Routine approach to qualitatively screening 300 pesticides and quantification of those frequently detected in fruit and vegetables using liquid chromatography tandem mass spectrometry (LC-MS/MS).

This paper describes an efficient and effective analytical scheme to first screen for 300 pesticides in fruit and vegetables samples using liquid chromatography tandem mass spectrometry (LC-MS/MS) with a commercially enhanced product ion method. Then presumed positive extracts are analysed using a quantitative and confirmatory LC-MS/MS method optimized for 55 pesticides. A quick, easy, cheap, effective, rugged, and safe (QuEChERS) method with acetate buffering (AOAC Official Method 2007.01) was used for sample preparation, which has been previously shown to yield high-quality results for hundreds of pesticide residues in foods. The advantages and disadvantages of both the qualitative screening and quantitative/confirmatory methods and their combination are critically discussed. No false-negatives for the 55 pesticides occurred above 10 ng g(-1) for extracts analysed by both LC-MS/MS methods, and the no false-positives were encountered from the screening analysis (after analyst review) because all presumptive identifications were confirmed in the second analysis. The monitoring scheme was applied during a one-year period on 200 fruit and vegetable samples from Hungarian markets. No pesticide residues were found in half the samples, and twelve violations of European maximum residue limits were detected.

Analysis of pesticide residues in eggs by direct sample introduction/gas chromatography/tandem mass spectrometry.

Direct sample introduction (DSI) or "dirty sample injection" is a rapid, rugged, and inexpensive approach to large volume injection in gas chromatography (GC) for semivolatile analytes such as pesticides. DSI of complex samples such as eggs requires a very selective detection technique, such as tandem mass spectrometry (MS-MS), to determine the analytes among the many semivolatile matrix components that also appear. In DSI, the nonvolatile matrix components that normally would contaminate the GC system in traditional injection methods remain in a disposable microvial, which is removed after every injection. For example, 3 microg of nonvolatile residue typically remained in the microvial after an injection of egg extract using the DSI method. This analytical procedure involves the following: (i) weighing 10 g of egg in a centrifuge tube and adding 2 g of NaCl and 19.3 mL of acetonitrile (MeCN); (ii) blending for 1 min using a probe blender; (iii) centrifuging for 10 min; and (iv) analyzing 10 microL (5 mg of egg equivalent) of the extract using DSI/GC/MS-MS. No sample cleanup or solvent evaporation steps were required to achieve quantitative and confirmatory results with <10 ng/g detection limits for 25 of 43 tested pesticides from several chemical classes. The remaining pesticides gave higher detection limits due to poor fragmentation characteristics in electron impact ionization and/or degradation. Analysis of eggs incurred with chlorpyrifos-methyl showed a similar trend in the results as a more traditional approach.

Optimization and evaluation of low-pressure gas chromatography-mass spectrometry for the fast analysis of multiple pesticide residues in a food commodity.

A fast method of analysis for 20 representative pesticides was developed using low-pressure gas chromatography-mass spectrometry (LP-GC-MS). No special techniques for injection or detection with a common quadrupole GC-MS instrument were required to use this approach. The LP-GC-MS approach used an analytical column of 10 m x 0.53 mm I.D., 1 microm film thickness coupled with a 3 m x 0.15 mm I.D. restriction capillary at the inlet end. Thus, the conditions at the injector were similar to conventional GC methods, but sub-atmospheric pressure conditions occurred throughout the analytical column (MS provided the vacuum source). Optimal LP-GC-MS conditions were determined which achieved the fastest separation with the highest signal/noise ratio in MS detection (selected ion monitoring mode). Due to faster flow-rate, thicker film, and low pressure in the analytical column, this distinctive approach provided several benefits in the analysis of the representative pesticides versus a conventional GC-MS method, which included: (i) threefold gain in the speed of chromatographic analysis; (ii) substantially increased injection volume capacity in toluene; (iii) heightened peaks with 2 s peak widths for normal MS operation; (iv) reduced thermal degradation of thermally labile analytes, such as carbamates; and (v) due to larger sample loadability lower detection limits for compounds not limited by matrix interferences. The optimized LP-GC-MS conditions were evaluated in ruggedness testing experiments involving repetitive analyses of the 20 diverse pesticides fortified in a representative food extract (carrot), and the results were compared with the conventional GC-MS approach. The matrix interferences for the quantitation ions were worse for a few pesticides (acephate, methiocarb, dimethoate, and thiabendazole) in LP-GC-MS, but similar or better results were achieved for the 16 other analytes, and sample throughput was more than doubled with the approach.

Method for the analysis of thyreostats in meat tissue using gas chromatography with nitrogen phosphorus detection and tandem mass spectrometric confirmation.

An analytical method not requiring a mercury column cleanup step is described for the isolation and detection of four thyreostatic agents in meat tissue. The use of these growth promotants in livestock has been banned by regulatory agencies. The meat tissue is homogenized with acetonitrile-water, centrifuged, and the supernatant is partitioned with petroleum ether. The acetonitrile-water is concentrated and then passed through a silica-gel column. The solvent is then removed and the residue derivatized with N-methyl-N-(trimethylsilyl)-trifluoroacetamide. The total amount of organic solvent used for the analysis is merely 35 mL. The derivatized thyreostats are detected and quantitated by gas chromatography (GC) equipped with a nitrogen-phosphorus detector. Percent recoveries from fortified meat tissue (n = 6) at the 0.1-microg/g (parts per million) level are 93.5 +/- 2.9 for 2-thiouracil, 90.3 +/- 3.0 for tapazole, 87.5 +/- 2.9 for 6-methyl-2-thiouracil, and 85.1 +/- 5.8 for 6-n-propyl-2-thiouracil. For the confirmation of analyte identities, GC-tandem mass spectrometry with an ion-trap instrument is used. The estimated minimum level for a reliable measurement is 0.050 microg/g in meat tissue.

Analysis of pesticide residues in mixed fruit and vegetable extracts by direct sample introduction/gas chromatography/tandem mass spectrometry.

Direct sample introduction (DSI), or "dirty sample injection," was investigated in the determination of 22 diverse pesticide residues in mixed apple, green bean, and carrot extracts by benchtop gas chromatography/tandem mass spectrometry (DSI/GC/MS-MS). The targeted pesticides, some of which were incurred in the samples, included chlorpyrifos, azinphos-methyl, parathion-methyl, diazinon, terbufos, p,p'-DDE, endosulfan sulfate, carbofuran, carbaryl, propargite, bifenthrin, dacthal, trifluralin, metalaxyl, pendimethalin, atrazine, piperonyl butoxide, diphenylamine, vinclozolin, chlorothalonil, quintozene, and tetrahydrophthalimide (the breakdown product of captan). The analytical DSI method entailed the following steps: (1) blend 30 g sample with 60 mL acetonitrile for 1 min in a centrifuge bottle; (2) add 6 g NaCl and blend 30 s; (3) centrifuge for 1-2 min; (4) add 5 mL upper layer to 1 g anhydrous MgSO4 in a vial; and (5) analyze 11 microL extract, using DSI/GC/MS-MS. Sample cleanup is not needed because GCIMS-MS is exceptionally selective for the targeted analytes, and nonvolatile coextracted matrix components do not contaminate the injector or the GC/MS-MS system. Average recoveries of the pesticides were 103 +/- 7% with relative standard deviations of 14 +/- 5% on average, and limits of detection were <2 ng/g for nearly all pesticides studied. The DSI/GC/ MS-MS approach for targeted pesticides is quantitative, confirmatory, sensitive, selective, rugged, rapid, simple, and inexpensive.

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.

Potential artifact formation of dioxins in ball clay during supercritical fluid extraction.

Earlier surveys indicate that meat, fish and dairy products are the principal source of polychlorinated dibenzo-p-dioxin (PCDD) exposure in the diet. A recent finding by others of PCDDs in chickens that consumed a feed containing PCDD led to the finding of ball clay, an anti-caking agent, as the source. Supercritical fluid extraction (SFE) was studied as a means to isolate PCDDs from commercial ball clays using GC-electron capture detection (muECD) as a means to screen for these contaminants. The finding of ng/g amounts and recoveries >100% in several samples of ball clay containing octachlorodibenzo-p-dioxin (OCDD) suggested that PCDD may form artifactually as a result of analysis. Studies on pentachlorophenol (PCP) fortified ball clay were carried out by SFE and soxhlet extraction and the results compared. The values obtained by SFE were considered more problematic. The results obtained from ball clay suggest that precautions need to be exercised when using SFE to analyze for dioxins in solid samples containing chlorophenols.

Supercritical fluid extraction of pesticides in foods.

This article summarizes research findings involving the supercritical fluid extraction (SFE) of pesticides in food and other tissue matrices. Emphasis is placed on multiresidue analysis of pesticides in nonfatty foods, including some previously unpublished aspects of SFE in this application. Brief overviews of pesticides and traditional multiresidue methods are given, followed by discussion of results for SFE applications in the pesticide residue analysis of foods.

Evaluation of a fibrous cellulose drying agent in supercritical fluid extraction and pressurized liquid extraction of diverse pesticides.

A fibrous cellulose powder (CF-1) was investigated as a drying agent for supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE), also known as accelerated solvent extraction. Analysis of fifty-eight diverse pesticides was performed using gas chromatography-ion-trap mass spectrometric detection (GC-ITD). Extraction efficiencies were correlated versus pesticide polarity with samples of different water-CF-1 ratios. The effect of water was much more pronounced in SFE using CO2 than PLE using acetonitrile. Pesticide recoveries and limits of detection of fortified tomato samples mixed with CF-1 were determined. PLE gave recoveries > 80% for nearly all pesticides, and SFE gave similar recoveries except for the most polar and non-polar pesticides. SFE typically gave lower detection limits than PLE due to fewer matrix interferants.

Evaluation of hydromatrix and magnesium sulfate drying agents for supercritical fluid extraction of multiple pesticides in produce.

The simultaneous extraction of relatively polar and nonpolar pesticides has been problematic in multiresidue analysis using supercritical fluid extraction (SFE) with carbon dioxide. In fruit and vegetable samples, which typically contain 80-95% water, moisture acts to increase SFE recoveries of many polar pesticides, but a drying agent should be used to control water in SFE. Hydromatrix, a prevalent drying agent, has many desirable characteristics, but it reduces recovery of certain important pesticides, such as methamidophos, acephate, and omethoate. MgSO4 has been shown previously to be applicable for the extraction of methamidophos and six other pesticides, but MgSO4 has practical disadvantages in its use. In this study, properties and SFE results with the individual drying agents and their combination were evaluated. Simultaneous recoveries for polar and nonpolar pesticides were achieved for 71 pesticides fortified in apple using a mixture of 2 + 1 + 2 MgSO4-H2O-Hydromatrix-sample for extraction. The advantages of each drying agent were maintained by their combination. The analysis of real samples, however, showed that more study was needed to improve recoveries of nonpolar pesticides.

Evaluation of different solid-phase traps for automated collection and clean-up in the analysis of multiple pesticides in fruits and vegetables after supercritical fluid extraction.

This study was designed to determine which combination of sorbent-trap and elution solvent provided the most efficient automated method of collection in supercritical fluid extraction (SFE), elution of analytes, and clean-up of orange, sweet potato and green bean extracts for analysis of 56 diverse pesticides using GC-ion-trap MS. The solid-phase traps evaluated consisted of octyldecylsilane (ODS), diol, Tenax and Porapak-Q, and the elution solvents compared were acetone, ethyl acetate, acetonitrile and methanol. SFE collection by bubbling into each organic solvent was also compared. Recoveries, elution volumes, limits of detection and clean-up aspects were determined for each combination of commodity, trap and solvent tested. High trapping efficiencies were achieved in each case, and acetone usually eluted the pesticides in the least volume (< 1 ml) from the traps. The few matrix components that interfered in GC-ion-trap MS continued to interfere in all trap/solvent pairs, and limits of detection were independent of trap/solvent combination. The use of the ODS trap and acetone elution solvent gave the most consistently high recoveries of the traps and solvents tested.

Development of a method of analysis for 46 pesticides in fruits and vegetables by supercritical fluid extraction and gas chromatography/ion trap mass spectrometry.

A multiresidue method using supercritical fluid extraction (SFE) and gas chromatography/ion trap mass spectrometry (GC/ITMS) was developed for analysis of 46 pesticides in fruits and vegetables. The SFE procedure used 2 commercial instruments that trapped the extracts on solid-phase material. Silica gel chemically bound to octadecylsilane (ODS) collected the extracted pesticides efficiently, and elution of the trap with acetonitrile gave high recoveries. Extracts thus obtained were sufficiently clean for subsequent GC/ITMS analysis. The SFE conditions were 320 atm and 60 degrees C (0.85 g/mL CO2 density) and 1.6 mL/min CO2 flow rate for 6 extraction vessel volumes. Trapping on 1 mL ODS occurred at 10 degrees C, and a 0.4 mL/min flow rate of acetonitrile at 40 degrees-50 degrees C was used to elute the pesticides. Quantitative and qualitative analyses of the 46 pesticides were performed simultaneously by GC/ITMS. Studies of fortified samples gave > 80% recoveries for 39 pesticides, and recoveries of > 50% for the other pesticides, except methamidophos and omethoate. Grapes, carrots, potatoes, and broccoli were used as samples during method development, and a blind experiment involving incurred and fortified samples was used to test the approach. Results of the blind study compared satisfactorily with results from 7 laboratories using traditional GC detectors and solvent-based extractions.

Supercritical fluid extraction and gas chromatography/ion trap mass spectrometry of pentachloronitrobenzene pesticides in vegetables.

An analytical approach using supercritical fluid extraction (SFE) followed by gas chromatography/ion trap mass spectrometry (GC/ITMS) was developed for the analysis of the fungicide pentachloronitrobenzene and several analogues in vegetables. The method was tested in the analysis of carrots, potatoes, green beans, celery, and radishes fortified with pentachloronitrobenzene, tetrachloronitrobenzene, pentachloroanisole, pentachlorothioanisole, pentachlorobenzene, hexachlorobenzene, and pentachloroaniline. An incurred carrot sample analyzed by the method was shown to contain hexachlorobenzene at 7 +/- 3 ng/g, which agreed with the concentration (8 +/- 4 ng/g) determined using a traditional solvent-based method. The SFE method consisted of the following steps: (1) homogenizing a 50 g vegetable sample and weighing a 3 g subsample; (2) mixing 2 g sorbent (Hydromatrix) with the subsample to absorb moisture and packing a 10 mL extraction vessel; (3) extracting with 40 mL CO2 at 200 atm, 40 degrees C, and a flow rate of 3 mL/min; and (4) collecting the extract on a 1 g alumina basic trap at 25 degrees C and flushing with 8 mL isooctane. Collection of the extract on alumina efficiently removed chlorophyll and other matrix interferences. GC/ITMS in the electron-impact mode confirmed and quantitated the analytes at concentrations as low as 1 ng/g.

Evaluation of commercial immunoassays for the detection of alachlor in milk, eggs and liver.

For regulatory purposes, there is a need for rapid, uncomplicated, and inexpensive methods to monitor pesticide residues in food. Commercial immunoassay kits from 3 manufacturers were evaluated for the detection of alachlor in cow milk and urine, and one kit was chosen for assay of chicken eggs and livers. Milk and urine were analyzed after a 1:2 dilution in water, and a rapid extraction procedure was developed for eggs and liver samples. Assays of incurred samples were performed after dosing a cow and several chickens with alachlor. Alachlor was detected in milk and eggs, but not in livers from hens dosed up to 12 mg/kg body weight. The ELISA detection limits were 0.3 ng/mL in milk, 2 ng/g in eggs, and 3 ng/g in liver. The major drawback with the kits was the low cross-reactivity of the antibodies for some of the alachlor metabolites.