László Tölgyesi LC/MS scientist EFSC Demo Group. International Symposium on Food Safety and Quality: Applications of Nuclear and Related Techniques
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1 Minimizing matrix effects in multiresidue pesticide analysis using a new sensitivity-enhanced triple quadrupole instrument in combination with extensive sample dilution László Tölgyesi LC/MS scientist EFSC Demo Group International Symposium on Food Safety and Quality: Applications of Nuclear and Related Techniques IAEA Vienna /0/04
2 Pesticides screening regulation and residue situation EU regulation - Maximum residue levels regulated in commision regulation (EC) 96/005 appendix II IV regulates > maximum residue limits (matrix-pesticide combinations) - European and national monitoring programs are in place also regulated by (EC) 96/005 - notifications related to pesticide residues in tea have been entered in the Rapid Alert System for Food and Feed (RASFF) IAEA Vienna /0/04
3 Why do you want a 6495? Utmost sensitivity is required for the analysis of a large number of relevant compounds with a single injection dilution of samples for reduced matrix effects, better accuracy and improved robustness improved LOQs for pesticides in complex matrices highest precision Experimental setup Small validation with tomato, orange, black tea QuEChERS extraction Dilution of black tea samples with acetonitrile prior injection Quantitation with solvent calibration one calibration for all matrices? IAEA Vienna /0/04
4 x UHPLC/MS/MS Setup 90 Infinity UHPLC column: Zorbax RRHD Eclipse Plus C8 A: H O / 0.% HCOOH / 5mM NH 4 COOH B: MeOH / 0.% HCOOH / 5mM NH 4 COOH 6495 LC/MS system single run, fast polarity switching, dynamic MRM 4 Aminocarb Oxamyl Thiabendazole Acetamiprid Thiacloprid Tricyclazole Pirimicarb Carbofuran Pyracarbolid Prometon Methabenzthiazuron DEET Clomazone Furalaxyl Isoxaben Triazophos Metolachlor Beflubutamid Benalaxyl Pencycuron Triflumizole Prosulfocarb Picolinafen Pyriproxyfen Spiromesifen Carbosulfan Etofenprox Ivermectin Ba Fenazaquin Spiked and diluted tea extract (0. ng/ml) Counts vs. Acquisition Time (min) IAEA Vienna /0/04 4
5 Proven ifunnel Technology Agilent Jet Stream Hexabore Capillary Dual Ion Funnel Thermal confinement of ESI plume Efficient desolvation Creates an ion rich zone Six capillary bores Samples x6 times more ion rich gas from the source Removes the gas but captures the ions Removes neutral noise IAEA Vienna /0/04 5
6 6495 QQQ Technologies Continued Development Proven ifunnel Technology Agilent Jet Stream Hexabore Capillary Dual Ion Funnel Increased ion generation Enhanced ion sampling New Enhanced Q Ion Optics Improved ion transmission New Tapered Hexapole Collision Cell Effective ion collection and transmission New Detector with High Energy Conversion Dynode (up to 0 kv) Improved NEG ion detection with low noise IAEA Vienna /0/04 6
7 New Enhanced Q Ion Optics Improved transmission of ions and system robustness. Octopole. New prefilter New optimized MS prefilter geometry for improved precursor ion transmission Improved peak area response and peak area %RSD, more sensitive and precise New optical lens elements for reduced contamination and easier autotune More reliable and robust performance IAEA Vienna /0/04 7
8 New Curved and Tapered Hexapole Collision Cell Effective collection and transmission of ions Curved and Tapered Hexapole Assembly for efficient collection and transmission of product ions Consistent collision cell pressure for higher quality MS/MS spectra Designed for consistent collision energies across all QQQ platforms IAEA Vienna /0/04 8
9 New Detector with 0 kv High Energy Conversion Dynode More efficient detection of ions with low noise characteristics Gain in Signal with Higher HED Voltage: Anti-EHR/neu mab Peptides Improved ion detection efficiency with High Energy Dynode (HED) voltage up to 0 kv - Improved peak area response and peak area %RSD in positive & negative ion mode - Improved sensitivity and precision for a wide m/z range of product ions Low noise level at 0 kv - Improved signal to noise 60 0% increase in response at 0kV IAEA Vienna /0/04 9
10 6495 QQQ Technologies Continued Development Proven ifunnel Technology Agilent Jet Stream Hexabore Capillary Dual Ion Funnel Increased ion generation Enhanced ion sampling New Enhanced Q Ion Optics Improved ion transmission New Tapered Hexapole Collision Cell Effective ion collection and transmission New Detector with High Energy Conversion Dynode Improved NEG ion detection with low noise Quantitative Applications Enhanced peak area response Improved peak area %RSD More sensitive and precise Lower Limits of Detection (IDL)and Quantitation (LLOQ) More reliable and robust IAEA Vienna /0/04 0
11 Bentazon: Superb Precision & Sub-fg IDL in Black Tea Amount measured Replicates %RSD t(99%) IDL pg/ml,.0 fg (LLOQ) n = 7 injections pg/ml, 0.7 fg IDL = t x (%RSD/00) x Amount =.4 x (.4/00) x.0 fg = 0.7 fg Injection volume = µl x0 -ESI MRM Frag=80.0V CID@6.0 (9. -> x0 -ESI MRM Frag=80.0V CID@6.0 (9. -> 8 pg/ml * pg/ml pg/ml 6 6 x ESI MRM Frag=80.0V CID@6.0 (9. -> pg/ml * x ESI MRM Frag=80.0V CID@6.0 (9. -> pg/ml Inj# Peak Area S/N (Peak to peak) x0 8 -ESI MRM Frag=80.0V CID@6.0 (9. -> pg/ml. 70. x0 8 -ESI MRM Frag=80.0V CID@6.0 (9. -> pg/ml Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) %RSD.4 Ave 6. Excellent peak area precision (%RSD) are observed at the lowest level in black tea matrix IAEA Vienna /0/04
12 Signal Improvements for High Relevance Pesticides 0ng/g in BlackTea_-0dil [Methomyl] x > 88. Area= > 06. Area= ng/g in BlackTea_-0dil [Methomyl] 0ng/g in BlackTea_-0dil [Methidathion] 0ng/g in BlackTea_-0dil [Methidathion] x > 88. Area=55 x > 45.0 Area=50 x > 45.0 Area= > 06. Area= > 85. Area= > 85. Area=56 Methomyl Area Gain.5x Methidathion Area Gain.7x 0ng/g in BlackTea_-0dil [Monocrotophos] x > 7.0 Area= > 9. Area=77 0ng/g in BlackTea_-0dil [Monocrotophos] x > 7.0 Area= > 9. Area=56 MonocrotophosArea Gain.9x 0ng/g in BlackTea_-0dil [Buprofezin] x > 0. Area= > 6. Area=958 0ng/g in BlackTea_-0dil [Buprofezin] x > 0. Area= > 6. Area= Buprofezine Area Gain 4.x ng/g in BlackTea_-0dil [Thiabendazol] x > 75. Area= >. Area= ng/g in BlackTea_-0dil [Thiabendazol] x > 75. Area= >. Area= ng/g in BlackTea_-0dil [Spiromesifen] x > 7.0 Area= > 55.0 Area= Area Gain.x Thiabendazole ng/g in BlackTea_-0dil [Spiromesifen] x > 7.0 Area= > 55.0 Area= Spiromesifen Area Gain.x Improved sensitivity (peak area gain of x) are observed on the new 6495 vs. the 6490 IAEA Vienna /0/04
13 Improved Precision & Lower LLOQs in Black Tea Comparison of Area %RSD for 50 Pesticides at MRL # of pesticides detected at MRL % of pesticides were easily detected with a %RSD < 0 with :00 dilution This means LLOQs < 0 pg/ml (ppt) %RSD < 0 meets SANCO guidelines Improved precision (%RSD) are observed, particularly at the lowest levels (LLOQs) The enhanced peak area response and improved precision (%RSD) means pesticides achieve lower LLOQs ( MRL level) using the new IAEA Vienna /0/04
14 Dilution allows More Efficient Ionization of Pesticides Alanycarb, 0 µg/kg in Black Tea No Dilution : 5 : 0 : 0 : 50 : 00 Oxamyl, 0 µg/kg in Black Tea No Dilution : 5 : 0 : 0 : 50 : 00 IAEA Vienna /0/04 4
15 Recovery (%) in Black Tea with Different Dilutions Pesticides No dilution (n = 5) Dilution :5 (n = 5) Dilution :0 (n = 5) Dilution :0 (n = 5) Dilution :50 (n = 5) Dilution :00 (n = 5) Acetamiprid 9.4 ± ± ± ± ± ±.4 Alanycarb 0.4 ±. 7.9 ±. 8.5 ± ± ± ± 0.8 Aldicarb 6.9 ± ± ± ± ± ± 4. Carbaryl 56.9 ± ± ± ± ± ± 9.6 Dimethoate.9 ± ± ± ± ± ± 7.5 Diuron 79.7 ± ± ± ± ± ±.5 Flufenoxuron 95.4 ± ± ±.8 9. ± ± ±.9 Monocrotophos 4.6 ± 0..9 ± 0..8 ± ± ± ± 5.7 Oxamyl 0.8 ± ± ± ±.0 9. ± ± 5. Thiamethoxam 40.0 ± ± ±.8 5. ± ± ±.0 Cells shaded in green shows %recovery of 70 0, which are in compliance with SANCO requirements. Pesticides show full recovery and no signal suppression with :00 dilution Neat solvent calibration curve can be used for pesticides quantitation with :00 dilution IAEA Vienna /0/04 5
16 Summary - The method benefits from the highly sensitive Agilent 6495 Triple Quadrupole LC/MS System. - It takes full advantage of the low delay volumes of the Agilent 90 Infinity LC System. - Enhanced sensitivity allowed for more flexibility in the degree of sample dilution. - Extensive dilution of sample extracts was applied to minimize matrix effects - With any dilution, a lower matrix amount is introduced into the LC/MS system IAEA Vienna /0/04 6
17 Thank you for your attention! IAEA Vienna /0/04 0
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