Determination of amitraz and its degradation products and monitoring degradation process in quince and cucumber

Amitraz is a non-systemic acaracide and insecticide. Current maximum residue limits for amitraz are stated as ‘Amitraz including the metabolites containing the 2,4-dimethylaniline moiety’. Therefore, determination of amitraz and its all degradation products are important. In this study, we develop a gas chromatography/mass spectrometry (GC/MS) method for determination of amitraz and its degradation products 2,4 dimethylaniline (DMA), 2,4 dimethylformamidine (DMF) and N-(2,4-dimethyl phenyl)-N’-methylformamidine (DMPF) in cucumber and quince. The mechanism of the degradation process was monitored at different temperatures. Amitraz and its degradation products were extracted using the QuEChERS method. To determine amitraz and its degradation products, we used GC/MS. Quantification was carried out by using selected ion monitoring, and total ion chromatogram was used to monitor additional degradation products. The method was validated by studying linearity, limit of detection (LOD) and limit of quantification (LOQ), recovery and precision. The mechanism of the degradation process was monitored at different temperatures. Degradation of amitraz mainly to three degradation products, namely DMA, DMF and DMPF, increased with temperature. Besides these three main degradation products, two other new degradation products were detected.     

Determination of amitraz and its transformation products in pears by ethyl acetate extraction and liquid chromatography–tandem mass spectrometry

A method has been developed for identification and quantification of the acaricide amitraz and its transformation products, 2,4-dimethylaniline (DMA), 2,4-dimethylformamidine (DMF) and N-2,4-dimethylphenyl-N-methylformamidine (DMPF) in pears. The analytes were extracted using ethyl acetate and anhydrous sodium sulphate. Analysis was performed by liquid chromatography–electrospray tandem mass spectrometry (LC–ESI-MS/MS) in the positive ion mode using a triple quadrupole (QqQ) instrument. Two precursor-product ion transitions were monitored for each compound in the selected reaction monitoring (SRM) mode. The method was validated with pears taken from the orchard before the amitraz treatment and spiked at the limit of quantification (LOQ), 10 times the LOQ and the maximum residue limit (MRL). Recoveries were between 70 and 106% and relative standard deviations were below 19% (n = 5 at each spiked level). Excellent sensitivity resulted in limits of detection (LODs) for all the compounds below 10 μg kg⁻¹. Quantification was carried out using matrix-matched standards calibration, response was a linear function of the concentration from the LOQs to, at least, three orders of magnitude. Recoveries and standard deviations were comparable to those obtained after hydrolysis of amitraz and its metabolites to DMA. Occurrence of amitraz and its metabolites in field-treated pears showed that, seven days after the treatment, DMPF and DMF are the main degradation products. This work reports for the first time the use of a conventional pesticide multiresidue method and LC–ESI-MS/MS for determining amitraz and its metabolites in pears.     

高效液相色谱-串联质谱法测定蔬菜水果中双甲脒及其代谢产物

建立了高效液相色谱-串联质谱（HPLC-MS/MS）检测蔬菜水果中双甲脒（AMZ）及其降解或代谢产物单甲脒（DMPF）、2，4-二甲基苯基甲酰胺（DMF）和2，4-二甲基苯胺（DMA）的分析方法。样品经0.1 mol/L NaOH溶液稀释、正己烷-异丙醇（2：1，v/v）混合提取液提取，然后采用HPLC-MS/MS对目标物进行定性确证和定量分析。采用Phenomenex Kinetex C18色谱柱（100 mm×4.6 mm，2.6 μm）分离，以0.1%（v/v）甲酸水溶液和甲醇为流动相进行梯度洗脱，采用电喷雾电离（ESI）源、正离子监测模式进行检测。在1.0~200.0 μg/L范围内，4种目标化合物呈良好的线性关系，相关系数（r）大于0.99；方法的定量限为0.01~0.4 μg/kg。4种目标化合物在0.5、5.0和20.0 μg/kg 3个水平下的加标回收率为62.5%~105.0%，RSD为7.5%~17.6%。该法简便快捷，准确可靠，灵敏度高，定量限满足目前国内外残留限量要求。     

基于增强型脂质去除固相小柱净化结合液相色谱-串联质谱法测定猪肉和猪肝中的双甲脒农药及其代谢物

建立了采用增强型脂质去除(EMR-Lipid)固相小柱净化结合液相色谱-串联质谱(LC-MS/MS)测定猪肉和猪肝中双甲脒 (AMZ) 及其3种代谢物2,4-二甲基苯胺(DMA)、单甲脒(DMPF)和2,4-二甲基苯基甲酰胺(DMF)残留量的方法。 样品经乙腈蛋白沉淀及盐析提取,通过Captiva EMR-Lipid 过滤小柱净化,滤膜过滤后,采用Agilent ZORBAX Eclipse Plus C₁₈柱(50 mm×2.1 mm,1.8 μm)分离,以0.1%甲酸水溶液与0.1%甲酸乙腈为流动相进行梯度洗脱,采用电喷雾离子源正离子方式扫描,多反应监测模式进行检测。 结果表明,在猪肝与猪肉中AMZ、DMA线性范围为1～200 μg/kg,DMPF和DMF的线性范围为0.1～200 μg/kg,相关系数(R²)均大于0.991;AMZ、DMA、DMPF和DMF的方法定量限(S/N=10)分别为0.6、0.6、0.05、0.05 μg/kg;对空白猪肉和猪肝进行0.1、1、5、50 μg/kg 4个浓度水平的加标实验,回收率在60.2%～127.4%之间,相对标准偏差均低于12%。 该方法简便、快捷,适用于猪肉和猪肝中双甲脒及其代谢物残留量的同时测定。     

固相支撑液液萃取/气相色谱-串联质谱法同时测定血液中双甲脒、杀虫脒及其代谢产物

建立了气相色谱-串联质谱法同时测定血液中双甲脒、杀虫脒及其代谢产物的分析方法。优化了样品前处理方法及气相色谱-串联质谱的分析条件,样品经固相支撑液液萃取柱(SLE)净化,乙腈-二氯甲烷(体积比1∶1)洗脱后,在多反应监测模式(MRM)下检测。结果表明,2,4-二甲基苯胺、4-氯邻甲苯胺和双甲脒在1.0~1 000 ng/m L范围内,其余目标物在2.0~1 000 ng/m L范围内线性关系良好,相关系数均大于0.99;定量下限为1.0~2.0 ng/m L,加标回收率为88.6%~113%。该方法简便、快捷、样品用量小,结果准确可靠,灵敏度高,适用于血液中6种目标物的同时检测。     

液相色谱-串联质谱法测定蔬菜和水果中硝苯菌酯的残留量

建立了一种通过检测硝苯菌酯(2,4-dinitro-6-(1-methylheptyl) phenyl crotonate, 2,4-DNOPC)相应的水解产物2,4-二硝基-6-(1-甲基庚基)苯酚(2,4-dinitro-6-(1-methylheptyl) phenol, 2,4-DNOP)来测定蔬菜和水果中2,4-DNOPC残留量的液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)检测方法。蔬菜和水果样品用丙酮-甲醇-盐酸的混合液进行液-液分配提取,在碱性条件下超声水解后再进行液-液分配提取,然后进行LC-MS/MS测定。2,4-DNOPC在6种蔬菜和水果中的回收率试验结果表明,2,4-DNOPC在甘蓝中的添加回收率为89.7%～93.3%,相对标准偏差(RSD)为6.3%～8.5%;在黄瓜中的添加回收率为87.7%～95.1%, RSD为5.8%～10.4%;在番茄中的添加回收率为89.3%～96.0%, RSD为6.8%～9.2%;在苹果中的添加回收率为92.0%～98.3%, RSD为5.1%～10.3%;在梨中的添加回收率为89.0%～95.0%, RSD为5.3%～10.2%;在葡萄中的添加回收率为81.2%～95.8%, RSD为5.8%～10.4%。2,4-DNOPC在6种蔬菜和水果的最低检测浓度均为0.01 mg/kg。该检测方法样品前处理简单、快速,分析时间短,灵敏度、准确度和精密度均符合农药残留检测要求,适用于蔬菜和水果中硝苯菌酯残留量的检测。     

Characterization and monitoring of amitraz degradation products in honey

Capillary gas chromatography in combination with atomic emission and electron impact-chemical ionizaion mass spectrometry detectors have been used to detect amitraz degradation products in honey storage, characterize their structure, and evaluate their occurrence over a 100 day peroid. To this end, honey samples were extracted with an 8:2 v/v n-hexane/acetone mixture. Amitraz was found to be rapidly decomposed into five related compounds, of which N-(2,4-dimethylphenyl)formamide was the most abundant and persistent.     

特丁二甲硅烷基衍生化气相色谱-质谱法检测尿中4种苯氧羧酸类除草剂

建立了尿中2,4-滴(2,4-D)、2,4-滴丙酸(2,4-DP)2、甲4氯(MCPA)和2甲4氯丙酸(MCPP)4种苯氧羧酸类除草剂的气相色谱-质谱(GC-MS)分析方法。尿样加氯化钠饱和,酸化后用乙醚萃取,萃取物进行特丁二甲硅烷基(TBDMS)衍生化后分析。尿中4种除草剂的浓度在3~3 000 ng/mL范围内工作曲线的线性关系良好,检出限在1 ng/mL以下,3、100和1 000 ng/mL水平加标回收率在97.0%~102.2%之间,精密度在6.2%~14.2%之间。该法灵敏,可用于中毒者和职业接触者尿中苯氧羧酸类除草剂的分析。     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) method for the measurement of 2,4-dichlorophenoxyacetic acid in human urine

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoxyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline containing 0.05% Tween and 0.02% sodium azide, with analysis by a 96-microwell plate immunoassay format. No clean up was required as dilution step minimized sample interferences. Fifty urine samples were received without identifiers from a subset of pesticide applicators and their spouses in an EPA pesticide exposure study (PES) and analyzed by the ELISA method and a conventional gas chromatography/mass spectrometry (GC/MS) procedure. For the GC/MS analysis, urine samples were extracted with acidic dichloromethane (DCM); methylated by diazomethane and fractionated by a Florisil solid phase extraction (SPE) column prior to GC/MS detection. The percent relative standard deviation (%R.S.D.) of the 96-microwell plate triplicate assays ranged from 1.2 to 22% for the urine samples. Day-to-day variation of the assay results was within ±20%. Quantitative recoveries (>70%) of 2,4-D were obtained for the spiked urine samples by the ELISA method. Quantitative recoveries (>80%) of 2,4-D were also obtained for these samples by the GC/MS procedure. The overall method precision of these samples was within ±20% for both the ELISA and GC/MS methods. The estimated quantification limit for 2,4-D in urine was 30 ng/mL by ELISA and 0.2 ng/mL by GC/MS. A higher quantification limit for the ELISA method is partly due to the requirement of a 1:5 dilution to remove the urine sample matrix effect. The GC/MS method can accommodate a 10:1 concentration factor (10 mL of urine converted into 1 mL organic solvent for analysis) but requires extraction, methylation and clean up on a solid phase column. The immunoassay and GC/MS data were highly correlated, with a correlation coefficient of 0.94 and a slope of 1.00. Favorable results between the two methods were achieved despite the vast differences in sample preparation. Results indicated that the ELISA method could be used as a high throughput, quantitative monitoring tool for human urine samples to identify individuals with exposure to 2,4-D above the typical background levels.     

Comparative evaluation of liquid chromatography-mass spectrometry versus gas chromatography-mass spectrometry for the determination of hexabromocyclododecanes and their degradation products in indoor dust

Domestic and office dust samples (n=37) were analyzed for hexabromocyclododecanes (HBCDs) using gas chromatography-electron-capture negative ionization-mass spectrometry (GC-ECNI/MS) and liquid chromatography-electrospray tandem mass spectrometry (LC-ESI/MS/MS). To determine the best method to quantify HBCDs using GC-ECNI/MS, BDE 128 was used as internal standard (I.S.) in all samples, while 13C-labeled alpha-HBCD was used as I.S. in some samples. Total HBCD concentrations (sum of alpha-, beta-, and gamma-HBCD diastereomers) were calculated using response factors (RFs) for alpha- and gamma-HBCD as individual diastereomers and using an average RF for both diastereomers. Statistical comparison showed that concentrations obtained via GC-ECNI/MS were statistically indistinguishable (p>0.05) from those obtained using LC-ESI/MS/MS. The closest match between the two techniques was obtained using [13C]alpha-HBCD as I.S. and the average RF for alpha- and gamma-HBCDs. Excellent linear correlations (Pearson coefficient values r>0.9) were obtained between the GC-ECNI/MS and LC-ESI/MS/MS results, with slopes ranging from 0.76 to 1.36. Pentabromocyclododecenes (four isomers) and tetrabromocyclododecadienes (two isomers) were detected in the studied samples and were identified as degradation products of HBCDs after separation from the parent compound on the basis of both retention time and mass spectrum. This finding suggests that the elimination of HBr is the major degradation pathway for HBCDs in dust.     

固液萃取-高效液相色谱-串联质谱法同时测定土壤中阿特拉津及其降解产物

建立了高效液相色谱-串联质谱法(HPLC-MS/MS)同时检测土壤中阿特拉津及其降解产物残留的分析方法。样品以甲醇-水(4∶1,V/V)作为提取溶剂,使用涡旋振荡提取,采用HPLC-MS/MS法进行测定,外标法定量。在0.01、0.2和5.0mg/kg三个添加浓度水平下,阿特拉津及其降解产物的平均回收率在73.7%~104.7%之间,相对标准偏差为0.4%~5.1%;阿特拉津,羟基阿特拉津在土壤样品中的方法检出限均0.045μg/kg,而脱乙基阿特拉津、脱乙基脱异丙基阿特拉津及脱异丙基阿特拉津在土壤样品中的方法检出限则分别为0.090、0.45和0.90μg/kg。本方法的灵敏度较高,且简便、快速,能较好的解决目标物极性差别大及样品基质对检测结果的干扰等问题,可以满足土壤中阿特拉津及其降解产物残留检测的需要。     

Voltammetric analysis of the acaricide amitraz and its degradant, 2,4-dimethylaniline

Amitraz is a formamide acaracide used in the control of ticks and mites in livestock. An electrochemical method for the determination of total amitraz residues and its final breakdown product, 2,4-dimethylaniline, is presented. Cyclic voltammetry at a glassy carbon electrode showed the irreversible oxidation of amitraz and of 2,4-dimethylaniline. A linear current response was obtained with an extrapolated limit of detection of 2 × 10⁻⁸ M for amitraz and 1 × 10⁻⁸ M for 2,4-dimethylaniline. The biological degradation of amitraz and subsequent formation of 2,4-dimethylaniline was readily monitored in spent cattle dip. Amitraz and 2,4-dimethylaniline was also monitored in milk and honey samples.     

Kinetics and mechanism of amitraz hydrolysis in aqueous media by HPLC and GC-MS

Degradation processes of amitraz in aqueous media have been studied by spectrophotometry, HPLC and GC-MS. Amitraz undergoes hydrolysis reactions at any pH, but towards the acidic pH range hydrolysis proceeds at a faster rate. Depending on the pH value, different products of the hydrolysis have been identified. The main degradation products are 2,4-dimethylaniline at very acidic pH values (pH<3), N-(2,4-dimethylphenyl)-N'-methylformamidine and 2,4-dimethylphenylformamide at less acidic media (pH 3-6) and 2,4-dimethylphenylformamide at basic pH. The mechanisms of the different hydrolysis processes have been elucidated.     

气相色谱-串联质谱法快速筛查测定中药材中144种农药残留

利用气相色谱-串联质谱(GC-MS/MS)检测技术,采用QuEChERS法作为样品前处理方法,建立了能应用于11种中药材中144种农药残留的检测方法。探究了样品前处理过程中提取溶剂、缓冲盐体系、净化剂组成和用量对样品提取、净化等方面的影响,最终确定了用乙腈提取,甲苯复溶,以混合净化剂净化,过有机膜后经GC-MS/MS测定,外标法定量。144种农药在10~2000 μg/kg之间线性关系良好,相关系数(r²)>0.983;除乙酰甲胺磷、灭虫威、西玛津、克菌丹、异狄氏剂、异菌脲外,其余农药的定量限(LOQ)均低于20 μg/kg;在20、50、200 μg/kg的添加水平下,除乙酰甲胺磷、艾氏剂和双甲脒回收率偏低外,其余141种农药的平均回收率在74.3%~111.8%之间,相对标准偏差(RSD)为0.5%~14.6%。与已有的标准方法对比,此方法不仅检测结果一致,而且高效、快速,准确性好,灵敏度高,适用于中药材中144种农药残留的快速筛查与定量分析。     

Gas chromatography/ion trap mass spectrometry applied for the analysis of triazine herbicides in environmental waters by an isotope dilution technique

A gas chromatography/ion trap mass spectrometry method was developed for the analysis of simazine, atrazine, cyanazine, as well as the degradation products of atrazine, such as deethylatrazine and deisopropylatrazine in environmental water samples. Isotope dilution technique was applied for the quantitative analysis of atrazine in water at low ng/l levels. One liter of water sample spiked with stable isotope internal standard atrazine-d₅ was extracted with a C₁₈ solid-phase extraction cartridge. The analysis was performed on an ion trap mass spectrometer operated in MS/MS method. The extraction recoveries were in the range of 83-94% for the triazine herbicides in water at the concentrations of 24, 200, and 1000 ng/l, while poor recoveries were obtained for the degradation products of atrazine. The relative standard deviation (R.S.D.) were within the range of 3.2-16.1%. The detection limits of the method were between 0.75 and 12 ng/l when 1 l of water was analyzed. The method was successfully applied to analyze environmental water samples collected from a reservoir and a river in Hong Kong for atrazine detected at concentrations between 3.4 and 26 ng/l.     

Potential of short-column liquid chromatography with tandem mass spectrometric detection for the rapid study of pesticide degradation

Summary The applicability of solid-phase extraction-LC using two short columns (SPE-LC) and/or single-short-column liquid chromatography (SSC) combined on-line with tandem mass spectrometry (MS) was demonstrated for the rapid study of pesticide degradation. A fast analytical procedure was developed to provide preliminary information concerning experimental conditions, approximate rates of degradation and identity of the degradation products. Surface water samples were spiked at relevant concentration levels with well-known microcontaminants and photolysis was used to transform parent compounds into their degradation products. In general, the strategy was as follows: at 30-min intervals 10-mL samples were on-line enriched, separated by short-column LC and recorded in full-scan MS to obtain information on the disappearance of the parent compound and the appearance of breakdown products. To obtain structural information, product-ion spectra of selected compounds appearing in the full-scan MS chromatogram were recorded; this enabled the identification of several degradation products. Total analysis time of enrichment/separation and detection was about 10–15 min.     

气相色谱-串联质谱法测定皮革和纺织品中的富马酸二甲酯

建立了气相色谱-串联质谱(GC-MS/MS)准确、快速测定皮革和纺织品中富马酸二甲酯(DMF)的分析方法。样品经乙酸乙酯提取、浓缩及固相萃取柱净化处理,通过VF-5ms色谱柱分离、串联质谱对DMF进行定性和定量分析。实验结果表明: 优化样品预处理条件、固相萃取条件、质谱分析条件后,该方法可进一步消除杂质干扰,结果准确可靠,灵敏度、精密度、线性关系良好,回收率较高。3种浓度水平的加标回收率(n=6)保持在84%～93%之间,相对标准偏差小于7.2%;DMF在8种皮革和纺织品中的检出限 (S/N=3)为0.012～0.039 mg/kg, 0.05～100 mg/L质量浓度范围内线性相关系数为0.9990,适用于皮革和纺织品中DMF的日常检测。     

Dispersive liquid-liquid microextraction based on solidification of floating organic droplet followed by high-performance liquid chromatography with ultraviolet detection and liquid chromatography-tandem mass spectrometry for the determination of triclosan and 2,4-dichlorophenol in water samples

A novel, simple and efficient dispersive liquid-liquid microextraction based on solidification of floating organic droplet (DLLME-SFO) technique coupled with high-performance liquid chromatography with ultraviolet detection (HPLC-UV) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of triclosan and its degradation product 2,4-dichlorophenol in real water samples. The extraction solvent used in this work is of low density, low volatility, low toxicity and proper melting point around room temperature. The extractant droplets can be collected easily by solidifying it at a lower temperature. Parameters that affect the extraction efficiency, including type and volume of extraction solvent and dispersive solvent, salt effect, pH and extraction time, were investigated and optimized in a 5 mL sample system by HPLC-UV. Under the optimum conditions (extraction solvent: 12 μL of 1-dodecanol; dispersive solvent: 300 of μL acetonitrile; sample pH: 6.0; extraction time: 1 min), the limits of detection (LODs) of the pretreatment method combined with LC-MS/MS were in the range of 0.002-0.02 μg L(-1) which are lower than or comparable with other reported approaches applied to the determination of the same compounds. Wide linearities, good precisions and satisfactory relative recoveries were also obtained. The proposed technique was successfully applied to determine triclosan and 2,4-dichlorophenol in real water samples.     

Identification and characterization of stressed degradation products of metoprolol using LC/Q-TOF-ESI-MS/MS and MS(n) experiments

A rapid, specific and reliable isocratic high-performance liquid chromatography combined with quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed and validated for the identification and characterization of stressed degradation products of metoprolol. Metoprolol, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified conditions. The drug showed extensive degradation under oxidative and hydrolysis (acid and base) stress conditions. However, it was stable to thermal, neutral and photolysis stress conditions. A total of 14 degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on a C(18) column (4.6 × 250 mm, 5 μm). To characterize degradation products, initially the mass spectral fragmentation pathway of the drug was established with the help of MS/MS, MS(n) and accurate mass measurements. Similarly, fragmentation pattern and accurate masses of the degradation products were established by subjecting them to LC-MS/QTOF analysis. Structure elucidation of degradation products was achieved by comparing their fragmentation pattern with that of the drug. The degradation products DP(2) (m/z 153) and DP(14) (m/z 236) were matched with impurity B, listed in European Pharmacopoeia and British Pharmacopoeia, and impurity I, respectively. The LC-MS method was validated with respect to specificity, linearity, accuracy and precision.     

Rapid screening of amitraz and its metabolite residues in honey using a quick,easy, cheap,effective, rugged,and safe extraction method coupled with UHPLC and Q Exactive

A method for determining amitraz and 2,4‐dimethylaniline in honey was established by using ultra‐high‐performance liquid chromatoghaphy and Q Exactive after applying quick, easy, cheap, effective, rugged, and safe extracting process. A suitable extraction method was designed to extract the amitraz and 2,4‐dimethylaniline after a suitable amount of honey samples was dissolved. A Thermo Syncronis C₁₈ column (100 × 2.1 mm, 1.7 μm) was used for chromatographic separation of the samples. Then the two compounds were quantitatively analyzed via a program of Q Exactive. The linearity of amitraz and 2,4‐dimethylaniline was good in the concentration range of 0.5–100 μg/L, and the correlation coefficient R² was >0.99. The average recovery and relative standard deviation of each component were 81.3–90.0% and 5.1–7.2%. The 24‐ and 48‐h test results showed that the sample needed to be tested within 24 h. The limit of detection was 0.1 μg/kg for amitraz and 2,4‐dimethylaniline, whereas for both the limit of quantitation was 0.3 μg/kg.