Key Takeaways & Executive Findings
- •• • 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.
Abstract
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.
1. Introduction
Neonicotinoids are the most widely used class of insecticides globally, yet their pervasive presence in human matrices raises concerns about chronic low-dose exposure and potential health effects. Existing analytical methods often suffer from insufficient sensitivity, complex sample preparation, or limited scope, hindering comprehensive biomonitoring. The challenge is particularly acute for simultaneous quantification of multiple neonicotinoids and their metabolites in both urine and serum, which is essential for accurate exposure assessment and toxicokinetic studies.
This study addresses these bottlenecks by developing a robust liquid-liquid extraction (LLE) protocol coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The method integrates enzymatic deconjugation to release conjugated metabolites, followed by ethyl acetate extraction, achieving high enrichment and cleanup. With detection limits in the sub-ng/L range and excellent recoveries, the method enables reliable quantification of nine analytes in paired urine and serum samples. Its application to a cohort of 123 Guangzhou residents demonstrates its practical utility, revealing significant differences in analyte distribution between matrices and providing a validated tool for future epidemiological investigations.
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JIA Xiangyu, DENG Fenfang, TIAN Hemi, YUAN Jun, PENG Rongfei, PAN Xinhong, LI Juntao, TAN Lei (2026). 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. Environmental Chemistry. https://doi.org/10.7524/j.issn.0254-6108.2025032801
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Frequently Asked Questions
What is the limit of detection (LOD) and limit of quantification (LOQ) for neonicotinoids in serum and urine, and how do these compare to typical environmental exposure levels?
In urine, LODs range from 0.001 to 0.010 μg·L−1 and LOQs from 0.004 to 0.035 μg·L−1. In serum, LODs are 0.0004–0.02 μg·L−1 and LOQs 0.0014–0.07 μg·L−1. These are sufficiently low to detect background levels found in the general population, as evidenced by detection rates of 94.3–100% in urine and 74.8–99.2% for four analytes in serum.
How does the method ensure accurate quantification of conjugated metabolites, and what is the recovery and precision?
Samples undergo enzymatic hydrolysis to release conjugated forms before extraction. Average recoveries are 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, indicating high accuracy and reproducibility.
What is the significance of the correlation between urine and serum concentrations for specific analytes?
Significant positive correlations were found for clothianidin, thiamethoxam, imidacloprid, and N-desmethyl-acetamiprid, with N-desmethyl-acetamiprid showing the strongest correlation. This suggests that urine levels can reliably reflect systemic exposure for these compounds, supporting the use of urine as a non-invasive biomonitoring matrix.
How does the method's performance compare to existing techniques in terms of sensitivity and throughput?
The method achieves LODs in the sub-ng/L range, comparable to or better than many published methods. The use of liquid-liquid extraction is simple and cost-effective, and the UPLC-MS/MS analysis allows simultaneous quantification of nine analytes in a single run, offering high throughput suitable for large-scale biomonitoring studies.
What are the implications of the finding that urinary concentrations are significantly higher than serum concentrations?
This indicates that urine is a more sensitive matrix for detecting recent exposure, as neonicotinoids are rapidly metabolized and excreted. The higher detection rates and concentrations in urine (P<0.05) underscore its utility for exposure assessment, while serum may better reflect steady-state internal dose. The method's ability to measure both matrices provides complementary information for toxicokinetic modeling.
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