A rapid and sensitive LC-MS/MS method for the simultaneous determination of chlorfenapyr and its major metabolite...

Chlorfenapyr (CFN), widely used as an insecticide, is a pro-insecticide that acts after being metabolized to the active metabolite tralopyril (TLP). In clinical practice, CFN poisoning is characterized by atypical symptoms, delayed toxicity and high mortality. The therapeutic importance of assessing TLP levels in the management of CFN poisoning has been demonstrated in recent clinical and toxicokinetic studies. Unfortunately, the simultaneous quantification of CFN and TLP poses technical challenges and is still lacking in clinical practice. In this study, a novel LC-MS/MS method was developed for rapid and accurate simultaneous analysis of CFN and TLP in human plasma within 3 min. Samples were extracted by acetonitrile protein precipitation. Stable CFN anionic adducts [M + HCOO]^- were selected to provide satisfactory MS signals. To improve the signal and peak shape, the mobile phase was preferred to consisting of 2 mM ammonium formate containing 0.01 % ammonia and acetonitrile. The optimized multiple reaction monitoring transitions of m/z 451.0 → 346.9, 349.9 → 78.9 and 307.1 → 161.1 were selected in the negative ionization mode to detect CFN, TLP and IS (warfarin), respectively. The method demonstrated satisfactory linearity with low LLOQ (5 ng/mL for CFN and 1 ng/mL for TLP, respectively), and the precision and accuracy were acceptable. Both compounds showed good stability under relevant conditions. Furthermore, the method was successfully applied to 72 clinical plasma samples from 20 patients with CFN poisoning. The results showed that the concentration of TLP in plasma was significantly higher than that of CFN (P < 0.0001), and all five deaths demonstrated high TLP concentrations (>1000 ng/mL). These data demonstrate the potential relationship between toxins concentration and toxicity in CFN poisoning patients.