Comparative pharmacokinetics of difethialone stereoisomers in male and female rats and mice: development of an intra- and inter-species model to predict the suitable formulation mix.

The ecotoxicity of anticoagulants used for rodent pests' management is a major concern, particularly with second generation anticoagulants, which are more persistent in the body of rodents and therefore more likely to cause secondary exposure in their predators. One of the solutions envisaged to mitigate this risk is to use stereoisomers of these anticoagulants, each of which has particular pharmacokinetics. However, the few studies published to date have considered only one species and one sex. Here, we study the pharmacokinetics of the 4 stereoisomers of 3.4 mg/kg of difethialone in rats (Rattus norvegicus) and 3 mg/kg in mice (Mus musculus) in both sexes and propose a model to choose the optimal stereoisomer efficacy/ecotoxicity mixture for the management of all these animals. Our results show that while the most persistent stereoisomer (E3-cis) is common to both species and sexes, the pharmacokinetics of the other stereoisomers show marked differences between sexes and species. Thus, the area under curve (AUC) of E4-trans in male rats is four times lower than in females or mice, making it a priori unusable in male rats. Conversely, our modeling seems to show that the E1-trans stereoisomer seems to offer the best compromise AUC persistence. In conclusion, we highlight that studies on anticoagulants must necessarily integrate research on the effect of gender and species both on efficacy and with regard to the ecotoxicity of these molecules.

Evaluating the effects of anticoagulant rodenticide bromadiolone in Wistar rats co-exposed to vitamin K: impact on blood-liver axis and brain oxidative status.

The aim of this study was to investigate the beneficial effects of vitamin K relate to protection against detrimental effects of bromadiolone. Wistar rats (n = 30) were divided in three groups (n = 10): control group and two groups treated with bromadiolone (0.12 mg/kg) and bromadiolone + vitamin K (0.12 mg/kg + 100 mg/kg) over the period of four days. The main findings in the bromadiolone-exposed rats, such as damaged hepatocytes, high levels of globulin, total proteins and lymphocytes, and altered albumin/globulin ratio, collectively indicate an acute inflammatory process. Morphological changes in erythrocytes include microcytosis, hypochromia, hyperchromia, hemolysis, stomatocytosis, and spherocytosis. Significantly low values of RBC, Hct, and hemoglobin concentrations indicate impairments of the hematopoietic pathway causing combined anemia. The selected dose of bromadiolone caused a non-significant increase of catalase activity and a significant increase of the total protein content in brain tissue homogenates. Vitamin K supplementation reduced many of the harmful effects of bromadiolone. The cytoprotective role of vitamin K was proved to be of great importance for the preservation of structural changes on the membranes of hepatocytes and erythrocytes, in addition to the known role in the treatment of coagulopathies. The results of the study suggest valuable properties of vitamin K in the prevention and treatment of various types of anemia caused by bromadiolone toxicity. Future research is necessary to determine the adequate dose and treatment duration with vitamin K in disorders caused by the cumulative action of bromadiolone and possibly other pesticides.

Water vole management - Could anticoagulant rodenticides stereochemistry mitigate the ecotoxicity issues associated to their use?

Cyclic water vole population explosions can be controlled in some European countries with anticoagulant rodenticides leading sometimes to wildlife poisonings due to the toxin's tissue persistence. Here, we analyzed the pharmacokinetics of rodenticide residues in voles and we explored potential ways of improving the mass application of these agents based on the concept of stereoisomers. We demonstrated the dramatic persistence of bromadiolone in vole tissues with a hepatic half-life of about 10-30 days, while the tissue persistence of chlorophacinone is rather short with a hepatic half-life of about one day. The dramatic persistence of bromadiolone is due to the trans-isomer group (the major compound in bromadiolone), while the cis-isomer group has a short half-life. Because of resistance to chlorophacinone, the cis-bromadiolone isomers may constitute an excellent compromise between efficacy and ecotoxicological risk to control voles. A mathematical model is proposed to favor the development of baits mixed with cis-isomer groups.

Differences in teratogenicity of some vitamin K antagonist substances used as human therapeutic or rodenticide are due to major differences in their fate after an oral administration.

All vitamin K antagonist active substances used as rodenticides were reclassified in 2016 by the European authorities as active substances "toxic for reproduction", using a "read-across" alternative method based on warfarin, a human vitamin K antagonist drug. Recent study suggested that all vitamin K antagonist active substances are not all teratogenic. Using a neonatal exposure protocol, warfarin evokes skeletal deformities in rats, while bromadiolone, a widely used second-generation anticoagulant rodenticide, failed to cause such effects. Herein, using a rat model we investigated the mechanisms that may explain teratogenicity differences between warfarin and bromadiolone, despite their similar vitamin K antagonist mechanism of action. This study also included coumatetralyl, a first-generation active substance rodenticide. Pharmacokinetic studies were conducted in rats to evaluate a potential difference in the transfer of vitamin K antagonists from mother to fetus. The data clearly demonstrate that warfarin is highly transferred from the mother to the fetus during gestation or lactation. In contrast, bromadiolone transfer from dam to the fetus is modest (5% compared to warfarin). This difference appears to be associated to almost complete uptake of bromadiolone by mother's liver, resulting in very low exposure in plasma and eventually in other peripheric tissues. This study suggests that the pharmacokinetic properties of vitamin K antagonists are not identical and could challenge the classification of such active substances as "toxic for reproduction".

[Study on the pharmacokinetics of bromadiolone in oral poisoning rabbits].

Objective: To investigate the variations of bromadiolone concentration in blood and its metabolism in rabbits after oral administration of bromadiolone, and to provide reference for the study of bromadiolone metabolism. Methods: Designed absolute alcohol (1 g/kg) reagent control group, high dose (0.3 mg/kg) and low dose group (0.05 mg/kg) , there were 6 rabbits in each group. Blood samples were collected from the rabbit central auricular artery at regular intervals as 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, 24 h, 36 h, 48 h, 72 h, 168 h, 336 h, 504 h after oral administration. The samples were centrifuged within 1 h. Prothrombin time (PT) , activated partial thromboplastin time (APTT) and concentrations of bromadiolone in plasma were tested. Metabolic kinetics data was analyzed by DAS 3.0.2 software. Results: Bromadiolone had no significant effect on the body weight of the experimental rabbits during the experimental period (P>0.05) . PT and APTT were significantly abnormal in different dose groups, but for occurrence of exception, PT was earlier than APTT. The concentration of bromadiolone in plasma reached the peak value 12 h after gavage in both high-dose and low-dose groups. The absorption time of t(1/2Ka) in high-dose group was 4.34 h, the clearance time of t(1/2) was 81.52 h, the absorption time of t(1/2Ka) in low-dose group was 6.90 h, and the elimination time of t(1/2) was 56.38 h. The atrioventricular model of bromadidone was three compartment model in rabbits. Conclusion: Bromadiolone can be absorbed rapidly by oral administration, but its metabolism is slow. The change of bromadiolone in vivo accords with the three compartment model.

Determination of anticoagulant rodenticides in faeces of exposed dogs and in a healthy dog population.

BACKGROUND: Exposure to anticoagulant rodenticides (ARs) in dogs is among the most common causes of poisoning in small animal practice, but information about toxicokinetic of these rodenticides in dogs is lacking. We analysed blood and faeces from five accidentally exposed dogs and 110 healthy dogs by reversed phase ultra-high performance liquid chromatography-tandem mass spectrometry. The aim of the study was to estimate elimination of brodifacoum, bromadiolone and difenacoum after acute exposure, calculate the half-lives of these rodenticides in dogs, estimate faecal elimination in a litter of puppies born, and further to identify the extent of AR exposure in a healthy dog population. RESULTS: Three dogs were included after single ingestions of brodifacoum; two dogs ingested bromadiolone and one dog ingested difenacoum. Maximum concentrations in faeces were found after day 2-3 for all ARs. The distribution half-lives were 1-10 days for brodifacoum, 1-2 days for bromadiolone and 10 days for difenacoum. Brodifacoum and difenacoum had estimated terminal half-lives of 200-330 days and 190 days, respectively. In contrast, bromadiolone had an estimated terminal half-life of 30 days. No clinical signs of poisoning or coagulopathy were observed in terminal elimination period. In blood, the terminal half-life of brodifacoum was estimated to 8 days. Faeces from a litter of puppies born from one of the poisoned dogs were examined, and measurable concentrations of brodifacoum were detected in all samples for at least 28 days after parturition. A cross-sectional study of 110 healthy domestic dogs was performed to estimate ARs exposure in a dog population. Difenacoum was detected in faeces of one dog. Blood and faecal samples from the remaining dogs were negative for all ARs. CONCLUSIONS: Based on the limited pharmacokinetic data from these dogs, our results suggest that ARs have a biphasic elimination in faeces using a two-compartment elimination kinetics model. We have shown that faecal analysis is suitable and reliable for the assessment of ARs exposure in dogs and a tool for estimating the AR half-lives. Half-lives of ARs could be a valuable indicator in the exposed dogs and provides important information for veterinarians monitoring AR exposure and assessment of treatment length in dogs.

Mass spectrometry characterization of anticoagulant rodenticides and hydroxyl metabolites.

RATIONALE: Anticoagulant rodenticides (ARs) are used worldwide for rodent population control to protect human health and biodiversity, and to prevent agricultural and economic losses. Rodents may develop a metabolic resistance to ARs. In order to help understand such metabolic resistance, mass spectrometry was used to position the hydroxylated group of hydroxyl metabolites of second-generation ARs (SGARs). METHODS: Most AR pesticides are derived from the 4-hydroxycoumarin/thiocoumarin family. We used low-resolution and high-resolution mass spectrometry to understand the fragmentation pathways of the ARs and their respective metabolites, and to better define the structure of their tandem mass spectrometry product ions. RESULTS: Seven specific product ions were evidenced for five ARs, with their respective chemical structures. Those ions were obtained as well from the mass spectra of the hydroxyl metabolites of four SGARs, difenacoum (DFM), brodifacoum (BFM), difethialone (DFTL) and flocoumafen (FLO), with different positions of the hydroxyl group. CONCLUSIONS: The differences in chemical structure between DFM on the one hand and BFM, FLO and DFTL on the other could explain the differences in bioavailability between these two groups of molecules. The defined product ions will be used to investigate the part played by the metabolic issue in the field resistance of SGARs.

Effects of vitamin K1 treatment on plasma concentrations of long-acting anticoagulant rodenticide enantiomers following inhalation of contaminated synthetic cannabinoids.

Background: An outbreak of synthetic cannabinoid (SC)-associated coagulopathy and bleeding in Illinois, USA was determined to be due to inhalation of SC contaminated with brodifacoum (BDF), difenacoum (DiF), and bromadiolone (BDL), highly potent long-acting anticoagulant rodenticides (LAARs). Treatment with high-dose vitamin K1 (VK1) prevented mortality; however, plasma LAAR levels were not measured risking recurrence of coagulopathy and bleeding due to premature discontinuation. The goal of this study was to determine if plasma LAAR levels were reduced following standard of care treatment to normalize coagulopathy.Methods: Blood samples were collected from a cohort of 32 patients, and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis used to quantify plasma LAAR levels including enantiomers.Results: BDF was detected in 31 samples; 30 also contained DiF and 18 contained BDL. Initial plasma levels were 581 ± 87, 11.0 ± 1.9, and 14.9 ± 5.9 ng/mL for BDF, DiF, and BDL, respectively (mean ± SE). At discharge plasma, BDF levels remained elevated at 453 ± 68 ng/mL. Plasma half-lives for BDF, DiF, and BDL were 7.5 ± 1.3, 7.2 ± 1.9, and 1.8 ± 0.3 days, respectively. The half-life for trans-BDF enantiomers (5.7 ± 0.8 days) was shorter than for cis-enantiomers (7.6 ± 1.9 days). BDF half-lives were shorter, and coagulopathy normalized faster in patients receiving intravenous VK1 as compared to oral VK1. Patients prescribed VK1 at discharge had fewer re-admittances.Conclusions: These results demonstrate that plasma LAAR levels at discharge were elevated in poisoned patients despite normal coagulation, and that the route of VK1 administration affected LAAR pharmacokinetics and INR normalization. We propose plasma LAAR levels and coagulation be monitored concomitantly during follow-up of patients with LAAR poisoning. KEY POINTSIn patients treated with high-dose vitamin K1 for LAAR poisoning, plasma levels remained 40-fold above safe levels upon discharge from hospital.LAAR half-lives, normalization of coagulopathy, and readmittances were reduced by treatment with intravenous vitamin K1.

Factors associated to adequate time in therapeutic range with oral vitamin K antagonists in Tunisia.

INTRODUCTION:   The quality of chronic anticoagulation and predictor factors of poor anticoagulant control in patients under acenocoumarol were unknown in North Africa. METHODS: It is an observational study, carried out between November 2015 and November 30, 2016. The international normalized ratio (INR) values were prospectively obtained, and TTR was calculated using the Rosendaal method. RESULTS: Overall, 215 patients were included in this study, with a mean age of 63±0,8 years. The prevalence of poor anticoagulation control was 78.1%; 95% CI [72.2-83.2] (168 patients with TTR less than 65%). The median TTR with the Rosendaal method was 44.4%. After multivariate adjustment, variables significantly associated with adequate anticoagulation level were: history of ischemic stroke (Adjusted OR equal to 4.3, 95% CI: 1.4-12.9), associated prescription of antiplatelet therapy (Adjusted OR equal to 3.5, 95% CI: 1.1-11.2), daily prescribed dose of coumarins less than 6 mg (Adjusted OR equal to 6.4, 95% CI: 1.1- 36) and lower risk of bleeding assessed as HAS-BLED score (Adjusted OR: 0.5, 95% CI: 0.3-0.8). CONCLUSION: The quality of anticoagulation management with VKA among outpatients who received acenocoumarol was suboptimal. Strategies should be undertaken by clinicians and patients to improve the quality of anticoagulation, to address challenges to adverse cardiovascular outcomes in individuals treated with chronic anticoagulation.

The relative toxicity of brodifacoum enantiomers.

Brodifacoum (BDF) is a potent, long-acting anticoagulant rodenticide that can cause fatal poisoning in humans. The chemical structure of BDF includes 2 chiral carbons, resulting in 2 pairs of diastereomers, BDF-cis (R/S and S/R) and BDF-trans (R/R and S/S). However, the relative potency of these molecules is not known. The purpose of this study was to compare the in vitro and in vivo toxic effects of the 2 BDF diastereomer pairs. In adult Sprague-Dawley rats BDF-cis was significantly more toxic than BDF-trans (LD50 values of 219 versus 316 µg/kg, respectively) while racemic BDF had intermediate potency (266 µg/kg). In adult New Zealand white rabbits, BDF-cis had a longer half-life than BDF-trans which could contribute to its observed increased toxicity. Lastly, BDF-cis (10 µM), but not BDF-trans, damaged cultured SH-SY5Y human neuroblastoma cells by attenuating mitochondrial reductive capacity. Taken together, these data suggest that different toxic manifestations of BDF poisoning in mammals could be attributed, in part, to differences in relative enantiomer concentrations present in racemic formulations of this commercially-available toxicant.

Should Cytochrome P450 Inducers be Used to Accelerate Clearance of Brodifacoum from Poisoned Patients?

A recent multi-state outbreak of life-threatening bleeding following inhalation of synthetic cannabinoids has been attributed to contamination with the long-acting anticoagulant rodenticide (LAAR) brodifacoum, a second-generation, highly potent, long-acting derivative of the commonly used blood thinner warfarin. While long-term treatment with high-dose vitamin K1 restores coagulation, it does not affect brodifacoum metabolism or clearance, and, consequently, brodifacoum remains in the human body for several months, thereby predisposing to risk of bleeding recurrence and development of coagulation-independent injury in extrahepatic tissues and fetuses. This has prompted the evaluation of pharmacological measures that accelerate brodifacoum clearance from poisoned patients. Since the induction of certain cytochrome P450 (CYP) enzymes accelerates warfarin metabolism, using CYP inducers, such as phenobarbital, to accelerate brodifacoum clearance seems plausible. However, unlike warfarin, brodifacoum does not undergo significant metabolism in the liver, nor have the effects of phenobarbital on vitamin K1 metabolism been previously determined. In addition, the safety of phenobarbital in brodifacoum-poisoned patients has not been established. Therefore, we propose that CYP inducers should not be used to accelerate the clearance of brodifacoum from poisoned patients, but that alternative approaches such as reducing enterohepatic recirculation of brodifacoum, or using lipid emulsions to scavenge brodifacoum throughout the body, be considered.

Recovery of brodifacoum in vomitus following induction of emesis in dogs that had ingested rodenticide bait.

AIM: To assess the benefit of inducing emesis in dogs that have ingested rodenticide bait containing brodifacoum (BDF), by determining the amount of BDF in bait recovered from the vomitus relative to the estimated amount consumed. METHODS: Between 2014 and 2015 samples of vomitus from seven dogs that ingested rodenticide baits containing BDF were submitted by veterinarians in New Zealand. All seven dogs had been given apomorphine by the veterinarian and vomited within 1 hour of ingesting the bait. Some or all of the bait particles were retrieved from each sample and were analysed for concentrations of BDF using HPLC. Based on estimations of the mass of bait consumed, the concentration of BDF stated on the product label, and the estimated mass of bait in the vomitus of each dog, the amount of BDF in the vomited bait was calculated as a percentage of the amount ingested. RESULTS: For five dogs an estimation of the mass of bait ingested was provided by the submitting veterinarian. For these dogs the estimated percentage of BDF in the bait retrieved from the vomitus was between 10-77%. All dogs were well after discharge but only one dog returned for further testing. This dog had a normal prothrombin time 3 days after ingestion. CONCLUSIONS AND CLINICAL RELEVANCE: The induction of emesis within 1 hour of ingestion can be a useful tool in reducing the exposure of dogs to a toxic dose of BDF. The BDF was not fully absorbed within 1 hour of ingestion suggesting that the early induction of emesis can remove bait containing BDF before it can be fully absorbed.

Accumulation of anticoagulant rodenticides (chlorophacinone, bromadiolone and brodifacoum) in a non-target invertebrate, the slug, Deroceras reticulatum.

Anticoagulant rodenticides (ARs) are used worldwide to control populations of agricultural and urban rodents, but these pesticides may be accumulated in and poisoned non-target species of wildlife. Slugs may feed on rodenticide bait following field applications. Thus, it can be assumed that their predators are exposed to rodenticides through food chain transfer. However, AR exposure in the slugs has not been systematically studied. We investigated the accumulation of three ARs (chlorophacinone, bromadiolone or brodifacoum) in the slug Deroceras reticulatum exposed for a period of 5days followed by depuration time of 4days in the laboratory. Moreover, we studied the exposure of slugs to brodifacoum in the field. In the laboratory exposure, the slugs consumed rodenticide baits, but no mortality was observed. After 1day, their concentrations were stable over the time and no differences were detected between the concentrations of the three ARs. After 5days of exposure, mean concentrations in slugs were 1.71, 1.91 and 0.44mg/kg wet weight for chlorophacinone, bromadiolone and brodifacoum respectively. A significant decrease of bromadiolone and brodifacoum in slugs was observed in the post exposure period. In the field study, brodifacoum was detected in >90% of analyzed slugs after application of brodifacoum baits. Then, based on a toxicity-exposure ratio approach, we found that slug consumption may represent a risk of secondary poisoning for three of their predators under acute, repeated or subchronic exposure scenarios. These results suggest that the slugs are not only the potential subject to primary exposure, but also the source of secondary exposure for their predators following application of rodenticide baits.

Effect of ramadan fasting on acenocoumarol-induced antocoagulant effect.

Eating patterns, food intake and type of alimentation vary greatly during the month of ramadan. Furthermore, fasting, which practiced during the month of ramadan, can have an impact on drug's metabolism. These two factors, fasting and eating habits changes during the month of ramadan, may impact acenocoumarol anticoagulant effect, translated by variations of INR values. The aim of our study was to see ramadan fasting effects on INR variations in patients treated by acenocoumarol. A prospective monocentric study was conducted during the ramadan month on fasting outpatients that were treated by acenocoumarol. Baseline INR values (e.i. most recent available value before the month of ramadan) were compared to INR values obtained during the month of ramadan. All patients were monitored for signs of secondary haemorrhagic complications linked to treatment by anti-vitamin K (AVK). Thirty patients were included in the study with a sex ratio 1. Patients mean age was 65 years. Around two thirds of the patients were treated by AVK for atrial fibrillation. The majority of patients (94%) have been treated by AVK for more than a year. Mean INR was significantly higher during the month of ramadan than baseline (3.51 vs 2.52; p< 0.0001). There were also more overdoses during the month of ramadan than baseline (9 vs. 0; p=0.014). The increased INR values highlights the need of a close monitoring of INR values during the month of ramadan, particularly in patients with a high haemorrhagic risk.

The Long-Lasting Rodenticide Brodifacoum Induces Neuropathology in Adult Male Rats.

Superwarfarins are very long-lasting rodenticides effective in warfarin-resistant rodents at extremely low doses. The consequences of chronic superwarfarin levels in tissues, due to biological half-lives on the order of 20 days, have not been examined. We now characterized the neurological effects of brodifacoum (BDF), one of the most widely used superwarfarins, in adult male Sprague Dawley rats. Dosing curves established the acute oral lethal dose for BDF as 221 ± 14 µg/kg. Measurement of tissue BDF levels showed accumulation throughout the body, including the central nervous system, with levels diminishing over several days. Immunocytochemical staining showed that both astrocyte and microglial activation was increased 4 days after BDF administration, as were levels of carbonylated proteins, and neuronal damage assessed by fluorojade B staining. Direct toxic effects of BDF on neurons and glia were observed using enriched cultures of cerebellar neurons and cortical astrocytes. Proteomic analysis of cerebellar lysates revealed that BDF altered expression of 667 proteins in adult rats. Gene ontology and pathway analysis identified changes in several functional pathways including cell metabolism, mitochondria function, and RNA handling with ribosomal proteins comprising the largest group. In vitro studies using primary astrocytes showed that BDF suppressed de novo protein synthesis. These findings demonstrate that superwarfarin accumulation increases indices of neuroinflammation and neuropathology in adult rodents, suggesting that methods which minimize BDF toxicity may not address delayed neurological sequelae.

Development of an Ecofriendly Anticoagulant Rodenticide Based on the Stereochemistry of Difenacoum.

Difenacoum, an antivitamin K anticoagulant, has been widely used as rodenticide to manage populations of rodents. Difenacoum belongs to the second generation of anticoagulant, and, as all the molecules belonging to the second generation of anticoagulant, difenacoum is often involved in primary poisonings of domestic animals and secondary poisonings of wildlife by feeding contaminated rodents. To develop a new and ecofriendly difenacoum, we explored in this study the differences in properties between diastereomers of difenacoum. Indeed, the currently commercial difenacoum is a mixture of 57% of cis-isomers and 43% of trans-isomers. Cis- and trans-isomers were thus purified on a C18 column, and their respective pharmacokinetic properties and their efficiency to inhibit the coagulation of rodents were explored. Tissue persistence of trans-isomers was shown to be shorter than that of cis-isomers with a half-life fivefold shorter. Efficiency to inhibit the vitamin K epoxide reductase activity involved in the coagulation process was shown to be similar between cis- and trans-isomers. The use of trans-isomers of difenacoum allowed to drastically reduce difenacoum residues in liver and other tissues of rodents when the rodent is moribund. Therefore, secondary poisonings of wildlife should be decreased by the use of difenacoum largely enriched in trans-isomers.

A global quality control system to check PT-INR portable monitor for Antivitamin K antagonists.

INTRODUCTION: Although most guidelines for quality assessment of INR PMs recommend specific procedures, no clear regulation or methodology is required for outpatients in our country. We have developed a specific INR portable monitor (PM) quality control system within our telemedicine organization to check over time quality performances and plan corrective actions. METHODS: Based on current guidelines for laboratory QC, the following aspects were assessed: suitability of PM, defined in terms of imprecision and accuracy; intra-assay imprecision, defined according to monthly revision of Levey-Jennings cards with data from each peripheral healthcare unit (PHU), using an internal QC provided by the manufacturer (CV ± 20% considered as acceptable); quarterly accuracy study, for assessing agreement between analytical instruments, based on duplicate analysis of three samples with INR values reflecting different therapeutic ranges (differences ± 0.5 considered as acceptable); external quality assessment (NEQAS). RESULTS: In the nine PHU, 18 portable monitors were used to perform 22 929 test during year 2010. Analytical imprecision was low, showing CVs always <5%. Accuracy check showed two of 216 results out of range (0.92%), thus providing timely indication for instrument replacement. The external QC NEQAS showed optimal performance. CONCLUSION: The current protocol for INR PMs quality assessment was effective to establish and maintain a reliable control of devices, ensuring the quality of analytical data over time. National authorities should be prompted to guarantee and apply correct protocols for INR-PM use.

Novel transition metal complexes of 4-hydroxy-coumarin-3-thiocarbohydrazone: pharmacodynamic of Co(III) on rats and antimicrobial activity.

A new series of stable transition metal complexes of the formula M(L)X·S, where M = Cu(II), Ni(II), Co(III), Cr(III) and Fe(III) and L is the deprotonated ligand of 4-hydroxy-coumarin-3-thiocarbohydrazone, X = Cl(-), NO(3)(-) or CH(3)COO(-) and S = H(2)O and/or EtOH. The HL ligand was prepared by the reaction of 3-formyl-4-hydroxy-coumarine with thiocarbohydrazide in the molar ratio 1:1. The HL ligand and its metal complexes were characterized by elemental analysis, (1)H NMR, IR and electronic spectra, and molar conductance and magnetic measurements and thermal gravimetric analysis (TGA). The HL ligand acts as a monobasic tridentate ONS donor in all metal complexes, and coordinated through the phenolic OH, azomethine nitrogen and thione sulfur. Electronic spectra with magnetic moments suggested varieties of geometries around the central metal atoms. Thermal gravimetric analysis indicates that the complexes are stable up to 300°C, and release the uncoordinated and/or coordinated H(2)O/solvent molecules, which is accompanied by a color change. The formed complexes after releasing the solvent were investigated and their structures are suggested to have square planar or octahedral arrangement. Pharmacodynamic of cobalt(III) complex on some biochemical parameters and histological studies in serum and heart tissue in rats have been studied. Although the complexes demonstrated a significant effect at low dose than the high dose, the ligand showed significant good effects in both high and low doses on the biochemical analysis in serum and heart tissue. Cobalt complex was screened in order to evaluate its antifungal activity against the filamentous fungi Aspergillus niger, Aspergillus fumigatus, and Aspergillus flavus, and antibacterial activity against the Candida albicans, Escherichia coli, Klebseilla pneumoniae and Pseudomonas aeruginosa.

An HPLC method for the determination of bromadiolone plasma kinetics and its residues in hen eggs.

Cereal-based bromadiolone anticoagulant is often used for rodent control, and because these baits are attractive for poultry they may be accidentally ingested. Thus, the aim of this study was to develop a new high-performance liquid chromatography (HPLC) method for the determination of bromadiolone residues in hens' eggs and its plasma kinetics. Laying hens (n = 48) were divided into four groups of 12 animals each. Groups I and II received orally a single dose of bromadiolone 10 mg/kg, group III received a single dose of bromadiolone 60 mg/kg, and group IV was the control. Eggs were collected from groups I, III, and IV, whereas plasma was collected from groups II and IV. The HPLC method developed was reproducible, sensitive, accurate, and linear within the range 0.1-20 µg/g. The final HPLC conditions were as follows: mobile phase MeOH-ammonium acetate (0.5 M) triethylamine buffer (pH 5, 51:49, v/v); analytical column Luna C18 ODS2; wavelength 260 nm; flow rate of 1.5 mL/min; and warfarin as internal standard (5 µg/mL). Recoveries for bromadiolone were in the range of 72-80% with RSD lower than 10%. Pharmacokinetic behavior of bromadiolone in hens results faster than that reported in other animals and humans. Following 10 and 60 mg/kg treatment bromadiolone was not detected in albumen but was present in yolk from day 4 to 5 and from day 2 to 9. In conclusion, the bromadiolone amount found in eggs was well below the toxic dose of this anticoagulant for humans, and no anticoagulant effect should be observed.