关键词: 尿样, 拟除虫菊酯, 代谢产物, 超高效液相色谱-串联质谱, 液液萃取

Abstract:

Pyrethroids are extensively employed in agricultural pest management and household sanitation practices. However， their widespread use has raised concerns as they pose a multitude of health risks to humans. Consequently， the development of precise， highly sensitive， and efficient biomonitoring techniques for evaluating internal exposure levels of pyrethroids across different populations has emerged as a paramount goal in the field of environmental exposure and health effect research. This study developed a method using ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） for the simultaneous determination of five common pyrethroid metabolites in urine， with sample pretreatment involving hydrochloric acid hydrolysis and liquid-liquid extraction. The method was optimized for mass spectrometric acquisition parameters and liquid chromatography separation conditions. Chromatographic separation was successfully accomplished utilizing a BEH C₁₈ column （100 mm×2.1 mm，1.7 μm）. Mass spectrometric data were acquired in negative ion mode under multiple reaction monitoring （MRM） conditions. Among the analytes， trans-3-（2，2-dichlorovinyl）-2，2-dimethylcyclopropanecarboxylic acid （trans-DCCA） and cis-3-（2，2-dichlorovinyl）-2，2-dimethylcyclopropanecarboxylic acid （cis-DCCA） are isomers， as are 2-phenoxybenzoic acid （2-PBA） and 3-phenoxybenzoic acid （3-PBA）， and they share identical MS acquisition parameters. Identification was based on reference standards and retention times. The mobile phase consisted of 0.1% acetic acid in water （A） and acetonitrile （B）. The gradient elution program was as follows： 0-0.5 min， 10%B； 0.5-4.5 min， 10%B-70%B； 4.5-5 min， 70%B-100%B； 5-7 min， 100%B； 7-8 min， 100%B-10%B； 8-10 min， 10%B. The optimization of urine sample pretreatment conditions was divided into two parts： hydrolysis and extraction. Using the recoveries of target analytes as the evaluation metric， parameters including the dosage of hydrolysis reagent， hydrolysis temperature and duration， as well as the type， dosage， and extraction time of the extraction solvent were systematically optimized. The optimized pretreatment protocol is delineated as follows： Initially， 40 µL of the 2-PBA internal standard working solution was precisely added to 1 mL of urine sample， followed by thorough mixing to ensure homogeneity. Subsequently， 150 µL of hydrochloric acid （2 mol/L） was introduced to facilitate hydrolysis， which was allowed to proceed at ambient room temperature for a duration of 30 min. Finally， extraction was carried out using 2 mL of ethyl acetate， accompanied by vigorous shaking for 30 min to maximize extraction efficiency. Following centrifugation， the organic phase was separated， evaporated to near dryness， and reconstituted with 1.0 mL of acetonitrile prior to instrumental analysis. The matrix effects were evaluated using pure solvent and matrix-matched standards. Among the five analytes， three exhibited a moderate matrix effect， while two showed a weak matrix effect. The recovery performance using 2-PBA as an internal standard was also assessed. Consequently， a combination of the working curve and the internal standard method was selected for the quantification of the target analytes. Based on this， the methodological parameters of the method were validated. The results indicate that the five pyrethroid metabolites exhibited good linearity， with correlation coefficients of the calibration curves all exceeding 0.995. The limits of detection （LODs） ranged from 0.13 ng/mL to 1.32 ng/mL， and the limits of quantification （LOQs） ranged from 0.44 ng/mL to 4.39 ng/mL. The average recoveries of the samples at three spiked levels of 20， 50， and 80 ng/mL ranged from 91.0% to 102.0%. The intra-batch precision was between 1.1% and 8.1%， while the inter-batch precision was between 1.1% and 4.6%. Sample stability was demonstrated for at least one week when stored at 4 ℃. The established method was applied to analyze 18 urine samples from the general population. Neither cis-DCCA nor 4-fluoro-3-phenoxybenzoic acid （4F-3PBA） was detected in any sample. The mass concentration of 3-PBA ranged from 0.69 ng/mL to 1.59 ng/mL， with a detection rate as high as 88.9%. These results are largely consistent with screening studies on human pyrethroid metabolite levels reported in domestic and international literature in terms of both the detection rate and mass concentration range of 3-PBA. The presence of 3-PBA may originate from household insecticide exposure or dietary sources. The method demonstrates simple and efficient sample pretreatment， strong cost-effectiveness， low LODs， and high accuracy and precision. It can therefore serve as a reliable technical reference for monitoring and exposure assessment in various populations， particularly sensitive groups such as the general population， pregnant women， and children.

Key words: urine, pyrethroids （PYRs）, metabolites, ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）, liquid-liquid extraction （LLE）