Physicochemical properties of otic products for Canine Otitis Externa: comparative analysis of marketed products.

BACKGROUND: Otitis externa is a commonly diagnosed dermatological disorder in canines. The pathogens primarily involved in canine otitis externa (COE) include Staphylococcus pseudintermedius, Pseudomonas aeruginosa, Proteus mirabilis, and Malassezia pachydermatis. As COE tends to be superficial, medications delivered topically are often effective and practical in managing the condition. As such, there is a wide variety of approved topical products currently available in the market. The efficacy of topical dosage forms can be dependent on various factors such as the pharmacology of active constituents and the physicochemical properties of the formulation, including pH, viscosity, spreadability, and bio-adhesion. Currently, there is a lack of published literature available on the optimal properties of topical COE products. In this study, we compared the physicochemical properties of nine commercially available otic veterinarian products in Australia used clinically to manage COE. RESULTS: Based on our comparative analysis, the pH (6.26 ± 0.04) of an aqueous-based product was similar to a healthy dog's external auditory canal. Products containing polymers exhibited higher viscosity and bio-adhesion. Spreadability was inversely related to viscosity and Osurnia ® a product with high viscosity demonstrated the lowest spreadability. Aqueous-based otic products showed better syringebility whereas oil-based systems required higher force to expel the products. Variability in droplet size was noted. Derm Otic, Baytril Otic, and Aurizon Ear Drops had the lower standard deviation which indicates they would give a more consistent dose. CONCLUSIONS: Findings from this work provide considerations for industry researchers or formulation scientists working in the area of otic dosage formulations.

Comparison of analyte identification criteria and other aspects in triple quadrupole tandem mass spectrometry: Case study using UHPLC-MS/MS for regulatory analysis of veterinary drug residues in liquid and powdered eggs.

Ultrahigh-performance liquid chromatography (UHPLC) coupled with triple quadrupole tandem mass spectrometry (MS/MS) is one of the most powerful tools for the multiclass, multiresidue analysis of veterinary drugs, pesticides, mycotoxins, and other chemical contaminants in foods and other sample types. Until approximately 2010, commercial MS/MS instruments using multiple reaction monitoring (MRM) were generally limited to minimum dwell (and inter-dwell) times of 10 ms per ion transition. To achieve the needed accuracy and detection limits for hundreds of targeted analytes, older UHPLC-MS/MS methods typically acquired only two ion transitions per analyte (yielding only one ion ratio for qualitative identification purposes), which is still the norm despite technological advancements. Newer instruments permit as little as 1 ms (inter-)dwell times to afford monitoring of more MRMs/analyte with minimal sacrifices in accuracy and sensitivity. In this study, quantification and identification were assessed in the validation of 169 veterinary drugs in liquid and powdered eggs. Quantitatively, an "extract-and-inject" sample preparation method yielded acceptable 70-120% recoveries and < 25% RSD for 139-141 (82-83%) of the 169 diverse drug analytes spiked into powdered and liquid eggs, respectively, at three levels of regulatory interest. Qualitatively, rates of false positives and negatives were compared when applying three different regulatory identification criteria in which two or three MRMs/drug were used in each case. Independent of the identification criteria, rates of false positives remained <10% for 95-99% of the drugs whether 2 or 3 ions were monitored, but the percent of drugs with >10% false negatives decreased from 25-45 to 10-12% when using 2 vs. 3 MRMs/analyte, respectively. Use of a concentration threshold at 10% of the regulatory level as an identification criterion was also very useful to reduce rates of false positives independent of ion ratios. Based on these results, monitoring >2 ion transitions per analyte is advised when using MS/MS for analysis, independent of SANTE/12682/2019, FDA/USDA, or 2002/657/EC identification criteria. (Quant)identification results using all three criteria were similar, but the SANTE criteria were advantageous in their greater simplicity and practical ease of use.

Trace analysis of progesterone and 21 progestins in milk by ultra-performance liquid chromatography coupled with high-field quadrupole-orbitrap high-resolution mass spectrometry.

A method for rapid screening and quantification of progesterone and progestins in milks by ultrahigh-performance liquid chromatography coupled with quadrupole-high field Orbitrap high-resolution mass spectrometry (UHPLC QE HF HRMS) was established. Milks samples were extracted by acetonitrile + hexane (80 + 20), purified by prime HLB SPE and analyzed by UHPLC QE HF HRMS. The detection limit of progesterone and 21 progestins in milk is between 0.05 µg/kg -0.3 µg /kg, the correlation coefficient of progesterone and progestins in the corresponding concentration range is more than 0.99, recoveries for milk samples are between 80.7% and 108.3% with the relative deviation is less than 15%.The method fulfils the requirements of veterinary drug residue detection validation of EU and China, and successfully applied to detecting the µg/kg level of progesterone and monitoring residual of progestins in real milk.

Sensitive and Fast Determination of Ceftiofur in Honey and Veterinary Formulation by HPLC-UV Method with Pre-column Derivatization.

A novel, sensitive and rapid high performance liquid chromatography (HPLC) method for the determination of ceftiofur by pre-column derivatization with 1,2-naphthoquinone-4-sulfonate. Analysis was performed within 5 min on a Kinetex C18 column based on core-shell technology. The mobile phase composed of acetonitrile-water (50:50, v/v) pumped isocratically at a flow rate of 1.0 mL/min under UV detection at 254 nm. The factors affecting the derivatization reaction and separation conditions were carefully evaluated and optimized. The method was linear over the concentration range of 45-450 ng/mL with a limit of detection of 3.29 ng/mL and limit of quantitation of 10.97 ng/mL. The new method was successfully applied for the analysis of ceftiofur in the veterinary formulation and honey with average recoveries of 100.78% and 98. 83%, respectively. The present method is suitable and favorable for the analysis of ceftiofur on account of its sensitivity, rapidity and cost-effectiveness. In addition, it could have significant application for the determination of ceftiofur in other food products.

A Structural Analysis of the FDA Green Book-Approved Veterinary Drugs and Roles in Human Medicine.

The FDA Green Book is a list of all drug products that have been approved by the FDA for use in veterinary medicine. The Green Book, as published, lacks structural information corresponding to approved drugs. To address this gap, we have compiled the structural data for all FDA Green Book drugs approved through the end of 2019. Herein we discuss the relevance of this data set to human drugs in the context of structural classes and physicochemical properties. Analysis reveals that physicochemical properties are highly optimized and consistent with a high probability of favorable drug metabolism and pharmacokinetic properties, including good oral bioavailability for most compounds. We provide a detailed analysis of this data set organized on the basis of structure and function. Slightly over half (51%) of vet drugs are also approved in human medicine. Combination drugs are biologics are also discussed.

Perspectives of photodynamic therapy in biotechnology.

Photodynamic therapy (PDT) is a current and innovative technique that can be applied in different areas, such as medical, biotechnological, veterinary, among others, both for the treatment of different pathologies, as well as for diagnosis. It is based on the action of light to activate photosensitizers that will perform their activity on target tissues, presenting high sensitivity and less adverse effects. Therefore, knowing that biotechnology aims to use processes to develop products aimed at improving the quality of life of human and the environment, and optimizing therapeutic actions, researchers have been used PDT as a tool of choice. This review aims to identify the impacts and perspectives and challenges of PDT in different areas of biotechnology, such as health and agriculture and oncology. Our search demonstrated that PDT has an important impact around oncology, minimizing the adverse effects and resistance to chemotherapeutic to the current treatments available for cancer. Veterinary medicine is another area with continuous interest in this therapy, since studies have shown promising results for the treatment of different animal pathologies such as Bovine mastitis, Malassezia, cutaneous hemangiosarcoma, among others. In agriculture, PDT has been used, for example, to remove traces of antibiotics of milk. The challenges, in general, of PDT in the field of biotechnology are mainly the development of effective and non-toxic or less toxic photosensitizers for humans, animals and plants. We believe that there is a current and future potential for PDT in different fields of biotechnology due to the existing demand.

Formulations for Allergen Immunotherapy in Human and Veterinary Patients: New Candidates on the Horizon.

Allergen immunotherapy is currently the only causal treatment for allergic diseases in human beings and animals. It aims to re-direct the immune system into a tolerogenic or desensitized state. Requirements include clinical efficacy, safety, and schedules optimizing patient or owner compliance. To achieve these goals, specific allergens can be formulated with adjuvants that prolong tissue deposition and support uptake by antigen presenting cells, and/or provide a beneficial immunomodulatory action. Here, we depict adjuvant formulations being investigated for human and veterinary allergen immunotherapy.

Detection of azithromycin residue in broiler feathers by liquid chromatography-tandem mass spectrometry.

In the study, a sensitive and reproducible method for the quantitative analysis of azithromycin in broiler feather samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Feather samples were rinsed after being wrapped in medical gauze, then chopped and then added to 5% (v/v) ammonia in methanol solution for ultrasonic extraction. The extract was purified by the combination of commercial polymeric microparticles (Oasis MCX) and Fe3O4 nanoparticles. The LC separation was performed on an Agilent Eclipse plus C18 column. Multiple reaction monitoring was used for the selective detection of azithromycin. The good linearity curve of azithromycin in feather sample was in the range from 1.0 µg kg-1 to 100.0 µg kg-1 with 0.9935 of correlation coefficient. And the limit detection and limit of quantification was 0.5 µg kg-1 and 2.0 µg kg-1 in spiked feather samples. The recoveries of azithromycin were 85.2-94.7% with the relative standard deviation less than 10%. The established method is simple, rapid, sensitive and specific, and could meet the need of government and enterprises to monitor the illegal use of azithromycin in livestock and poultry breeding.

Determination of 62 veterinary drugs in feedingstuffs by novel pressurized liquid extraction methods and LC-MS/MS.

A fast and simple method for the determination of 62 veterinary drugs in feedingstuffs was developed, optimized, validated, and applied to real samples. Sample preparation was based on a pressurized liquid extraction method using a hard cap coffee machine, which was compared to a commercial pressurized liquid extraction system. Extraction was performed with diatomaceous earth, acetonitrile (20%), and formic acid (0.1%). A central composite design was used to optimize the composition of the extraction solvent. The extracts were analyzed using two chromatographic modes (reversed phase with C18 and HILIC). Analytical limits were set to 25 (limit of detection) and 75 µg kg-1 (limit of quantitation). For banned substances, a salting-out step was included, achieving LOQ lower as 1 µg kg-1 for ractopamine. Other figures of merit such as precision, trueness, decision limit (CCα), method capability (CCß), matrix effects, stability, recovery, and measurement uncertainty were also reported for analytical validation. The method was successfully applied to hundreds of real samples demonstrating its fitness-for-purpose for the analysis of sulfonamides, tetracyclines, fluoroquinolones, avermectins, quinolones, beta-agonists, beta-lactams, amphenicols, benzimidazoles, coccidiostats, lincosamides, macrolides, nitrofurans, quinoxalines, melamine, and trimethoprim.

Validated spectrophotometric determination of maduramicin ammonium using three charge transfer complexation reactions.

Direct spectrophotometric determination of Maduramicin ammonium (MAD) represents an analytical challenge since it is a weak UV-absorbing and lacking a strong chromophore. This work represents the first spectrophotometric determination of MAD as no direct spectrophotometric or colorimetric determination methods for MAD are available in the literature. The present study illustrates the development of three simple, rapid and inexpensive colorimetric methods for the routine quality control analysis of MAD based on the formation of colored charge transfer complexes with three electron acceptors namely p-chloranilic acid (p-CA), 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) and picric acid (PA). The color products of MAD with p-CA, DDQ and PA were measured at 519, 588 and 405nm respectively. The proposed methods were validated in terms of linearity, ranges, precision, accuracy, robustness and limits of detection and quantification. MAD was effectively determined over concentration ranges of 100-1000, 25-250 and 30-150µg/mL using p-CA, DDQ and PA, respectively with good linearity as shown by the values of correlation coefficients not less than 0.9991. The developed methods were successfully implemented in the assay of MAD powder pharmaceutical formulation for veterinary use.

Poultry Farms as a Potential Source of Environmental Pollution by Pharmaceuticals.

Industrial poultry breeding is associated with the need to increase productivity while maintaining low meat prices. Little is known about its impact on the environment of soil pollution by pharmaceuticals. Breeders routinely use veterinary pharmaceuticals for therapeutic and preventive purposes. The aim of this work was to determine the influence of mass breeding of hens on the soil contamination with 26 pharmaceuticals and caffeine. During two seasons-winter and summer 2019-15 soil samples were collected. Liquid extraction was used to isolate analytes from samples. Extracts were analyzed using ultra-high performance liquid chromatography coupled with tandem mass spectrometry detection (UPLC-MS/MS). The results showed the seasonal changes in pharmaceutical presence in analyzed soil samples. Ten pharmaceuticals (metoclopramide, sulphanilamide, salicic acid, metoprolol, sulphamethazine, nimesulide, carbamazepine, trimethoprim, propranolol, and paracetamol) and caffeine were determined in soil samples collected in March, and five pharmaceuticals (metoclopramide, sulphanilamide, sulphamethazine, carbamazepine, sulfanilamid) in soil samples collected in July. The highest concentrations were observed for sulphanilamide, in a range from 746.57 ± 15.61 ng/g d.w to 3518.22 ± 146.05 ng/g d.w. The level of bacterial resistance to antibiotics did not differ between samples coming from intensive breeding farm surroundings and the reference area, based on antibiotic resistance of 85 random bacterial isolates.

Evaluation of the mucoadhesive strengths of Abelmoschus esculentus and Irvingia gabonensis gums for possible application in veterinary vaccine delivery: the effect of extraction methods.

This study evaluates the effects of different gum extraction methods on the mucoadhesive strengths of Abelmoschus esculentus (AE) and Irvingia gabonensis (IG) gums and the release of vaccine antigen in vaccine-gum formulations. AE and IG gums were extracted employing previously documented methods with acetone or sodium chloride (NaCl) and either oven-dried or freeze-dried. Gum extracts were analyzed for mucoadhesive strengths using a modified rotational cylinder method on animal mucosa. The time taken to detach from the mucosa was taken as the Peak Adhesion Time (PAT). The gum extracts were charged with Peste des petits ruminant vaccine and the antigen release was evaluated using agar gel immunodiffusion technique. The means of the PATS were analyzed using Mann-whitney t-test at p < .05. The NaCl extracted and freeze-dried IG gum showed sustained mean PATs of 1766 ± 73 s; 2116 ± 101 s; 7044 ± 117 s, while the oven-dried IG gum and both AE gums showed short-lived average PATs. Vaccine-gum formulations of IG at ratios 2:1, 1:1 & 1:2 had strong positive reactions while only that of AE at 2:1 showed a strong positive reaction. This study shows that NaCl extracted and freeze-dried IG gum has immunomodulatory potential for mucoadhesive vaccine delivery in ruminants.

Broad spectrum detection of veterinary drugs with a highly stable metal-organic framework.

The broad spectrum detection of veterinary drugs is very important for rapid and large-scale safe screen of animal-derived foods. Metal-organic frameworks (MOFs), as a kind of emerged functional porous materials are quite promising in the chemical sensing and molecular detection. In this work, we report the high-performance broad spectrum detection of 15 commonly-used veterinary drugs through the fluorescence quenching in a newly-designed chemically stable Al-based MOF, Al3(µ3-O)(OH)(H2O)2(PPTTA)3/2 (BUT-22). To the best of our knowledge, this is the first systematic investigation for the application of MOFs in the detection/sensing of veterinary drugs through fluorescence quenching method. The quenching efficiencies of the tested veterinary drugs on BUT-22 are all beyond 82%, and the limits of detection (LOD) are low at parts per billion (ppb) levels. Interestingly, BUT-22 also enables the selective detection of nicarbazin (NIC) through the clearly-observed red shift of its maximum fluorescence emission wavelength. Moreover, the fluorescence quenching mechanism was explored with the help of theoretical calculations. Our work indicates that MOFs are favorable materials for the detection of veterinary drugs, being potentially useful in monitoring drug residues of animal-derived foods.

Toxicological effects of some antiparasitic drugs on equine liver glutathione S-Transferase enzyme activity.

Benzimidazoles are antiparasitic drugs having an extensive application field like agriculture, medicine, and especially in veterinary medicine. In this study, we report the effect of some benzimidazole drugs such as ricobendazole (RBZ), thiabendazole (TBZ), albendazole (ALBA) and oxfendazole (OFZ) on glutathione s-transferase (GST) enzyme activity. The kinetics studies, IC50 and Ki values of the tested drugs on GSTs enzyme activity were investigated. The obtained ranking of IC50 values were found to be approximately RBZ (53.31 µM, r2: 0.9778) < OFZ (57.75 µM, r2: 0.9630) < ALBA (63.00 µM, r2: 0.9443) < TBZ (69.30 µM, r2: 0.9491). And the obtained ranking of Ki values of the tested drugs (RBZ, TBZ, ALBA, and OFZ) for GSTs enzyme activity was found to be approximately 26.37 ±â€¯2.96, 44.01 ±â€¯5.74, 39.82 ±â€¯3.98 and 30.14 ±â€¯3.03 µM, respectively. Experimental results showed that tested the benzimidazoles drugs have some significant inhibitory effect on GSTs enzyme activity. And also, it was determined that RBZ, ALBA, OFZ are competitive inhibition, but TBZ is non-competitive inhibitors on GSTs enzyme activity. RBZ drug showed the best inhibitory effect with the lowest Ki value.

Background Signal-Free Magnetic Bioassay for Food-Borne Pathogen and Residue of Veterinary Drug via Mn(VII)/Mn(II) Interconversion.

Paramagnetic ion-mediated sensors can greatly simplify current magnetic sensors for biochemical assays, but it remains challenging because of the limited sensitivity. Herein, we report a magnetic immunosensor relying on Mn(VII)/Mn(II) interconversion and the corresponding change in the low-field nuclear magnetic resonance (LF-NMR) of the transverse relaxation rate (R2). The fact that the NMR R2 of the water protons detected in Mn(II) aqueous solution is much stronger than Mn(VII) aqueous solution enables the modulation of the LF-NMR signal intensity of R2. By employing immunomagnetic separation and enzyme-catalyzed reaction, this Mn(VII)/Mn(II) interconversion allows the development of a background signal-free magnetic immunosensor with a high signal-to-background ratio that enables detection of ractopamine and Salmonella with high sensitivity (the limits of detection for ractopamine and Salmonella are 8.1 pg/mL and 20 cfu/mL, respectively). This Mn-mediated magnetic immunosensor not only retains the good stability but also greatly improves the sensitivity of conventional paramagnetic ion-mediated magnetic sensors, offering a promising platform for sensitive, stable, and convenient bioanalysis.

Prediction of Transformation Products of Monensin by Electrochemistry Compared to Microsomal Assay and Hydrolysis.

The knowledge of transformation pathways and identification of transformation products (TPs) of veterinary drugs is important for animal health, food, and environmental matters. The active agent Monensin (MON) belongs to the ionophore antibiotics and is widely used as a veterinary drug against coccidiosis in broiler farming. However, no electrochemically (EC) generated TPs of MON have been described so far. In this study, the online coupling of EC and mass spectrometry (MS) was used for the generation of oxidative TPs. EC-conditions were optimized with respect to working electrode material, solvent, modifier, and potential polarity. Subsequent LC/HRMS (liquid chromatography/high resolution mass spectrometry) and MS/MS experiments were performed to identify the structures of derived TPs by a suspected target analysis. The obtained EC-results were compared to TPs observed in metabolism tests with microsomes and hydrolysis experiments of MON. Five previously undescribed TPs of MON were identified in our EC/MS based study and one TP, which was already known from literature and found by a microsomal assay, could be confirmed. Two and three further TPs were found as products in microsomal tests and following hydrolysis, respectively. We found decarboxylation, O-demethylation and acid-catalyzed ring-opening reactions to be the major mechanisms of MON transformation.

Discovery of the Marker Residue of Olaquindox in Pigs, Broilers, and Carp.

The excretion, metabolism, distribution, and residue depletion of olaquindox (OLA), an antibacterial and growth-promoting agent used in food-producing animals for decades without a clear understanding of metabolic fate, was completely studied in pigs, broilers, carp, and rats using a radio-tracing approach combined with liquid chromatography-ion trap/time-of-flight mass spectroscopy to define the scientific marker residue (MR). After a single gavage of [3H]OLA, over 92% of the dose was excreted via urine. OLA was transformed into eight metabolites (O1-O8) in pigs and broilers, four metabolites (O1, O2, O4, and O7) in carp, and nine metabolites (O1-O9) in rats. O2 was the major residue in edible tissues of four species and persisted for the longest time in the kidneys with the longest half-life of 3.52-4.6 d. Bisdesoxyolaquindox (O2) is designated to be the MR, and the kidneys are considered to be the target tissue for OLA in food producing animals. Monitoring for this metabolite would improve the food safety evaluation and residue control of this drug.

Effects of veterinary antibiotics on the fate and persistence of 17ß-estradiol in swine manure.

The fate and persistence of natural estrogens from livestock manure and the interactions of these compounds with veterinary antibiotics (VAs) have not been well studied. We therefore employed 14C-labeling to explore the mineralization, degradation, and residual distribution of 17ß-estradiol (E2) in swine manure in the absence and presence of six categories of VAs at concentrations of 10 and 100 mg/kg. After 16 days of incubation, 94% of the E2 dissipated, of which 28% was mineralized to 14CO2, 18% was transformed into organic-extractable E1 (9%) and other unknown metabolites (9%), and 48% into non-extractable residues (NER). VAs inhibited, enhanced or had no effect on E2 mineralization or its degradation to E1 and other metabolites. Principal component analysis showed that the overall effect of VAs was not necessarily related to their physicochemical properties or concentrations. However, high doses of macrolides inhibited E2 mineralization in manure and increased the retention of E2 and its metabolites in both free and NER forms. Our study demonstrates that considerable amounts of E2 and NER are retained in manure, despite nearly complete mineralization. Thus, VAs administered to livestock may increase the persistence of natural estrogens in manure and, accordingly, the environmental risks posed by these compounds.

Benzoylphenyl ureas as veterinary antiparasitics. An overview and outlook with emphasis on efficacy, usage and resistance.

Six benzoylphenyl ureas are currently used in formulations approved as veterinary medicines: diflubenzuron for fly control mainly on cattle, lice and blowfly strike control on sheep, and lice control on farmed salmonids; lufenuron for flea control on dogs and cats and for lice control on farmed salmonids; triflumuron for lice and blowfly strike control on sheep; fluazuron for tick control on cattle; teflubenzuron for lice control on farmed salmon; and novaluron for fly and tick control on cattle and for flea control on dogs. Resistance to diflubenzuron and triflumuron has already been reported for sheep body lice and blowflies, and to fluazuron in cattle ticks. These and other minor veterinary usages, as well as the current status of resistance, are reviewed and perspectives for future opportunities are discussed based on unexplored potentials and threats posed by future resistance development.

Development and validation of a fully automated solid phase microextraction high throughput method for quantitative analysis of multiresidue veterinary drugs in chicken tissue.

This paper presents the development and validation of a fully automated, high-throughput multiclass, multiresidue method for quantitative analysis of 77 veterinary drugs in chicken muscle via direct immersion solid phase microextraction (DI-SPME) and ultra-high pressure liquid chromatography-electrospray ionization - tandem mass spectrometry (UHPLC-ESI-MS/MS). The selected drugs represent more than 12 different classes of drugs characterized by varying physical and chemical properties. A Hydrophilic-lipophilic balance (HLB)/polyacrylonitrile (PAN) extraction phase, prepared using HLB particles synthesized in-house, yielded the best extraction/desorption performance among four different SPME extraction phases evaluated in the current work. The developed SPME method was optimized in terms of SPME coating and geometry, desorption solvent, extraction and rinsing conditions, and extraction and desorption times. Multivariate analysis was performed to determine the optimal desorption solvent for the proposed application. The developed method was validated according to the Food and Drug Administration (FDA) guidelines, taking into account Canadian maximum residue limits (MRLs) and US maximum tolerance levels for veterinary drugs in meat. Method accuracy ranged from 80 to 120% for at least 73 compounds, with relative standard deviation of 1-15%. Inter-day precision ranged from 4 to 15% for 70 compounds. Determination coefficients values were higher than 0.991 for all compounds under study with no significant lack of fit (p > 0.05) at the 5% level. In terms of limits of quantitation, the method was able to meet both Canadian and US regulatory levels for all compounds under study.