cncerning the fllwing EURL-SRM - Analytical Observatins Reprt Cmpund(s): Pymetrzine Cmmdities: Varius cmmdities f plant rigin Methd(s): QuEChERS, adjustment f ph Instrumentatin: LC-MS/MS Analysis f Pymetrzine by the QuEChERS Methd - Impact f ph n Recvery Rate Reprted by: EURL-SRM Versin 1 (last update: 18.04.2016) Backgrund infrmatin / Initial Observatins: When using the citrate buffered QuEChERS methd (EN15662), recvery rates f pymetrzine are ften < 70 %, mstly in the range between 45 t 65 %. Sme examples are shwn in Table 1: Table 1: Recvery rates fr pymetrzine using citrate buffered QuEChERS (EN15662) Cucumber 5 0.01 62 % 5 % Avcad 5 0.01 59 % 10 % Wheat 5 0.1 58 % 6 % Grapes 5 0.1 55 % 4 % Lemn 4 0.1 46 % 6 % Withut buffering, recvery rates are mre strngly influenced by the natural ph f the extracted cmmdities, with recveries frm neutral cmmdities like lettuce being higher than thse frm acidic nes. Pymetrzine, being weakly basic at the pyridine grup (pk a f the crrespnding acid ~4.4) and weakly acidic at the triazinne grup (pk a ~11.4), exhibits an ambiphilic character 1. At ph values < 4.4 the prtnated frm predminates. At ph values <2 even a duble prtnated frm cmes int play (pk a ~0.7 at the triazinn grup). As the ph decreases the lgd value (a measure f lipphilicity) drps frm ~-0.25 at ph 5 t ~-0.5 at ph 4 and further t ~-0.7 at ph 3. This explains the lwer recvery rates at lwer ph. 1 pka values calculated by www.chemicalize.cm; in the EFSA peer review n pymetrzine (http://www.efsa.eurpa.eu/sites/default/files/scientific_utput/files/main_dcuments/3817.pdf) the pka is given as 4.06 Page 1
Between ph 6 and 10 pymetrzine is almst exclusively present in its - still quite plar - neutral frm (lgp ~ -0.2), and has a mre favrable transfer int the acetnitrile phase. Using the citrate buffering salts f EN 15662 the ph value during extractin is arund 4. Althugh the citrate salt mixture in itself buffers in the ph range f 5-5.5, the presence f MgSO 4 causes the ph t drp as Mg 2+ ins frm a cmplex with citrate, thus releasing prtns. As a result the recvery f pymetrzine remains well belw 70%. Facts at a glance: Pymetrzine Mde f actin Lg D Insecticide selective against Hmptera like aphids and whiteflies [1] Partitin cefficient www.chemicalize.rg lg P/w = -0.19 (ph 5, 25 C) lg P/w = -0.19 (ph 7, 25 C) lg P/w = -0.20 (ph 9, 25 C) [3] 320 mg/l (25 C; ph 5) 270 mg/l (25 C; ph 7) 270 mg/l (25 C; ph 9), all 99.7% [3] Water slubility Dissciatin cnstant Residue definitin EU Registratin Status ADI 0.03 mg/kg bw/day [3] ARfD 0.1 mg/kg bw [3] pka,1 = 4.06 (basic, prtnatin at nitrgen atm f the pyridine ring) [1,3] pka,2 < 1 (basic, prtnatin at nitrgen atm f triazinne ring) [1,3] pka,3 = 11.4 (acidic, deprtnatin at nitrgen f triazinne ring) [chemicalize.rg] Pymetrzine Fr milk: pymetrzine, 6-hydrxymethylpymetrzine and its phsphate cnjugate, expressed as pymetrzine [2] Apprved within the EU: AT, BE, CY, CZ, DE, DK, EE, EL, ES, FI, FR, HR, HU, IE, IT, LT, LU, LV, NL, PL, PT, RO, SE, SI, UK Page 2
Observatins and experiments cnducted: Materials and instrumentatin (exemplary 2 ): Pymetrzine (purity 99.9%), purchased frm Riedel-de-Haen (stck slutin at 1 mg/ml was prepared in methanl) Magnesium acetate tetrahydrate, min. 99% frm Sigma-Aldrich Sdium acetate p.a. anhydrus, min. 99% frm Merck Table 2: Instrumentatin details LC Agilent 1290 MS/MS ABSCIEX 4000, run in ESI psitive mde MRMs 218.2 / 105.0 (target); 218.2 / 78.0 (qualifier) Clumn Phenmenex Synergi 4 µ Hydr-RP; 150x2 mm Pre-clumn Aqua C18 125A 4 mm x 2 mm Mbile Phase A: 5 mml NH 4 -frmiate in purified H2O withut additin f methanl (use brwn bttles t avid frmatin f algae) B: 5 mml NH 4 frmate in methanl Gradient Mbile Phase A Mbile Phase B Flw Time (min) (%) (%) (ml/min) 0 100 0 0.1 3 30 70 0.1 6 15 85 0.3 9 10 90 0.3 18.5 10 0 0.3 19 100 0 0.3 28 100 0 0.3 Clumn temperature 40 C Injectin vlume 3 µl, autmatically diluted with 10 µl f mbile phase A during injectin prcedure Internal Standard Nte: Shuld the pssibility fr an autmated dilutin f the slutins in the instrument injectr nt exist, the sample extracts shuld be manually diluted with mbile phase A (e.g. 1+1 t 1+4) Chlrpyrifs-ethyl D10 Direct injectin f QuEChERS extracts nt traditinal C-18 clumns ften results in peak distrtin r even splitting. Diluting the extracts with the aqueus mbile phase A prir r during injectin, imprves the situatin. The use f gradients starting with highly aqueus eluent cmpsitin facilitates reversed phase interactins f highly plar cmpunds such as pymetrzine with the statinary phase. Plar end-capped RP clumns are helpful in this respect as they can tlerate purely aqueus eluents withut the hydrcarbn-chains cllapsing. 2 Disclaimer: Names f cmpanies are given fr the cnvenience f the reader and d nt indicate any preference by the EURL-SRM twards these cmpanies and their prducts Page 3
Impact f extractin ph n pymetrzine recveries T study the influence f varius salt mixtures n the ph varius cmbinatins f salts (used instead the citrate buffering salts described in EN15662) were tested using cucumber as neutral and range juice as acidic cmmdity: The resulting ph values in the extracts are shwn in Table 3. Table 3: ph values f QuEChERS extracts using different buffering salt cmbinatins Orange juice Cucumber Orange juice Cucumber Citrate buffering salts EN15662 3.88 4.20 55 % 65 % 4 g MgSO 4. 1 g NaCl 2.55 4.84 22 % 80 % 4 g MgSO 4. 1 g NaCl. 0.5 g Na-Acetate 5.21 6.55 94 % 86 % 4 g MgSO 4. 0.5 g Mg-Acetate 5.33 6.63 Nt analyzed Nt analyzed 4 g MgSO 4. 1 g Mg-Acetate 5.54 6.63 89 % 97 % The resulting ph values indicate that magnesium r sdium acetate is necessary t elevate the ph value f acidic cmmdities abve ph=5 in rder t ensure that pymetrzine lays almst entirely in its less plar neutral frm. The recveries achieved with the different variatins are shwn belw: Table 4: Recvery figures fr pymetrzine using different QuEChERS salt cmpsitins (Chlrpyrifs D10 was used as internal standard) Frtificatin level 0.1 mg/kg (n=5) Recvery % CV % Recvery % CV % 4 g MgSO 4, 1 g Mg-Acetate 86 3 86 2 4 g MgSO 4, 0.5 g Na-Acetate, 1 g NaCl 86 2 89 3 Using 10 ml water and the salt mixtures listed abve, the vlume f the rganic phase was measured. In the case f 4 g MgSO 4 + 1 g Mg-Acetate the vlume f the rganic phase was 10.8 ml and in the case f 4 g MgSO 4 + 0.5 g Na-Acetate + 1 g NaCl it was 10.0 ml. In principle, such vlume deviatins can be cmpensated by internal standards, hwever if n internal standard is used the secnd ptin is favred. T prve that the pr recveries f pymetrzine are nt due t degradatin range juice was spiked and extracted a) immediately and b) after 1 hur delay. The mean recveries did nt decrease indicating that pymetrzine is nt particularly sensitive t degradatin at lw ph. Page 4
Overall Cnclusins: Pymetrzine shws ph-dependent recvery rates using the QuEChERS methd. ph shuld ptimally exceed 5 fr the pymetrzine recvery rates t be well abve 70%. ph can be increased by replacing the citrate salts with acetate salts. The fllwing mixtures were fund t wrk well fr 10 g sample: 4 g MgSO 4 +1 g NaCl + 0.5 g sdium acetate and 4 g MgSO 4 +1 g Mg-Acetate. [1] The Pesticide Manual 14 th Editin. C D S Tmlin. BCPC 2006 [2] Regulatin (EU) 2015/401 [3] EFSA Jurnal 2014;12(9):3817. Cnclusin n the peer review f the pesticide risk assessment f the active substance pymetrzine Histry Actin When Versin Cnductin f experiments June 2015 February 2016 Methd placed n-line April 2016 V1 Page 5