Determination of Neonicotinoid Insecticides and Their Metabolites in Serum and Urine by Liquid-Liquid Extraction Coupled with Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry and Application to Human Biomonitoring

• • The method achieves LODs as low as 0.0004 μg·L−1 in serum and 0.001 μg·L−1 in urine, enabling trace-level detection of neonicotinoids critical for biomonitoring at environmentally relevant concentrations. • • Average recoveries of 83.0%–115.0% in serum and 89.3%–115.0% in urine with RSDs ≤9.5% demonstrate high accuracy and precision, essential for regulatory and clinical decision-making. • • In 123 paired samples, urinary concentrations of all nine analytes were significantly higher than serum (P<0.05), with clothianidin median at 1.89 μg·L−1, indicating urine as a superior matrix for exposure assessment. • • Significant positive correlations between urine and serum for CLO, THM, IMI, and N-dm-ACE (strongest for N-dm-ACE) support the use of urine as a non-invasive proxy for systemic exposure, facilitating large-scale epidemiological studies.