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Anti-coagulant rodenticides (ARs) are commonly utilized for controlling rodent populations; however, non-target companion and wildlife animals are also exposed. A method was developed for quantitation of seven ARs (chlorophacinone, coumachlor, bromadiolone, brodifacoum, difethialone, diphacinone and warfarin) and dicoumarol (a naturally occurring anti-coagulant) in animal serum. Analytes were extracted with 10% (v/v) acetone in methanol and analyzed by reverse phase high-performance liquid chromatography-tandem mass spectrometry using electrospray ionization (negative mode) combined with multiple reaction monitoring. In-house method validation in the originating laboratory using non-blinded samples revealed method limits of quantitation at 2.5 ng/mL for all analytes. The inter-assay accuracy ranged from 99% to 104%, and the relative standard deviation ranged from 3.5% to 20.5%. Method performance was then verified in the originating laboratory during an exercise organized by an independent party using blinded samples. The method was successfully transferred to two naïve laboratories and further evaluated for reproducibility among three laboratories by means of Horwitz ratio (HorRat(R)) values. Such extensive validation provides a high degree of confidence that the method is rugged, robust, and will perform as expected if used by others in the future., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In this work, a method for the extraction of dicoumarol from plant sample utilizing selective adsorbent based on molecularly imprinted polymer was developed. Molecularly imprinted polymer (MIP) was synthesized by bulk polymerization, and on the surface of magnetic particles using dicoumarol as a template, methacrylic acid as functional monomer, chloroform as a porogen, and ethyleneglycol dimethacrylate as a cross-linker. Prepared polymeric materials were evaluated on the base of capacity, selectivity, and morphology. The maximal specific adsoption capacities of the sorbents were 45 μg (for bulk MIP) and 36 μg (for MIP on the magnetic particles) of dicoumarol per 1 g of polymer. An efficiencies of sorption processes of dicoumarol on prepared sorbents were evaluated under various conditions (type of sample solvent, pH, types of washing and elution solvents). The MIP based sorbents were used for solid phase extraction of dicoumarol. Applicability of MIP-SPE coupled to HPLC-DAD was tested for the selective extraction of dicoumarol from sample of sweet clover. The method was linear over concentration range from 1 to 100 μg mL -1 (the correlation coefficient 0.9984) with limit of detection 0.2 μg mL -1 . Accuracy of the method was assessed for spiked sample at three concentration levels and recovery values were higher than 84% with relative standard deviation lower than 3.2%., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Eight of 600 Holstein heifers and cows died after ingestion of sweet clover silage (Melilotus sp) that contained excessive concentrations of dicumarol caused by mold infestation. The cattle developed subcutaneous hemorrhages and bled from the vagina, became weak, were unable to move, and died. To the best of our knowledge, this is the first report of sweet clover poisoning in cattle from California and is discussed in light of previous findings in the Midwest and Canada. Sweet clover poisoning is caused by dicumarol, a fungal metabolite produced from substrates in sweet clover, and is a common livestock problem in the Northern Plains and Canada. Sweet clover poisoning should be considered in livestock animals with clinical evidence of hemostatic dysfunction, prolonged coagulation times, subcutaneous hemorrhages, and hemorrhagic abortions. Definite diagnosis of moldy sweet clover poisoning can be accomplished by analysis of serum and feed samples for dicumarol concentrations.

Nine medicinal plants known to be the ingredients of the traditional herbal medicinal elixir, and seven popular commercial alcoholic herb elixirs were investigated for the content of dicumarol by using high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC) methods. Umbelliferae (Conioselinum Univittatum) were the only medicinal plants found to contain dicumarol 0.04 mg/dl. Dicumarol content was also found in three out of seven brands of commercial alcoholic herb elixirs with the concentration of 0.58, 1.86 and 6.00 mg/dl. These findings indicated that the traditional herbal medicinal elixirs containing dicumarol in varying amount may play a role in inducing bleeding diathesis in breast-fed infants of mothers known to consume the elixir.

The dicumarol concentration in 272 cured sweet clover samples from 4 counties in North Dakota was determined. Dicumarol concentrations ranged from 0 to 164.7 mg/kg of sweet clover, with 64.6% of the 272 samples containing less than 10 mg/kg. Round bales were significantly (P less than 0.05) higher in dicumarol concentration (mean of 22.9 +/- 3.10 mg/kg) than were stacks or silage (means of 1.8 +/- -6.3 mg/kg and 0.6 +/- 2.1 mg/kg, respectively). The outer section of core samples taken from round bales contained significantly (P less than 0.0021) higher concentrations of dicumarol than did the inner section of the core, with means of 30.8 mg/kg and 16.6 mg/kg, respectively. A significant (P less than 0.0001) correlation existed between crude protein and dicumarol values (r = 0.44).

This collaborative study was undertaken to determine if the anticoagulants acenocoumarol, phenprocoumon, and potassium warfarin could be analyzed by the automated analysis system described in the collaborative study for the analysis of sodium warfarin and dicumarol. Collaborators were supplied with a composited tablet sample of each anticoagulant. Results agreed well with the National Formulary methods for phenprocoumon and potassium warfarin, and an unpublished method for acenocoumarol. For acenocoumarol, coefficients of variation on individual sets of data ranged from 0.30 to 1.94% For phenprocoumon, coefficients of variation ranged from 0.52 to 1.20%. For potassium warfarin, coefficients of variation ranged from 0.54 to 1.79%. The results of this study show that acenocoumarol, phenprocoumon, and potassium warfarin can be analyzed by the official AOAC method for the analysis of sodium warfarin and dicumarol tablets.

A liquid chromatographic method for the determination of coumarin anticoagulants in tablets was collaboratively studied by 7 laboratories. The method uses an octadecylsilane-bonded microparticulate column, tetrahydrofuran-methanol-water-acetic acid mobile phase, and photometric detection at 311 nm. Each collaborator received samples of warfarin sodium, phenprocoumon, and dicumarol as a synthetic composite and as commercial individual and composited tablets. Pooled average assay values for synthetic and commercial tablet samples of warfarin sodium were 101.6 and 99.5%, respectively, with a combined reproducibility SD of 2.38% (CV = 2.37%) and combined repeatability SD of 1.49% (CV = 1.49%). Pooled average (SD) assay values for dicumarol and phenprocoumon commercial samples were 98.0 (2.27) and 101.3% (4.00), respectively. The content uniformity determinations of 2 mg warfarin sodium and 25 mg dicumarol tablets indicated average tablet contents (range) of 99.5% (91.0-116.0) and 98.0% (89.8-108.8), respectively. The method has been approved interim official first action.

An outbreak of a haemorrhagic diathesis in cattle fed home produced hay is described. A similar syndrome was reproduced experimentally in calves by feeding them the hay. The experimental disease was characterised by increased prothrombin and partial thromboplastin times while the leucocyte and erythrocyte counts remained normal until the terminal haemorrhage. The calves ate well and grew well until the rapid onset of progressive weakness, stiff gait, mucosal pallor, tachycardia, tachypnoea and haematomata ending in sudden death. The absence of blood coagulation was seen at necropsy while petechial, ecchymotic and free haemorrhages were found in most organs. Particularly striking were massive ecchymotic haemorrhages on the peritoneal surface of the rumen, a bloody, gelatinous mass enveloping each kidney and extensive bruising, haemorrhage and haematomata in the subcutis of the limbs. In a second feeding trial the effects of various preparations of vitamin K1 and vitamin K3 were investigated. Oral administration of large quantities of vitamin K1 reduced the elevated prothrombin time; vitamin K3 acted less consistently. Analysis of the hay for trichothecene mycotoxins was negative but floral analysis revealed that sweet vernal grass (Anthoxanthum odoratum) comprised about 80 per cent of the hay. Dicoumarol was detected in the hay and in the serum and ruminal contents of the experimental calves. The diagnosis, treatment, control and importance of this syndrome in the United Kingdom are discussed.

After intravenous administration of dicumarol-14C to rats, the bile excreted over the next 24 hr contained from 32 to 46% of the administered radioactivity. At least three primary metabolites and a small amount of unchanged dicumarol were present in the bile. Over 91% of the primary metabolites was converted to dicumarol and 7-hydroxydicumarol by hydrolysis with beta-glucuronidase. Some primary metabolites were hydrolyzed simply by acidification to pH 3 or by treatment under the acidic conditions utilized in the enzymatic hydrolysis. The three primary metabolites contain carboxylic acid groups, as indicated by their electrophoretic mobility-pH profiles, and some are simple glucuronides of dicumarol and 7-hydroxydicumarol. The possibility that others are derivatives of these compounds in which a coumarin lactone ring is opened cannot be ruled out. When the metabolites released by either acidification or enzymatic hydrolysis were chromatographed in n-butanol-3 M ammonia, artifacts were produced, presumably as a result of decomposition of 7-hydroxydicumarol. The question is raised whether a previously reported metabolite (B055) is an artifact.