos land iguanas along with DBS as part of a large-scale health assessment. In plasma versus DBS concentration comparisons, Fe, Cu, Se and Mn correlated well with R^2 values of 0.799, 0.818, 0.896 and 0.899, respectively, and slopes ranging 0.88 - 1.3. Co and Zn showed greater scatter. Mo had insufficient points above its reporting limit and offered advantages for toxicity assessments. Bland-Altman diagrams showed flat scatter between 2x standard deviation boundaries with no undue trends except for Mn which had few points above its reporting limit. Bias, defined as the average difference [DBS - plasma] divided by the average value, was relatively low throughout, with values of - 19.3 % (Fe), - 48.7 % (Co), - 19.6 % (Cu), - 6.9 % (Zn), - 21.4 % (Se) and + 40.7 % (Mn). Normal distribution assessment of iguana Cu, Zn, Se and Fe plasma values showed unanticipated divergences between two species. CONCLUSIONS: The DBS approach for nutritional element analysis offers a suitable methodology for determining crucial elements Mn, Fe, Co, Cu, Zn, Se, and Mo in veterinary samples. Analyses of samples from Conolophus species revealed interesting divergences particularly for Cu, Zn, Se and Fe, elements generally associated with defense against oxidative stress.

Synthesis and characterization of d5 -barbarin for use in barbarin-related research.

Based on structural similarities and equine administration experiments, Barbarin, 5-phenyl-2-oxazolidinethione from Brassicaceae plants, is a possible source of equine urinary identifications of aminorex, (R,S)-5-phenyl-4,5-dihydro-1,3-oxazol-2-amine, an amphetamine-related US Drug Enforcement Administration (DEA) controlled substance considered illegal in sport horses. We now report the synthesis and certification of d5 -barbarin to facilitate research on the relationship between plant barbarin and such aminorex identifications. D5 -barbarin synthesis commenced with production of d5 -2-oxo-2-phenylacetaldehyde oxime (d5 -oxime) from d5 -acetophenone via butylnitrite in an ethoxide/ethanol solution. This d5 -oxime was then reduced with lithium aluminum hydride (LiAlH4 ) to produce the corresponding d5 -2-amino-1-phenylethan-1-ol (d5 -phenylethanolamine). Final ring closure of the d5 -phenylethanolamine was performed by the addition of carbon disulfide (CS2 ) with pyridine. The reaction product was purified by recrystallization and presented as a stable white crystalline powder. Proton NMR spectroscopy revealed a triplet at 5.88 ppm for one proton, a double doublet at 3.71 ppm for one proton, and double doublet at 4.11 ppm for one proton, confirming d5 -barbarin as the product. Further characterization by high resolution mass spectrometry supports the successful synthesis of d5 -barbarin. Purity of the recrystallized product was ascertained by High Performance Liquid Chromatography (HPLC) to be greater than 98%. Together, we have developed the synthesis and full characterization of d5 -barbarin for use as an internal standard in barbarin-related and equine forensic research.

Single oral or intravenous administration of voriconazole achieved recommended therapeutic minimum inhibitory concentrations against Aspergillus in the common raven (Corvus corax).

OBJECTIVE: To determine the pharmacokinetics of voriconazole after single IV or orally administered boluses in common ravens (Corvus corax). ANIMALS: 8 healthy common ravens. PROCEDURES: Voriconazole (5 mg/mL, 10 mg/kg IV) was administered to 8 birds, and then plasma voriconazole concentrations were measured at various time points by high-pressure liquid chromatography with mass spectrometry. Starting 6 months later in a randomized 3-treatment 3-period regimen, birds received a single oral dose of voriconazole suspension (10 mg/mL; 6, 12, and 24 mg/kg PO). The study period was May 2015 to March 2016. RESULTS: Voriconazole (10 mg/kg IV) achieved an initial plasma concentration of 6.31 µg/mL when measured over 21 hours. After oral administration of voriconazole at 6, 12, and 24 mg/kg, the relative bioavailability was 67.5%, 209%, and 183%, respectively. For the 6-mg/kg dose, the maximum plasma concentration was reached at 30 minutes after administration and remained in the therapeutic range of 0.5 to 1 µg/mL for approximately 15 hours. The 12- and 24-mg/kg doses resulted in concentrations in a potentially toxic range. CLINICAL RELEVANCE: Voriconazole was well tolerated. All 4 doses resulted in plasma concentrations of voriconazole > 0.5 µg/mL, which is the minimum inhibitory concentration recommended for pathogenic species of Aspergillus fungi known to affect birds. A single dose of voriconazole administered as 10 mg/kg IV or 6 mg/kg PO resulted in recommended target plasma concentrations. Administration of voriconazole 6 mg/kg PO 2 to 3 times daily may be adequate for treatment without exceeding the toxic range.