[Determination of brodifacoum in rat plasma by HPLC].

OBJECTIVE: A determination method of brodifacoum in rat plasma with bromadiolone as an internal standard was developed. METHODS: A volume of 10 microl internal standard (bromadiolone) was added into rat plasma, and then extracted by 0.5 ml of acetonitrile by shaking for 2 min. The residue was dissolved with 200 microl of mobile phase after centrifugation for 10 min, and evaporation to dryness by Nitrogen blowing. A C18 column and PDA detector were used for separating and detecting. The wavelength was 254 nm, the flow rate was 1.0 ml/min, and the injection volume was 20 microl. RESULTS: The liner range was 1.0-20 microg/ml, and the correlation coefficient was 0.9992. The detection limit was 0.3 microg/ml in plasma (S/N=3). The intra-assay and inter-assay coefficients of variation were 1.89%-2.45% and 2.51%-3.61% respectively. The recoveries in plasma at levels of low, middle and high concentrations were (80.8 +/- 3.1)%, (81.8 +/- 2.7)% and (87.9 +/- 3.6)% (n=6), respectively. The accuracies were 84.1%-91.5% and 86.7%-93.2%, respectively. CONCLUSION: This method is simple, fast and accurate for the determination of brodifacoum in rat plasma.

Chiral liquid chromatography-tandem mass spectrometry analysis of superwarfarin rodenticide stereoisomers - Bromadiolone, difenacoum and brodifacoum - In human plasma.

Superwarfarins are second-generation long-acting anticoagulant rodenticides that can cause unintended human and wildlife toxicity due, in part, to their prolonged half-lives. Commercially available superwarfarin rodenticides are synthesized as racemates with two asymmetric carbons, producing four stereoisomers. To support studies of human plasma half-lives of individual superwarfarin stereoisomers, a method was developed based on LC-MS/MS to separate and quantify stereoisomers of the commercially important superwarfarins bromadiolone, difenacoum and brodifacoum. Human plasma samples were prepared using protein precipitation and centrifugation. Chiral-phase HPLC separation was carried out on-line with tandem mass spectrometric quantitative analysis of the eluting stereoisomers using selected-reaction monitoring with positive ion electrospray on a triple quadrupole mass spectrometer. All four stereoisomers of each superwarfarin were resolved within 12.5 min with calibration curves spanning 2-3 orders of magnitude and lower limits of quantitation between 0.87 and 2.55 ng/mL. This method was used to determine the half-lives of superwarfarin stereoisomers in plasma from patients who had inhaled synthetic cannabinoid products contaminated with superwarfarins. These data may be used to guide the development of safer next generation anticoagulant rodenticides stereoisomers.

Determination of bromadiolone residues in fox faeces by LC/ESI-MS in relationship with toxicological data and clinical signs after repeated exposure.

In many countries, the fox (Vulpes vulpes), predator of small mammals, is particularly affected by anticoagulant rodenticides such as bromadiolone due to secondary poisoning. Nevertheless, to date, no method of exposure monitoring is applicable in the field over large areas, and no toxicological data are available concerning sensitivity of foxes to bromadiolone. The aim of this work was to compare excretion kinetics of bromadiolone in fox faeces with clinical and haemostatic effects after repeated exposure to intoxicated voles. A sensitive method for the quantification of bromadiolone excretion in fox faeces and plasma was developed, using liquid chromatography combined with electrospray ionisation mass spectrometry (LC/ESI-MS). The LoD was 0.9microg/kg and 0.15microg/L, and the LoQ was 3.0microg/kg and 0.5microg/L, in faeces and in plasma, respectively. Four captive foxes were fed for 2 or 5 days with water voles (Arvicola terrestris Sherman) spiked with bromadiolone at concentrations close to those measured in the field. Faeces and blood were collected for bromadiolone titration, and blood-clotting tests were performed to monitor fox health daily during 10 days and then every 3-4 days until the end of the experiment (D28). Then, after euthanasia, a complete necropsy was performed, and levels of bromadiolone residues in the liver were determined. Bromadiolone residues were detected in faeces 15h after the first exposure. They increased dramatically during the exposure period and then gradually decreased, but they remained detectable at the end of the experiment, i.e., 26 days after the last exposure. Bromadiolone residues in plasma showed a similar pattern but were no longer detectable 7-24 days after the last exposure. Two foxes presented very severe external haemorrhages, requiring the administration of the antidote vitamin-K1. Bromadiolone residues in faeces and their relationships with exposure and other direct-markers that were measured are discussed. Liver residues and the toxicity data of our study will help to interpret data from fox carcasses collected by wildlife disease surveillance networks. These findings provide a basis for programs aiming to monitor the exposure of wild fox populations to bromadiolone using non-invasive methods based on standard sampling and analysis of residues in faeces.

Sudden nasal bleeding and brodifacoum: A case of accidental exposure or attempted homicide?

A 50-year-old man was admitted to the emergency department with abrupt massive epistaxis. An accurate anamnesis and physical evaluation could not reveal any other anomalies, while coagulation tests showed potentially life threatening prolonged prothrombin time, with activated partial thromboplastin and thrombin time, with fibrinogen and antithrombin III within limits. Despite the prompt pharmacological and compressive local treatment, bleeding continued and the patient was therefore hospitalized. Highly specific coagulation and toxicological testing-among others high-performance liquid chromatography assessment on plasma-were performed, leading to the unexpected identification of brodifacoum. Police and criminal justice authorities revealed the source of exposure to brodifacoum after several months of investigation, residing in his everyday life. Brodifacoum is a long-lasting anticoagulant, acting as a vitamin K antagonist, and belongs to the family of superwarfarins. Brodifacoum use is authorized as rodenticide in many countries worldwide, but has been reported as cause of severe coagulopathies in humans, both intentional or involuntary, even consumed as a contaminant of herbal drugs, such as cannabis. The original contribution of this case to the knowledges of human brodifacoum intoxication resides in the multidisciplinary approach and the collaborative interplay of clinical and toxicology experts as well as judicial authorities.

Enantiomeric fraction evaluation of the four stereoisomers of difethialone in biological matrices of rat by two enantioselective liquid chromatography tandem mass spectrometry methods: Chiral stationary phase or derivatization.

The need for the control of rodent populations with anticoagulant rodenticides remains actual, and enantioselective analytical methods are mandatory to understand ecotoxicity issues of those chiral pesticides. This study presents two enantioselective methods to achieve the residue levels and differentiated persistence of the four stereoisomers of difethialone (called in this work E1-trans, E2-cis, E3-cis and E4-trans), which is one of the most toxic second generation anticoagulant rodenticide. Their enantiomeric fraction evaluation in biological matrices of rats was determined by two LC-MS/MS methods. The first one (chiral-LC-MS/MS) combined a chiral column employed in reversed-phase mode (with acetonitrile-water mobile phase) to be compatible with mass spectrometry detection. The second one was also a LC-MS/MS method but with a reversed phase column after a derivatization step with (1S)-(-)-camphanic chloride. Extraction process combined Solid-Liquid extraction and sorbent cartridges. The methods were fully validated. The chiral column was chosen as a reference method for our laboratory because it was quicker and cheaper, and enantioresolution and sensitivity were better. This chiral-LC-MS/MS method was used to measure the enantiomeric fraction of the four stereoisomers of difethialone in rodent biological matrices (liver, plasma, blood and feces) of female rats treated with 3.5 mg/kg of difethialone. The results showed that metabolism is not the same for all the stereoisomers: cis-E3-difethialone was the most persistent, and E4-trans-difethialone was the most quickly eliminated. This chiral-LC-MS/MS method will be used to study the pharmacokinetics of the four stereoisomers of difethialone, and for ecotoxicological surveillance to evaluate the specific persistence of each stereoisomer of difethialone in case of secondary exposure of wildlife non-target species.

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.

Bromadiolone toxicokinetics: diagnosis and treatment implications.

INTRODUCTION: Ingestion of bromadiolone can lead to prolonged and life-threatening coagulopathy. Traditional treatment of bromadiolone intoxication relies on the coagulation profile. Currently, there is scanty information on bromadiolone elimination kinetics and half-life. CASE REPORT: We report a case of bromadiolone poisoning in a 40-year old female who, by history, ingested four 42.5-gram bags of rat poison (0.005% bromadiolone), equivalent to 8.5 mg bromadiolone (0.17 mg/kg body weight), four days prior to admission. On admission, her prothrombin time was 92.0 seconds, international normalized ratio was 5.7, and activated partial thromboplastin time was 50.2 seconds with no bleeding on clinical examination. The first plasma bromadiolone level (5 days post-ingestion) was 92 ng/mL. Serial measurement of plasma bromadiolone levels confirmed the diagnosis and demonstrated that bromadiolone obeys the elimination kinetic of a two-compartment model with a rapid, fairly steep decline phase (half-life 3.5 days) followed by a slower termination phase (half-life 24 days). Plasma bromadiolone level of less than 10 ng/mL in our patient was associated with a consistently normal coagulation profile without vitamin K1 therapy. CONCLUSIONS: There is a lack of information on the toxicodynamics and toxicokinetics of bromadiolone in humans; further studies are needed before the plasma bromadiolone level can serve as one of the logical and safe therapeutic endpoints for vitamin K1 therapy.

[Guidelines for certification of International Normalized Ratio (INR) for vitamin K antagonists monitoring according to the EN ISO 22870 standards]. / Recommandations pour l'accréditation de l'International normalized ratio (INR) pour la surveillance des traitements par antivitamine K en biologie délocalisée selon la norme EN ISO 22870.

Point of care testing (POCT) must comply with regulatory requirements according to standard EN ISO 22870, which identify biologists as responsible for POCT. INR for vitamin K antagonists (VKAs) monitoring is a test frequently performed in haemostasis laboratories. Bedside INR is useful in emergency room, in particular in case of VKAs overdosage but also for specific populations of patients like paediatrics or geriatrics. INR POCT devices are widely used at home by the patients for self-testing, but their use in the hospital by the clinical staff for bedside measurement is growing, with devices which now comply with standard for POCT accreditation for hospital use. The majority of point of care devices for INR monitoring has shown a good precision and accuracy with results similar to those obtained in laboratory. With the aim to help the multidisciplinary groups for POCT supervision, the medical departments and the biologists to be in accordance with the standard, we present the guidelines of the GFHT (Groupe français d'étude sur l'hémostase et la thrombose, subcommittee "CEC et biologie délocalisée") for the certification of POCT INR. These guidelines are based on the SFBC guidelines for the certification of POCT and on the analysis of the literature to ascertain the justification of clinical need and assess the analytical performance of main analysers used in France, as well as on a survey conducted with biologists.

Brodifacoum poisoning with toxicokinetic data.

The case of a 46-year-old woman who survived after a brodifacoum poisoning is presented. The patient was admitted due to a severe coagulopathy. Initial prothrombin time and activated partial thromboplastin time were both greater than 110 seconds and the patient suffered severe gastric and pulmonary hemorrhage requiring fresh frozen plasma transfusion and parenteral phytonadione administration (up to 100 mg per day). Serum brodifacoum levels were determined by HPLC during seven months. Five days after admission, serum brodifacoum level was 1302 ng/ml. Serum brodifacoum levels decreased till day 209 when became not detectable. Brodifacoum elimination showed a first order kinetic and a 56-day half-life. Investigation of superwarfarin should be considered in any patient with vitamin K dependent coagulation disorder. It would be also useful to obtain periodic brodifacoum levels and build the corresponding elimination curve to help direct phytonadione therapy in poisoned patients.

The rapid determination of bromadiolone in liver and blood plasma by in-injector pyrolysis gas chromatography - Ion trap tandem mass spectrometry.

The unintentional poisoning of off-target animals by bromadiolone, a second generation anticoagulant rodenticide, is an undesirable outcome requiring sensitive analytical methods. In this study, a rapid and sensitive method for the determination of bromadiolone in liver and blood plasma by means of gas chromatography coupled with tandem mass spectrometry without need for derivatization was developed. The method is based on the in-injector pyrolysis of bromadiolone and subsequent gas chromatography coupled with ion trap tandem mass spectrometry with electron ionization. Sample preparation includes extraction with methanol, evaporation under nitrogen stream, and dissolution in toluene. The pyrolysis of bromadiolone was carried out in an injector at 390°C. Chromatographic separation of the pyrolytical fragment of bromadiolone was achieved using a VF-5ms column with helium as the mobile phase. Tandem in-time mass spectrometry of the separated pyrolytical fragment of bromadiolone was carried out using an ion trap mass spectrometer after electron ionization. Recovery ranged from 94 to 98%. The method showed good linearity up to 1000µgkg-1 for liver and 1000µgL-1 for plasma. The limit of detection was 0.38µgkg-1 for liver and 0.26µgL-1 for plasma. The developed method was used successfully in several animal poisoning cases.

A negative association between bromadiolone exposure and nestling body condition in common kestrels: management implications for vole outbreaks.

BACKGROUND: Vole outbreaks have been extensively described, along with their impacts on humans, particularly in agricultural areas. The use of rodenticides is a common legal practice to minimise crop damage induced by high vole density for biocidal use. However, rodenticides can have negative direct and indirect impacts on non-target species that feed on voles. We studied whether the use of a second-generation anticoagulant rodenticide (SGAR), bromadiolone, can be detected in the blood of fledglings of wild common kestrels Falco tinnunculus in two areas of central Spain, exploring its possible indirect effects. RESULTS: We found that 16.9% of fledglings had a detectable concentration of bromadiolone in their blood, with an average concentration of 0.248 ± 0.023 ng mL-1 . Fledglings with bromadiolone in their blood, regardless of the concentration, had 6.7% lower body mass than those without detectable bromadiolone. CONCLUSION: The use of bromadiolone was detectable in the blood of alive non-target species. Detected bromadiolone in blood may reduce the body condition of nestlings, potentially reducing their fitness. The source of bromadiolone found in nestlings needs to be determined in future studies to derive accurate management advice. However, we urge the discontinuation of official SGAR distribution to farmers and their use in agrarian lands to minimise damage of voles on crops, particularly where common kestrels breed, and encourage the use of alternative effective practices. © 2016 Society of Chemical Industry.

Acute bromadiolone intoxication.

A 55-year-old man came to the hospital with a bleeding wound on his tongue. The coating of his tongue was green, and his sputum was red. Because an increased international normalized ratio-value was measured, a blood sample was sent to our laboratory with the suspicion of coumarin intoxication. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis confirmed the poisoning was by bromadiolone, with its maximum serum concentration at 440 microg/L. The analysis of further samples resulted in a calculated elimination half-life of 140 h. The analytical method described was developed for the determination and quantitation of bromadialone using LC-MS. This method is suitable for the simultaneous identification and quantitation of 10 indirect anticoagulants in human serum, which include five superwarfarins (brodifacoum, bromadiolone, difenacoum, difethialone, and flocoumafen) as rodenticides licenced in Germany and five other vitamin K antagonists (acenocoumarol, coumatetralyl, coumachlor, phenprocoumon, and warfarin). The method is based on an acidic (pH 4.2) liquid-liquid extraction followed by LC-ESI-MS analysis. Analytical separation was carried out using an Atlantis C18 column (2.1 x 20 mm, 3 microm). The mobile phase consisted of methanol/0.1% formic acid; the flow rate was 0.6 mL/min, and the time needed for analysis was 5 min. The lower limit of quantitation was 5 microg/L (signal-to-noise > 10).

Assessment of ruminal degradation, oral bioavailability, and toxic effects of anticoagulant rodenticides in sheep.

OBJECTIVE: To assess the rate and extent of ruminal degradation of warfarin, chlorophacinone, and bromadiolone in vitro and determine the oral availability and clinical and hemostatic effects of each anticoagulant rodenticide in adult sheep. ANIMALS: 3 Texel sheep. PROCEDURE: Samples of ruminal fluid were incubated with each of the anticoagulants to assess the kinetics of ruminal degradation over 24 hours. To determine the plasma kinetics of the anticoagulants, each sheep received each of the anticoagulants IV or via a rumenimplanted cannula at 2-month intervals (3 rodenticide exposures/sheep). At intervals during a 240- to 360- hour period after treatment, prothrombin time (PT) was measured, plasma anticoagulant concentration was assessed, and clinical signs of rodenticide poisoning were monitored. In plasma and rumen extracts, anticoagulant concentrations were determined via high-performance liquid chromatography. RESULTS: In the rumen extracts, anticoagulants were slightly degraded (< 15%) over 24 hours. In vivo, oral availability of warfarin, chlorophacinone, and bromadiolone was estimated at 79%, 92%, and 88%, respectively. Although maximum PT was 80 seconds after chlorophacinone and bromadiolone treatments, no clinical signs of toxicosis were detected; PT returned to baseline values within 2 weeks. CONCLUSIONS AND CLINICAL RELEVANCE: In sheep, warfarin, chlorophacinone, and bromadiolone were not degraded in the rumen but their bioavailabilities were high after oral administration; the kinetics of these compounds in sheep and other mammals are quite similar. These data suggest that the lack of susceptibility of ruminants to these anticoagulant rodenticides cannot be explained by either ruminal degradation or the specific toxicokinetics of these anticoagulants.

Plasma metabolic profiling analysis of toxicity induced by brodifacoum using metabonomics coupled with multivariate data analysis.

Brodifacoum is one of the most widely used rodenticides for rodent control and eradication; however, human and animal poisoning due to primary and secondary exposure has been reported since its development. Although numerous studies have described brodifacoum induced toxicity, the precise mechanism still needs to be explored. Gas chromatography mass spectrometry (GC-MS) coupled with an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was applied to characterize the metabolic profile of brodifacoum induced toxicity and discover potential biomarkers in rat plasma. The toxicity of brodifacoum was dose-dependent, and the high-dose group obviously manifested toxicity with subcutaneous hemorrhage. The blood brodifacoum concentration showed a positive relation to the ingestion dose in toxicological analysis. Significant changes of twenty-four metabolites were identified and considered as potential toxicity biomarkers, primarily involving glucose metabolism, lipid metabolism and amino acid metabolism associated with anticoagulant activity, nephrotoxicity and hepatic damage. MS-based metabonomics analysis in plasma samples is helpful to search for potential poisoning biomarkers and to understand the underlying mechanisms of brodifacoum induced toxicity.

Case management and plasma half-life in a case of brodifacoum poisoning.

Brodifacoum is a readily available, second-generation anticoagulant rodenticide (superwarfarin) that causes extended depletion of vitamin K1-dependent clotting factors. Brodifacoum ingestions are being reported with increasing frequency. For the first time, we compare plasma brodifacoum concentration to prothrombin levels over time in a case of brodifacoum poisoning. Brodifacoum was eliminated according to a two-compartment model, with an initial half-life of 0.75 days and a terminal half-life of 24.2 days. On admission, the brodifacoum level was 731 micrograms/L and the patient suffered severe urinary tract hemorrhage, requiring transfusion of blood products. Persistently increased prothrombin times necessitated treatment with phytonadione up to 80 mg/d for 4 months, until the brodifacoum level reached 10 micrograms/L. These data may help project the duration of phytonadione treatment required in future cases of brodifacoum poisoning. Superwarfarin exposure must be suspected in an otherwise unexplained vitamin K1-deficient coagulopathy.

A study of the relationship between the pharmacokinetics and the pharmacodynamics of the 4-hydroxycoumarin anticoagulants warfarin, difenacoum and brodifacoum in the rabbit.

The pharmacokinetics and pharmacodynamics of the 4-hydroxycoumarin anticoagulants, brodifacoum, difenacoum, and warfarin have been studied in the rabbit. Sensitive (50 ng ml-1) and specific high performance liquid chromatography assays have been developed for the determination of plasma concentrations of warfarin, brodifacoum and difenacoum. After administration of a single intravenous dose (20 mumol kg-1), plasma concentrations of warfarin underwent mono-exponential decay, with a terminal half-life of 5.6 +/- 0.7 h (mean +/- s.e. mean), whereas plasma concentrations of brodifacoum and difenacoum underwent bi-exponential decay with terminal half-lives of 60.8 +/- 1.9 h and 83.1 +/- 10.3 h respectively. The plasma half-life of brodifacoum in a single patient poisoned with the compound was 487 h. The pharmacological response to the anticoagulants was measured as changes in prothrombin complex activity, from which the rate of clotting factor synthesis was determined. Clotting factor synthesis recovered in a monophasic fashion after a single intravenous dose of warfarin, compared with a more complex biphasic, pattern of recovery of clotting factor synthesis after administration of either brodifacoum or difenacoum. The slope (m) of the intensity of effect-log (amount of drug in the body) curve was derived for each anticoagulant. There was no significant difference in the value of m after single intravenous doses of racemic, R-, and S-warfarin, difenacoum and brodifacoum, which is consistent with the hypothesis that all the 4-hydroxycoumarin anticoagulants produce their anticoagulant effect by acting at the same receptor site, vitamin K epoxide reductase. Determination of the minimum plasma concentration of each anticoagulant that corresponded with the complete inhibition of clotting factor synthesis indicated that racemic warfarin, R-warfarin and brodifacoum have similar potencies in the rabbit and are less potent than S-warfarin and difenacoum.

A gas chromatographic method for the estimation of phenprocoumon, 3-(1-phenyl-propyl)-4-hydroxycoumarin (Marcoumar, Liquamar), in human serum or plasma.

A rapid and reliable gas-chromatographic method is described for the estimation of phenprocoumon from human serum or plasma. No interference was found from other usual oral anticoagulant drugs. The method can be used for therapeutic and toxic levels. The appearance of two volatile derivatives of phenprocoumon (in constant ratio) is discussed.

Superwarfarin and glass ingestion with prolonged coagulopathy requiring high-dose vitamin K1 therapy.

A 23-year-old man was brought to the emergency department after eating four boxes of brodifacoum-containing rodenticide over a 4-day interval and pieces from approximately two bottles of glass over the previous 2 weeks. He was asymptomatic but his prothrombin time was markedly elevated with an international normalized ratio (INR) of 37.8. A plain abdominal film showed diffuse radiopaque foreign bodies, presumably glass, in the large and distal small intestines. Treatment for ingested glass consisted of stool softeners and bulk-forming laxatives. The patient developed mild gingival bleeding and received fresh frozen plasma (FFP) infusions and vitamin K1 orally. At a vitamin K1 dosage of 300 mg/day, the INR corrected to less than 2.0 and the patient was discharged taking that dosage. He returned 26 days later with hematuria and flank pain, and his INR was 189. He was administered FFP and packed red blood cells, and his vitamin K1 dosage was increased to 800 mg/day; his INR returned to baseline. Compliance with taking the vitamin K1, which required ingestion of 60-160 tablets/day, was a serious problem, requiring numerous follow-up calls and visits to the patient at home and work. At 5-month follow he was doing well. Compliance with large daily doses of vitamin K1 for treatment of "superwarfarin" ingestion may be poor because of the duration of treatment and large number of pills required. A more concentrated formulation may be advantageous for management of patients with brodifacoum poisoning.

The anticoagulant activity of some selected warfarin analogues.

The anticoagulant activities of 6-, 7-, 8-, 4'-hydroxy, 6-chloro- and 6-bromowarfarin were determined in rabbits after intraperitoneal administration of 16.2 mumol kg-1 over 96 h. Substitution on the 4-hydroxycoumarin moiety resulted in reduction of the anticoagulant activity. 6-Chlorowarfarin was more potent than 6-bromowarfarin suggesting that the molecular size of 4-hydroxycoumarin moiety may be crucial for biological activity.