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
A highly sensitive and accurate method using liquid-liquid extraction (LLE) coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for simultaneous quantification of nine neonicotinoid insecticides and their metabolites in human urine and serum. Samples underwent enzymatic hydrolysis followed by ethyl acetate extraction, effectively enriching target analytes. Gradient elution and optimized mass spectrometry conditions enabled simultaneous determination. The method exhibited excellent linearity with correlation coefficients >0.999. In urine, limits of detection (LOD) ranged from 0.001 to 0.010 μg·L−1 and limits of quantification (LOQ) from 0.004 to 0.035 μg·L−1. In serum, LODs were 0.0004–0.02 μg·L−1 and LOQs 0.0014–0.07 μg·L−1. Average spiked recoveries were 89.3%–115.0% in urine and 83.0%–115.0% in serum, with relative standard deviations (RSD) of 0.5%–8.0% and 2.5%–9.5%, respectively. Analysis of paired urine and serum samples from 123 Guangzhou residents revealed detection rates of 94.3%–100% for the nine analytes in urine, with clothianidin showing the highest median concentration (1.89 μg·L−1). In serum, detection rates for clothianidin, thiacloprid, acetamiprid, imidacloprid-olefin, and 5-hydroxy-imidacloprid were below 60%, while the remaining four analytes ranged from 74.8% to 99.2%. Urinary concentrations of all nine analytes were significantly higher than serum concentrations (P<0.05). Significant positive correlations between urine and serum concentrations were observed for clothianidin, thiamethoxam, imidacloprid, and N-desmethyl-acetamiprid, with N-desmethyl-acetamiprid showing the strongest correlation. The LLE-UPLC-MS/MS method efficiently and accurately detects neonicotinoids and metabolites in urine and serum, providing a reliable tool for human exposure assessment.