色谱 ›› 2022, Vol. 40 ›› Issue (12): 1087-1094.DOI: 10.3724/SP.J.1123.2022.02012

• 研究论文 • 上一篇    下一篇

固相萃取净化-超高效液相色谱-高分辨质谱法测定尿液中百草枯和敌草快残留

潘胜东1,*(), 王立1, 邱巧丽1, 何仟2,*()   

  1. 1.宁波市疾病预防控制中心, 浙江省微量有毒化学物健康风险评估技术研究重点实验室, 浙江 宁波 315010
    2.中国疾病预防控制中心, 职业卫生与中毒控制所, 北京 100050
  • 收稿日期:2022-02-26 出版日期:2022-12-08 发布日期:2022-07-07
  • 通讯作者: 潘胜东,何仟
  • 基金资助:
    浙江省自然科学基金项目(LQ19B050001);宁波市自然科学基金项目(2018A610404);宁波市自然科学基金项目(2016A610178);宁波市医学重点学科(2022-B18);宁波市领军和拔尖人才工程

Determination of paraquat and diquat residues in urine samples based on solid-phase extraction and ultra performance liquid chromatography-high resolution mass spectrometry

PAN Shengdong1,*(), WANG Li1, QIU Qiaoli1, HE Qian2,*()   

  1. 1. Key Laboratory of Health Risk Appraisal for Trace Toxic Chemicals of Zhejiang Province, Ningbo Municipal Center for Disease Control and Prevention, Ningbo 315010, China
    2. National Institute of Occupational Health and Poison Control, Chinese Municipal Center for Disease Control and Prevention, Beijing 100050, China
  • Received:2022-02-26 Online:2022-12-08 Published:2022-07-07
  • Contact: PAN Shengdong, HE Qian
  • Supported by:
    Zhejiang Provincial Natural Science Foundation(LQ19B050001);Ningbo Municipal Natural Science Foundation(2018A610404);Ningbo Municipal Natural Science Foundation(2016A610178);Ningbo Municipal Key Medical Discipline(2022-B18);Ningbo Municipal Program for Leading and Top-Notch Talents

摘要:

尿液样品中百草枯(PQ)和敌草快(DQ)的检测是理化检验工作的难点。PQ和DQ具有分子极性大和水溶性好等特点,常规反相色谱柱难以保留;现有文献方法多采用亲水相互作用色谱法(HILIC)进行保留,但文献方法需采用高浓度缓冲盐作为流动相,增加了质谱仪的污染。基于上述问题,研究建立了弱阳离子交换(WCX)固相萃取净化-超高效液相色谱-高分辨质谱法(UPLC-HRMS)快速准确测定尿液样品中PQ和DQ残留的检测方法。尿液样品经混合磷酸盐缓冲液(pH=6.86)稀释和WCX固相萃取净化后,在Syncronis HILIC色谱柱(100 mm×2.1 mm, 1.7 μm)上进行梯度洗脱分离,采用正离子电喷雾离子化模式(ESI+)和一级全扫描-数据依赖二级质谱扫描模式(Full mass-ddMS2)进行定量分析。研究通过对色谱条件的不断优化,将HILIC模式下流动相中甲酸铵缓冲盐的浓度降低至10 mmol/L,并系统优化了样品前处理过程中影响PQ和DQ准确性的因素。在最优条件下,PQ和DQ线性关系良好(r2>0.998),在4个加标水平下(1.0、20.0、100.0和200.0 μg/L), PQ和DQ的平均加标回收率分别为85.8%~101%和80.3%~86.9%,精密度(RSD)分别为0.8%~5.1%和0.9%~4.2%。方法的检出限(S/N≥3)和定量限(S/N≥10)分别为0.2 μg/L和0.6 μg/L。将建立的方法用于中毒病人临床治疗过程尿液中DQ含量的跟踪监测。该方法具有快速、简便、灵敏和准确等优点,适用于临床中毒病例尿液样品中PQ和DQ的检测。

关键词: 超高效液相色谱-高分辨质谱, 弱阳离子交换固相萃取, 百草枯, 敌草快, 尿液样品, 基质效应

Abstract:

Determining the presence of paraquat (PQ) and diquat (DQ) in urine samples through physical and chemical testing is challenging. As PQ and DQ have characteristics such as high molecular polarity and good water solubility, they are difficult to be retained by conventional reversed-phase columns. Most of the methods in the literature use hydrophilic interaction chromatography (HILIC) for the retention of PQ and DQ, but they often require high concentrations of buffer salts as the mobile phase, which increase the contamination of the mass spectrometer. In view of the above problems, a rapid and accurate analysis method was developed for the determination of PQ and DQ residuals in urine samples based on weak cation exchange (WCX) solid-phase extraction (SPE) and ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) in this study. Urine samples were first diluted with phosphate buffer (pH=6.86) and pretreated using the WCX SPE method. Chromatographic separation was performed on a Syncronis HILIC column (100 mm×2.1 mm, 1.7 μm). An electrospray ion source in the positive (ESI+) mode and full mass-data dependent MS2 (full mass-ddMS2) mode was used for quantification by matrix-matched external standard method. In this study, the concentration of ammonium formate in the mobile phase in the HILIC mode was effectively reduced to 10 mmol/L by the continuous optimization of the chromatographic conditions. MS optimization results indicated that the molecular ion (M) of PQ and DQ had the strongest response. In addition, sample pretreatment conditions were also optimized. The obtained results indicated that the hydrophobic polytetrafluoroethylene (PTFE) filter membrane, acetonitrile-water (1∶1, v/v) as a fixing solution, and polypropylene vials were suitable for PQ and DQ analysis. Under the optimal conditions, the linearity of PQ and DQ was good with correlation coefficients (r2) greater than 0.998. The limits of detection (LODs, S/N≥3) and limits of quantification (LOQs, S/N≥10) were 0.2 μg/L and 0.6 μg/L, respectively. Mean spiked recoveries of PQ and DQ at the four spiked levels (1.0, 20.0, 100.0, and 200.0 μg/L) were in the range of 85.8%-101% and 80.3%-86.9%, with the RSDs of 0.8%-5.1% and 0.9%-4.2%. The established method was employed for the analysis and confirmation of PQ and DQ for clinical poisoning cases. In one case, a 23-year-old male who had taken approximately 20 mL of pesticide orally was confirmed as DQ poisoning by the developed method. DQ concentration monitoring of the urine samples was conducted for this case during the clinical treatment process. The patient was successfully discharged from the hospital after five times of blood perfusion and other treatments until the DQ concentration was low in the urine samples. In conclusion, the method developed in this study based on WCX SPE-UPLC-HRMS can be used for the confirmation of poisoning cases and concentration monitoring during clinical treatment, providing strong technical support for clinical precision treatment. The method is rapid, simple, sensitive, and accurate, and it is suitable for the detection of PQ and DQ in urine samples.

Key words: ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS), weak cation exchange (WCX) solid-phase extraction (SPE), paraquat (PQ), diquat (DQ), urine samples, matrix effect

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