LC-MS/MS法检验土壤中的磺酰脲类和磺酰胺类除草剂
宋丽娟, 许满军
山东省公安厅物证鉴定研究中心,济南 250001

作者简介: 宋丽娟(1983—)女,山东潍坊人,工程师,博士,研究方向为毒物毒品检验鉴定。 E-mail: ljsong@iccas.ac.cn

摘要

目的 磺酰脲类和磺酰胺类除草剂属内吸传导性药剂,此类除草剂具有用量少、高效、广谱、低毒和高选择性等特点,在农业生产中被广泛使用。其中,苯磺隆、苄嘧磺隆、氯磺隆、双氟磺草胺被用于防除阔叶杂草。使用此类制剂时,若剂量不当,容易对当茬及后茬轮种作物造成一定的药害。正是由于某些作物对此类除草剂的敏感性,犯罪分子实施不法行为的相关案件频繁发生。鉴于此,本文以苯磺隆、苄嘧磺隆、氯磺隆和双氟磺草胺为代表,对土壤中此类除草剂的提取、检验方法以及鉴定时效性进行系统研究。方法 分别利用常见溶剂甲醇、乙腈、丙酮和水对土壤中此类除草剂进行提取,计算提取率。并通过对同一溶剂提取物的重复测定,考查目标物在不同提取溶剂中的分解速度。兼顾分析物的提取率和稳定性,确定最佳提取试剂。液相色谱和质谱的基本条件如下:色谱柱:Agilent Eclipse XDB-C18(4.6mm×150mm,5 μm)。色谱条件:进样量10μL;流动相A为甲醇,流动相B为0.1%甲酸(V/V)的水溶液。质谱条件:离子源:ESI;CUR:20 psi;GS1:65 psi; GS2:55 psi,气体均为N2;加热头温度650℃。喷雾电压IS:5500 V(+)/4500(-)。MRM模式选取离子对:苯磺隆:396.2/199.0、396.2/181.2、396.2/154.9、396.2/135.2;苄嘧磺隆:411.2/182.1、411.2/149.0、411.2/139.1、411.2/119.1;氯磺隆:355.9/139.1、355.9/124.3、355.9/107.2、355.9/82.2;双氟磺草胺:358.1/167.2、358.1/152.0、358.1/131.9、358.1/104.2。通过优化液相洗脱模式、流动相组成、不同化合物的去簇电压 (DP)和不同特征碎片离子的碰撞能 (CE),确定目标物的最佳定性、定量分析方法。在此基础上,通过向空白土壤中添加一定浓度的标准物质,以24 h为间隔依次进行提取分析,并通过峰面积定量(定量离子对:苯磺隆396.2/154.9;苄嘧磺隆411.2/149.0;氯磺隆355.9/139.1;双氟磺草胺358.1/167.2),初步考查此类除草剂在土壤中的降解趋势。结果 甲醇、乙腈、丙酮、水四种常见溶剂对土壤中此类除草剂的提取率差异很大。甲醇对所有目标物提取效果均比较好,乙腈的提取效果与之相近,优于丙酮,且显著优于水。不同分析物在提取溶剂中的稳定性明显不同:15 h内,氯磺隆、双氟磺草胺在四种溶剂中均未出现显著分解,苯磺隆、苄嘧磺隆在甲醇、乙腈、丙酮中的分解速率大约为7.0%/h,而在水中高达10%/h。兼顾目标物的提取率及其在不同溶剂中的稳定性,甲醇成为最优提取溶剂,对四种分析物的平均提取率均可达到67%以上。液相色谱洗脱梯度的设置和溶剂强度的选择对分析物的保留时间、分离度及峰形均有较大影响。当使用甲醇和0.1%甲酸水溶液进行等度洗脱(各50%)时,双氟磺草胺峰形较差,且由于溶剂强度较弱,苯磺隆、苄嘧磺隆、氯磺隆均无法被有效洗脱。采用梯度洗脱模式并保持相关参数,用乙腈代替甲醇作为有机相时,目标物的保留和峰形均较好,但分离选择性变差。最终,采用梯度洗脱模式,并利用甲醇和0.1%甲酸水溶液作为流动相。当流动相的流速在400~600 μL/min之间变化时,随流速变大,目标物保留时间缩短,但峰强度显著降低。为保证检测灵敏度,设置流速为400 μL/min。通过优化电离模式、DP、CE等质谱条件,苯磺隆和苄嘧磺隆在正离子模式下或氯磺隆和双氟磺草胺在负离子模式下均可得到良好的检测。通过此类除草剂的稳定性考查实验发现,经过30天,土壤中分析物含量衰减至初始含量的20%~40%。为确保鉴定的有效性,此类案件相关的检材提取、送检以及检验均应及时、迅速。通过系列土样测定,得到苯磺隆、苄嘧磺隆和氯磺隆、双氟磺草胺的工作曲线线性范围分别为0.25~5 μg/g和0.05~5 μg/g,相关系数均大于0.99,方法检出阈为1.0~20 ng/g。结论 利用固液萃取和液相色谱-串联质谱法,建立了土壤中磺酰脲类和磺酰胺类除草剂的提取及鉴定方法。该方法提取过程简单,检测灵敏度高,可用于实际案件中此类除草剂的检验。鉴于此类药剂对防除对象的作用机制及其自身性质,时效性在案件侦办中极为重要,办案及鉴定人员应着重注意。

关键词: 法医毒物学; 除草剂检测; 固液萃取; 液相色谱-串联质谱; 土壤; 磺酰脲类; 磺酰胺类
中图分类号:DF795.1 文献标志码:A 文章编号:1008-3650(2015)05-0382-06 doi: 10.16467/j.1008-3650.2015.05.009
Detection of Sulfonylurea and Sulfamide Herbicide in Soil by LC-MS/MS
SONG Lijuan, XU Manjun
Institute of Forensic Science, Shandong Provincial Public Security Bureau, Jinan 250001, China

Author: Song Lijuan, Ph.D, focusing on the detection of toxic and drugs. E-mail: ljsong@iccas.ac.cn

Abstract

Objective To establish a method for extraction and detection of sulfonylurea and sulfamide herbicides in soil using tribenuron methyl, bensulfuron methyl, chlorsulfuron and florasulam as test samples. Methods Solid-liquid extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for analysis of the target samples. In order to reduce sample loss in the procedure of solid-liquid extraction, the polarity-different solvents of methanol, acetonitrile, acetone and water were tested to determine the recovery of each analyte. The optimized conditions were chosen on consideration of both the extraction ratio and sample stability. Characterized ion pairs (396.2/199.0, 396.2/181.2, 396.2/154.9, 396.2/135.2 for tribenuron methyl, 411.2/182.1, 411.2/149.0, 411.2/139.1, 411.2/119.1 for bensulfuron methyl, 355.9/139.1, 355.9/124.3, 355.9/107.2, 355.9/82.2 for chlorsulfuron, and 358.1/167.2, 358.1/152.0, 358.1/131.9, 358.1/104.2 for florasulam) acquired from tandem mass spectrometry were taken for qualitative and quantitative analysis of the herbicides. On basis of the previous researches, degradation tendency of the analytes spiked into soil was determined by day to day experiment. Results The average recovery, determined by a known amount of herbicide standards spiked into soil samples, was above 67% with limits of detection ranging from 1.0 to 20 ng/g and decomposition velocity lower than 7.3%/h when methanol was taken as the extraction solvent. The working curves were constructed between peak area and concentration with all of the correlation coefficients greater than 0.99. Linear ranges were 0.25~5.0 μg/g for tribenuron methyl and bensulfuron methyl, and 0.05~5.0 μg/g for chlorsulfuron and florasulam. In 30 days, tribenuron methyl, bensulfuron methyl, chlorsulfuron and florasulam standards, spiked into soil samples, degraded to 20%, 20%, 40% and 30% of their original amounts, respectively. Conclusions The proposed method is facilitating, fast, sensitive and applicable to the qualitative and quantitative analysis of sulfonylurea and sulfamide herbicides left in soil. The strategy can be readily implemented in criminal cases relating with crop damage. According to the results of decomposition tendency, a special attention should be paid that such analytes are quite sensitive to environment. For actual application, sample preparation and determination should be performed in short time so as to avoid false negative response caused by degradation.

Keyword: forensic toxicology; herbicide detection; solid-liquid extraction; LC-MS/MS; soil; sulfonylurea; sulfamide
1 Introduction

Sulfonylurea and sulfamide herbicides are widely used in agriculture because of their certain characteristics as low dosage, low mammalian toxicity, high efficiency and high selectivity [1]. Among them, tribenuron methyl, bensulfuron methyl, chlorsulfuron and florasulam are often applied for the suppression and eradication of broad-leaved weeds. However, the residues of such herbicides left in soil are highly toxic to the on-growing and following crops when over-dosage happens in practice [2, 3, 4]. It is for this reason that crimes relating with sulfonylurea and sulfamide herbicides occurred frequently during the past years. In order to provide efficient assistance in the investigation of this kind of cases, such herbicides in soil were systematically studied on their extraction, detection and decomposition tendency through their representatives in this research.

2 Materials and Methods
2.1 Chemicals and Reagents

Tribenuron methyl, bensulfuron methyl, chlorsulfuron and florasulam were obtained from Aladdin (Shanghai, China). Methanol, acetonitrile, dichloromethane and formic acid, all of HPLC grade, were purchased from TEDIA (OH, USA). Acetone, analytical reagent grade, was from Beijing Chemical Work (Beijing, China), and the ultra-pure water was produced in our laboratory by a PureLab Ultra system from ELGA (High Wycombe, UK).

2.2 Sample Preparation

Stock standard solution containing tribenuron methyl, bensulfuron methyl, chlorsulfuron and florasulam was prepared in dichloromethane at concentration of 1.0 mg/mL. For the selection of extraction solvent, standard mixture of tribenuron methyl, bensulfuron methyl, chlorsulfuron and florasulam was firstly added into four blank soil samples (2.0 g, pH≈ 7.0) with a known amount of 0.5 μ g for each herbicide. After intensive mixing, 2.0 mL of methanol, acetonitrile, acetone or water was separately used for the subsequent extraction. For stability test, standard mixture of target compounds was firstly added into thirty blank soil samples (5.0 g, pH≈ 7.0) with a known amount of 5.0 μ g for each herbicide. After intensive mixing, all the samples were kept open to simulate the real circumstances of field. And following extraction was carried out one by one at a 24-hour interval using 5.0 mL methanol. For the test of linearity and limit of detection (LOD), blank soil samples (pH≈ 7.0) in aliquot of 2.0 g were spiked with the standard mixture of target compounds at concentrations of 0.001, 0.005, 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1.0, 2.0 and 5.0 μ g/g, and then extracted individually with 2.0 mL methanol. For case sample preparation, 5.0 g of soil from case was extracted with 5.0 mL methanol. For extraction procedure in all of the experiments, the samples were sonicated for 10 min, centrifugated at 10, 000 r/min for 10 min, and followed with each of the supernatant being subjected to LC-MS/MS analysis after filtration through a membrane of 0.22 μ m pores.

2.3 LC-MS/MS Analysis

Analysis was performed on a Qtrap 3200 triple quadrupole mass spectrometer (AB Sciex, USA) coupled to a 1100 LC Series (Agilent, USA). Chromatographic separation was carried out by an Eclipse XDB-C18 column (150mm × 4.6mm, particle size 5 μ m, Agilent, USA) at room temperature. The injection volume was 10 μ L. The mobile phase was consisted of two eluents: methanol as eluent A and 0.1% formic acid in water as eluent B. A gradient was run at a flow rate of 0.4 mL/min, starting from 20% A to 60% A increased linearly in 8 min, holding for 4 min, then continuing to increase to 90% A in 2 min and keeping for 6 min before returning to initial condition in 0.1 min. An equilibration time of 2.9 min was allowed prior to the next injection.

Ionization was performed using an electrospray ionization source (ESI) operated in positive or negative ionization mode. The following parameters were chosen for all substances: spray voltage 5.5 kV (positive ionization mode) and -4.5 kV (negative ionization mode); curtain gas=20 psi; GS1=65 psi; GS2=55 psi; temperature=650oC; dwell time=200 ms. Other instrument parameters as declustering potential and collision energy were optimized by direct infusion of 1.0 μ g/mL freshly prepared standard solutions in methanol/ultrapure water (50/49.9, v/v) containing 0.1% formic acid at a flow rate of 5 μ L/min. The precursor ion for each analyte was mass-selected by the first quadrupole and fragmented through a combination of the second quadrupole with collision energies to obtain the produced ions. Analysis was achieved by multiple-reaction monitoring (MRM) mode. For target compounds to increase the specificity of the method, four produced ions were used for qualitative analysis and one ion for quantification. The instrument control and data acquisition were carried out by the operating software of Analyst 1.4.2. Each analyte’ s LC-MS/MS parameters including retention time (RT), ionization mode (IM), precursor ion (Q1), product ion (Q3), declustering potential (DP) and collision energy (CE) were summarized in Table 1.

3 Results and Discussion
3.1 Optimization of the LC-MS/MS Conditions

Optimization of the LC separation parameters was conducted with the analyzed standards dispersing in pure solvent. Commonly, such factors as elution gradient, solvent strength and flow rate predominately affect the retention time, peak shape and resolution of analytes [5]. In this research, both eluting modes of isocratic and gradient were evaluated for their separation of target herbicides. In the case of isocratic elution, a typical mobile phase of methanol/water with 0.1% formic acid provided much broader peak for florasulam and too strong retention for tribenuron methyl, bensulfuron methyl and chlorsulfuron. In gradient elution, the use of acetonitrile produced proper retention and good peak shape yet poor selectivity of separation for all of the analytes (the migration order of tribenuron methyl and bensulfuron methyl was reversed in positive ionization mode while co-migration of chlorsulfuron and florasulam appeared in negative ionization mode). In order to compromise among the resolution, running time and peak shape, the gradient elution was adopted with methanol/0.1% formic acid in water as its mobile phase for subsequent experiments.

Further study showed that the sensitivity to detect all the analytes decreased with the flow rate of mobile phase changing from 400 μ L/min to 600 μ L/min despite the obvious improvement of running speed. Under such conditions, the common choice is to set the flow rate at a certain value that can yield running speed as fast as possible. Nevertheless, when sensitivity is the dominant issue in analyses such as the trace one, a lower flow rate should be selected to ensure high sensitivity. Thus, the flow rate of 400 μ L/min was our final selection in this study.

Parameters for MS/MS tuning were optimized for maximum sensitivity in different modes. Tribenuron methyl and bensulfuron methyl presented greater ionization efficiency in positive ionization mode than that in the negative one, while for chlorsulfuron and florasulam only the negative mode provided normal response. The declustering potential for a precursor and the collision energy for effective fragmentations of the selected precursor ions were separately optimized over the range of 0~200 V and 5~130 V according to the analytes (Table 1). Typical LC-MS/MS chromatograms of tribenuron methyl and bensulfuron methyl in positive ionization mode were shown in Fig.1 and those of chlorsulfuron and florasulam in negative ionization mode in Fig.2.

Table 1 LC-MS/MS parameters for herbicides

Fig.1 MRM LC-MS/MS chromatograms of tribenuron methyl and bensulfuron methyl. Typical MRM LC-MS/MS chromatogram of a mixed standard solution containing tribenuron methyl and bensulfuron methyl (a), positive ion MS/MS spectra from MRM LC-MS/MS analysis of tribenuron methyl (b), and bensulfuron methyl (c).

Fig.2 MRM LC-MS/MS chromatograms of florasulam and chlorsulfuron. Typical MRM LC-MS/MS chromatogram of a mixed standard solution containing florasulam and chlorsulfuron (a), negative ion MS/MS spectra from MRM LC-MS/MS analysis of florasulam (b), and chlorsulfuron (c).

3.2 Selection of Extraction Solvent

Proper selection of extraction solvent plays a crucial role in sample preparation. Four commonly used solvents were tested in this study (Table 2). Selection procedures were involved with the usage of soil samples spiked with analytes at the concentration of 0.25 μ g/g and recovery calculation which was performed by comparison of the response of soil extracts with that of standard solutions. The results presented in Table 2 revealed methanol providing higher recovery for most of the herbicides except for florasulam which had similar recoveries against different solvents being used.

Table 2 Comparison of extraction results using different solvents

In the following experiments, it was observed that solvent characteristics have a great influence on stability of the analytes. To ensure efficient extraction and identification of herbicides, decomposition velocity of target compounds in different extracting solvents was carefully investigated (Table 2), thus water was ruled out because of faster decomposition of analytes in it. On consideration of both the extraction efficiency and analyte’ s stability, methanol was chosen in this research.

3.3 Stability of Herbicides in Soil

For verifiable analysis, it is essential that the sample must be a representative of the whole from which it was taken. Sulfonylurea and sulfamide are translocated herbicides, and their inhibitory effect on broad-leaved weeds commonly appears in one week or even longer time after application [4]. Besides, such kinds of herbicide are quite sensitive to temperature, humidity and pH magnitude. Their concentrations in soil can change with time due to sample degradation from chemical interactions, volatility and exposure to air or light. Consequently, research on degradation trend of herbicides is extremely important in terms of effective detection.

A stability test was designed to explore the length of time that analytes can be still detected under simulated agriculture circumstances. These experiments were carried out with blank soil spiked with the herbicide standards as described in Section 1.2. In soil samples, concentrations of all the analytes reduced significantly along with the storage time. It was observed that tribenuron methyl, bensulfuron methyl, chlorsulfuron and florasulam degraded to 20%, 20%, 40% and 30% of their initial content after 30 days, respectively (Fig.3). Therefore, soil samples relating with criminal cases should be collected and analyzed as soon as possible.

Fig.3 Decaying trend of tribenuron methyl and bensulfuron methyl (a)and florasulam and chlorsulfuron in soil (b).

3.4 Validation of Quantification

In order to validate the established method and to further reveal its features, its quantification property was investigated through determination of the standard samples spiked into soil. Working equations of herbicides were constructed between peak area (y) and concentration (x) as listed in Table 3, with linear correlation coefficients all above 0.99. The LOD (S/N =3) obtained from the analysis of soil samples spiked with low concentrations of standard analytes was between 1.0 and 20 ng/g.

Table 3 Working equation and detection limits of the analytes
4 Method Application

The developed method had ever successfully played its role in criminal cases relating to crop damage. For example, on a December 9, the police was told that the leaves of a tomato seedling in a farmer’ s greenhouse became yellow and the whole plant was withering gradually, with addition of the similar phenomena and damage suffering to another six greenhouses belonged to different farmers. Just with this method, tribenuron methyl was finally detected from the soil samples collected from the involved greenhouses (Fig.4).

Fig.4 MRM LC-MS/MS chromatograms of (a) blank soil, (b) case soil and (c) Tribenuron Methyl standard

5 Conclusions

The open environmental usage and complexity of farm chemicals make the cases of crop damage hard to crack. Although such cases had less serious social influence than homicide ones, the economic losses usually lead to huge burden for the relevant farmers. Therefore, we should develop effective analytic method to meet the demand in practice. In this study, analysis methods for sulfonylurea and sulfamide herbicides were established by solid-liquid extraction and LC-MS/MS. The average recoveries were all above 67%, and the limits of detection were in the range of 1.0~20 ng/g. On consideration of the acting mechanism and inherent characteristics of such chemicals, the degradation trend of them in soil was systematically investigated, indicating that efficient collection and detection of the samples are quite important for forensic identification. The conducted experiment hereof and its practical use in real cases demonstrated that our method is facilitating, fast, sensitive and applicable to the qualitative and quantitative analysis of sulfonylurea and sulfamide herbicides.

The authors have declared that no competing interests exist.

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