Development and full validation of a multiresidue method for the analysis of a wide range of pesticides in processed fruit by UHPLC-MS/MS.

A reliable 16-min analytical method for the simultaneous determination of 250 pesticides in processed fruit using ultra-performance liquid chromatography, coupled to tandem mass spectrometry (UHPLC-MS/MS), was developed and validated according to SANTE 11813/2017 guidelines and accredited successfully based on ISO 17025. Extraction was achieved using a modified QuEChERS method, without any clean-up, including a dilution to obtain good peak shapes and to reduce matrix effects. Pesticides were quantified using matrix-matched calibration, and the method was validated in terms of relative retention time window, linearity (6-167 µg kg-1 and 0.6-16.7 µg kg-1, coefficient R2 ≥ 0.98), trueness (recovery of 70-120%), selectivity, precision (RSD ≤ 20%), limits of quantification (LOQs = 0.6-6.0 µg kg-1) and uncertainty. Finally, the method was applied to the routine analysis of 103 samples of processed fruits, detecting the presence of several pesticide residues, such as fluopyram, spinosad or cyprodinil (0.006-0.22 mg kg-1).

Solid phase microextraction and gas chromatography coupled to magnetic sector high resolution mass spectrometry for the ultra-trace determination of contaminants in surface water.

With the aim of monitoring water quality according to the regulations established by the European Union it would be necessary to implement analytical methodologies capable of simultaneously determining a broad range of organic pollutants at ultra-trace levels, allowing for increased sample throughput. In addition, the high number of samples to be analyzed requires a particular focus on setting up fully automated analytical methodologies. In view of that, this study is aimed at the development of a complete automated procedure for the ultra-trace determination of certain pesticides, polycyclic aromatic hydrocarbons (PAHs), brominated diphenyl ethers (BDEs) and polychlorinated biphenyls (PCBs) in surface waters. The proposed method is based on an on-line combination of solid phase microextraction (SPME) and gas chromatography coupled to double-focusing magnetic sector high resolution mass spectrometry (GC-HRMS). SPME as well as GC-HRMS conditions were optimized to achieve maximum extraction efficiency and sensitivity, which was reinforced by using multiple ion detection (MID) as acquisition mode. Using only 19mL of water and with minimum sample manipulation, the method allowed for the determination of 53 compounds exhibiting good linearity (R2>0.99), recoveries between 84 and 118% and relative standard deviation (RSD) values <20% for intra-day and inter-day precision. In addition, the method provides quantification limits (LOQs) between 0.1-50ngL-1, lower than the Environmental Quality Standards (EQS) fixed by Directive 2013/39/EC. Finally, the method was successfully applied to determine target contaminants in Almería surface water compartments, detecting dioxin-like PCBs, BDEs and some pesticides.

Simultaneous analysis of chlorophenols, alkylphenols, nitrophenols and cresols in wastewater effluents, using solid phase extraction and further determination by gas chromatography-tandem mass spectrometry.

An analytical methodology has been developed for the simultaneous extraction of 13 phenolic compounds, including chlorophenols (CPs), nitrophenols (NTPs), cresols and alkylphenols (APs) in different types of wastewater (WW) effluents. A solid-phase extraction (SPE) method has been optimized prior to the determination by gas chromatography coupled to triple quadrupole tandem mass spectrometry (GC-QqQ-MS/MS). Due to the complexity of the matrix, a comparison study of matrix-matched-calibration (MMC) and standard addition calibration (SAC) was carried out for quantification purposes. The optimized procedure was validated using the SAC approach since it provided the most adequate quantification results (in terms of recovery and precision values). Recoveries were in the range 60-135% (0.5 µg L(-1)), 70-115% (1 µg L(-1)), and 78-120% (5 µg L(-1)), with precision values (expressed as relative standard deviation, RSD) ≤ 30% (except for 2-nitrophenol) involving intra-day and inter-day precision studies. Limits of detection (LODs) and quantification (LOQs) were also evaluated, and LOQs ranged from 0.03 µg L(-1) to 2.5 µg L(-1). The proposed method was applied to the analysis of 8 real WW effluent samples, finding some phenolic compounds (e.g. 2-chlorophenol, 2,4,6-trichlorophenol and 4-tert-octylphenol) at concentrations higher than the established LOQs.

Simultaneous determination of pesticides, biopesticides and mycotoxins in organic products applying a quick, easy, cheap, effective, rugged and safe extraction procedure and ultra-high performance liquid chromatography-tandem mass spectrometry.

A method for the simultaneous determination of pesticides, biopesticides and mycotoxins from organic products was developed. Extraction of more than 90 compounds was evaluated and performed by using a modified QuEChERS-based (acronym of Quick, Easy, Cheap, Effective, Rugged, and Safe) sample preparation procedure. The method was based on a single extraction with acidified acetonitrile, followed by partitioning with salts, avoiding any clean-up step prior the determination by ultra-high performance liquid chromatography/tandem quadrupole mass spectrometry (UHPLC-MS/MS). Validation studies were carried out in wheat, cucumber and red wine as representative matrixes. Recoveries of the spiked samples were in the range between 70 and 120% (with intra-day precision, expressed as relative standard deviation, lower than 20%) for most of the analysed compounds, except picloram and quinmerac. Inter-day precision, expressed as relative standard deviation, was lower than 24%. Limits of quantification were lower than 10µg kg⁻¹ and the developed method was successfully applied to the analysis of organic food products, detecting analytes belonging to the three types of compounds.

Determination of pesticide transformation products: a review of extraction and detection methods.

Pesticides are widely applied and they can produce a variety of transformation products (TPs), through different pathways and mechanisms. Nowadays there is a growing interest related to the determination of pesticide TPs in several matrices (environmental, food and biological samples), due to these compounds can be more toxic and persistent than parent compounds, and some of them can be used as markers of exposure to different pesticides. Although solid-phase extraction (SPE) is mainly used for the extraction of TPs, alternative techniques such as solid-phase microextraction (SPME) and liquid-phase extraction (LPE) can be used. These TPs are mainly determined by liquid chromatography (LC) due to the recent developments in this technique, especially when it is coupled to mass spectrometry (MS) detectors, allowing the determination of known and/or unknown TPs. Furthermore, MS is a very valuable tool for the structural elucidation of unknown TPs. This review discusses all phases of analytical procedure, including sample treatment and analysis, indicating the main problems related to the extraction of TPs from several matrices due to their high polarity, as well as the different alternatives found for the simultaneous determination of parent compounds and TPs, using chromatographic techniques coupled to MS detection.

Comparison of tandem-in-space and tandem-in-time mass spectrometry in gas chromatography determination of pesticides: application to simple and complex food samples.

Gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) is one of the most powerful techniques in pesticide residue analysis. MS/MS can be conceived in two ways: tandem in space (e.g. triple quadrupole, QqQ) or in time (e.g. ion trap, IT). QqQ and IT are commonly interfaced to GC; however, there has not been any direct comparison between them in pesticide residue analysis so far. In the present work, the performance of GC coupled to these two analyzers (GC-QqQ-MS/MS and GC-IT-MS/MS) was studied and compared for pesticide residue analysis as well as its application in food analysis. The large volume injection (LVI) technique together with programmed-temperature vaporization (PTV) was applied. For this purpose, 19 pesticides, including organochlorine and organophophorus pesticides and pyrethroids, were analyzed in both systems. Mass spectrometric data, performance characteristics (linearity, intra-day and inter-day precision) and the influence of the matrix nature on the analysis of low concentrations were compared. The target compounds were analyzed in solvent and in two representative food matrices such as cucumber (high water content) and egg (high fat content). MS data and intra-day precision were similar in QqQ and IT, whereas inter-day precision was significantly worse in QqQ. Linearity (expressed as determination coefficient, R(2)) in the range 10-150 microg L(-1) was adequate in both systems; however, better R(2) values were obtained with the QqQ analyzer in high and low concentration ranges (1-50 and 1-750 microg L(-1), respectively). The influence of the matrix nature on the analysis of low concentrations of each analyzer was also evaluated. The QqQ and IT performance was similar in cucumber and solvent. However, QqQ provided better sensitivity in egg working in selected reaction monitoring (SRM).

Multiresidue method for fast determination of pesticides in fruit juices by ultra performance liquid chromatography coupled to tandem mass spectrometry.

A new analytical method for the simultaneous determination of 90 pesticides in fruit juices by ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) has been developed and validated. Extraction was performed with acetonitrile, applying QuEChERS methodology, and the extracts were analyzed without any further clean-up step, providing better results than solid phase extraction (SPE) procedure. Before chromatographic step, extracts were diluted with water (1:1) in order to obtain good peak shapes. Several chromatographic conditions were evaluated in order to achieve a fast separation in Multiple Reaction Monitoring (MRM) mode, obtaining a run time of only 11 min. Matrix effect was studied for different types of fruit juices (peach, orange, pineapple, apple and multifruit), indicating that multifruit juice can be selected as representative matrix for routine analysis of these food commodities. Pesticides were quantified using matrix-matched calibration with recoveries between 70.4 and 108.5% and relative standard deviation lower than 20%. Limits of quantification were lower than 5 microg L(-1) in all the cases. The developed procedure was applied to commercial fruit juices, detecting carbendazim, cyprodinil and thiabendazol in a few samples.

High-throughput determination of pesticide residues in food commodities by use of ultra-performance liquid chromatography-tandem mass spectrometry.

A rapid, simple, and sensitive multiresidue method for analysis of 53 pesticides in fruit and vegetables by ultra-performance liquid chromatography (UPLC) coupled to triple-quadrupole tandem mass spectrometry (MS-MS) has been developed and validated. Prior to analysis, analytes were extracted by use of buffered QuEChERS (quick, easy, cheap, effective, rugged, safe) methodology without further cleanup for non fatty matrices. Chromatographic conditions were optimised in order to achieve a fast separation in multiple reaction monitoring (MRM) mode. Indeed, more than 50 pesticides can be separated in less then 10 min. Four common representative matrices (cucumber, orange, strawberry, and olive) were selected to investigate the effect of different matrices on recovery and precision. Mean recoveries ranged from 70 to 109% with relative standard deviations lower than 20% for all the pesticides assayed in the four selected matrices. The method has been applied to the analysis of 200 vegetable samples, and imidacloprid was the pesticide most frequently found, with concentrations ranging from 0.01 to 1.00 mg kg(-1). This methodology combines the advantages of both QuEChERS and UPLC-MS-MS producing a very rapid, sensitive, and reliable procedure which can be applied in routine analytical laboratories.

Simultaneous determination of selected veterinary antibiotics in gilthead seabream (Sparus Aurata) by liquid chromatography-mass spectrometry.

A method was optimised and validated for simultaneous monitoring of several drugs of different classes of antibiotics such as quinolones (oxilinic acid and flumequine), tetracyclines (oxytetracycline), sulfonamides (sulfadiazine) and trimethoprim in fish muscle and skin. The method is based on solid-liquid extraction without further sample clean up followed by liquid chromatography-mass spectrometry (LC-MS) determination with electrospray ion source (ESI) in positive mode. The limits of quantification (LOQs) were lower than 20 microg/kg for all compounds and repeatability, expressed as relative standard deviations (RSD), were lower than 15%. Therefore, the LC-MS method was successfully applied for the quantitative determination of antibiotics in gilthead sea bream muscle and skin and oxytetracycline in medicated fishes.

Characterization of recovery profiles using gas chromatography-triple quadrupole mass spectrometry for the determination of pesticide residues in meat samples.

The assessment of the recovery factor with the analyte concentration in meat samples has been studied for the determination of organochlorine and organophosphorus pesticides in meat by gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS). For that purpose, recent IUPAC recommendations, which distinguishes between two terms, recovery factor and apparent recovery, have been followed. Besides, the systematic error due to the matrix effect has been evaluated by a new term recently proposed, calibration recovery. Recovery profiles were obtained analyzing spiked blank matrix, where the analytes were added before and after the extraction procedure. In a first step, the quantification of the compounds was carried out using a solvent calibration curve. The systematic errors due to the matrix effect during the quantification step and the error due to the sample treatment have been evaluated. Both apparent and calibration recovery components depend on the actual analyte concentration in the sample while the recovery factor remains constant except for analyte concentration close to quantification limit. In addition, the concentration limits, from which an acceptable recovery value (70-110%) can be obtained, are given. If spiked samples are quantified by matrix-matched calibration, the matrix effect is minimized and the calibration recovery component is 100%, and apparent recovery only depends on the recovery factor. The obtained values indicate recovery factor does not depend on the analyte concentration, except for those values closed to quantification limit.

Validation of a gas chromatography/triple quadrupole mass spectrometry based method for the quantification of pesticides in food commodities.

A new multiresidue method has been validated in cucumber matrix for the routine analysis of 130 multiclass pesticide residues by gas chromatography/triple quadrupole mass spectrometry. The pesticides were extracted with ethyl acetate. A first identification of the pesticides was based on a tandem mass spectrometric (MS/MS) screening method, which monitors a single transition for each target compound, in less than 12 min. After that, potentially non-negative samples were analyzed again by the MS/MS confirmation/quantification method, which monitors two or three MS/MS transitions for each compound, also in less than 12 min. Performance characteristics, such as trueness, precision, linear range, detection limit (LOD) and quantification limit (LOQ), for each pesticide were calculated. The average recoveries obtained ranged between 70 and 120% at three different fortification levels (25, 200 and 500 microg/kg) with precision, expressed as relative standard deviation (RSD), values lower than 15%. The calculated LOD and LOQ were typically <3.2 and 9.6 microg/kg, respectively. Such limits were much lower than the maximum residue levels (MRLs) established by European legislation. The proposed methodology was applied to the determination of pesticides in real vegetable samples from Almería (Spain).

Determination of multiclass pesticides in food commodities by pressurized liquid extraction using GC-MS/MS and LC-MS/MS.

Pressurized liquid extraction (PLE) was applied to the simultaneous extraction of a wide range of pesticides from food commodities. Extractions were performed by mixing 4 g of sample with 4 g of Hydromatrix and (after optimization) a mixture of ethyl acetate:acetone (3:1, v/v) as extraction solvent, a temperature of 100 degrees C, a pressure of 1000 psi and a static extraction time of 5 min. After extraction, the more polar compounds were analyzed by liquid chromatography (LC), and the apolar and semipolar pesticides by gas chromatography (GC); in both cases LC and GC were coupled with mass spectrometry in tandem (MS/MS) mode. The overall method (including the PLE step) was validated in GC and LC according to the criteria of the SANCO Document of the European Commission. The average extraction recoveries (at two concentration levels) for most of the analytes were in the range 70-80%, with precision values usually lower than 15%. Limits of quantification (LOQ) were low enough to determine the pesticide residues at concentrations below or equal to the maximum residue levels (MRL) specified by legislation. In order to assess its applicability to the analysis of real samples, aliquots of 15 vegetable samples were processed using a conventional extraction method with dichloromethane, and the results obtained were compared with the proposed PLE method; differences lower than 0.01 mg kg(-1) were found.

Analysis of biogenic volatile organic compounds in zucchini flowers: identification of scent sources.

An analytical method has been applied to determine volatile organic compounds in zucchini flowers. In a first step, the analytical method was applied to characterize the main scents emitted by whole male and female living flowers of three main commercial cultivars of zucchini (Tosca, Chapin, and Consul). In a second step, the compounds were quantified in different parts of the living flowers to identify the contribution of nectar, petals, anther, and stigma to the aroma profile of the flower. The analytical method is based on headspace solid-phase microextraction coupled on-line with GC and tandem MS detection (HS-SPME-GC-MS/MS). A reference compound is added to samples as part of the field quality control procedure to check for likely analyte losses or sample decomposition. The reference compound also acts as an internal standard for quantification purposes. Results have been statistically studied applying principal component analysis (PCA), which shows that three components explain more than 91% of the variance. PCA emphasizes the great importance of nectar as being the main source of 1,4-dimethoxybenzene and 1,2,4-trimethoxybenzene, which influence the aroma profile of flowers. The remaining components can be accounted for by emissions from petals and sexual organs (adroecium and gynoecium anthers or stigmas).

Determination of pesticides and some metabolites in different kinds of milk by solid-phase microextraction and low-pressure gas chromatography-tandem mass spectrometry.

A new analytical method is proposed to determine more than 40 multiclass pesticides in different kinds of processed (whole, skimmed and powdered) and unprocessed (goat and human) milk samples using solid-phase microextraction (SPME). A comparative study between headspace (HS) and direct immersion (DI) was carried out. The effect of milk dilution and the use of acid to reduce the influence of the matrix in DI-SPME mode were also evaluated. DI of the SPME fiber into previously diluted and acidified milk samples achieved the best sensitivity results. Pesticides were determined using low-pressure gas chromatography-tandem mass spectrometry (LP-GC-MS/MS). Both of the selected techniques have been shown to be effective at reduce fat interference and can determine analytes present at very low concentrations (limits of quantification between 0.02 and 1.00 microg L(-1)). Performance characteristics such as linearity, recovery, precision, and lower limits, together with an estimation of the measurement uncertainty using validation data, are presented for each pesticide. All of the pesticides presented recovery rates of between 81 and 110% and precision values lower than 12% (expressed as the relative standard deviation). The overall uncertainty of the method was estimated at three different concentrations (10, 25 and 50 microg L(-1)) and was lower than 25.5% in all cases. The proposed analytical methodology was applied to the analysis of target pesticides in 35 samples: 15 commercial, 3 human and 17 goat milk samples. The metabolite p,p'-DDE was the compound most frequently found in both the breast and goat milk samples, at concentration levels < 20 microg L(-1). However, pesticide residues were not found in any of the other 15 commercial milk samples (skimmed, powdered and whole milk) analyzed.

Setting up of recovery profiles: A tool to perform the compliance with recovery requirements for residue analysis.

The use of the recovery term has presented some confusion in Analytical Chemistry. Recent IUPAC recommendations propose to distinguish between two terms: recovery or recovery factor, Re, and apparent recovery, Re*. Apparent recovery includes recovery factor and a new recovery term proposed in this paper, named calibration recovery, Re(C), which depends of the type of systematic error due to the matrix effect (constant and/or proportional) and is related to the applied calibration methodology. This paper highlights the dependence of the calibration recovery on the sample analyte concentration and, for extension, of the apparent recovery, defines the recovery profile, and makes evident the need to determine a "fit for purpose" analyte concentration interval to comply with a regulated recovery requirements. An approach to estimate the calibration recovery and its associated uncertainty in relation to the above-mentioned dependence is presented. The usefulness of the proposed methodology has been shown in the quantification of a pesticide by GC-ECD for assessing dermal exposure.

Assessment of potential (inhalation and dermal) and actual exposure to acetamiprid by greenhouse applicators using liquid chromatography-tandem mass spectrometry.

New analytical methods based on liquid chromatography with electrospray tandem mass spectrometry (LC-MS/MS) have been developed and validated for assessing the exposure of greenhouse workers to acetamiprid. Both ambient (potential inhalation and dermal exposure) and internal dose (biological monitoring of urine samples) measurements were carried out. Potential inhalation exposure was assessed using Chromosorb 102 cartridges connected to air personal samplers. Potential dermal exposure was estimated by using whole body dosimetry. The measurement of actual exposure was done by analyzing the parent compound in urine samples of the applicators, after a solid-phase extraction (SPE) step. The methods showed a good accuracy (72-92%), precision (2-13%) and lower limits (few microg l(-1)). The validated approaches have been applied to assess potential and actual exposure of agricultural workers spraying acetamiprid in greenhouses. The results shown the need to wear personal protective equipment (suits) in order to reduce the absorbed dose of acetamiprid.

Screening method for pesticides in air by gas chromatography/tandem mass spectrometry.

A multiresidue method for determining more than 70 pesticides in air has been validated using a single injection with gas chromatography/tandem mass spectrometry (GC/MS/MS). The method validation considered both stages of sampling and analysis. The sampling method, based on active sampling using sorption in sorbent cartidges, was validated by generating standard atmospheres. Performance parameters of the method were evaluated, with a reduction in the limits of quantification by injecting a higher volume of sample extract, and increase of selectivity by the use of MS/MS detection mode. The method was based on solid-phase extraction, which permits a degree of automation. The best adsorbents were found to be Chromosorb 106 and Tenax TA. The retention capacity of these sampling sorbents allows up to 1440 L of air to be sampled without any breakthrough for most of the compounds. Data were generated for assessing the potential exposure of bystanders. The application of the method to the analysis of the air in urban locations near agricultural areas showed that pesticides were present in most of the samples.

Determination of fifteen priority phenolic compounds in environmental samples from Andalusia (Spain) by liquid chromatography-mass spectrometry.

This work describes the optimisation of a method to determinate fifteen phenolic compounds in waters, sediments and biota (green marine algae) by liquid chromatography coupled to mass spectrometry (LC-MS) with atmospheric pressure chemical ionisation (APCI) in the negative mode. The LC separations of the studied compounds and their MS parameters were optimised in order to improve selectivity and sensitivity. Separation was carried out with a C(18) column using methanol and 0.005% acid acetic as mobile phase in gradient mode. The molecular ion was selected for the quantitation in selective ion monitoring (SIM) mode. A solid-phase extraction (SPE) method was applied in order to preconcentrate the target analytes from water samples. However, extraction of the compounds from sediment and biota samples was carried out by liquid-solid extraction with methanol/water after studying the influence of other organic solvents. In addition, a clean-up step by SPE with HLB Oasis cartridges was necessary for sediments and biota. The proposed analytical methodology was validated in the target environmental matrices by the analysis of spiked blank matrix samples. Detection limits were 10-50 ng L(-1) for water, 1-5 microg kg(-1) for sediments and 2.5-5 microg kg(-1) for biota samples. Good recoveries and precision values were obtained for all matrices. This methodology has been successfully applied to the analysis of incurred water, sediment and biota samples from Andalusia (Spain).

Reduction of analysis time in gas chromatography. Application of low-pressure gas chromatography-tandem mass spectrometry to the determination of pesticide residues in vegetables.

An alternative to conventional capillary gas chromatography (GC) is evaluated as a new approach to determine pesticide residues in vegetables. Low-pressure gas chromatography-tandem mass spectrometry (LP-GC-MS-MS) is proposed after a fast and simple extraction of the vegetable samples with dichloromethane and without clean up. The use of the above-mentioned GC technique reduced the total time required to determine 72 pesticides to less than half the present time (31 min), increasing the capability of a monitoring routine laboratory. The use of guard column and plug of carbofrit into the glass liner in combination with LP-GC was evaluated. The method was validated with limits of quantitation low enough to determine the pesticide residues at concentrations below the maximum residue levels stated by legislation. In order to assess its applicability to the analysis of real samples, 25 vegetable samples previously determined using conventional-capillary GC-MS-MS were analysed by LP-GC-MS-MS. The results obtained with the compared techniques showed differences lower than 0.01 mg kg(-1).

A comparative study of the correction of systematic errors in the quantitation of pyrethroids in vegetables using calibration curves prepared using standards in pure solvent.

A comparative study of two mathematical approaches was performed in order to correct systematic errors due to the presence of the unexpected interferences which appear when the quantitation of the analyte in real samples is carried out with calibration curves built using standards in pure solvent. These methods consisted in the establishment of different mathematical expressions which transform the concentration (Cs) obtained using calibration graphs built using pure solvent into the corrected concentration (C(M)) that should be obtained if the quantitation is carried out with calibration curves built using standards dissolved in blank matrix extracts. In the two approaches the correction is performed from the results of an intermediate precision study which was carried out using both calibration graphs (prepared using pure solvent and blank matrix extract). By using ANCOVA to compare the slope of both solvent-based and matrix-matched calibration graphs, matrix effect was found in the determination of deltamethrin in tomato and acrinathrin in tomato and pepper. In these cases, both approaches led to good results.