Simultaneous determination of twelve neonicotinoids and six metabolites in human urine with isotope-dilution UPLC-Q-Orbitrap HRMS

The extensive global use of Neonicotinoid insecticides (NNIs) has led to widespread human exposure, necessitating the development of effective methods for large-scale biomonitoring. However, current methods are inadequate in simultaneously and accurately detecting various NNIs or their metabolites (m-NNIs). In this study, we aimed to establish a robust method using solid-phase extraction (SPE)-ultra high performance liquid chromatography tandem Q-Orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) for the simultaneous determination of 12 NNIs and 6 m-NNIs in human urine. Samples were prepared using Oasis HLB 96 well plate with Isopropanol: methanol (7:3, v/v) as the elution solvent. The target compounds were separated using the Accucore RP-MS column and subsequently analyzed under parallel reaction monitoring mode. NTN32692 (m/z=255.06433) was confirmed to be the specific metabolites of cycloxaprid for the further detection. Satisfactory recoveries (81.6-122.4%) of the NNIs and m-NNIs were observed, with intra- (n = 3) and inter-day (n = 9) relative standard deviation (RSD) ranging from 0.9% to 13.7% and from 1.1% to 18.6%, respectively. Good linearity (R2>0.99) was achieved for all analytes. The limits of detection (LODs) for all target analytes ranged from 0.01 ng/mL to 0.65 ng/mL. This method was applied to urine samples collected from 10 children recruited from an agricultural area in China. Our study provides an effective method to identify and assess human exposure to NNIs and their metabolites.