Margaret Johnson

Diagnostic Analysis of Veterinary Dried Blood Spots for Toxic Heavy Metals Exposure

Dried blood spots (DBS) on filter paper have been used in human medicine since the 1960s, predominantly for screening in-borne metabolic disorders and more recently, for toxicology. Despite its 50-year existence, this technology has not been adopted by veterinarians for routine diagnoses and research. We have validated a novel DBS analytical procedure for the routine measurement of toxic heavy metals using 50 µL of whole blood on a single DBS by inductively coupled plasma mass spectrometry (ICP-MS). Targeted heavy metals are arsenic, cadmium, mercury, lead, selenium and thallium. The limits of quantitation (LOQ) on DBS are: arsenic 1.7 µg/L, cadmium 4.0 µg/L, mercury 13.7 µg/L, lead 13.3 µg/L, selenium 6.3 µg/L and thallium 1.5 µg/L. These LOQs suffice for routine diagnoses of heavy metal intoxication in domesticated and wildlife species as well as for basic, applied and epidemiological studies. The technique is ideal for population studies involving investigations of wildlife exposure to heavy metals and other environmental pollutants. The small blood volume involved (50 µL) makes it feasible to study small animals (birds, reptiles, amphibians and small mammals) that were previously excluded, or difficult to study due to the relatively large sample volumes required by current gold standard blood collection techniques.

Examination of Eurasian Griffon Vultures (Gyps fulvus fulvus) in Israel for Exposure to Environmental Toxicants Using Dried Blood Spots

The griffon vulture (Gyps fulvus) is one of seven species of Old World Gyps vultures found over a wide range from the Iberian peninsula in the west through the Balkans, Turkey, and the Middle East to India in the east. The population of the griffon vultures in Israel has suffered a dramatic decrease, and in recent years productivity has been severely reduced. In this study, whole-blood samples taken from 25 apparently healthy griffon vultures at various stages of maturity were examined to investigate whether the vultures are being excessively exposed to environmental contaminants that might deleteriously affect their reproduction. Five groups of environmental contaminants, comprising toxic elements, organochlorine pesticides, polychlorinated biphenyls, polybrominated diphenyl ethers, and perfluorinated compounds, were monitored in dried blood spots. Results of the analyses showed low levels of exposure of griffon vultures to environmental contaminants compared with the sparse data available on griffon vultures and other diurnal raptors in other countries.

Liquid chromatographic-electrospray mass spectrometric determination of 1-methyl-4-phenylpyridine (MPP+) in discrete regions of murine brain.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is widely used as a neurotoxin in several models of Parkinson’s disease in mice. MPTP is metabolized to 1-methyl-4-phenylpyridinium (MPP+), which is a mitochondrial toxicant of central dopamine (DA) neurons. There are species, strain, and age differences in sensitivity to MPTP. Simultaneous measurement of the MPTP active metabolite MPP+ and dopamine (DA) in the brain would be helpful in mechanistic studies of this neurotoxin. The objective of this study was to develop a liquid chromatography–mass spectrometry (LC/MS) method for analysis of MPTP and MPP+ in brain tissue and correlate these in the same sample with changes in DA measured via HPLC coupled with electrochemical detection. Twenty-five C57BL/6J7 8-week old female mice were used in the study. Mice were given a single subcutaneous injection of MPTP (20 mg/kg) and were sacrificed 1, 2, 4, or 8 h later. Zero time control mice received an injection of 0.9% normal saline (10 ml/kg) and were killed 1 h later. Brains were rapidly harvested and quickly frozen, and microdissected brain regions were placed in 0.1 M phosphate-citric acid buffer containing 20% methanol (pH 2.5). A new LC/MS method was successfully developed that utilized selected reaction monitoring (SRM) of MPP+ m/z 170→127, 170→128, and 170→154 fragmentation for quantitation and area ratios (m/z 127)/(m/z 128) and (m/z 154)/(128) for identity confirmation. A similar SRM strategy from m/z 174 was unable to detect any significant levels of MPTP down to 0.4 ppb. According to this method, MPP+ was detected in the nucleus accumbens (NA) and the striatum (ST), with the levels in the NA being 3-times higher than those in the ST. The advantage of this approach is that the tissue buffer used in this procedure allowed concurrent measurement of striatal DA, thus enabling direct correlation between accumulation of tissue MPP+ and depletion of DA concentrations in discrete regions of the brain.

Determination of Organochlorine Pesticides in Wildlife Liver and Serum Using Gas Chromatography Tandem Quadrupole Mass Spectrometry

A single laboratory validation of a quantitative capillary gas chromatography tandem quadrupole mass spectrometer (GC-MS/MS) method utilizing a Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) approach for the extraction of 24 organochlorine analytes in liver and blood serum is presented here. The QuEChERS approach utilizes an acetonitrile extraction, partitioning facilitated by the addition of salts and a dispersive solid phase extraction cleanup. This method simultaneously monitored 24 organochlorine pesticide residues representing four different classes, including chlordecone, cyclodienes, dichlorodiphenylethanes, and hexachlorocyclohexanes. Calculated limits of detection (LOD) varied from 0.002 to 2.4 ppb and limits of quantification (LOQ) varied from 0.01 to 7.4 ppb. This multi-residue method proved to be a sensitive approach to the measurement of persistent organic pollutants in biological matrices.

Rapid and Reliable Identification of Ionophore Antibiotics in Feeds by Liquid Chromatography–Tandem Mass Spectrometry

Veterinary diagnostic laboratories are constantly required to be innovative to develop quick, reliable, and cost-effective methods for use in diagnosis of intoxications. Livestock intoxication with ionophore antibiotics is sometimes encountered. A rapid qualitative method based on liquid chromatography–tandem mass spectrometry was developed to screen for the presence of monensin, lasalocid, salinomycin, and narasin antibiotics in feeds. This new method has high specificity and high sensitivity, and yet minimum sample preparation was employed. The analytes were extracted in methanol–water (90:10) and analyzed without further purification. Because the test uses a single extraction procedure and no cleanup is necessary, its turnaround time is 4 hr. The method applies the concept of „identification points” (IP) score to identify suspected ionophore antibiotics. The total IP score used to identify each of the ionophore antibiotics by this method was 7.0, which exceeds the minimum score of 4.0 required for acceptable identification of unknown compounds. The specificity of the method was achieved using retention time and multiple reaction monitoring with a total IP score of 7.0.

Determination of methomyl in the stomach contents of baited wildlife by gas chromatography-mass spectrometry.

The poisoning of wildlife with fly-bait containing the active ingredient methomyl is an intentional and illegal act in many jurisdictions. A case of 2 animals poisoned by methomyl through consumption of tainted bait at multiple stations is described. Although thermally and ultraviolet-labile, methomyl can be identified by gas chromatography–mass spectrometry and is detected in abundance in bait samples; however, it is not readily observed in tissues, owing to its rapid metabolism and elimination. The application of derivatizing functionalities, such as trimethylsilyl groups, stabilizes the methomyl-oxime metabolite to facilitate its detectability during exposure to the relatively harsh conditions of gas chromatography. This brief communication reports on the analytical detection of methomyl in baits and biological samples from poisoned wildlife. Essential to the case were the added determination of a fly-bait coactive ingredient, (Z)-9-tricosene, and identification of a chemical indicator, caffeine, to confirm both the type of pesticide product involved in the poisoning incident and the vehicle used to perpetrate its delivery.

Rapid quantification of ionophores in feeds by liquid chromatography– tandem mass spectrometry

Ionophores are widely used in veterinary medicine as coccidiostats and for improving nutrient utilization in livestock production. Because of widespread use, ionophores sometimes cause poisoning in livestock. Quantifying concentration of these compounds in feeds for diagnostic purposes is needed. A method with a single step of solvent extraction was developed for rapid simultaneous quantification of monensin, lasalocid, salinomycin, and narasin in feeds by liquid chromatography–tandem mass spectrometry (LC-MS/MS). The ionophores were extracted using methanol:water (90:10). With the high specificity and high sensitivity of tandem mass spectrometry, the extract was introduced for measurement without further processing. The effect of particle size of feeds on extraction efficiency was also investigated. It was found that feeds passing through a 1-mm filter or sieve show better quantitative extraction. Nigericin was used as internal standard for the measurement. The method was validated by fortification of the selected ionophore compounds in horse feed at different concentrations. The typical recovery rate was 69–122%. Meanwhile, various interlaboratory proficiency test samples of different matrices were also quantified as part of the procedure for method validation. A good agreement was found between results and the suggested values. The method is very sensitive, with detection limits between 0.018 µg/g and 0.056 µg/g for the compounds tested. Results showed that the lower limit of quantification was 0.2 µg/g for the ionophore compounds, which is much lower than the contents of the ionophores in medicated feeds, which is generally approximately 10–100 µg/g feed.

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

Abstract 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.

Characterization of Bromethalin and its Degradation Products in Veterinary Toxicology Samples by GC–MS-MS

Bromethalin is a neurotoxicant with unusual instability and chromatographic behavior that make it difficult to analyze by gas chromatography (GC) in forensic examination of non-target animal deaths. Physicochemical breakdown of bromethalin produced multiple unique products with discernible mass spectra. This paper describes an investigation of the GC electron impact-mass spectrometric properties of bromethalin and its capacity to generate up to twenty heat- or light-induced breakdown products. Two principal breakdown products are isomeric with one another and involve release of both fluorine and methyl groups to develop dehydrofluorodesmethylbromethalin products. These compounds have proven to be excellent surrogate markers in screening forensic samples for bromethalin exposure, particularly in veterinary samples in which the active metabolite desmethylbromethalin has not yet accumulated to any appreciable extent, such as baits and animal stomach contents. The compounds as well as their parent bromethalin were easily monitored by GC interfaced with a tandem-quadrupole mass spectrometer using multiple-reaction monitoring (MRM) modes. Unusual gas chromatographic properties of bromethalin included: (i) specific requirements for a maximum oven temperature; (ii) non-linear increases in detector response on increased injection volumes, hypothesized to result from variable diffusion coefficients. We report here the development of GC strategies that facilitate detection of bromethalin and its breakdown products, as well as their MRM analysis by tandem-quadrupole mass spectrometry. The developed approaches are applicable to feed, baits and stomach contents as well as extracted tissue samples such as liver and kidney.

Development of a Quantitative Gas Chromatography–Tandem Mass Spectrometry Method for the Determination of Pentobarbital in Dog Food

In 2017, a commercially available dog food was found by our laboratory to be adulterated with the euthanasia drug pentobarbital. An FDA class 1 voluntary recall by the company ensued. Since there is no set tolerance for pentobarbital in food or feed, a sensitive method for its detection was required. We describe a simple, yet efficient, method for the extraction and quantitative analysis of the barbiturate in dog food. 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 quantitative analysis by gas chromatography-tandem mass spectrometry (GC/MS/MS) using pentobarbital- d5 as an internal standard. This procedure improves upon other GC/MS methodologies in that derivatization of pentobarbital or its deuterated internal standard is unnecessary, and sensitivity to a calculated limit of detection (LOD) of 0.6 ppb and a limit of quantitation (LOQ) of 2 ppb is achieved.