Establishment and Validation of Anticoagulant Rodenticides in Animal Samples by HPLC-MS/MS, Focusing on Evaluating the Effect of Modified QuEChERS Protocol on Matrix Effect Reduction

[Image: see text] A rapid, accurate, and selective analytical method to simultaneously quantify 13 anticoagulant rodenticides in animal biological samples was developed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) coupled with electrospray ionization (ESI) in negative mode. Samples were extracted and purified based on a modified QuEChERS (quick, easy, cheap, effective, rugged, safe) sample preparation technique. The sample pH and the type of extraction solvent and cleanup sorbent used to estimate the procedure’s effectiveness were optimized. To improve the matrix effects and obtain acceptable recoveries for 13 rodenticides, 0.1 mL/g biological sample and 1 mL acetonitrile (or acetonitrile: EtOAc = 1:1/(v:v)) extraction followed by Florisil/HC-C18/anhydrous Na(2)SO(4) (NaCl) cleanup under alkaline conditions was fully validated and shown to be selective, precise, accurate, and linear in the range from 1 to 100 ng/mL (g). The mean recoveries were between 52.78 and 110.69%, while the limits of detection and quantification ranged from 0.05 to 0.5 and 0.1–1 ng/mL (μg/kg), respectively. Ideal soft matrix effects (≤20%) were observed for the vast majority of rodenticides (>95%) showing either suppression or enhancement. This method meets international criteria and is capable of simultaneously identifying and quantifying anticoagulant rodenticides in animal blood and tissues and can be suitable for the detection of poisoning cases in the field of forensic or public health.