Improved sample preparation and HPLC/MS determination of pesticides in fat-rich vegetables Frank Michel1, E. Barrey2, K. Stenerson2, O.Shimelis2, M.Ye2 1Sigma-Aldrich, Taufkirchen, GER, 2Supelco/Sigma-Aldrich, Bellefonte/PA, USA Dispersive Solid Phase Extraction (dSPE, known as QuEChERS) is the mostly used clean-up for the analysis of pesticides in food prior to LC/MS [1-3]. Fruits and vegetables with a high content of fat usually still exhibit a challenge, because the fat can interfere with analytes in the HPLC/MS determination. In this work we present a new adsorbent (zirconia-coated silica) for QuEChERS method. This new particle has got a higher capacity to retain lipids and pigments than the usually used C18 adsorbent. • Supel™ QuE Z-Sep/C18 • Supel QuE Z-Sep+ • Mixture of of Discovery® DSC-18 and Z-Sep (zirconia-coated silica) Results (continued) Using the zirconia-coated silica higher recovery of several pesticides was achieved and ion-suppression by lipids was reduced (Fig. 4). HPLC conditions on request. 160 Recoveries in Olives 140 Z-Sep/C18 C18/PSA 120 PSA 100 % Introduction The usual approach to apply QuEChERS to fat-rich samples is either to add C18 silica to PSA adsorbent in the purification step or to freeze out the fat. Fat interferes by either co-extracting non-polar pesticides compromising recovery or by co-elution in chromatogram. Zirconia-coated silica is an interesting approach for lipid removal, because the d-electron deficient Zirconium acts a Lewis acid that interacts strongly with Lewis bases such as phospholipids (Fig. 1). 80 60 40 20 • Dual Bonded C18 and Z-Sep 0 • Use for cleanup of LC-MS samples ZrO ZrO C18 ZrO ZrO ZrO ZrO C18 C18 ZrO C18 + ZrO ZrO C18 ZrO C18 C18 ZrO C18 ZrO Fig. 4: Recoveries of different pesticides from olives at 50 ppb (n=3). Quantification against calibration curve standards made in solvent. C18 C18 C18 C18 Fig. 1: Graphic of Z-Sep/C18 and Z-Sep+ Particles Results Zirconia-coated silica works well for the removal of oleins from in acetonitrile solution (Fig. 2). 140 100% 120 90% 100 80% C18/PSA Z‐Sep(+)/PSA Z‐Sep+ 80 70% 60% 60 50% 40 40% The mixture of zirconia-coated silica and C18 was applied in the QuEChERS method for sample prep of olives. The work-up using this adsorbent was compared to PSA and PSA/C18. Details of the method can be found in Fig. 3. Weigh 10 g olive sample into 50 mL centrifuge tube Add 10 mL acetonitrile, mix Add citrate buffer, MgSO4 and NaCl, shake for 1 min Centrifuge at 3200 rpm for 5 min Add aliquot 0.7 mL of supernatant to adsorbent tubes: PSA , PSA/C18, Z-Sep/C18 Shake for 1 min and centrifuge at 5000 rpm for 5 min Dilute (if appropriate), analysis by LC/MS Fig. 3.: Conditions for extraction and clean-up of olive samples. Deltamethrin Cypermethrin Coumaphos 4,4'‐DDT Cyfluthrin isomers Fig. 2: 25 mg of sorbent was mixed with 1 mL solution of oleins (400 mg/mL total concentration) in acetonitrile. The remaining mono-, di- and trioleins were quantified by LC-ELSD and removal was calculated. Methoxychlor Silica Endosulfan I LRA Chloropyrifos alumina Malathion NH2 Metolachlor PSA Heptachlor C18 Qunitozene ZSep/C18 gamma‐BHC Z-Sep+ Simazine 0% Dimethoate 10% Hexachlorobenzene 0 20% Trifluralin 20 30% alpha‐BHC Removal A similar work-up using Z-Sep+, PSA/Z-Sep+ and PSA/C18 was applied for the sample preparation of avocado (details on the method on request). The dualbonded Z-Sep+ resulted in the best recoveries of several pesticides (Fig. 5). HPLC conditions on request. Fig 5.: Comparison of recoveries of pesticides from avocado using different adsorbents in QuEChERS clean-up. Conclusion • For fatty vegetables using zirconia-coated silica in QuEChERS method improved clean-up compared to other adsorbents. • In olives, Z-Sep/C18 cleaned extracts exhibited better pesticide recoveries than PSA and PSA/C18. This is possibly due to ion supression effects from lipids in the PSA and PSA/C18 cleaned extracts. • Z-Sep+ provided higher recoveries for the extraction of pesticides from avocado. • Zirconia-based adsorbents retain more oleins than traditional cleanup sorbents. • More matrix was removed by Z-Sep+ than other cleanup sorbents (data not shown). References: 1. M. Anastassiades, S.J.Lehotay. et al., J. AOAC International 86(22) (2003) 412-431 2. EN15662:2008 3. AOAC Official Method 2007.01