Second generation anticoagulant rodenticides (SGARs), pesticides used worldwide to control rodent populations, exist in two diastereoisomer chemical species because they own two stereogenic centers. A core-shell LC-MS/MS multi-residue method for comprehensive quantitative analysis of the diastereoisomers of five SGARs as well as three first generation anticoagulant rodenticide molecules has been fully validated in liver of rats according to a bioanalytical guideline. A core-shell column (superficially porous particles) has been chosen for its ability to separate the diastereomers of bromadiolone, difenacoum, brodifacoum, flocoumafen and difethialone and for its robustness to rat liver extracts. The highly selective chromatographic separation of the diastereoisomers contributes to good signal to noise ratios and then enhances the sensitivity of the method compared to the ones of fully porous columns. An elution gradient has been optimized with 10mM ammonium acetate and acetonitrile as aqueous/organic mobile phase respectively. Triple quadrupole mass detector has been used to achieve specifity and LLOQ from 0.92 to 2.2ng/g for each diastereoisomer, or first generation anticoagulant rodenticides. Then we evidenced diastereoisomeric ratios in liver of rats issued from not controlled exposure of wild rats (Rattus norvegicus) trapped in a French Parisian park through a campaign of rodent eradication. We compared them to diastereoisomeric ratios in SGARs commercial baits that contain both isomers, and showed that one of the two diastereoiomers had nearly disappeared in liver of rats. The proportions of cis-bromadiolone and trans-difenacoum were really lowered compared to the baits: 5/7 and 9/12 rats had only trans-bromadiolone and cis-difenacoum hepatic residues respectively. Liver persistence of the two diastereoisomers of bromadiolone and difenacoum was different due to differences in their pharmacokinetics in wild rats. The new core-shell LC-MS/MS method is particularly well adapted for further exploration of diastereoisomers ratios in rodent and predatory wildlife biological samples in order to evaluate ecological consequences of actual baits, to explore new formulated baits with a good balance between efficacity (ability to kill rodents) and diastereoisomers persistence, and hopefully to mitigate exposure of non-target species.

Second-generation anticoagulant rodenticides (SGARs) have been used since the 1980s for pest management. They are highly efficient even in warfarin-resistant rodents. Nevertheless, because of their tissue persistence, nontarget poisoning by SGARs is commonly described in wildlife. Due to this major problem, a new generation of anticoagulants must be developed to limit this risk. This study proposes a method of developing a new generation of anticoagulant rodenticides by revisiting the old SGARs based on the concept of stereochemistry. Each current SGAR is a mixture of diastereomers. Diastereomers of each compound were purified, and their biologic properties were compared by determining their ability to inhibit vitamin K epoxide reductase (VKOR) activity involved in the activation of vitamin K-dependent clotting factors and their toxicokinetic properties. Systematically, for each SGAR, both diastereomers are as effective in inhibiting VKOR activity. However, their toxicokinetic properties are very different, with one of the two diastereomers always more rapidly cleared than the other one. For all SGARs except flocoumafen, the less persistent diastereomer is always the less predominant isomer present in the current mixture. Therefore, the development of baits containing only the less persistent diastereomer would avoid the ecotoxicological risk associated with their use without decreasing their efficacy.

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