Determination of rodenticides and related metabolites in rabbit liver and biological matrices by liquid chromatography coupled to Orbitrap high resolution mass spectrometry.

An analytical method based on ultra-high performance liquid chromatography (UHPLC) coupled to Orbitrap high resolution mass spectrometry was developed for the determination of rodenticides (bromadiolone, brodifacoum, difenacoum, chlorophacinone, diphacinone, coumachlor and warfarin) in liver matrix. Different extraction conditions were tested, obtaining the best results when the "dilute and shoot" method (acidified acetonitrile as extraction solvent) and a clean-up step with primary secondary amine (PSA) were used. The optimized method was validated, obtaining recoveries ranging from 60 to 120%. Repeatability and reproducibility were evaluated obtaining values lower than 20%, except for brodifacoum at 10µg/kg. Limits of quantification (LOQs) ranged from 0.1 to 0.5µg/kg, except for brodifacoum, which was 100µg/kg. Six liver samples were analyzed and diphacinone and chlorophacinone were detected in three samples at concentrations ranging from 4µg/kg to 13µg/kg. Moreover a retrospective screening of rodenticide metabolites in those samples and in animal forensic samples was developed based on Orbitrap capabilities. Brodifacoum was detected in three samples, and warfarin alcohol, which is a metabolite of warfarin, was also detected in one sample.

[Recurrent pyoderma gangrenosum-like ulcers induced by oral anticoagulants]. / Ulcérations récidivantes à type de pyoderma grangrenosum induites par les antivitamines K.

BACKGROUND: Other than the classic skin necrosis induced by oral anticoagulants (OAC) in patients with protein C and S deficiencies, other types of OAC induced-skin ulcers are little known. Herein, we describe an original case of recurrent pyoderma gangrenosum (PG)-like ulcers induced by OAC. PATIENTS AND METHODS: A 70-year-old female heart-transplant recipient presented deep, hyperalgesic and quickly-spreading necrotic ulceration of the right leg 6 weeks after starting oral anticoagulant therapy with fluindione. Histological analysis revealed dermal infiltrate containing polynuclear neutrophils, which accords with the histopathological diagnosis of leukocytoclastic vasculitis or PG. Infectious, autoimmune and thrombophilic causes were ruled out. Fluindione was withdrawn and the ulcer healed completely within a month. Six months later, right leg ulceration recurred two weeks after the patient resumed fluindione but healed within 1 month of discontinuation of the drug. An OAC from another chemical family (warfarin) was then introduced, with further recurrence of ulceration after 2 weeks of treatment. DISCUSSION: The chronology of events and the negativity of aetiological explorations allowed a diagnosis to be made of OAC-induced skin ulcer, a rare complication of which the pathophysiology is unclear. This is the first case of PG-like ulcers induced by OAC.

Characterization and stability of ternary solid dispersions with PVP and PHPMA.

The effect of adding a third polymer to immiscible binary solid dispersions was investigated. The model actives griseofulvin (GF), progesterone (PG) and phenindione (PD) were selected because they exemplify a key property of many poorly soluble molecules of having at least one hydrogen bonding acceptor moiety while not having any hydrogen bond donating moieties. Ternary solid dispersions of the drug, PVP (polyvinylpyrrolidone) (proton acceptor) and PHPMA (poly[2-hydroxypropyl methacrylate]) (proton acceptor and donor) were prepared by spray drying. Stability results showed that binary solid dispersions (API and PVP) of GF and PVP crystallized quickly while the amorphous form was not possible to prepare for PG and PD. The amorphous form was prolonged upon the incorporation of PHPMA in the solid dispersion (API, PHPMA and PVP). Based on measuring the melting points, the energy of mixing the drug with the polymer was calculated using the Flory-Huggins theory. The results showed that GF had the lowest free energy followed by PG and finally PD which agreed well with the stability results. These results suggest that the addition of a third polymer to immiscible binary solid dispersions can significantly improve the stability of the amorphous form.

An enhancement of europium-/gadolinium- diphacinone -DL-histidine- cetylpyridine bromide system for the determination of diphacinone in serum.

As one of the first generation anticoagulant rodenticide, diphacinone (DPN) was extensively used in China, and often occurred accidental and intentional intoxications. To meet the needs of clinical emergency rescue, a simple, rapid and sensitive method was necessary for the determination of DPN in serum. In this paper, a fluorimetric method using a ternary europium-gadolinium-DPN-DL-histidine-cetylpyridine bromide system was developed. The fluorescence intensity was measured with a 1 cm quartz cell at 335 nm excitation wavelength and 612 nm emission wavelength. The fluorescence intensity of the europium-DPN- DL- histidine complex system was further enhanced by using gadolinium ion (Gd(3+)) in a cetylpyridine bromide (CPB) colloid-type suspension. By adding Gd(3+) at pH 8.5 to 9.5 in the presence of an ammonia-ammonium chloride buffer solution, about a 100-fold enhancement of the fluorescence intensity was achieved. The enhanced fluorescence intensity showed a good linear relationship with the DPN concentration from 0.01 mg/L to 5.0 mg/L. The limit of detection was 0.001 mg/L in serum. The established method was used to determine DPN in real human serum in clinical specimens. The luminescence mechanism was discussed in detail. In the fluorescence system of the europium-gadolinium-DPN-DL-histidine-CPB, the CPB not only acted as the surfactant but also acted as the energy donor.