Monitoring of Polar Pesticides by QuPPE in different vegetal matrices from routine samples by LC-MS/MS in Italian Official Laboratory. Angela Paoloni, Irene Diamanti, Ivan Pecorelli. Laboratorio Contaminanti Ambientali, Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche (IZSUM), via Salvemini 1, 06126 Perugia Italy; email: i.pecorelli@izsum.it ISTITUTO ZOOPROFILATTICO SPERIMENTALE DELL’UMBRIA E DELLE MARCHE-PERUGIA Introduction Highly polar Pesticides have been excluded for a long time from the routine scope of laboratory investigations due to a lack of simple methods that are able to cover more than one pesticide at the same time. With the QuPPe (Quick Polar Pesticides) method the EURL – SRM has filled this gap[1]. Analysis of non QuEChERS amenable pesticides is not performed by many laboratories all over EU, as a consequence there are not many data regarding the contamination of food of vegetable origin by such compounds yet. Analysis of very polar pesticides in vegetables, cereals and processed products thereof are now performed by Quick Polar Pesticides Method (QuPPe) proposed by EURL-SRM. The methods used in our laboratory analyzed 13 molecules at the same time: Ethephon, Glufosinate, N-Acetyl-glufosinate, MPPA, Glyphosate, AMPA, Phosphonic acid, N-Acetyl-AMPA, Fosetyl, Maleic-hydrazide, Perchlorate, Chlorate, Chlormequat and Mepiquat by LC-MS/MS (QqQ) in Multiple Reaction Monitoring (MRM) at two different electrospray ionization (ESI) polarities. Materials and Methods Residues were extracted from the sample following water adjustment and the addition of acidified methanol. The mixture was centrifuged, filtered and directly analyzed by LC-MS/MS (Applied Biosystems API 3200 QTRAP, operating in MRM mode, coupled with Shimadzu Nexera X2). Separation was carried out on a Thermo Hypercarb column (100 x 2,1 mm; 5 µm) with in-line pre-column with same phase, using acidified methanol and water as mobile phase (Fig. 1). Method proposed by EURL-SRM was slightly modified to prevent splitting of peaks by pH adjustment to 4 before LC-MS/MS injection in all matrices except low water content commodity group (Fig. 2). Sample preparation Table 1: Result for vegetables, fruits and cereals in routine samples for QuPPE and QuEChERS determination. Vegetables and Fruits N° of Samples Commodity Pesticides (QuPPe) [c] mg/Kg Pesticides (QuEChERS) 1 Apples -- Non detected (<0.01 mg/Kg)* or Non detected (<0.05 mg/Kg)** Non detected (<0.01 mg/Kg) 2 Artichokes Phosphonic Acid 1.9 3 Broccoli 0.061 4 0.021 5 6 7 Cabbage 8 9 Cauliflower 10 11 Dry tomatoes 0.047 12 Pears 1.1 13 Peas Mepiquat 0.031 14 Pepper Imidacloprid 0,075 mg/Kg 15 Cichory 16 Radishes 6.3 17 Spinach Chlorate 0.033 18 Strawberries 0.11 19 4.5 0.017 20 0.49 Boscalid 0.022 mg/Kg Methiocarb 0.061 mg/Kg Myclobutanyl 0.027 mg/Kg Spiromesifen 0.12 mg/Kg 21 0.29 22 Table grapes 11.0 Fosetyl 0.022 23 0.080 Chlorpyrifos 0.030 mg/Kg lambda-Cyhalothrin 0.048 mg/Kg Penconazole 0.037 mg/Kg Pirymethanil 0.86 mg/Kg 24 Tomato puree 0.069 25 0.087 26 0.024 27 0.025 0.010 28 29 0.088 30 0.17 31 1.4 32 0.2 33 Tomatoes Cyprodinil 0.011 mg/Kg 34 35 36 37 38 Tomatoes (concentrate) 0.39 39 Wine grapes Cereals Pesticides Baby food 0.25 0.12 Barley 0.059 Lentils Oats Spelt Wheat Figure 1: Schematic rappresentation of sample extraction Filter 1 mL of supernatant with PTFE syringe filter 0.45 μm Centrifuge at 4000 rpm for 10 min. Add 10 mL MeOH containing 1% formic acid Shake thoroughly for 1 min. Weigh 10 g (or 5 g for cereals) sample homogenate in 50 mL centrifuge tube pH adjustment for vegetable samples LC-MS/MS analysis Figure 2: A: Matrix calibration standard of Chlorate at 0.1 µg/mL without pH adjustment. B: with ph adjustment. A B (*) Chlorate; Chlormequat; Ethephon; Mepiquat; MPPA; N-Acetyl AMPA; N-Acetyl Glufosinate and Perchlorate: Non detected (<0.01 mg/Kg). (**) Glufosinate; Glyphosate; AMPA; Phosphonic acid; Fosetyl; Maleic-Hydrazide: Non detected (<0.05 mg/Kg) Results and Conclusions In table 1 and 2 results for all commodities monitored in the last few months in our laboratory from 50 samples collected during routine control program from Umbria Region in central Italy are reported. Approximately 50% of all analyzed samples contained detectable residues. Among all pesticides, Phosphonic acid and Chlorate, were the most frequently identified, in all types of matrix investigated, even if their concentrations were below MRL values established by the EU Regulation (EC) N° 396/2005. Residues of Phosphonic acid were detected in a wide range of samples (see table 1 and 2), with concentration ranging from 0.02 mg/Kg to 6.0 mg/Kg. The aim of this work was to compare the results obtained by QuPPe method and QuEChERS multiresidue method in the same samples of vegetable origin collected during routine control program in the Umbria Region, central Italy. Despite the number of analytes potentially detectable is very different (13 by QuPPe and 250 by QuEChERS) the number of findings by QuPPe is very high compared to the other method. Results for QuPPE amendable pesticides were compared with data presented by EURL-SRM[2,3]., In our laboratory, incidence of finding is of the same order of magnitude compared to EURL-SRM data (see Table 2) despite the low number of samples analized. Table 2: Positive findings in our lab and SRM data. IZSUM LAB COMMODITY Number of samples Samples containing phosphonic acid Samples containing chlorate Fruits 11 8 (72 %) 1 (10 %) Vegetables 31 14 (45 %) 4 (13 %) Cereals 2 (18 %) EURL-SRM 749 412 (55 %) 70 (9 %) 813 203 (25 %) 150 (18 %) -- Reference: [1] Quick Method for the Analysis of numerous Highly Polar Pesticides in Foods of Plant Origin via LC-MS/MS involving Simultaneous Extraction with Methanol (QuPPe-Method)-Version 9. [2] News on SRMs. Presentation of M.ANASTASSIADES Joint EURL Workshop 30 Sept-02 Oct 2015 in Stuttgart . http://www.eurl- pesticides.eu/userfiles/file/2015_JointWorkshop/6_Anastassiades%20News%20on%20SRM_Workshop2015d_pptx_NoPictures.pdf [3] Phosphonic Acid: Pesticide or “Foliar Fertilizier”? Residues in Organic and Conventional Samples from the German Market. E-Mail: Marc.Wieland@cvuas.bwl.de. EPRW 2014 book of abstract (PM033) Conclusion xxxxxxxxxxxxxxxxxxxxxxxxxx