Determination Of Bromadiolone In Biomaterials By Ionic Liquid-dispersive Liquid-liquid Micro-extraction And High-performance Liquid Chromatography

Background and Objective Bromadiolone, belongs to the second generationanticoagulant rodenticides, is widely used in China for its remarkable efficacy on micekilling, low toxicity to human and animals. Because of the improper use anddeficiency of management, the bromadiolone poisoning accidents increasedcontinuously. Therefore, it is imperative to establish a fast and efficient method for thedetermination of bromadiolone in biological samples.Pretreatment of samples in forensic toxicological analysis is very important; itdirectly affects the detection efficiency of toxicants. The traditional sample preparationmethods, such as solid-phase extraction and liquid-liquid extraction, are cumbersome,time-costing, solvent-consuming and lower extraction efficiency. Dispersive liquid-liquid micro-extraction （DLLME） is a novel method of sample preparation, reportedby Rezaee and his coworkers in2006. This method integrates sampling, extracting andconcentrating into one step. It became very popular because of its simplicity, celerity,low cost, high enrichment factor, low organic solvent consumption and environmentalfriendliness.Methods This study introduced the ionic liquid–DLLME technology into thepretreatment of bromadiolone in urine or plasma etc. biological samples. Throughinvestigating the volume of extraction and disperser solvent, sample pH, saltconcentration, extraction time and centrifuging time, the pretreatment conditions havebeen optimized and the performance was satisfactory. And, the treatment-sample wasdetected by HPLC.Results The research showed,1-Hexyl-3-methy limidazoliumhexafluorophosphate（[C₆MIM][PF₆]） and methanol were used respectively as extraction solvent anddispersant to extract bromadiolone in biological samples. The optimized extractionconditions were as follows: extraction solvent,[C₆MIM][PF₆],50μL; dispersant, methanol,100μL; sample pH,5.0; extraction time,5min; centrifuging time,8min.This study established a method for detecting the bromadiolone by highperformance liquid chromatography （HPLC）. The separation was performed on aSymmetry C18column at30℃using methanol（A） and25mmol/L phosphatebuffer（pH=3）（B） as mobile phase by isocratic elution, A+B=85+15（v/v）, and the flowrate was1.0mL/min. The detection was performed at λ=260nm. Bromadiolone hadgood linearity in the range of1⁵00μg/mL, the relative standard deviations（RSDs）were less than5%. This developed method is of high sensitivity and strongreproducibility.Under the optimized experimental conditions, the detection limit of bromadiolonein urine was1.1ng/mL（S/N>3）. Linearity was observed in the range of0.01⁵μg/mL,r=0.9997. Recoveries at three different spiked levels were82.4%,93.0%,95.6%,RSDs（n=6） were6.63%,3.89%,3.86%, respectively. The detection limit ofbromadiolone in plasma was1.1ng/mL（S/N>3）. Linearity was in the range of0.01⁵μg/mL, r=0.9988. Recoveries at three different spiked levels were76.4%,82.6%and92.1%, RSDs（n=6） were4.17%,2.99%and1.67%, respectively.Conclusion The results show, this proposed method has been successfully appliedfor the determination of bromadiolone in biological samples.