Detection of allopurinol and oxypurinol in canine urine by HPLC/MS-MS: Focus on veterinary clinical pharmacokinetics.

Allopurinol is the most commonly used drug for the treatment of hyperuricemia in people, and in view of the risks of fatal hypersensitivity in patients with renal dysfunction, doses based on the glomerular filtration rate are proposed. In veterinary medicine, allopurinol is used in the treatment of canine leishmaniasis (CanL) caused by Leishmania infantum owing to the drug action of inhibiting the parasite's RNA synthesis. However, renal dysfunction frequently ensues from disease progression in dogs. The purpose of the present study was to standardize and validate a sensitive high-performance liquid chromatography-mass spectrometric (HPLC-MS/MS) method to determine the concentration of allopurinol and its active metabolite oxypurinol in canine urine for clinical pharmacokinetic investigation. Urine samples of eleven (11) dogs with naturally occurring CanL and in the maintenance phase of the treatment with alopurinol were used. For the chromatographic analysis of urine, the mobile phase consisted of a solution of 0.1 % formic acid (88 %) in 10 mM ammonium acetate. Separation of allopurinol and oxypurinol occurred in a flow of 0.8 mL/min on a C8 reverse phase column 5 µm, and acyclovir was the internal standard. The HPLC-MS/MS method was validated by reaching the limits of detection and quantification, reproducibility and linearity. The lower limit of quantification achieved by the method was 10 µg/mL for both allopurinol and oxypurinol. Calibration curves were prepared in blank urine added with allopurinol at concentrations of 10-1000 µg/mL, and oxypurinol at 10-200 µg/mL. Coefficients of variation of less than 15 % between intracurrent and intercurrent accuracy values were observed for both allopurinol and oxypurinol. Urine test samples remained stable after being subjected to freeze-thaw cycles and remaining at room temperature for 4 h. The method proved to be adequate to quantify allopurinol and oxypurinol in urine samples from dogs under treatment.

Antibiotic use in critical illness.

OBJECTIVE: To provide a review on the current use of antimicrobials with a discussion on the pharmacokinetic and pharmacodynamic profiles of antimicrobials in critically ill patients, the challenges of drug resistance, the use of diagnostic testing to direct therapy, and the selection of the most likely efficacious antimicrobial protocol. ETIOLOGY: Patients in the intensive care unit often possess profound pathophysiologic changes that can complicate antimicrobial therapy. Although many antimicrobials have known pharmacodynamic profiles, critical illness can cause wide variations in their pharmacokinetics. The two principal factors affecting pharmacokinetics are volume of distribution and drug clearance. Understanding the interplay between critical illness, drug pharmacokinetics, and antimicrobial characteristics (ie, time-dependent vs concentration-dependent) may improve antimicrobial efficacy and patient outcome. DIAGNOSIS: Utilizing bacterial culture and susceptibility can aid in identifying drug resistant infections, selecting the most appropriate antimicrobials, and hindering the future development of drug resistance. THERAPY: Having a basic knowledge of antimicrobial function and how to use diagnostics to direct therapeutic treatment is paramount in managing this patient population. Diagnostic testing is not always available at the time of initiation of antimicrobial therapy, so empiric selections are often necessary. These empiric choices should be made based on the location of the infection and the most likely infecting bacteria. PROGNOSIS: Studies have demonstrated the importance of moving away from a "one dose fits all" approach to antimicrobial therapy. Instead there has been a move toward an individualized approach that takes into consideration the pharmacokinetic and pharmacodynamic variabilities that can occur in critically ill patients.

Personalized medicine: going to the dogs?

Interindividual variation in drug response occurs in canine patients just as it does in human patients. Although canine pharmacogenetics still lags behind human pharmacogenetics, significant life-saving discoveries in the field have been made over the last 20 years, but much remains to be done. This article summarizes the available published data about the presence and impact of genetic polymorphisms on canine drug transporters, drug-metabolizing enzymes, drug receptors/targets, and plasma protein binding while comparing them to their human counterparts when applicable. In addition, precision medicine in cancer treatment as an application of canine pharmacogenetics and pertinent considerations for canine pharmacogenetics testing is reviewed. The field is poised to transition from single pharmacogene-based studies, pharmacogenetics, to pharmacogenomic-based studies to enhance our understanding of interindividual variation of drug response in dogs. Advances made in the field of canine pharmacogenetics will not only improve the health and well-being of dogs and dog breeds, but may provide insight into individual drug efficacy and toxicity in human patients as well.

Consensus on the Rational Use of Antithrombotics in Veterinary Critical Care (CURATIVE): Domain 4-Refining and monitoring antithrombotic therapies.

OBJECTIVES: To systematically review the evidence for therapeutic monitoring of antithrombotic drugs in small animals, develop guidelines regarding antithrombotic monitoring, and identify knowledge gaps in the field. DESIGN: First, a standardized, systematic literature review was conducted to address predefined PICO (Population/Patient, Intervention, Control, Outcome) questions, with categorization of relevant articles according to level of evidence and quality. Preliminary guidelines were developed by PICO worksheet authors and the domain chair. Thereafter, a Delphi-style survey was used to develop consensus on guidelines regarding therapeutic monitoring of antithrombotics in dogs and cats. SETTING: Academic and referral veterinary medical centers. RESULTS: PICO questions regarding the utility of therapeutic monitoring were developed for 6 different antithrombotic drugs or drug classes, including aspirin, clopidogrel, warfarin, unfractionated heparin, the low molecular weight heparins, and rivaroxaban, The majority of the literature pertaining to therapeutic monitoring of antithrombotic drugs was either performed in experimental animal models of disease or involved studies of drug pharmacokinetics and pharmacodynamics in healthy laboratory animals. There was a paucity of high level of evidence studies directly addressing the PICO questions, which limited the strength of recommendations that could be provided. The final guidelines recommend that therapeutic monitoring should be performed when using warfarin or unfractionated heparin in dogs and cats at risk of thrombosis. There is insufficient evidence to make strong recommendations for therapeutic monitoring of aspirin or low molecular weight heparin in dogs and cats at this time. CONCLUSIONS: As in other CURATIVE domains, significant knowledge gaps were highlighted, indicating the need for substantial additional research in this field. Ongoing investigation of the role of therapeutic monitoring of antithrombotic therapies will undoubtedly facilitate improved outcomes for dogs and cats at risk of thrombosis.

Residue Distribution and Depletion of Ractopamine in Goat Tissues After Exposure to Growth-Promoting Dose.

The objectives of the present study was to investigated the ractopamine (RAC) distribution and depletion process in various tissues of goat including liver, kidney, spleen, lung, heart, fat, bile, brain and the eyes. The experiment was carried out on 21 goats (18 treated and 3 controls). Treated goats were orally administered RAC in a dose of 1 mg/kg body mass per day for last 28 days and randomly sacrificed on withdrawal days of 0.25, 1, 3, 7, 14 and 21. RAC in all matrices were determined by ultra-high performance liquid chromatography-quadrupole orbitrap high resolution mass spectrometry. After 21 days treatment discontinuation, the levels of RAC in bile reached at 13.48 ± 3.36 mg/L, which was significantly higher than that in the other tissues. The concentrations of RAC were followed by kidney, the excretory organ and liver, the major metabolic organ (4.49 ± 0.16 mg/kg for kidney and 1.81 ± 0.11 mg/kg for liver, respectively). The residual concentration of the drug in the eyes of goat was less than that in bile, kidney, liver, lung and spleen on withdrawal days 0.25. RAC residues was higher than the limits of detection = 0.15 µg/mL in liver on Day 21. These findings demonstrated that liver can serve as an alimentary matrix and as a matrix for the control of RAC abuse hypothetically except for urine.

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.

Techniques for Monitoring Drug Efficacy.

The efficacy of drugs can vary greatly between species and individuals. Establishing efficacious drug doses for a species requires integration of population pharmacokinetic and pharmacodynamic data into a dose-response curve. Unfortunately, these data sets are rarely available for exotic species. The use of alternative monitoring techniques is required to determine drug efficacy and safety. This article discusses methods to integrate efficacy monitoring into clinical practice, including the use of diagnostic testing and therapeutic drug monitoring.

Simple and Fast Gas-chromatography Mass Spectrometry Assay to Assess Delta 9-Tetrahydrocannabinol and Cannabidiol in Dogs Treated with Medical Cannabis for Canine Epilepsy.

BACKGROUND: To date, an increasing number of pet owners, especially in the USA, are using cannabis-derived products containing generally delta 9-tetrahydrocannabinol (THC) and cannabidiol (CBD) to help their animals' health. Unfortunately, studies on the clinical use of cannabinoids in veterinary medicine are still limited, and the application of analytical methodologies for the determination of cannabinoids in animal (especially dog) biological matrices such as plasma, is still missing. METHODS: A reliable, fast, accurate, simple gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the quantification of THC and CBD in plasma samples of eight dogs under therapeutic treatment for epilepsy and receiving oral administration of medical cannabis (Bediol). RESULTS: The method was linear for both the analytes under investigation with coefficients of determination (r2) of at least 0.99. Absolute analytical recovery (mean ± SD) ranged from 80.6 ± 6.2% for THC and 81.7 ± 4.3% for CBD. The matrix effect showed less than 10% analytical suppression due to endogenous substances for both the analytes. The intra-assay and inter-assay precision values ranged from 4.9% to 12.7%, and from 5.2% to 8.7% respectively. The intra-assay and inter-assay accuracy values ranged from 2.3% to 9.6% and from 3.4% to 13.0%, respectively. CONCLUSION: The validated method was successfully applied to real samples; moreover, to assess the potential of the method applicability and robustness in future veterinary clinical studies on cannabinoids therapy, we attempted to follow the kinetic of THC and CBD in the plasma of two dogs under therapy at different times after Bediol administration.

Hair Analysis to Monitor the Illegal Use of Salbutamol in Beef Cattle.

This study measured the concentrations of salbutamol residues in red and white hair of cattle during and after salbutamol administration. Three Chinese Simmental beef cattle received an oral administration of 150 µg/kg body weight/d salbutamol for 21 consecutive days. Salbutamol concentrations were determined on Days 1, 7, 14, and 21 of administration and on Days 7, 14, 28, 42, and 70 following the last administration dose using ultra-performance liquid chromatography-tandem mass spectrometry. The concentrations of salbutamol that eluted from hair were determined. The results revealed that salbutamol concentrations were higher in red hair than in white hair on the same sampling day (P < 0.01). In red hair, salbutamol concentrations increased from 29.82 ± 1.8 ng/g on Day 1 of administration to 442.55 ± 250.29 ng/g on Day 21 of administration, and decreased to 33.36 ± 19.22 ng/g on Day 70 after the last administration. In white hair, salbutamol concentrations changed from 4.25 ± 0.32 ng/g on Day 1 of administration to 33.81 ± 6.44 ng/g and 12.25 ± 2.51 ng/g on Days 14 and 70, respectively, after the last administration. The concentrations of salbutamol that eluted from white hair on Days 1 and 7 were 22.94 ± 2.00 ng/g and 92.94 ± 22.49 ng/g, respectively. Our findings revealed that hair is an appropriate biological matrix for assessing the illegal use of salbutamol in animal husbandry.

Electroencephalogram-based indices applied to dogs' depth of anaesthesia monitoring.

Hypnotic drug administration causes alterations in the electroencephalogram (EEG) in a dose-dependent manner. These changes cannot be identified easily in the raw EEG, therefore EEG based indices were adopted for assessing depth of anaesthesia (DoA). This study examines several indices for estimating dogs' DoA. Data (EEG, clinical end-points) were collected from 8 dogs anaesthetized with propofol. EEG was initially collected without propofol. Then, 100 ml h⁻¹ (1000 mg h⁻¹) of propofol 1% infusion rate was administered until a deep anaesthetic stage was reached. The infusion rate was temporarily increased to 200 ml h⁻¹ (2000 mg h⁻¹) to achieve 80% of burst suppression. The index performance was accessed by correlation coefficient with the propofol concentrations, and prediction probability with the anaesthetic clinical end-points. The temporal entropy and the averaged instantaneous frequency were the best indices because they exhibit: (a) strong correlations with propofol concentrations, (b) high probabilities of predicting anaesthesia clinical end-points.

Evaluation of a hydrophilic gingival dental sealant in beagle dogs.

A liquid solution, gingival sealant containing polymers that form a barrier film upon application was evaluated in dogs. It is a non-toxic, low viscosity, hydrophilic barrier sealant that dries in approximately 10 to 15-seconds after subgingival application. It was designed as a preventative to be applied immediately following a professional oral hygiene procedure in order to block plaque and calculus formation in the sulcus and aid in the prevention of periodontal disease in companion animals. Additionally, the polymer was designed to promote an aerobic environment in the sulcus by oxygen and water transport through engineered pores within the polymer. A 30-day split-mouth, blinded study in two groups of 15 beagle dogs was used. Plaque was significantly (p < 0.05) reduced on the side receiving the sealant by 30.0% and 50.5% (average = 40.3%) in groups 1 and 2, respectively. Calculus was significantly (p < 0.05) reduced on the side receiving the sealant by 27.2% and 20.0% (average = 23.6%) in groups 1 and 2, respectively. Gingival inflammation was monitored to assess product safety. Sides receiving sealant showed no statistically significant differences in gingival index score. No adverse events were observed in the study. This study demonstrates that this gingival sealant can be used as another valuable tool for aiding in the prevention of periodontal disease in dogs.

Antiepileptic drug therapy and monitoring.

Over the past 2 decades, the number of antiepileptic drugs (AEDs) available to veterinarians has grown exponentially. Coupled with this increase is the ability to rapidly and accurately diagnose underlying brain disease with readily accessible magnetic resonance imaging. As a result, the veterinary community is attuned to the need for early treatment intervention. As more treatment choices become available, the unrelenting questions still arise are when should treatment begin, which initial drug therapy is best for our patients, when should treatment changes be considered, and finally, what are the advantages that newer drugs provide for our patients. The purpose of this chapter is to review decision-making strategies for AED therapy, provide an overview of the applicability of current AED available, and present information on the therapeutic advances in epilepsy.

Accuracy of a clinical PET/CT vs. a preclinical µPET system for monitoring treatment effects in tumour xenografts.

PURPOSE: Small animal imaging is of growing importance for preclinical research and drug development. Tumour xenografts implanted in mice can be visualized with a clinical PET/CT (cPET); however, it is unclear whether early treatment effects can be monitored. Thus, we investigated the accuracy of a cPET versus a preclinical µPET using (18)F-FDG for assessing early treatment effects. MATERIALS AND METHODS: The spatial resolution and the quantitative accuracy of a clinical and preclinical PET were evaluated in phantom experiments. To investigate the sensitivity for assessing treatment response, A431 tumour xenografts were implanted in nude mice. Glucose metabolism was measured in untreated controls and in two therapy groups (either one or four days of antiangiogenic treatment). Data was validated by γ-counting of explanted tissues. RESULTS: In phantom experiments, cPET enabled reliable separation of boreholes≥5mm whereas µPET visualized boreholes≥2mm. In animal studies, µPET provided significantly higher tumour-to-muscle ratios for untreated control tumours than cPET (3.41±0.87 vs. 1.60±.0.28, respectively; p<0.01). During treatment, cPET detected significant therapy effects at day 4 (p<0.05) whereas µPET revealed highly significant therapy effects even at day one (p<0.01). Correspondingly, γ-counting of explanted tumours indicated significant therapy effects at day one and highly significant treatment response at day 4. Correlation with γ-counting was good for cPET (r=0.74; p<0.01) and excellent for µPET (r=0.85; p<0.01). CONCLUSION: Clinical PET is suited to investigate tumour xenografts≥5mm at an advanced time-point of treatment. For imaging smaller tumours or for the sensitive assessment of very early therapy effects, µPET should be preferred.

Novel insights in the faecal egg count reduction test for monitoring drug efficacy against gastrointestinal nematodes of veterinary importance.

The faecal egg count reduction test (FECRT) is the method of choice to monitor anthelmintic efficacy against gastro-intestinal nematodes in livestock. Guidelines on how to conduct a FECRT are made available by the World Association for the Advancement of Veterinary Parasitology (WAAVP). Since the publication of these guidelines in the early 1990 s, some limitations have been noted, including (i) the ignorance of host-parasite interactions that depend on animal and parasite species, (ii) their feasibility under field conditions, (iii) appropriateness of study design, and (iv) the high detection limit of the recommended faecal egg count (FEC) method. Therefore, the objective of the present study was to empirically assess the impact of the level of excretion and aggregation of FEC, sample size and detection limit of the FEC method on the sensitivity and specificity of the FECRT to detect reduced efficacy (<90% or <95%) and to develop recommendations for surveys on anthelmintic resistance. A simulation study was performed in which the FECRT (based on the arithmetic mean of grouped FEC of the same animals before and after drug administration) was conducted under varying conditions of mean FEC, aggregation of FEC (inversely correlated with k), sample size, detection limit and 'true' drug efficacies. Classification trees were built to explore the impact of the above factors on the sensitivity and specificity of detecting a truly reduced efficacy. For a reduced-efficacy threshold of 90%, most combinations resulted in a reliable detection of reduced and normal efficacy. For the reduced-efficacy threshold of 95% however, unreliable FECRT results were found when sample sizes <15 were combined with highly aggregated FEC (k=0.25) and detection limits ≥ 5 EPG or when combined with detection limits ≥ 15 EPG. Overall, an increase in sample size and mean preDA FEC, and a decrease in detection limit improved the diagnostic accuracy. FECRT remained inconclusive under any evaluated condition for drug efficacies ranging from 87.5% to 92.5% for a reduced-efficacy-threshold of 90% and from 92.5% to 97.5% for a threshold of 95%. The results highlight that (i) the interpretation of this FECRT is affected by a complex interplay of factors, including the level of excretion and aggregation of FEC and (ii) the diagnostic value of FECRT to detect small reductions in efficacy is limited. This study, therefore, provides a framework allowing researchers to adapt their study design according to a wide range of field conditions, while ensuring a good diagnostic performance of the FECRT.

Utility of capsule endoscopy for evaluating anthelmintic efficacy in fully conscious dogs.

The current accepted standard for evaluating the efficacy of gastrointestinal anthelmintic drugs is necropsy of infected animals followed by a comparison of worm counts between treated and non-treated groups. In this study capsule endoscopy, a minimally invasive method of imaging the small intestine of humans, is evaluated as a possible alternative to necropsy for the purposes of worm quantification in dogs. Eighteen Beagle dogs were included in this study. These dogs were part of a separate trial intended to determine the efficacy of various candidate parasiticides against Ancylostoma caninum via the necropsy standard. Dogs were inoculated with A. caninum L3s 4 weeks prior to treatment with one of the candidate compounds; a control group (n=8) received no treatment. Capsule endoscopy was performed 6-14 days post-treatment, followed by necropsy the following day. Seventeen dogs had complete examinations, i.e. the capsule traversed the small intestine and reached the colon within the battery life of the capsule. A strong correlation (r(s)=0.87, P<0.0001) was observed between the worm counts acquired by capsule endoscopy and necropsy. There was no clear relationship between the ability of the capsule endoscope to detect hookworms and either visibility of the intestinal lumen or small intestinal transit time. Generation of a virtual spatial record of hookworm location from the capsule endoscopy data revealed a temporal trend, with the majority of worms present in the proximal small intestine in the morning versus the central to distal small intestine in the afternoon. Worm distribution as determined by capsule endoscopy closely resembled post-mortem findings. In conclusion, capsule endoscopy shows promise as an alternative to necropsy for the enumeration of A. caninum in the canine small intestine, although further work is required to improve completion rates and optimise intestinal examination.

Drug residues.

The use of veterinary drugs in animal production is necessary for the prevention and treatment of disease; however, such use may result in residues. Regulatory authorities administer legislative frameworks which ensure that foods derived from animals treated with approved veterinary drugs are safe for human consumption. A human food safety evaluation is conducted as follows: it estimates the risk to human health and safety--based on scientific assessment of the available information and data--formulates measures for controlling the risks identified, and communicates the findings and implications of the risk assessment to interested parties. Foods derived from animals are monitored for the presence of drug residues. The reported incidence of illegal residues from these programmes is very low. These findings reassure the public that veterinary drugs are effectively regulated and that food obtained from treated animals does not contain residues that might constitute a health hazard to consumers. Non-regulatory organizations, including the veterinary pharmaceutical industry, producer organisations, veterinarians and food processors, all contribute to a safe food supply. The food safety risk analysis framework is continually refined to ensure that the health of all consumers is protected.

Simple and sensitive monitoring of sulfonamide veterinary residues in milk by stir bar sorptive extraction based on monolithic material and high performance liquid chromatography analysis.

A simple, rapid, and sensitive method for the quantitative monitoring of five sulfonamide antibacterial residues (SAs) in milk was developed by stir bar sorptive extraction (SBSE) coupling to high performance liquid chromatography with diode array detection. The analytes were concentrated by SBSE based on poly (vinylimidazole-divinylbenzene) monolithic material as coating. The extraction procedure was very simple, milk was diluted with water then directly sorptive extraction without elimination of fats and protein in samples was required. To achieve optimum extraction performance for SAs, several parameters, including extraction and desorption time, desorption solvent, ionic strength and pH value of sample matrix were investigated. Under the optimized experimental conditions, low detection limits (S/N=3) quantification limits (S/N=10) of the proposed method for the target compounds were achieved within the range of 1.30-7.90 ng/mL and 4.29-26.3 ng/mL from spiked milk, respectively. Good linearities were obtained for SAs with the correlation coefficients (R(2)) above 0.996. Finally, the proposed method was successfully applied to the determination of SAs compounds in different milk samples and satisfied recoveries of spiked target compounds in real samples were obtained.