Miniaturized Salting-Out Assisted Liquid-Liquid Extraction Combined with Disposable Pipette Extraction for Fast Sample Preparation of Neonicotinoid Pesticides in Bee Pollen

As the main source of nutrients for the important pollinator honeybee, bee pollen is crucial for the health of the honeybee and the agro-ecosystem. In the present study, a new sample preparation procedure has been developed for the determination of neonicotinoid pesticides in bee pollen. The neonicotinoid pesticides were extracted using miniaturized salting-out assisted liquid-liquid extraction (mini-SALLE), followed by disposable pipette extraction (DPX) for the clean-up of analytes. Effects of DPX parameters on the clean-up performance were systematically investigated, including sorbent types (PSA, C18, and silica gel), mass of sorbent, loading modes, and elution conditions. In addition, the clean-up effect of classical dispersive solid-phase extraction (d-SPE) was compared with that of the DPX method. Results indicated that PSA-based DPX showed excellent clean-up ability for the high performance liquid chromatography (HPLC) analysis of neonicotinoid pesticides in bee pollen. The proposed DPX method was fully validated and demonstrated to provide the advantage of simple and rapid clean-up with low consumption of solvent. This is the first report of DPX method applied in bee pollen matrix, and would be valuable for the development of a fast sample preparation method for this challenging and important matrix.     

Determination of organochlorine and pyrethroid pesticides in fruit and vegetables using solid phase extraction clean-up cartridges

A method to determine six organochlorine and three pyrethroid pesticides in grape, orange, tomato, carrot and green mustard based on solvent extraction followed by solid phase extraction (SPE) clean-up is described. The pesticides were spiked into the sample prior to analysis, extracted with ethyl acetate, evaporated and reconstituted with a solvent mixture of acetone:n-hexane (3:7). Three different sorbents (Strong Anion Exchanger/Primary Secondary Amine (SAX/PSA), Florisil and C18) were used for the clean-up step. Pesticides were eluted with 5mL of acetone:n-hexane (3:7, v/v) and determined by gas chromatography and electron-capture detection (GC-ECD). SAX/PSA was the sorbent, which provided chromatograms with less interference and the mean recoveries obtained were within 70-120% except for captafol. The captafol recoveries for grape were within acceptable range with C18 clean-up column.     

Multiresidue analysis of pesticides in animal liver by gas chromatography using triple quadrupole tandem mass spectrometry

Two methods for extracting organochlorine (OCs) and organophosphorus (OPs) pesticides from animal liver have been developed. The determination was carried out by gas chromatography with electron impact ionization tandem mass spectrometry (GC-(EI-)MS/MS) using a triple quadrupole (QqQ) analyzer. First, a liquid-solid extraction performed with a high-speed homogenizer (Polytron) using ethyl acetate as solvent, and a subsequent clean-up by gel permeation chromatography (GPC) was applied, determining 34 pesticides. Secondly, a matrix solid phase dispersion (MSPD) extraction with octadecylsilyl (C(18)) sorbent combined with a Florisil clean-up and ethyl acetate elution was performed, analyzing 25 compounds. These methodologies have been tested and compared in the sample pre-treatment due to the fatty nature of the matrix. The GPC method was finally selected and validated, yielding recoveries in the range 70-115%, with precision values expressed as relative standard deviation (RSD) lower or equal to 20%, at the spiking levels of 25 and 50 microg kg(-1), and limits of quantification (LOQs) lower than the maximum residue levels (MRLs) set by the European Union in animal products, except for isofenphos. Linearity was also studied ranging between 5 and 300 microg kg(-1) for most of pesticides. This method was applied to the analysis of real liver samples of chicken, pork and lamb.     

Matrix solid-phase dispersion extraction of organophosphorous pesticides from beeswax

Beeswax is a complex mixture of lipophilic compounds and other components such as aliphatic alcohols and carotenoids. Then, extraction and clean-up for pesticide analysis in beeswax is a challenge. In this work, a multiresidue method for the analysis of dichlorvos (DCV), diazinon, malathion, methyl parathion and coumaphos (CMF) in beeswax was developed. The proposed approach is based on matrix solid-phase dispersion extraction. The adsorbent for sample clean-up was studied and a simplex-centroid cubic statistical design was applied to evaluate pure solvents and their binary and ternary mixtures to elute the analytes. Finally, Florisil and ethyl acetate were chosen as solid support and eluting solvent, respectively. After extraction, pesticides were separated and detected by gas chromatography/mass spectrometry. The method achieved acceptable recoveries (70–85%; except for DCV, 24–38%) with relative standard deviations below 5%. The repeatability of the method was lower than 8% and interday variability was below 12%. The limit of detection (LOD) for the analytes varies between 0.2 and 2.6 µg?kg^?1 and limit of quantification from 0.93 to 8.8 µg?kg^?1. LOD reached for CMF was below the maximum residue limit allowed by the legislation of the United States and Canada.     

Application of SiO2 hollow fibers for sorptive microextraction and gas chromatography–mass spectrometry determination of organochlorine pesticides in herbal matrices

A method involving simultaneous extraction and sample clean-up procedure: hollow fiber sorptive microextraction, coupled with gas chromatography–mass spectrometric detection for quantification of seven organochlorine pesticides in Radix et Rhizoma Rhei is described. SiO₂ hollow fiber with porous structure was synthesized for the first time. The internal diameter of SiO₂ hollow fiber is 380 μm and average wall thickness is 100 μm. Aggregated SiO₂ particles deposited on the surface of the hollow fiber in a regular array lead to porous structure. SiO₂ hollow fiber was applied to the determination of organochlorine pesticides in Radix et Rhizoma Rhei to avoid sample clean-up and minimize the matrix effects. Extraction solvent, extraction temperature and equilibration time were optimized. Fiber to fiber repeatability over the concentration ranges were less than 10%. Recoveries were satisfactory (between 63% and 115%) for most of organochlorine pesticides at spiking levels. Furthermore, the proposed method was also applied to determine seven organochlorine pesticides in 43 commercial Radix et Rhizoma Rhei samples, in which the selected pesticides were found in eight samples. The results have been further confirmed by solvent extraction methods according to China Pharmacopoeia (2005).     

Sample preparation for planar chromatography

High-performance thin-layer chromatography has favorable properties for high-throughput separations with a high matrix tolerance. Sample preparation, however, is sometimes required to control specific matrix interferences and to enhance the detectability of target compounds. Trends in contemporary applications have shifted from absorbance and fluorescence detection to methods employing bioassays and mass spectrometry. Traditional methods (shake-flask, heat at reflux, Soxhlet, and hydrodistillation) are being challenged by automated instrumental approaches (ultrasound-assisted and microwave-assisted solvent extraction, pressurized liquid extraction, and supercritical fluid extraction) and the quick, easy cheap, efficient, rugged, and safe extraction method for faster and streamlined sample processing. Liquid-liquid extraction remains the most widely used approach for sample clean-up with increasing competition from solid-phase extraction. On-layer sample, clean-up by planar solid-phase extraction is increasingly used for complex samples and in combination with heart-cut multimodal systems. The automated spray-on sample applicator, the elution head interface, biological detection of target and non-target compounds, and straightforward mass spectrometric detection are highlighted as the main factors directing current interest toward faster and simpler sample workflows, analysis of more complex samples, and the determination of minor contaminants requiring high concentration factors.     

An evaluation method for determination of non-polar pesticide residues in animal fat samples by using dispersive solid-phase extraction clean-up and GC-MS

A rapid and easy method has been proposed, optimized and evaluated for quantitative determination at trace level of a representative group of non-polar pesticides in fat samples. The method includes n-hexane-saturated acetonitrile extraction, fat precipitation by cooling pre clean-up followed by dispersive solid-phase extraction (d-SPE) based on QuEChERS procedure clean-up. Determination was performed by gas chromatography?Cmass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. Efficiency of the d-SPE clean-up step was evaluated by comparison with fat oxidation treatment and gel permeation chromatography. Different combinations of d-SPE extraction reagents and sample amounts were tested in order to minimize matrix co-extractives and interferences. Best recoveries were obtained with 1200?mg of MgSO₄, 400?mg of end-capped C₁₈, 400?mg of PSA and 1?g of sample amount. SIM method, matrix effect, precision, and accuracy were evaluated with spiked pork fat samples for 38 representative pesticides. Results of this study showed that this technique is applicable in routine analysis for its application into monitoring programs. It simplifies time-consuming clean-up steps and allows a satisfactory long-term chromatographic performance.     

对比3种不同的QuEChERS前处理方式在气相色谱-串联质谱检测分析烟草中上百种农药残留中的应用

以气相色谱-串联质谱(GC-MS/MS)技术为基础,建立了适合烟草中上百种农药残留分析的3种QuEChERS前处理方法:溶剂转换法、提取液稀释法和正己烷液液萃取法。以烟草中的有机磷、有机氯、拟除虫菊酯类、酰胺类、氨基甲酸酯类、二硝基苯胺类等共155种农药为研究对象,从基质效应、共萃取基质、色谱峰干扰、回收率和定量限等方面对3种前处理方式进行对比分析。经考察发现,3种方法各有优缺点,正己烷液液萃取法得到的提取液中共萃取基质含量最少,但只能保证约100种目标物的回收率在70%~120%;溶剂转换法和提取液稀释法对绝大部分目标物都能保证回收率在70%~120%,适合用于多农药残留分析检测。对不同种类农药进行对比,发现有机磷、酰胺类和氨基甲酸酯类农药的基质效应相对较强,而有机氯和拟除虫菊酯类目标物的基质效应相对较弱,因此,对有机磷农药单独分析时,建议使用提取液稀释法;对有机氯和拟除虫菊酯类农药单独分析时,建议使用正己烷液液萃取法。     

Modification and re-validation of the ethyl acetate-based multi-residue method for pesticides in produce

The ethyl acetate-based multi-residue method for determination of pesticide residues in produce has been modified for gas chromatographic (GC) analysis by implementation of dispersive solid-phase extraction (using primary–secondary amine and graphitized carbon black) and large-volume (20 μL) injection. The same extract, before clean-up and after a change of solvent, was also analyzed by liquid chromatography with tandem mass spectrometry (LC–MS–MS). All aspects related to sample preparation were re-assessed with regard to ease and speed of the analysis. The principle of the extraction procedure (solvent, salt) was not changed, to avoid the possibility invalidating data acquired over past decades. The modifications were made with techniques currently commonly applied in routine laboratories, GC–MS and LC–MS–MS, in mind. The modified method enables processing (from homogenization until final extracts for both GC and LC) of 30 samples per eight hours per person. Limits of quantification (LOQs) of 0.01 mg kg⁻¹ were achieved with both GC–MS (full-scan acquisition, 10 mg matrix equivalent injected) and LC–MS–MS (2 mg injected) for most of the pesticides. Validation data for 341 pesticides and degradation products are presented. A compilation of analytical quality-control data for pesticides routinely analyzed by GC–MS (135 compounds) and LC–MS–MS (136 compounds) in over 100 different matrices, obtained over a period of 15 months, are also presented and discussed. At the 0.05 mg kg⁻¹ level acceptable recoveries were obtained for 93% (GC–MS) and 92% (LC–MS–MS) of pesticide–matrix combinations.     

Automated hollow fiber-protected dynamic liquid-phase microextraction of pesticides for gas chromatography-mass spectrometric analysis

Dynamic liquid-phase microextraction (LPME) controlled by a programmable syringe pump was evaluated for extracting pesticides in water prior to GC-MS analysis. A conventional microsyringe with a 1.3-cm length of hollow fiber attached to its needle was connected to a syringe pump to perform the extraction. The microsyringe was used as both the microextraction device as well as the sample introduction device for GC-MS analysis. The attached hollow fiber served as the "holder" and protector" of 3 microl of organic solvent. The solvent was repeatedly withdrawn into and discharged from the hollow fiber by the syringe pump. Pesticides were extracted from 4-ml water samples into the organic solvent impregnated in the hollow fiber. The effects of organic solvents, plunger movement pattern, agitation and extraction time were investigated. Good repeatabilities of extraction performance were obtained, with the RSD values ranging from 3.0% (alachlor) to 9.8% (4-chlorophenol) for the 14 pesticides; most RSD values were under 5.0%. The method provided a 490-fold preconcentration of the target pesticides. The limits of detection were in the range of 0.01-5.1 microg/l (S/N = 3) in the GC-MS selected ion monitoring mode. In addition, sample clean-up was achieved during LPME because of the selectivity of the hollow fiber, which prevented undesirable large molecules from being extracted. A slurry sample (mixture of 40 mg soil/ml of water) containing seven pesticides was extracted using this method which also gave good linearity and precision (most RSDs <7.0%, n = 3).     

高效液相色谱-串联质谱法快速测定6类食品中增效醚的残留

建立了水果、蔬菜、粮谷、油料、动植物脂类、动物源性食品等6类食品中增效醚残留量快速测定的液相色谱-质谱/质谱(LC-MS/MS)方法。样品盐析并除水后,用三氯甲烷提取,氟罗里硅土填料固相萃取柱净化,丙酮-三氯甲烷混合溶剂洗脱,以乙腈-0.1%甲酸为流动相,在Zobax SB C18液相色谱柱完成分离,并于电喷雾正离子多反应监测模式下质谱测定。考察了提取溶剂、净化方法、柱容量、仪器条件、基质效应对分析结果的影响。增效醚质量浓度在0.5～100μg/L范围内,线性相关系数(r)为0.9976,方法定量限(LOQ)为10μg/kg。17种食品基质添加LOQ、低MRL、2倍MRL、高MRL 4个不同浓度水平时,回收率在80.3%～96.3%之间,相对标准偏差为2.7%～14%,方法可以满足多种食品基质中增效醚残留量的定性和定量检测要求。     

Multi-residue analysis of pesticides in tea by online SEC-GC/MS

A multi-residue method for the analysis of pesticides in tea was developed by online size exclusion chromatography (SEC)-GC/MS with full scan mode. The sample was fortified with chlorpyrifos-d(10) isotope internal standard and extracted by acetonitrile. After purification by primary secondary amine sorbent and solvent exchange by SEC mobile phase, the sample was detected by online SEC-GC/MS. The purification result of the online system was evaluated by comparing the correlation between Chinese cabbage and tea matrix. The factors for method optimization included sample preparation, matrix effects and the instrument parameters of each online component. Scatter plot was introduced in this study to directly illustrate the results of the condition optimization and matrix effects in the online system. For most of the pesticides, the average recoveries ranged from 70 to 130% and the RSD were below 15%. The feasibility of the application of full scan mode in multi-residue determination of trace amounts of pesticides (LODs below 0.01 mg/kg) in a complex matrix was discussed.     

Matrix solid phase dispersion–Soxhlet simultaneous extraction clean-up for determination of organochlorine pesticide residues in tobacco

A novel method combining matrix solid phase dispersion (MSPD) with Soxhlet simultaneous extraction clean-up (SSEC) was developed. Being a single-step extraction and clean-up procedure, it could be used instead of multistep solvent extraction and Florisol column clean-up. It not only reduces sample contamination during the procedure, but it also decreases the amount of organic solvent needed. The retention times of standards were used to qualitatively assess the method, and the external standard method was used to quantitatively assess it. Residues of organochlorine pesticides (OCP) in tobaccos were determined by gas chromatography–electron capture detection (GC–ECD), and their identities were confirmed by the standard addition method (SAM). The performance of the method was evaluated and validated: the detection limit was 0.01–0.02 μg g⁻¹, relative standard deviations were 5–26%, and recoveries were 72–99% at fortification levels of 0.10, 1.00 and 10.0 μg g⁻¹. The analytical characteristics of MSPD–SSEC compared very favorably with the results from the classical multistep solvent extraction and Florisol column clean-up method.     

Rapid method for the determination of organochlorine pesticides in milk

This method involved one step solvent extraction of milk with ethyl acetate-acetone-methanol by ultrasonication. The supernatants were further cleaned-up and enriched by solid-phase extraction using octadecyl (C18)-bonded silica cartridges, then assayed by capillary gas-liquid chromatography with electron capture detection. The recoveries of eleven organochlorine pesticides (OCPs) from raw milks were quantitative, ranging from 90-110% at 10 times the limit of detection (LOD). The LOD ranged from 0.5 micrograms/l whole milk for alpha-hexachlorocyclohexane to 2.5 micrograms/l whole milk for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane. The day-to-day variation of the method was evaluated over 7 days using 3 different pools of spiked cow milks (at the LOD, 5 and 10 times the LOD). The coefficient of variations (C.V.s) were 16 +/- 6, 10 +/- 2 and 9 +/- 3% (mean S.D.), respectively. The method showed no emulsion problems common with conventional non-polar solvent extraction, and the use of solid-phase extraction considerably reduced the sample clean-up process compared with the existing methods. The method also showed that OCPs in milk could be extracted quantitatively without extraction of total fat, and that OCPs spiked into cows milk could be used to construct calibration curves for human milk determinations.     

Determination of pesticides in seaweeds by pressurized liquid extraction and programmed temperature vaporization-based large volume injection–gas chromatography–tandem mass spectrometry

A rapid method for the simultaneous identification and quantification of pesticide residues in edible seaweed has been developed. Target analytes were three pyrethroid, a carbamate and two organophosphorus pesticides. The procedure consists of a pressurized liquid extraction (PLE) with integrated clean-up, followed by gas chromatography coupled to tandem mass spectrometry. Five PLE parameters were investigated using a screening design: temperature, static extraction time, number of cycles, percent of flush volume and quantitative composition of the n-hexane/ethyl acetate extraction solvent. The effect of the in-cell clean-up with Florisil^® and graphitized carbon black adsorbents was investigated using a Doehlert response surface design. Large volumes of sample extracts were injected using a programmed-temperature vaporizer (PTV-LVI) to improve both sensitivity and selectivity of measurements. Quantification was carried by the internal standard method with surrogate deuterated standards. The method showed excellent linearity (R² > 0.999) and precision (relative standard deviation, RSD ≤ 8%) for all compounds, with detection limits ranging from 0.3 pg g⁻¹ for chlorpyrifos-ethyl, to 3.0 pg g⁻¹ for carbaryl (23.1 pg g⁻¹ for deltamethrin). Recoveries in real seaweed samples were within the range 82–108%. The method was satisfactory validated for the analysis of wild and cultivated edible seaweeds. The presence of pyrethroid and organophosphorus pesticides in some of the samples was evidenced.     

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.     

Does further clean-up reduce the matrix enhancement effect in gas chromatographic analysis of pesticide residues in food?

Sample extracts of apples, peas, green beans, oranges, raspberries, clementines, carrots, and wheat obtained using the Food and Drug Administration (acetone extraction) and Canadian Pest Management Regulatory Agency (acetonitrile extraction) multiresidue methods for pesticides were subjected to clean-up using different solid-phase extraction (SPE) cartridges in an attempt to reduce or eliminate the matrix enhancement effect. The matrix enhancement effect is related to the blocking of active sites on the injector liner by matrix components, thereby increasing signal in the presence of matrix versus standards in solvent in which the pesticides themselves interact with the active sites. Graphitized carbon black (GCB) was often used in combination with various anion-exchange SPE cartridges. The extracts were then spiked with organophosphorus insecticides. These process standards were then compared to standards in acetone of the same concentration using gas chromatography with flame photometric detection or ion trap mass spectrometric detection. Sample matrix enhancement varied from little to no effect for some pesticides (e.g. chlorpyrifos, malathion) to >200% in the case of certain susceptible pesticides. The GCB removed color components but showed little effect in reducing matrix enhancement by itself. The anion-exchange cartridges in combination with GCB or not, substantially reduced the matrix enhancement effect but did not eliminate it.     

Analyses of pesticides and their metabolites in foods and drinks

The importance of matrix pretreatment, sample extraction and clean-up in multiresidue methods for pesticide analyses is discussed, with emphasis on alternative new techniques attempted worldwide such as accelerated solvent extraction, microwave-assisted extraction, solid-phase extraction, solid-phase micro-extraction, matrix solid-phase dispersion, supercritical fluid chromatography, ultrasonic extraction and gel permeation chromatography. Detection employing capillary gas chromatography and high-performance liquid chromatography in conjunction with mass spectrometry, capillary electrophoresis, immunoassay techniques and others is summarized.     

Determination of carbamates and organophosphorus pesticides by SDME–GC in natural water

Water contamination due to the wide variety of pesticides used in agriculture practices is a global environmental pollution problem. Analytical methods with low quantification limits are necessary. The application of a new extraction technique, solvent drop microextraction (SDME), followed by gas chromatography with a nitrogen-phosphorus detector, was assessed for determining carbamates and organophosphorus pesticides in natural water. Experimental parameters which control the performance of SDME such as selection of microextraction solvent, optimization of organic drop volume, effects of sample stirring, salt addition, and, finally, sorption time profiles were studied. Once SDME was optimized, analytical parameters such as linearity (r ²>0.99), precision (<13%), and detection limits (0.2 to 5 μg/L), plus matrix effects were evaluated (no matrix effects were found). SDME is a dynamic technique able to extract pesticides from water in 14 min; the use of organic solvents and water samples for SDME is negligible compared to other extraction techniques.     

A new multi-residue method for analysis of pesticide residues in fruit and vegetables using liquid chromatography with tandem mass spectrometric detection

A new multi-residue method for determination of pesticide residues in a wide variety of fruit and vegetables, using the National Food Administration (NFA) ethyl acetate extraction and determination by means of LC-MS/MS, is presented. The method includes pesticides normally detected by LC-UV or LC-fluorescence such as benzimidazoles, carbamates, N-methylcarbamates and organophosphorus compounds with an oxidisable sulphide group as well. After extraction with ethyl acetate, the extract is concentrated and an aliquot of the extract is evaporated to dryness and redissolved in methanol before injection on LC-MS/MS. The method has been validated for 57 different pesticides and metabolites. Representative species from different commodity groups were chosen as matrices in order to study the influence from different matrices on recoveries. The fortification levels studied were 0.01-0.5 mg kg(-1). Matrix effects were tested for all matrices by means of standard addition to blank extracts. The matrix effect, expressed as signal in solvent compared to signal in matrix, was in general found to be small. The obtained recoveries are, with a few exceptions, in the range 70-100%. The proposed method is quick and straightforward and no additional clean-up steps are needed. The method can be used for the analysis of all 57 pesticides in one single determination step at 0.01 mg kg(-1).