芹菜、草莓基质对甲胺磷等4种有机磷农药测定的影响

研究芹菜、草莓基质对甲胺磷、乙酰甲胺磷、氧化乐果、毒死蜱4种有机磷农药检测的影响。利用样品基质溶液配制标准样品,对比草莓、芹菜样品、丙酮溶液中4种农药的保留时间、峰形、峰面积。结果发现DB–5MS色谱柱上,丙酮溶液、芹菜基质、草莓基质中甲胺磷、乙酰甲胺磷、氧化乐果色谱峰出峰时间不完全重合,保留时间最大漂移0.1 min。丙酮溶剂、芹菜基质较草莓基质中的甲胺磷、乙酰甲胺磷、氧化乐果更容易出现峰拖尾现象,且芹菜基质溶液中相同浓度的甲胺磷、乙酰甲胺磷峰面积分别约是草莓基质溶液中的75%,90%。毒死蜱的保留时间、峰形、响应面积在草莓基质、芹菜基质、丙酮溶液中基本一样。     

Influence of plant growth regulating substances on transport and degradation of acephate and its metabolite methamidophos in tomato

The occurrence of pesticides in vegetables such as tomato poses an increasing attention to their potential risk to human health. In this study, acephate and its metabolite methamidophos were investigated for their transport and degradation in tomato cultivated in spiking nutrient solutions containing acephate at 1 mg/L. Acephate was taken up and transported via xylems with the concentrations of 41–145 μg/L in xylem sap at 2 and 8 cm above the roots in control treatment. The residue levels of methamidophos derived from acephate were in a range of 0–4.21 μg/L, being 0.00–3.73% of the parent acephate. The influence of three plant growth regulating substances, 1-naphthylacetic acid (NAA), glucose and phlorizin, on the transport and degradation of acephate and methamidophos in tomato was evaluated. All of these substances had positive effects on the transport of acephate and methamidophos in tomato xylem system. The NAA or glucose treatment promoted the degradation of acephate to methamidophos; however, with the addition of phlorizin, the concentrations of acephate were reduced in comparison to that under glucose treatment, suggesting that phlorizin would cause an inhibitory effect on the transport of acephate in tomato plant. These results may indicate an effective approach to reduce acephate and methamidophos residues in tomato.     

Multi-Residue Analysis of Some Polar Pesticides in Water Samples with SPE and LC–MS–MS

In the present study a multi-residue analytical method was developed for monitoring some polar pesticides such as acephate, methamidophos, carbofuran, isoproturon, dimethoate in water with SPE (solid-phase extraction) and LC–MS–MS. Acetochlor was taken as surrogate, and alachlor as internal standard. SPE with different types of columns was compared with LLE (liquid-liquid extraction). Further, the breakthrough volume for different pesticides was determined. The results showed that the selected pesticides can be determined very sensitively with LC–MS–MS. The minimum detectable quantity (MDQ) for each pesticide was about 1.0 ng. To date, SPE cartridge studies showed that the Oasis HLB cartridges were suitable for further studies. However, for Oasis HLB cartridge, different pesticide showed different breakthrough volume. The results showed that for acephate and methamidophos, the breakthrough volume was about 30 mL of water sample, much less than the breakthrough volume of other pesticides studied. Because of the higher vapor pressure and higher Henry's constant of methamidophos, dimethoate and carbofuran, much attention should be paid on their losses in the evaporation step of the experiment. This analytical method can be applied to determine pesticide contamination in environmental water samples. Revised: 12 September 2005 and 21 October 2005     

Enantioseparation and dissipation of acephate and its highly toxic metabolite methamidophos in pakchoi by supercritical fluid chromatography tandem mass spectrometry

Acephate is widely used in crops as racemate. However, the enantioselective dissipation of acephate enantiomers has not been investigated in pakchoi. A sensitive and effective approach was established for determining residues of acephate and its highly toxic metabolite methamidophos enantiomers by supercritical fluid chromatography tandem mass spectrometry. Baseline separations for their enantiomers were achieved by using a Chiralcel OD-H column. The optimal chromatographic conditions were obtained as follows: CO₂/ethanol (95/5) as mobile phase; flow rate, 3.0 mL/min; column temperature, 40°C. The mean recoveries (RSDs) of analytes were in the range of 77–83.1% (6.1–9.9%), 75.4–87.5% (9.3–13.2%), and 81.5–84.2% (7.1–13.4%) at three fortification levels (0.005, 0.05, and 0.5 mg/kg for each enantiomer) for interday assay (n = 18). The method was used to evaluate the enantioselective dissipation of acephate and methamidophos in pakchoi. S-acephate dissipated faster than R-acephate, while the concentration of R-methamidophos was higher than that of S-methamidophos during the entire study period. The results indicated that the R-enantiomer of acephate and methamidophos was preferentially enriched in pakchoi. The established analysis approach and the study data provided useful information for the rational use of acephate in agriculture.     

A method for routine quantitation of urinary 8-hydroxy-2'-deoxyguanosine based on solid-phase extraction and micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

8-Hydroxy-2'-deoxyguanosine (8OHdG), one of the major oxidative DNA lesions induced by radical agents, is commonly used as a biomarker for oxidative stress, nowadays preferably in urine. In the absence of a commercially available internal standard a micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (micro-HPLC/ESI-MS/MS) method, suitable for routine analysis of 8OHdG in human urine using external calibration, was developed. Evaluation of the matrix effect showed that the method allows highly sensitive and accurate quantitation despite the absence of an internal standard. HPLC analysis was performed using gradient elution at a flow rate of 10 microL min(-1) using a capillary reversed-phase column and an injection volume of 0.5 microL, with detection of 8OHdG in positive multiple reaction monitoring (MRM) mode. The absolute limit of detection was 0.35 fmol using m/z 168 as a quantifier (fragment) ion. A linear (R2> 0.999) calibration curve in urine was obtained over a range 0.2-10 ng mL(-1). This method is about 20 times more sensitive than previously described procedures, and is characterized by high accuracy (mean 90%) and good reproducibility (RSD <10%). The optimized method was applied to determination of 8OHdG in 18 urinary samples derived from three healthy volunteers. 8OHdG urinary excretion ranged from 3.0-7.9 microg/day, and a large intra-individual variation was found. This method, which effectively circumvents the need for isotopically labeled 8OHdG (internal standard), is suitable for routine monitoring of exposure to DNA-damaging factors in a large number of subjects.     

Development of a method for the determination of 9 currently used cotton pesticides by gas chromatography with electron capture detection

A reliable, sensitive, and reproducible method was developed for quantitative determination of nine new generation pesticides currently used in cotton agriculture. Injector temperature significantly affected analyte response as indicated by electron capture detector (ECD) chromatograms. A majority of the analytes had an enhanced response at injector temperatures between 240 and 260 degrees C, especially analytes such as acephate that overall had a poor response on the ECD. The method detection limits (MDLs) were 0.13, 0.05, 0.29, 0.35, 0.08, 0.10, 0.32, 0.05, and 0.59 ng/mL for acephate, trifuralin, malathion, thiamethozam, pendimethalin, DEF6, acetamiprid, brifenthrin, and lambda-cyhalothrin. This study provides a precision (0.17-13.1%), accuracy (recoveries=88-107%) and good reproducible method for the analytes of interest. At relatively high concentrations, only lambda-cyhalothrin was unstable at room temperature (20-25 degrees C) and 4 degrees C over 10 days. At relatively low concentrations, acephate and acetamiprid were also unstable regardless of temperature. After 10 days storage at room temperature, 30-40% degradation of lambda-cyhalothrin was observed. It is recommended that acephate, acetamiprid, and lambda-cyhalothrin be stored at -20 degrees C or analyzed immediately after extraction.     

A novel derivatization approach for simultaneous determination of glyoxal, methylglyoxal, and 3-deoxyglucosone in plasma by gas chromatography-mass spectrometry

A two-step derivatization approach has been developed to enable the simultaneous analysis of glyoxal, methylglyoxal, and 3-deoxyglucosone by the most efficient and widely applied GC-MS methodology. These three analytes are reactive carbonyl compounds associated with the formation of advanced glycation and lipoxidation end products, a process thought to contribute to uremic toxicity and referred to as "carbonyl stress". Effective analysis of these compounds would facilitate understanding these compounds' role in diabetes-related complications. Plasma samples were deproteinized by acetonitrile, followed by a two-step derivatization approach. Pooled plasma samples from healthy individuals were used as the "blank" for preparing calibration standards. The concentrations of the analytes in the "blank" were first determined by standard addition method. Calibration parameters were accordingly established and used to analyze these compounds in plasma samples collected from healthy individuals and diabetic patients. Analytical findings are comparable with those reported in the literature. Quantitation data can be further improved by making available and using isotopically labeled analogs of these analytes as the internal standards.     

气相色谱法测定茶叶中的噻嗪酮、甲胺磷、乙酰甲胺磷及三唑磷残留

建立了同时测定茶叶中噻嗪酮、甲胺磷、乙酰甲胺磷和三唑磷4种农药残留量的测定方法。在45 ℃加温条件下,用乙酸乙酯-正己烷混合溶剂提取及活性炭色谱柱净化,用不同配比的乙酸乙酯-正己烷混合液梯度洗脱待测组分,以DB-210毛细管色谱柱分离、氮磷检测器测定。结果表明,上述4种农药在10 min内能很好地分离;样品加标回收率(n=3)为73.4%~96.9%。方法的变异系数为2.49%~3.35%,茶叶（干重）中4种农药的定量检测下限为7.0~12.0 μg/kg。     

Quantitative LC-ESI-MS Analysis for Pesticides in a Complex Environmental Matrix Using External and Internal Standards

Abstract

Quantitative liquid chromatography electrospray mass spectrometry (LC-MS) analysis for pesticides in a complex environmental matrix using external and internal standard calibration was investigated. Various approaches to introducing different internal standard compounds to address quantitative errors associated with signal suppression were also examined. The study involved the analysis of pesticides in wheat hay matrix samples using three kinds of internal standard compounds: deuterium labeled (D₃), carbon-13 (singly labeled), and structural analogs (derivatives) of the target analytes. Introduction of the internal standard by volumetric addition and direct post-column infusion were also studied and compared.

Isotopically labeled internal standards (i.e. D₃- ¹³C-) were found to be effective in correcting quantitative errors associated with signal suppression. The application of singly labeled ¹³C compounds may result in nonlinear calibration due to mass interference with the target analyte species. The interference may be compensated by using quadratic curve-fitting or subtraction of the interfering component. Although ineffective as volumetric internal standards, structural analogs can be effective in compensating for signal suppression when introduced into the LC effluent by continuous post-column infusion. Furthermore, the post-column introduction method allows the application of a single internal standard compound for the quantification of each analyte in a multi-component mixture.

The use of internal standards can be effectively incorporated into residue analysis development methods for pesticides in environmental matrices. High accuracy and reproducibility can be achieved while improving method efficiency by reducing the need for comprehensive sample clean-up.     

Different quantitation approaches for xenobiotics in human urine samples by liquid chromatography/electrospray tandem mass spectrometry

The potential of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) for the determination of pesticide metabolites in human urine at the sub-ppb level is explored. Metabolites from two organophosphorous pesticides, 4-nitrophenol (from parathion and parathion-methyl) and 3-methyl-4-nitrophenol (from fenitrothion), are taken as model analytes to conduct this study. After direct injection of the urine sample (10 microL), different approaches were evaluated in order to achieve correct quantitation of analytes using an electrospray ionisation (ESI) interface. Thus, the feasibility of using external calibration was checked versus the use of different isotope-labeled internal standards. The advantages of applying coupled-column liquid chromatography (LC/LC) as an efficient clean-up without any type of sample manipulation are also discussed. The combination of LC/LC with ESI-MS/MS allows the direct analysis of free metabolites in urine, as the automated clean-up performed by the coupled-column technique is sufficient for the removal of interferences that suppress the ionisation of analytes in the ESI source. Using this procedure with external calibration, good precision and recoveries, and detection limits below 1 ng/mL are reached with analysis run times of around 8 min. The hyphenated technique LC/LC/ESI-MS/MS is proved to be a powerful analytical tool, allowing the rapid, sensitive and selective determination of 4-nitrophenol and 3-methyl-4-nitrophenol in human urine without any sample treatment.     

Multiresidue analysis of pesticides in vegetables and fruits by gas chromatography/mass spectrometry after gel permeation chromatography and graphitized carbon column cleanup

A multiresidue method for pesticides that enables quantitative, sequential analysis of a large number of vegetable and fruit samples by gas chromatography/mass spectrometry has been developed. First, 89 important target compounds were selected for monitoring, and then the appropriate internal standards for these pesticides, 14 stable isotopically labeled pesticides (surrogates), were used. The sample was extracted with acetonitrile, and the extract was cleaned up by a salting-out step followed by redissolution in ethyl acetate. Coextractives were removed automatically by gel permeation chromatography with a graphitized carbon column, and then by use of a tandem silica-gel/PSA cartridge column. Recoveries of 82 of the 89 pesticides from fortified spinach, tomato, apple, and strawberry were within a range from 70 to 120%, and the relative standard deviation values of 80 of the 89 pesticides were <5%. The method was applied to 188 commercial vegetable and fruit samples to demonstrate its use in routine analysis.     

Accurate quantification of the redox-sensitive GSH/GSSG ratios in the yeast Pichia pastoris by HILIC–MS/MS

A novel method for the simultaneous quantification of both glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) by hydrophilic interaction chromatography–MS/MS has been developed and is critically discussed. Internal standardization based on isotopically labeled standards for both analytes is an absolute prerequisite for accurate quantification of this redox pair. Hence, a highly efficient and selective miniaturized procedure for the synthesis of isotopically labeled GSSG from commercially available glutathione-(glycine-¹³C₂,¹⁵N) was established using H₂O₂ as oxidant and NaI as catalyst. Moreover, a tool is presented to monitor and hence uncover artifactual GSSG formation due to oxidation of GSH during sample preparation, which is the main source of systematic error in GSSG analysis. For this purpose, we propose to monitor the oxidation product formed by reaction of naturally occurring GSH with the isotopically labeled GSH used as internal standard. For the determination of GSH/GSSG ratios in yeast, different extraction methods based on (1) hot extraction with aqueous, acidic, or organic solvents, (2) mechanical cell lysis, and (3) extraction at subambient temperature were investigated in terms of recovery, extraction efficiency, and artifactual formation of GSSG. Total combined uncertainties of as low as 25–30 % (coverage factor?=?2) for the determination of GSH/GSSG ratios without derivatization were made possible by the addition of the internal standards early in the analytical procedure (before extraction) and immediate analysis of the analytes.     

Determination of ibuprofen and its major metabolites in human urine by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic assay for free and total ibuprofen and its major metabolites in human urine is described. Urine is acidified, drug and metabolites are extracted into hexane-propanol, back-extracted into sodium bicarbonate, neutralized and chromatographed. Ibufenac (4-isobutylphenylacetic acid) and 2-phenylpropionic acid were employed as internal standards. The extraction efficiencies were 94-100% for all compounds. The two metabolites and their internal standard were separated using an isocratic chromatographic system, followed by an abrupt step gradient to a second eluent for separation of ibuprofen and its internal standard with a total run time of 18 min. Detection was by a fixed-wavelength detector (214 nm). Sample-to-sample and day-to-day reproducibility studies yielded coefficients of variability of less than 9% for all compounds. The sensitivity was sufficient to determine 2.5 micrograms/ml free ibuprofen in 100 microliters urine.     

固相萃取-液相色谱-串联质谱法测定蔬菜中4种有机磷农药及其代谢产物

在蔬菜种植中经常使用的有机磷农药马拉硫磷、甲基对硫磷、敌百虫及乙酰甲胺磷可能转化的主要代谢产物分别为O,O-二甲基二硫代磷酸酯、对硝基酚、敌敌畏及甲胺磷。根据蔬菜色素等基质的含量不同采用不同的净化方法,色素含量高的蔬菜采用活性炭和弗罗里硅土串联固相萃取小柱净化,其他蔬菜采用弗罗里硅土固相萃取小柱净化。色谱分离选择ACQUITY UPLC BEH HILIC色谱柱,以乙腈和5 mmol/L乙酸铵水溶液为流动相进行梯度洗脱,得到的分离效果较好;质谱采用电喷雾正或负离子电离、多反应监测模式检测。液相色谱-质谱检测的基质效应为15.3%~45.1%;4种有机磷农药及其代谢物的方法回收率为76.9%~102.8%,相对标准偏差为5.92%~10.19%;定量限范围为0.001~0.01 mg/L;在0.01~1.00 mg/L范围内线性相关系数为0.9982~0.9999。方法具有良好的回收率、相对标准偏差、定量限及线性关系,适合蔬菜中有机磷及代谢物的检测,应用该检测方法对农贸市场购买的白菜、辣椒、西红柿及洋葱进行了检测。     

Multiresidue analysis of pesticides in fruits and vegetables using solid-phase extraction and gas chromatographic methods

A new extraction and cleanup procedure with gas chromatography was developed for the sensitive determination of acephate, dimethoate, malathion, diazinon, quinalphos, chlorpyrifos, profenofos, alpha-endosulfan, beta-endosulfan, chlorothalonil and carbaryl using 1-chloro-4-fluorobenzene as an internal standard in fruits and vegetables. Several extracting and eluting solvents for solid-phase extraction were investigated. The overall extracting solvent with a mixture of acetone:ethyl acetate:hexane (10:80:10, v/v/v) and a eluting solvent of 5% acetone in hexane used with the RPC18 cartridge gave the best recovery for all of the investigated pesticides, and minimized the interference from co-extractants. Under the optimal extraction and clean-up conditions, recoveries of 85 - 99% with RSD < 5.0% (n = 3) for most of the pesticides at the 0.02 - 0.5 mg/kg level were obtained. The limit of detection was between 0.005 - 0.01 mg/kg and the limit of quantification was 0.01 mg/kg. This analytical procedure was characterized with high accuracy and acceptable sensitivity to meet requirements for monitoring pesticides in crops.     

Development and Validation of a Dispersive Solid Phase Extraction Liquid Chromatography Mass Spectrometry Method with Electrospray Ionization for the Determination of Multiclass Pesticides and Metabolites in Meat and Milk

A dispersive solid phase extraction–liquid chromatography tandem mass spectrometry method with electrospray ionization was validated in food of animal origin for the determination of multiclass pesticide residues and their metabolites. A simple and low-cost sample preparation procedure using freezing as the clean-up step was used to identify and quantify analytes belonging to 39 different chemical classes in meat and milk matrices. Mean recoveries in the range of 70–120% with relative standard deviations <10% were obtained for the majority of the analytes. The limit of quantification of the method was 10 µg/kg. The matrix effects were statistically evaluated and the quantification of the analytes was conducted using calibration curves constructed with matrix matched calibration standards covering concentrations from 5 to 200 µg/kg. The proposed method was applied in 86 samples of animal origin taken from the Greek market, two of which were found positive for pesticides.     

Measurement of 4-nonylphenol and 4-tert-octylphenol in human urine by column-switching liquid chromatography-mass spectrometry

We report a method for determining 4-nonylphenol (NP) and 4-tert-octylphenol (OP) levels in human urine samples by column-switching liquid chromatography-electrospray mass spectrometry after enzymatic deglucuronidation. The method involves enzymatic deconjugation by β-glucuronidase and correction by the stable isotopically labeled internal standard, 4-(1-methyl)octylphenol-d₅. The compounds were separated by reversed-phase chromatography with a C₁₈ column, and detected by selected ion monitoring in the negative mode. After adding an internal standard to urine samples, a direct analysis was carried out. The average recoveries of OP and NP were above 85.0% with correction using the added internal standard. The quantitation limit in the urine samples was 0.3 ng ml⁻¹. The method enables the precise determination of standards and may be applied to the detection of trace amounts of OP and NP in human urine samples.     

Current role of liquid chromatography–mass spectrometry in clinical and forensic toxicology

This paper reviews multi-analyte single-stage and tandem liquid chromatography–mass spectrometry (LC-MS) procedures using different mass analyzers (quadrupole, ion trap, time-of-flight) for screening, identification, and/or quantification of drugs, poisons, and/or their metabolites in blood, plasma, serum, or urine published after 2004. Basic information about the biosample assayed, work-up, LC column, mobile phase, ionization type, mass spectral detection mode, and validation data of each procedure is summarized in tables. The following analytes are covered: drugs of abuse, analgesics, opioids, sedative-hypnotics, benzodiazepines, antidepressants including selective-serotonin reuptake inhibitors (SSRIs), herbal phenalkylamines (ephedrines), oral antidiabetics, antiarrhythmics and other cardiovascular drugs, antiretroviral drugs, toxic alkaloids, quaternary ammonium drugs and herbicides, and dialkylphosphate pesticides. The pros and cons of the reviewed procedures are critically discussed, particularly, the need for studies on matrix effects, selectivity, analyte stability, and the use of stable-isotope labeled internal standards instead of unlabeled therapeutic drugs. In conclusion, LC-MS will probably become a gold standard for detection of very low concentrations particularly in alternative matrices and for quantification in clinical and forensic toxicology. However, some drawbacks still need to be addressed and finally overcome. Photos of LC-MS apparatus and typical samples suitable for toxicological analysis     

Determination of herbicides and a metabolite in human urine by liquid chromatography-electrospray ionization mass spectrometry

A method was developed to determine simazine, atrazine and their metabolite, 2-chloro-4,6-diamino-1,3,5-triazine, in urine. The presence of these herbicides in urine may reflect possible exposure to pesticides. Sample preparation involved protein precipitation and solid-phase extraction. The samples were analyzed by high-performance liquid chromatography-mass spectrometry. The detection limits were 0.4 microg/l and the analytes have a linear response in the interval 6-800 microg/l. The precision of the method was reflected in the RSD of < 2.4% for the herbicides studied. Based on the detectable herbicide levels from spiked urine samples collected from unexposed volunteers, this method can be used to determine the low levels necessary for establishing reference values of the selected herbicides and the metabolite.     

Multiresidue determination of pesticides in acid-clay soils from Thailand

Tropical and temperate soils differ with respect to their chemical conditions and mineral composition. Consequently, assessment of the contamination of tropical soils with pesticides requires methods that provide exhaustive extraction from the specific soil matrix and reliable quantification. Our objective was to optimize the simultaneous extraction and determination of 32 representative pesticides (organophosphates, organochlorines, synthetic pyrethroids, triazines, acetamides, carbamates, diphenyl ethers, acylalanines, oxadiazoles, thiadiazoles, and phenoxy compounds) frequently used in Thailand. The compounds were added to a clayey Ultisol-topsoil (45% clay) from the Yom Region in Northern Thailand. Ten different solvent mixtures were tested; the use of a shake extraction with acetone-ethyl acetate-water (3 + 1 + 1, v/v/v) was most effective in providing exhaustive and reproducible extraction of pesticides both from freshly fortified soil and from fortified soil after incubation for 14-28 days. The pesticides were separated and quantified by using gas chromatography with mass selective detection. Except for methamidophos, which was almost completely lost during sample workup, the recoveries of all pesticides from soil ranged from 68.6 to 104% (mean standard error, 2.8%) for freshly spiked samples and from 45.9 to 107% (mean standard error, 2.3%) for spiked samples incubated for 14 days. The use of internal standards compensated for the losses of most pesticides during workup and increased the mean recovery of the pesticides from freshly fortified soil to 96%. The method was successfully applied to the determination of environmental concentrations of pesticides in a range of Thai agricultural soils.