Application of a tandem mass spectrometer and core-shell particle column for the determination of 151 pesticides in grains

A comparison of ultrahigh performance liquid chromatography (UHPLC) with a 2.6 μm core-shell particle column (Kinetex C(18)) and conventional liquid chromatography (LC) with a 3 μm porous particle column (Atlantis dC(18)), coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS), for the determination of 151 pesticides in grains is presented in this study. Pesticides were extracted from grain samples using a procedure known as QuEChERS (quick, easy, cheap, effective, rugged, and safe). Quantification, with an analytical range from 5 to 500 μg/kg, was achieved using matrix-matched standard calibration curves with isotopically labeled standards or a chemical analogue as internal standards. The method performance parameters that included overall recovery, intermediate precision, and measurement uncertainty were evaluated using a designed experiment, that is, the nested design. The UHPLC (Kinetex C(18)) was superior to conventional LC (Atlantis dC(18)) as it yielded a shorter analytical run time, increased method sensitivity, and improved method performance. For UHPLC/ESI-MS/MS (Kinetex C(18)), 90% of the pesticides studied had recoveries between 81 and 110%, 88% of the pesticides had intermediate precision ≤20%, and 84% of the pesticides showed measurement uncertainty ≤40%. As compared to UHPLC/ESI-MS/MS (Kinetex dC(18)), the LC/ESI-MS/MS (Atlantis dC(18)) showed a relatively lower sensitivity, less repeatability, and larger measurement uncertainty. UHPLC/ESI-MS/MS with 2.6 μm core-shell particle column and scheduled MRM proved to be a good choice for quantification or determination of pesticides in grains.     

Determination of five macrolide antibiotic residues in eggs using liquid chromatography/electrospray ionization tandem mass spectrometry

A method using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) for the determination of trace levels of five macrolide antibiotics (spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin) in eggs is presented. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two or three fragment ion transitions to provide a high degree of sensitivity and specificity for both quantification and confirmation. Matrix-matched standard calibration curves were used to achieve the best accuracy of the method. A fully nested experimental design was used to study the measurement uncertainty arising from intermediate precision and trueness or proportional bias. The overall recoveries, that is, those determined by the nested experiments, of spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin at fortified levels of 60, 100, 200, and 300 microg/kg were 96.8, 98.2, 98.3, 98.8, and 95.4%, respectively. The LC/ESI-MS/MS method detection limits (S/N > or = 3:1) of five macrolides were <1.0 microg/kg.     

Determination of the predominant catechins in Acacia catechu by liquid chromatography/electrospray ionization-mass spectrometry

A high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI-MS) method under selected ion monitoring mode (SIM) was developed to quantitate the predominant catechins, catechin, epicatechin, epicatechin-3-O-gallate, and epigallocatechin-3-O-gallate, in the medicinal plant catechu (Acacia catechu). Other major secondary products including caffeine, flavanol dimers, and flavonol glycosides were also identified by their molecular ion peaks and fragmentation peaks using LC/MS and LC/MS/MS. For the investigated ion concentration ranges of catechin, epicatechin, epicatechin-3-O-gallate, and epigallocatechin-3-O-gallate, good linearities (r2 > 0.99) were obtained for each calibration curve. Validation for this method showed an accuracy ranging from 1.06 to 11.76%, and the precision (relative standard deviation) varied between 1.60 and 9.36% for these four analytes. This is the first quantitative determination of all predominant catechins in catechu heartwood and leaves.     

A new LC/MS-method for the quantitation of acrylamide based on a stable isotope dilution assay and derivatization with 2-mercaptobenzoic acid. Comparison with two GC/MS methods

Acrylamide (AA) was found to form a stable thioether in reasonable yields (45-50%) when reacted with 2-mercaptobenzoic acid at 20 degrees C for 3 h. On the basis of this finding and using [(13)C(3)]-acrylamide as the internal standard, a sensitive and selective new stable isotope dilution analysis for AA quantitation in food samples was developed based on single stage LC/MS. Comparison of the quantitative results obtained by applying the new method to potato chips, crispbread or butter cookies with data obtained by two stable isotope dilution analysis, using direct measurement of AA by GC/MS, but differing in the workup procedure, revealed detection limits in the same order of magnitude (6.6 microg/kg). Quantitative data obtained by application of the three methods on the same samples of potato chips or cookies, respectively, were also in very good agreement. Quantitation of AA in crispbreads treated with an amylase/protease mixture did not show increased AA levels, thereby indicating that inclusion of AA in starch/protein gels is not very probable during breadmaking.     

Determination of seven glycidyl esters in edible oils by gel permeation chromatography extraction and liquid chromatography coupled to mass spectrometry detection

A method based on a gel permeation chromatography (GPC) extraction procedure combined with an additional cleanup by solid-phase extraction (SPE) on silica gel and liquid chromatography-mass spectrometry (LC-MS) detection has been validated for the analysis of seven glycidyl esters (GEs) including glycidyl laurate, myristate, palmitate, stearate, oleate, linoleate, and linolenate in various edible oils. This method was conjointly developed and validated by two different laboratories, using two different detection systems, a LC time of flight MS (LC-ToF-MS) and a LC triple-quadrupole MS (LC-MS/MS). The extraction procedure allowed targeting low contamination levels due to a highly efficient matrix removal from the 400 mg oil sample loaded on the GPC column and is suitable for routine analysis as 24 samples can be extracted in an automated and reproducible way every 12 h. GPC extraction combined with SPE cleanup and LC-MS/MS detection leads to a limit of quantification in oil samples between 50 and 100 μg/kg depending on the type of glycidyl ester. Recoveries ranged from 68 to 111% (average = 93%). Quantification was performed by automated standard addition on extracts to compensate matrix effects artifacts. To control recoveries of each sample four isotopically labeled GEs ((13)C(3) or (13)C(4)) were included in the method.     

Determination of pesticides and PCBs in virgin olive oil by multicolumn solid-phase extraction cleanup followed by GC-NPD/ECD and confirmation by ion-trap GC-MS

A multicolumn solid-phase extraction cleanup for the determination of organophosphorus (OP) and organochlorine (OC) pesticides plus PCB congeners in virgin olive oil is presented. The method involves dissolution of the olive oil in hexane, followed by a cleanup system using a diatomaceous earth column (Extrelut-QE) with reversed (C(18)) and normal (alumina) phase SPE columns. Determination of OPs was by GC-NPD, while the OCs and PCBs were analyzed using GC-ECD. Recovery assays for OPs varied from 81.7% to 105.3%, for OCs ranged between 74.3% and 99.4%, while for PCBs were from 60.1% to 119.2%. Quantitation limits ranged from 10 to 25 microg/kg olive oil for OPs, and from 1 to 6 microg/kg olive oil for OCs and PCBs. In the case of positive samples, the confirmation of pesticide identity was performed by ion-trap GC-MS/MS. The applicability of the method was assayed with 19 virgin olive oil samples collected from different olive mills of Aragón (Spain). Only one OP pesticide (acephate) was detected in one sample at a concentration of 10 microg/kg. Organochlorine pesticides were found in 5-47% of samples at very low levels ranging from 1.5 to 5.2 microg/kg. PCBs were found in 20-90% of samples, showing concentrations between 2.3 and 17.3 microg/kg.     

Quantitative analysis of tylosin in eggs by high performance liquid chromatography with electrospray ionization tandem mass spectrometry: residue depletion kinetics after administration via feed and drinking water in laying hens

Maximum residue limits (MRLs) have been established by the European Union when tylosin is used therapeutically. They are fixed at 200 microg/kg for eggs. A highly sensitive and selective quantitative liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) method suitable for monitoring tylosin residues in eggs to determine its depletion kinetics was developed and validated. For sample pretreatment all samples were liquid-liquid extracted with citrate buffer (pH 5.0) and acetonitrile. Liquid chromatographic separation was carried out on a reversed phase C18 column employing a 0.5% formic acid/acetonitrile gradient system. The tylosin recovery in eggs at a concentration range from 1.0-400 microg/kg was >82% with relative standard deviations between 1.5 and 11.0%. In two experimental studies administrating tylosin via feed (final dosage: 1.5 g/kg) or drinking water (final dosage: 0.5 g/L), no residues above the MRL were found during and after treatment. Moreover, all samples were well below the actual MRL of 200 microg/kg. Therefore, our residue data suggest that a withholding period for eggs is not required when laying hens are treated with tylosin in recommended dosages via feed or drinking water. Keywords: Tylosin; residue; depletion; laying hen; withholding period; mass spectrometry.     

Rugged LC-MS/MS survey analysis for acrylamide in foods

The described liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of acrylamide in food entails aqueous room temperature extraction, SPE cleanup, and analysis by LC-MS/MS. The method is applicable to a wide variety of foods. [(13)C(3)]acrylamide is the internal standard. The limit of quantitation is 10 ppb (microg/kg). Data were obtained in duplicate from >450 products representing >35 different food types. The variability in analyte levels in certain food types suggests that it may be possible to reduce acrylamide levels in those foods.     

Analysis of organophosphorus pesticides in dried ground ginseng root by capillary gas chromatography-mass spectrometry and -flame photometric detection

A method was developed to determine organophosphorus pesticides (OPs) in dried ground ginseng root. Pesticides were extracted from the sample using acetonitrile/water saturated with salts, followed by solid-phase dispersive cleanup, and analyzed by capillary gas chromatography with electron ionization mass spectrometry in selective ion monitoring mode (GC-MS/SIM) and flame photometric detection (GC-FPD) in phosphorus mode. The detection limits for most of the pesticides were 0.025-0.05 microg/g using GC-FPD but were analyte-dependent for GC-MS/SIM, ranging from 0.005 to 0.50 microg/g. Quantitation was determined from 0.050 to 5.0 microg/g with r 2 > 0.99 for a majority of the pesticides using both detectors. Recovery studies were performed by fortifying the dried ground ginseng root samples to concentrations of 0.025, 0.1, and 1.0 microg/g, resulting in recoveries of >90% for most pesticides by GC-FPD. Lower (<70%) and higher (>120%) recoveries were most likely from complications of pesticide lability or volatility, matrix interference, or inefficient desorption from the solid-phase sorbents. There was difficulty in analyzing the ginseng samples for the OPs using GC-MS at the lower fortification levels for some of the OPs due to lack of confirmation. GC-FPD and GC-MS/SIM complement each other in detecting the OPs in dried ground ginseng root samples. This procedure was shown to be effective and was applied to the analysis of OPs in ginseng root samples. One particular sample, a ground and dried American ginseng (Panax quinquefolius) root sample, was found to contain diazinon quantified at approximately 25 microg/kg by external calibration using matrix-matched standards or standard addition using both detectors. The advantage of using both detectors is that confirmation can be achieved using GC-MS, whereas the use of a megabore column in GC-FPD can be used to quantitate some of the nonpolar OPs without the use of matrix-matched standards or standard addition.     

Determination of acequinocyl and hydroxyacequinocyl on fruits and vegetables by HPLC-DAD

A method for determining residues of the new reduced-risk pesticide acequinocyl and its deacetylated derivative hydroxyacequinocyl on fruits and vegetables (grapes, lemons, pears, and tomatoes) by HPLC is described. The pesticides were extracted from the fruits and vegetables with hexane and ethyl acetate solution (1:1, v/v), determined by HPLC-DAD at 250 nm and confirmed by LC/MS. No cleanup was necessary. This method is characterized by recoveries (0.01-4 mg/kg) > 77%, while the coefficient of variation was determined to be less than 11%. The limit of quantitation for both acequinocyl and hydroxyacequinocyl was 0.01 mg/kg for all matrixes.     

Analysis of pesticides in honey by solid-phase extraction and gas chromatography-mass spectrometry

An analytical method for the simultaneous determination of 51 pesticides in commercial honeys was developed. Honey (10 g) was dissolved in water/methanol (70:30; 10 mL) and transferred to a C(18) column (1 g) preconditioned with acetonitrile and water. Pesticides were subsequently eluted with a hexane/ethyl acetate mixture (50:50) and determined by gas chromatography with electron impact mass spectrometric detection in the selected ion monitoring mode (GC-MS-SIM). Spiked blank samples were used as standards to counteract the matrix effect observed in the chromatographic determination. Pesticides were confirmed by their retention times, their qualifier and target ions, and their qualifier/target abundance ratios. Recovery studies were performed at 0.1, 0.05, and 0.025 microg/g fortification levels for each pesticide, and the recoveries obtained were >86% with relative standard deviations of <10%. Good resolution of the pesticide mixture was achieved in approximately 41 min. The detection limits of the method ranged from 0.1 to 6.1 microg/kg for the different pesticides studied. The developed method is linear over the range assayed, 25-200 microg/L, with determination coefficients of >0.996. The proposed method was applied to the analysis of pesticides in honey samples, and low levels of a few pesticides (dichlofluanid, ethalfluralin, and triallate) were detected in some samples.     

Evaluation of fumonisin contamination in cornflakes on the Belgian market by "flow-through" assay screening and LC-MS/MS analyses

A total of 205 cornflake samples collected in Belgian retail stores during 2003-2004 were surveyed for the natural occurrence of fumonisin B1 (FB1), B2 (FB2), and B3 (FB3). These cornflake samples, originating from conventional as well as from organic production, were analyzed using an intralaboratory-validated LC-MS/MS method. Additionally, 90 cornflake samples were subjected to rapid screening using a flow-through enzyme immunoassay method to demonstrate the practicability of a screening test coupled to a validated confirmatory LC-MS/MS method for the management of food safety risks. FB(1) concentrations ranged from not detected (nd) [LOD (FB1) = 20 microg/kg] to 464 microg/kg with mean and median concentrations of respectively 104 +/- 113 and 54 microg/kg. For FB2 and FB3, the concentration ranges varied respectively from nd [LOD (FB2) = 7.5 microg/kg] to 43 microg/kg and from nd [LOD (FB3) = 12.5 microg/kg] to 90 microg/kg. Mean concentrations for FB2 and FB3 were respectively 12 +/- 8 and 21 +/- 15 microg/kg, while the median concentration was 11 microg/kg for FB2 and 19 microg/kg for FB3. From the statistical tests (chi2 and ANOVA model III), it could be concluded that the agricultural practice did not have any significant effect on the fumonisin concentrations but that the variation between different batches was significant (p < 0.0001).     

Multiresidue analysis of pesticides in soil by gas chromatography with nitrogen-phosphorus detection and gas chromatography mass spectrometry

A rapid multiresidue method for the simultaneous determination of 25 fungicides and insecticides in soil was developed. Soil samples are extracted by sonication with a water-acetonitrile mixture, and the pesticides are partitioned into dichloromethane. Final determination was made by gas chromatography (GC) with nitrogen-phosphorus detection (NPD). Confirmation analysis of pesticides was carried out by GC-MS in the selected ion monitoring (SIM) mode. The identification of compounds was based on retention time and on comparison of the primary and secondary ions. The average recovery by the GC-NPD method obtained for these compounds varied from 68.5% to 112.1% with a relative standard deviation between 1.8% and 6.2%. The GC-NPD method presents good linearity over the range assayed 50-2000 microg/L, and the detection limit for the pesticides studied varied from 0.1 to 10.4 microg/kg. The proposed method was used to determine pesticide levels in soil samples from experimental greenhouse pepper cultivation.     

Characterization of triterpene alcohol and sterol ferulates in rice bran using LC-MS/MS

Ferulic acid esters of triterpene alcohols and sterols in rice bran oil have been extensively studied and reported to possess important pharmacological actions. Inconsistent results on the numbers and structures of ferulates have been reported, primarily because of the analytical procedures employed. Conventional methods for analysis of phytosterol content in oil are carried out by characterization of trimethylsilylated derivatives (TMS) using GC-EI-MS after saponification of oils or individual compound isolated from oils. This study developed an LC-MS/MS method for the direct analysis of triterpene alcohol and sterol esters in rice bran oil. In addition to verifying the results of previous research, nine new relatively polar triterpene alcohol and sterol esters were characterized by their retention behaviors in LC and ESI-MS data from both negative- and positive-ion mode. This is the first evidence for the presence of hydroxylated ferulate esters and caffeate esters as part of gamma-oryzanol in rice bran. The method enables rapid and direct on-line characterization of triterpene alcohol and sterol esters in oils. LC-MS/MS equipped with reverse-phase LC and ESI-MS should be well-suited for identification and quantification of the polar metabolites of phytosterols in biological fluids after consumption of rice bran oil or other oils.     

Simultaneous identification of soyasaponins and isoflavones and quantification of soyasaponin Bb in soy products,using liquid chromatography/electrospray ionization-mass spectrometry

A method has been developed for simultaneous identification of soyasaponins and soy isoflavones in soy products, based on liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Soy-based nutraceutical products were analyzed by LC/ESI-MS with detection of protonated and sodiated molecular ions, as well as characteristic fragment ions for these compounds. Soy isoflavones were characterized by a strong protonated molecular ion in addition to corresponding [aglycone + H](+) ions. Monitoring the soyasaponin-specific protonated aglycone and dehydrated aglycone ions throughout the chromatogram provided a unique fingerprint for soyasaponin content in the samples. This mass spectrometric fingerprint also allowed immediate classification of the soyasaponin analytes as group A or B soyasaponins, based on the unique masses of aglycone ions observed for each class. Quantification of soyasaponin B(b) in soy-derived materials, based on the use of a purified soyasaponin B(b) standard and a glycyrrhizin internal standard, has been accomplished.     

Quantification of imidacloprid uptake in maize crops

The systemic imidacloprid is one of the most used insecticides in the world for field and horticultural crops. This neurotoxicant is often used as seed-dressing, especially for maize, sunflower, and rape. Using a LC/MS/MS technique (LOQ = 1 microg/kg and LOD = 0.1 microg/kg), the presence of imidacloprid has been measured in maize from field samples at the time of pollen shed, from less than 0.1 microg/kg up to 33.6 microg/kg. Numerous random samples were collected throughout France from 2000 to 2003. The average levels of imidacloprid measured are 4.1 microg/kg in stems and leaves, 6.6 microg/kg in male flowers (panicles), and 2.1 microg/kg in pollen. These values are similar to those found previously in sunflower and rape. These results permit evaluation of the risk to honeybees by using the PEC/PNEC ratios (probable exposition concentrations/predicted no effect concentration). PEC/PNEC risk ratios were determined and ranged between 500 and 600 for honeybees foraging on maize treated with imidacloprid by seed dressing. Such a high risk factor can be related to one of the main causes of honeybee colony losses.     

Determination of oryzalin in water, citrus fruits, and stone fruits by liquid chromatography with tandem mass spectrometry

A new methodology is described for rapidly determining the herbicide oryzalin in water, citrus fruits, and stone fruits by liquid chromatography with negative ion electrospray ionization tandem mass spectrometry (LC/MS/MS). Oryzalin is extracted from water using a polymeric sorbent solid phase extraction (SPE) column and from fruit using methanol. The water samples require no further purification, but an aliquot of the fruit sample extracts is diluted with water and purified using a polymeric 96 well SPE plate. Purified extracts are concentrated prior to determination by LC/MS/MS at m/z 345 (Q1) and m/z 281 (Q3) using an external standard for calibration. The validated limits of quantitation were 0.05 microg/L in water (drinking water, surface water, and groundwater) and 0.01 microg/g in citrus fruits (oranges and lemons) and stone fruits (peaches and cherries). Recoveries averaged 102% for water samples and 85-89% for the various types of fruit samples. For all fortification levels combined, the relative standard deviations ranged from 4 to 6% for water and from 2 to 4% for fruit.     

Soil microbial activity is affected by Roundup WeatherMax and pesticides applied to cotton (Gossypium hirsutum)

Adoption of glyphosate-based weed control systems has led to increased use of the herbicide with continued use of additional pesticides. Combinations of pesticides may affect soil microbial activity differently than pesticides applied alone. Research was conducted to evaluate the influence of glyphosate-based cotton pest management systems on soil microbial activity. Soil was treated with commercial formulations of trifluralin, aldicarb, and mefenoxam + pentachloronitrobenzene (PCNB) with or without glyphosate (applied as Roundup WeatherMax). The soil microbial activity was measured by quantifying C and N mineralization. Soil microbial biomass was determined using the chloroform fumigation-incubation method. Soils treated with glyphosate alone exhibited greater cumulative C mineralization 30 days after treatment than all other treatments, which were similar to the untreated control. The addition of Roundup WeatherMax reduced C mineralization in soils treated with fluometuron, aldicarb, or mefenoxam + PCNB formulations. These results indicate that glyphosate-based herbicides alter the soil microbial response to other pesticides.     

Simplified multiresidue method for liquid chromatographic determination of N-methyl carbamate insecticides in fruits and vegetables

A simplified method is described for determining 7 N-methyl carbamates (aldicarb, carbaryl, carbofuran, methiocarb, methomyl, oxamyl, and propoxur) and 3 related metabolites (aldicarb sulfoxide, aldicarb sulfone, and 3-hydroxy carbofuran) in fruits and vegetables. Residues are extracted from crops with methanol; coextractives are then separated by gel permeation chromatography (GPC) or GPC with on-line Nuchar-Celite cleanup for crops with high chlorophyll and/or carotene content (e.g., cabbage and broccoli). Carbamates are separated on a reverse-phase liquid chromatography column, using a methanol-water gradient mobile phase. Separation is followed by postcolumn hydrolysis to yield methylamine, and the formation of a fluorophore with o-phthalaldehyde and 2-mercaptoethanol prior to fluorescence detection. Recovery data were obtained by fortifying 5 different crops (apples, broccoli, cabbages, cauliflower, and potatoes) at 0.05 and 0.5 ppm. Recoveries averaged 93% at both fortification levels except for the very polar aldicarb sulfoxide for which recoveries averaged around 52% at both levels. The coefficient of variation of the method at both levels is less than 5% and the limit of detection, defined at 5 times baseline noise, varies between 5 and 10 ppb, depending on the compound.     

Analysis of acrylamide,a carcinogen formed in heated foodstuffs

Reaction products (adducts) of acrylamide with N termini of hemoglobin (Hb) are regularly observed in persons without known exposure. The average Hb adduct level measured in Swedish adults is preliminarily estimated to correspond to a daily intake approaching 100 microg of acrylamide. Because this uptake rate could be associated with a considerable cancer risk, it was considered important to identify its origin. It was hypothesized that acrylamide was formed at elevated temperatures in cooking, which was indicated in earlier studies of rats fed fried animal feed. This paper reports the analysis of acrylamide formed during heating of different human foodstuffs. Acrylamide levels in foodstuffs were analyzed by an improved gas chromatographic-mass spectrometric (GC-MS) method after bromination of acrylamide and by a new method for measurement of the underivatized acrylamide by liquid chromatography-mass spectrometry (LC-MS), using the MS/MS mode. For both methods the reproducibility, given as coefficient of variation, was approximately 5%, and the recovery close to 100%. For the GC-MS method the achieved detection level of acrylamide was 5 microg/kg and for the LC-MS/MS method, 10 microg/kg. The analytic values obtained with the LC-MS/MS method were 0.99 (0.95-1.04; 95% confidence interval) of the GC-MS values. The LC-MS/MS method is simpler and preferable for most routine analyses. Taken together, the various analytic data should be considered as proof of the identity of acrylamide. Studies with laboratory-heated foods revealed a temperature dependence of acrylamide formation. Moderate levels of acrylamide (5-50 microg/kg) were measured in heated protein-rich foods and higher contents (150-4000 microg/kg) in carbohydrate-rich foods, such as potato, beetroot, and also certain heated commercial potato products and crispbread. Acrylamide could not be detected in unheated control or boiled foods (<5 microg/kg). Consumption habits indicate that the acrylamide levels in the studied heated foods could lead to a daily intake of a few tens of micrograms.