Core-shell LC–MS/MS method for quantification of second generation anticoagulant rodenticides diastereoisomers in rat liver in relationship with exposure of wild rats

Abstract

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.

Cis-bromadiolone diastereoisomer is not involved in bromadiolone Red Kite ( Milvus milvus) poisoning

“…Do the authors mean that LoQs were the same as in the previous study? In which case simply say "as also reported by Fourel et al, (2017)". Ok Table 1 legend.…”

“…Done Line 177. "….and are the same that the ones detailed for each isomer in Fourel et al, 2017. " It is not clear what this means.…”

“…Suggest rephrase to "…. difenacoum in their body (Damin-Pernik et al, 2016Fourel et al, 2017). Proportions of the minor isomers cis-bromadiolone and trans-difenacoum (minor isomers) in the livers liver of rats were very different…."…”

“…I deleted the first sentence but not the second one which was part of the original text. (Cort et al, 2012; DaminPernik et al, 2016 DaminPernik et al, , 2017Fourel et al, 2017). Both forms are present in all commercial baits in proportions that are controlled by the authorities…………."…”

“…In all the cases, the minor diastereoisomer forms (cisbromadiolone and trans-difenacoum) in hepatic residues were either not detected most of the time nil or at least those were present in a lower proportion than found in minor stereoisomers proportions were always substantially reduced compared to the ones of baits . Line 114.-123 ….." it was proposed by Damin et al, (2016Damin et al, ( , 2017Damin et al, ( ) 2016Damin et al, ( , 2017 proposed lowering to lower the proportion of the most persistent isomer (trans-bromadiolone) to produce obtain a new bait with the same ability to kill rodents (because both isomers have identical toxicity) but with decreased ecological risk for predators (because cisbromadiolone is less persistent in tissue). This proposal was hypothesis is based on pharmacological data from of laboratory and wild rats (Damin et al, 2016(Damin et al, , 2017Fourel et al, 2017), although while the pharmacokinetics of SGARs diastereoisomers in for other species (other rodents and non-target-species) is unknown.…”