CERTIFICATION REPORT The certification of the mass fraction of pesticides in cucumber: ERM-BC403

This report describes the production of ERM®-BC403, which is a cucumber material certified for the mass fraction of selected pesticides. This material was produced following ISO Guide 34:2009 and is certified in accordance with ISO Guide 35:2006. Cucumbers from a biodynamic farm were used as base material. The cucumbers were transformed into slurry, freeze-dried and cryogenically milled. The obtained product was partially reconstituted with water and a separate portion was spiked with a pesticide mixture. The contaminated portion was then mixed with the rest of the material, homogenised and accurately dispensed into 100-ml vials. A second freeze-drying cycle was applied with the vials placed directly in the freeze-dryer. Prior to analysis the resulting sponge of dry cucumber must be reconstituted with a specific volume of water. Between-unit homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006 [ ]. The material was characterised by an interlaboratory comparison of laboratories of demonstrated competence and adhering to ISO/IEC 17025. Technically invalid results were removed but no outliers were eliminated on statistical grounds only. Uncertainties of the certified values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) [ ] and include uncertainties related to possible inhomogeneity, instability and characterisation. The material is intended for the quality control / assessment of method performance. As with any reference material, it can be used for establishing control charts or used for validation of analytical methods. The CRM is available in sets of two glass vials containing each approximately 3.2 g of dried material. The vials were sealed under an atmosphere of argon. The minimum amount of sample to be used is 2.5 g of the reconstituted material.JRC.F.6-Reference Material

Nuevas aportaciones de LC-MS con analizadores de triple cuadrupolo y tiempo de vuelo en el análisis de residuos de plaguicidas y metabolitos en alimentos de origen vegetal.

A lo largo de esta Tesis Doctoral se ha estudiado el potencial analítico del acoplamiento instrumental cromatografía líquida-espectrometría de masas en tándem, haciendo uso de analizadores de triple cuadrupolo (QqQ) e híbrido cuadrupolo-tiempo de vuelo (QTOF), con el objetivo principal de investigar la presencia de residuos de plaguicidas y sus metabolitos en alimentos de origen vegetal. En ese sentido, la tesis aborda uno de los aspectos más importantes en el campo de la salud pública, ya que la presencia de plaguicidas y sus metabolitos en alimentos es un tema de interés toxicológico. La tesis está dividida en dos grandes bloques: un primer bloque centrado en el desarrollo de métodos analíticos mediante LC-QqQ para la determinación de residuos de plaguicidas y sus metabolitos con especial interés por sus características físico-químicas, que dificultan su inclusión en métodos multirresiduales de análisis; un segundo bloque dirigido a la investigación de las capacidades del analizador (Q)TOF tanto en el análisis de plaguicidas, como en la búsqueda e identificación de metabolitos de plaguicidas

Review of analytical methods for the determination of pesticide residues in grapes

This review paper presents an overview of analytical methods used for the analysis of pesticide residues in grapes and by-products in the last decade. The most widely used detection technique used for the determination of pesticides in grapes is mass spectrometry combined with gas and/or liquid chromatography. Multi-residue methods with selective sample treatment methodologies have been developed for this purpose. However, this review focuses not only on these common multi-residue methods but also on specific methodologies for the analysis of pesticides in grapes and by-products. Finally the limitations of multi-residue methods and future perspectives and trends for pesticide residue analysis in grapes are reviewed.JRC.D.5-Standards for Food Bioscienc

Investigation of pesticide metabolites in food and water by LC-TOF-MS

We illustrate the potential of liquid chromatography with (quadrupole) time-of-flight mass spectrometry [LC-(Q)TOF-MS] in investigating the presence of pesticide metabolites in food and water samples. The higher polarity of metabolites compared to their parent pesticides makes the combination of LC [both high-performance (HPLC) and ultra-performance (UPLC)] with TOF-MS one of the most appropriate techniques for their analysis, mainly from a qualitative point of view. Both target analysis and non-target analysis have been explored making use of this technique. Target analysis is typically applied in the inspection of maximum residue limits in food, when a relevant metabolite is included in the residue definition for toxicological reasons or its presence in significant amounts. Within this field, LC-TOF-MS, thanks to its intrinsic characteristics of high sensitivity in full-scan acquisition mode and elevated mass accuracy, has great potential for qualitative purposes, and it allows detection and reliable identification of a large number of metabolites in only one chromatographic run without the need for re-analysis. The latest generations of TOF instruments have also been successfully applied for quantitative purposes. By contrast with food analysis, for drinking water, most regulations of residues do not specify which pesticide metabolites must be included in analysis, and even less so for environmental waters. In such cases, analytical laboratories can focus analysis on a list of (normally) a few target metabolites, or on non-target analysis, where the objective is to identify (pre-defined) metabolites in samples without previously selecting them. We review the use of LC-TOF-MS and LC-(Q)TOF-MS in pesticide-metabolite analysis, considering different approaches:*multi-residue pesticide analysis, where several metabolites are included within the list of target analytes;*investigation of pesticide metabolites in samples that were positive for selected pesticides in previous target analysis;*investigation of metabolites in pesticide-metabolism or degradation studies, typically in laboratory or field experiments under controlled conditions; and,*non-target analysis, where there is no previous information or restrictions on the compounds to be searched in the samples. We show that LC-(Q)TOF-MS is at present one of the most advanced and efficient techniques for screening and identification of pesticide metabolites in food and water. © 2008 Elsevier Ltd. All rights reserved

Quantification, confirmation and screening capability of UHPLC coupled to triple quadrupole and hybrid quadrupole time-of-flight mass spectrometry in pesticide residue analysis

The potential of three mass spectrometry (MS) analyzers (triple quadrupole, QqQ; time of flight, TOF; and quadrupole time of flight, QTOF) has been investigated and compared for quantification, confirmation and screening purposes in pesticide residue analysis of fruit and vegetable samples. For this purpose, analytical methodology for multiresidue determination of 11 pesticides, taken as a model, has been developed and validated in nine food matrices for the three mass analyzers coupled to ultra high pressure liquid chromatography. In all cases, limits of quantification around 0.01 mg/kg were reached, fulfilling the most restrictive case of baby-food analysis. Regarding absolute sensitivity, the lower limits of detection were obtained, as expected, for QqQ (100 fg), whereas slightly higher limits (300 fg) were obtained for both TOF and QTOF. Confirmative capacity of each analyzer was studied for each analyte based on the identification points (IPs) criterion, useful for a comprehensive comparison. QTOF mass analyzer showed the highest confirmatory capacity, although QqQ normally led to sufficient number of IPs, even at lower concentration levels. The potential of TOF MS was also investigated for screening purposes. To this aim, around 50 commercial fruits and vegetables samples were analyzed, searching for more than 400 pesticides. TOF MS proved to be an attractive analytical tool for rapid detection and reliable identification of a large number of pesticides thanks to the full spectrum acquisition at accurate mass with satisfactory sensitivity. This process is readily boosted when combined with specialized software packages, together with theoretical exact mass databases. Several pesticides (e.g. carbendazim in citrus and indoxacarb in grape) were detected in the samples. Further unequivocal confirmation of the identity was performed using reference standards and/or QTOF MS/MS experiments

Use of ultra-high-pressure liquid chromatography–quadrupole time-of-flight MS to discover the presence of pesticide metabolites in food samples

In this paper we illustrate the use of two different methodologies to investigate the presence of pesticide metabolites in parent pesticide-positive food samples, using ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight (UHPLC–QTOF) mass spectrometry. First, a common fragmentation pathway between the parent pesticide and its metabolites has been considered to search for metabolites in two positive market samples (imazalil in lemon, chlorpyrifos in grape). Secondly, olive oil samples from field residue trials were used for automated application of comparative software (MetaboLynx), which was used with treated and untreated samples to search for expected and unexpected metabolites of phosmet. One of the main objectives when using these approaches was to avoid the tedious manual searching for potential metabolites within the huge amount of information contained in the total ion chromatogram acquired by TOF MS. The common fragmentation approach applied to TOF MS full-acquisition data, considering an enhanced fragmentation in the collision cell, has allowed the discovery of two metabolites of imazalil (1-[2-(2,4-dichlorophenyl)-2-hydroxyethyl]-1H-imidazole and 1-[2-(2,4-dichlorophenyl)-2-oxoethyl]-1H-imidazole) in a lemon positive sample, as well as another two metabolites of chlorpyrifos (chlorpyrifos-oxon and 3,5,6-trichloro-2-pyridinol) in a grape positive sample. Moreover, MetaboLynx application to TOF MS data, without promoting fragmentation, from treated and untreated olive oil samples has been helpful in detecting the metabolite phosmet-oxon. In both strategies, every metabolite detected by TOF MS was confirmed using QTOF and/or triple quadrupole instruments. Accurate masses given by TOF MS together with the valuable information on product ions given by QTOF MS/MS experiments were crucial for the unambiguous identification of metabolites

Optimisation and validation of a specific analytical method for the determination of thiram residues in fruits and vegetables by LC–MS/MS

Thiram is a non-systemic dithiocarbamate fungicide, which is easily degraded during sample preparation since it is affected by pH, matrix components and temperature. In this work, specific methodology for thiram analysis in vegetable (eggplant and lettuce) and fruit (strawberry) samples has been developed based on liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Minimising thiram degradation during standards storage and sample preparation was carefully studied. The effect of low temperature (about 5 °C), addition of a dehydrating agent (Na2SO4 anhydrous), pH regulator (NaHCO3), and enzymatic activity reduction (EDTA) during extraction was evaluated. The optimised procedure was validated for eggplants, lettuces, and strawberries. Satisfactory recoveries, between 80% and 106%, and relative standard deviations below 10% were obtained at 0.1 and 0.01 mg/kg fortification levels (n = 5). Limits of detection below 0.0012 mg/kg were achieved. The validated method has been applied to eggplant and lettuce samples collected from different field trials as well as several strawberry and apple samples

Development of a new Cucumber Reference Material for Pesticide Residue Analysis: Feasibility Study for Material Processing, Homogeneity and Stability Assessment

The feasibility of the production of a reference material for pesticide residue analysis in a cucumber matrix was investigated. Cucumber was spiked at 0.075 mg/kg with 15 pesticides, namely acetamiprid, azoxystrobin, carbendazim, chlorpyrifos, cypermethrin, diazinon, (α+β)-endosulfan, fenitrothion, imazalil, imidacloprid, iprodione, malathion, methomyl, tebuconazole and thiabendazole. Three different strategies were considered for material processing, based on the physicochemical properties of the vegetable and the target pesticides. As a result, a frozen spiked slurry of fresh cucumber, a spiked freeze-dried cucumber powder, and a freeze-dried cucumber powder spiked by spraying of the powder were studied. The effects of processing and aspects related to material reconstitution were evaluated by monitoring the pesticide levels in the three materials. Two analytical methods based on LC-MS/MS and GC-MS/MS respectively were developed and validated in-house. The spiked freeze-dried cucumber powder was selected as the most feasible material and more exhaustive studies of homogeneity and stability of pesticide residues in the matrix were carried out. Homogeneity between units was satisfactory at a sample intake of dried material as low as 0.1 g. A 9-week stability study was undertaken following an isochronous design at -20 ºC, 4 ºC and 18 ºC, with -70 ºC as reference temperature. The pesticides tested showed adequate stability at -20 ºC during the 9 weeks period as well as at -70 ºC for a period of 18 months. These results constitute a good basis for the development of a new candidate reference material for selected pesticides in a cucumber matrix.JRC.D.5-Standards for Food Bioscienc

Proficiency test on the determination of pesticide residues in grapes with multi-residue methods

This manuscript presents the results of the International Measurement Evaluation Programme 37 (IMEP-37) study, a proficiency test (PT) which was organized to assess the world-wide performance of food control laboratories on the determination of pesticide residues in grapes. This PT supports the implementation of Regulation (EC) No 396/2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin. Eighty-one participants reported results, forty from EU Member States and forty-one from outside the EU. The test item was a grape sample spiked with 20 selected pesticides. The results of the participants were rated with z- and zeta (ζ-) scores in accordance with ISO 13528 and ISO 17043. The standard deviation for the proficiency assessment, σ, of this PT was set at 25 % for the 20 measured pesticides based on previous experience with similar measurands. The results reported to IMEP-37 showed that the participants performed satisfactorily, ranging from 81 % (carbendazim) to 97 % (azoxystrobin, penconazole, pyrimethanil) of the participating laboratories. However, only 30 % of the participants managed to analyse all pesticides satisfactorily. Overall, the performance of the participants in this PT was good but there is room for improvement in the development of multi-residue methods for the simultaneous analysis of a large number of pesticides with an increased accuracy.JRC.D.5-Standards for Food Bioscienc

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

The applicability of LC-MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC-QTOF MS. © 2009 Elsevier Inc. All rights reserved