122220 APP 500+ Pesticides Fruit Veggies4.Indd

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122220 APP 500+ Pesticides Fruit Veggies4.Indd APPLICATION NOTE Liquid Chromatography / Mass Spectrometry Authors: Maria Laura Pati, Nicola Barbieri, Alfredo Fantastico, Piero Pontrelli S.A.Mer. Servizio Analisi Chimico-Merceologiche Bari, Italy Aristide Ganci Roberto Troiano PerkinElmer, Inc. Milan, Italy Ignazio Garaguso PerkinElmer, Inc. Rodgau, Germany Multi-residue Analytical Introduction Method for the Confirmation Pesticides are a group of compounds and Quantification of 500+ containing hundreds of listed listed substances, most of which are Pesticides in Fruit and Vegetables regulated by governmental agencies. Their function is to prevent, destroy, or control harmful organisms or diseases, as well as protect plants or plant products during production, storage and transport. Pesticides are primarily utilized in the agricultural sector, and contain one or more active substances. From the point of application, pesticides can be transported through various media, and ultimately be deposited on plants and animals humans consume. While some of these compounds have not been found to be harmful, others may have toxic properties to humans and animals, as well as pose a danger to our environment and ecosystems. The European Commission (EC) has set maximum residue limits (MRLs) for pesticide residues in or on food and feed of plant and animal origin, as detailed in legislative framework Regulation (EC) 396/2005.1 MRLs vary for given pesticides and food products, but generally, the MRLs are set at 0.01 mg/kg for many fruits and vegetables. For certain pesticides and matrices, different legally permitted concentrations have been set, mostly ranging from 0.001 – 100 mg/kg.2 For pesticides not listed in the regulation, a default MRL of 0.01 mg/kg applies.1 Rapid multi-residue analytical methods are needed to confirm Table 1. LX50 UHPLC Parameters. and quantitate the wide range of regulated pesticides, which are Column C18, 100x4.6 mm, 2.7 µm often present at very low levels in sample matrices with different chemistries. Based on regulatory requirements in recent years, Solvent A: 9 mM ammonium formate in water- Mobile acetonitrile (90:10, v/v) + 0.1 % formic acid liquid chromatography coupled to tandem mass spectrometry Phase Solvent B: 1 mM ammonium formate in acetonitrile- (LC/MS/MS) has become the technique of choice to perform water (90:10, v/v) + 0.1 % formic acid confirmatory analysis on these compounds. Owing to its ability Step Time [min] Flow Rate [ml/min] %A %B to detect hundreds of compounds in a single analysis, LC/MS/MS 1 0 0.4 100 0 offers unmatched sensitivity and selectivity based on large Gradient 2 10 0.4 0 100 numbers of multiple reaction monitoring (MRM) transitions. 3 15 0.4 0 100 4 16 0.4 100 0 For pesticide residue analysis in food, the European Commission’s 5 20 0.4 100 0 General-Directorate for Health and Food Safety (DG SANTE) has Injection published a guidance document on analytical quality control, 10 µL which details the performance requirements for the validation of Volume Column oven 40 °C analytical methods which should be followed by laboratories. The Temperature most recent guidance was published under SANTE/12682/2019,3 Autosampler 10 °C and includes procedures on acceptable retention time deviation, ion ratio (quantifier/qualifier ratio) range, and selectivity criteria to Table 2. QSight ESI Source Parameters. ensure reliable quantification results. Ionization Mode ESI with Polarity Switching Drying Gas 120 For sample preparation in the SANTE/12682/2019 method, QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) is the HSID Temperature 200 °C most commonly followed procedure and is based on extraction Nebulizer Gas 350 with acetonitrile, followed by a dispersive solid phase clean-up Spray Voltage 5000V / -4800V step.4 As acetonitrile is compatible with LC/MS/MS, this approach Source Temperature 340 °C has emerged as the method of choice for pesticide analysis in Detection Mode Time-managed MRM™ fruits and vegetables.5–7 In this application note, a fast, sensitive, and selective multi- Standard Preparation ® residue method, utilizing a QSight 220 triple quadrupole mass Pesticide standards were obtained from Lab Instruments (Bari, Italy). detector coupled to the LX50 ultra-high performance liquid Repeatability and recovery were evaluated by fortifications of chromatography (UHPLC) system, is described for the analysis pesticides prior to sample extraction at 10 μg/kg, 50 μg/kg and of 503 pesticides in fruit and vegetable matrices. 100 μg/kg, measured in replicates (n=10). Calibration curves were built by five levels of standards prepared in a neat solution Experimental (acetonitrile). Limit of detection (LOD) and limit of quantification Hardware/Software (LOQ) values were determined based on signal-to-noise (S/N) values Chromatographic separation of pesticides was conducted on a of 1:3 and 1:10, respectively. Two MRM transitions were monitored PerkinElmer UHPLC system, with subsequent analyte determination for each pesticide. A procedural internal standard (IS), triphenyl achieved utilizing a PerkinElmer QSight 220 triple quadrupole mass phosphate (TPP), was used for quantification of the obtained detector with a dual ionization source. All instrument control, data results, accounting for various sources of errors throughout all acquisition and data processing were performed using stages in the method, as well as possible matrix effects. Simplicity 3Q™ software. Sample Preparation Instrumental Parameters Samples were obtained from local farms, and prepared according to The UHPLC separation method and MS/MS source parameters are an established procedure (method EN 15662) using a QuEChERS shown in Table 1 and Table 2, respectively. A full list of the multiple based extraction technique (PerkinElmer part number: N9306901), reaction monitoring (MRM) mode transitions of the studied followed by a clean-up step (PerkinElmer part number: N9306920). pesticides are shown in Table 3. The table includes both positive The latter, depending on the vegetable matrix to be analyzed, can (460) and negative (43) analytes measured in ESI mode using fast include the use of 45 mg of porous graphitic carbon (PGC) for highly polarity switching times to avoid losing data points. To facilitate pigmented fruit and vegetables. In short, 10 g of sample was method development, the MS analysis parameters are homogenized and placed in a 50 ml tube. 100 µL of IS are added to automatically generated by selecting the pesticides of interest from the tube to arrive at a final concentration of 50 μg/kg, accounting the built-in compound library in the time-managed-MRM module for both extraction recoveries, as well as matrix effects in the mass of the Simplicity 3Q software. Depending on expected peak width, spectrometry analysis. 10 mL of acetonitrile was added, and the the cycle time of the instrument is set, and dwell times are solution was shaken and centrifuged. An aliquot of the supernatant optimized accordingly by the built-in algorithm. was transferred to the clean-up tube, shaken and centrifuged. The supernatant was transferred to a vial and 10 μL of extract was injected directly onto the QSight 220 LC/MS/MS system for quantification. 2 Results A total of 503 pesticides were investigated and analyzed in this influence the quality and precision of the measured data. In this study (Table 3) and measured in three matrices - apple, orange and work, automated time managed MRM experiments were used, and lettuce - to cover a range of both fruit and vegetable based samples an optimal dwell time calculation was automatically performed using of varying composition and acidity. Orange was chosen as an acidic a software integrated algorithm to obtain 9-12 data points across matrix, while apple as a neutral one for the group of fruits, and the peak for reliable and accurate quantification. Data points lettuce as a vegetable representative. Figure 1 shows an example of selected amongst early, mid and late eluting peaks are shown in overlayed MRM transitions for selected pesticides and their Figure 2 as representative examples for all measured analytes. chromatographic separation. Limit of Detection High resolution peak acquisition requires enough sampling points LOD and LOQ values were calculated by considering the signal of across the peaks of interest in order to obtain a precise and quantifier ions of matrix-matched standards with signal-to-noise reproducible result. MRM utilizes the retention time of measured ratio (S/N) > 3 and S/N > 10, respectively. The LOD values for 97% compounds, and overlapping windows ensure that only the of the 503 measured pesticides are equal or below 1 ng/ml, as compounds eluting at that time are measured. Dwell times of each detailed in Figure 3. The LOQ values for 90% of the 503 compounds transition and the associated acquisition speed influence the data are equal or below 2 ng/ml, and thus easily meet the required MRL points across the peak. Indeed, both factors are relevant and values as set by the EC 396/2005, as shown in Figure 4. Figure 1. Example of overlayed peaks showing separation of described pesticides (Quantifier MRM) at 10 ng/ml in neat solution. Figure 2. Representative examples for data points selected amongst peaks during early, mid and late eluting compounds in the chromatographic run. Figure 3. Distribution of LOD for tested pesticides. 96 % of all 503 pesticides tested had Figure 4. Distribution of LOQ for tested pesticides. 80 % of all 503 pesticides tested had a a LOD of equal or below 1 ppb with a S/N > 3. Only 1% of all pesticides had an LOD of LOQ of equal or below 1 ppb with a S/N > 10. Only 4% of all pesticides had an LOQ of above 5 ppb. above 5 ppb. 3 Linearity QuEChERS-method” in foods of plant origin. An overlay of validated The calibration curves for all compounds were created from their pesticides at 10 µg/kg in lettuce matrix is shown in Figure 6.
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