Development and Validation of Multiresidue Pesticide Methods at FDA/CFSAN Jon W. Wong U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition American Council of Independent Laboratories Meeting March 28, 2012
Multiresidue Analysis of Pesticides at FDA/CFSAN LC-HR-MS Accurate Quantitation Validated targeted analysis for 500 pesticides (resolution, mass accuracy, matrix effects, sensitivity, recovery) Developing non-targeted analysis Developing accurate mass product ion libraries for ~ 1,000 compounds LC-MS/MS and LC-MS Libraries Completed a 3-laboratory validation study (FDA, OME and NRCG) Completed single laboratory validation (~200 pesticides, 24 food matrices) Developing a LC-MS/MS method for 400 to 500 pesticides Developed pesticide libraries (including intra- and inter-laboratory studies) for additonal confirmation GC, GC-MS, GC-MS/MS Completed single laboratory validation for GC-MS/SIM and GC- MS/MS methods (~160 pesticides in fruits and vegetables) Developing a single laboratory validation for GC-MS/MS and botanical products (300 pesticides, 24 botanicals Unambiguous Identification
Method Validation 1) Accuracy 2) Precision 3) Linearity 4) Detection/Quantitation Limits 5) Ruggedness/Robustness (including Matrix effects) 6) Identification (false positive and negative) 7) Extension and applicability For example, to evaluate accuracy and precision recovery studies should be performed but What are the acceptable recovery ranges and RSD? How many replicates? How many fortification levels? At what concentration levels? How many food matrices? How many pesticides should be used?
Step-by-step procedures using QuEChERS Weigh sample Add solvent Shake Add salts Add internal standard Shake and centrifuge Transfer extract (top) for cleanup Analysis by LC-MS Shake and centrifuge Transfer (dilute or concentrate) to vials LC-GC, 2008, vol. 11 issue 1
Single Laboratory Method Development and Validation Recovery study: spiked at 10, 25, 100, and 500 ng/g, acceptable recovery: 70-120%, RSD<20% Sensitivity: LOD < 3 (LOQ<10) ng/g for the majority of pesticides Matrix effect: matrix matched vs. solvent calibration Application: Determination of pesticides on incurred samples Identification: RT, 2 ion transitions & relative ion ratio Quantitation: matrix matched calibration curve
LC-MS/MS of a QuEChERS Extract (~200 pesticides and Scheduled MRM ~400 transitions) XIC of +MRM (436 pairs): 238.1/163.0 amu Expected RT: 5.7 ID: 3-Hydroxycarbofuran.1 from Sample 13 (5.0 ppb-1) of Data20091211 blue b... Max. 1.4e4 cps. 2.6e5 2.5e5 2.4e5 2.3e5 2.2e5 2.1e5 2.0e5 1.9e5 1.8e5 1.7e5 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 0.0 LC time program 5.7 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 Time, min 95% B Flow rate 0.5 ml/min Injection volume 20 μl A: 4 mm NH 4 HCO 2 in H 2 O B: 4 mm NH 4 HCO 2 in MeOH Column: RESTEK Ultra Aqueous C18 (100 mm x 2.1mm x 3.0 μm) MS source temp: 500 o C Mode: ESI positive Spray voltage: 5000 volts Curtain gas: 30 Collision gas: medium Gas 1: 50 Gas 2: 50 5% B
Incurred Samples Analyzed using QuEChERS/LC-MS/MS 411.3 313.3 Ion ratio =0.3 732.5 142.2 Ion ratio =4.3 493 158.1 Ion ratio =1.3 404.1 372.1 Ion ratio =16 346.1 278.1 Ion ratio =16 202.1 145 Ion ratio =3.1 411.3 71.3 732.5 98.1 493 141.1 404.1 344.1 346.1 73 202.1 127 Spirodiclofen Spinosad Novaluron Azoxystrobin Triflumizole Carbaryl 256 209.1 230 199 190 163 324.1 262.1 Ion ratio =0.8 Ion ratio =1.3 Ion ratio =0.9 Ion ratio = 1.4 732.5 142.2 Ion ratio =3.3 256 175.1 230 125 190 136 324.1 242.1 324.1 242.1 732.5 98.1 Imidacloprid Dimethoate Tricyclazole Flutolanil Spinosad
Multiresidue Pesticide Methods for Fresh Produce Expand from ~200 pesticides to > 400 pesticides Extensive method validation studies (accuracy, precision, LOD/Q, linearity, ruggedness/robustness and identification) required Provide methods for FDA and non-fda (government, industry and private) laboratories Laboratories need to validate methods
Botanical Dietary Supplements A botanical is a plant or plant part valued for its medicinal or therapeutic properties, flavor, or scent. A botanical dietary supplement is a supplement to the diet, contains botanical products, intended to be taken by mouth as a pill, capsule, or liquid; and labeled as a dietary supplement. Diverse class of products (root, leaves, fruits, flowers, seeds, bark, husks, whole plant) Botanicals are difficult to analyze because they are dried and concentrated (pigments, lipids, tannins, polyphenols, lignin) No pesticide tolerance levels for many of these products
Multiresidue Pesticide Method for Botanicals using GC-MS/MS 1 g dry + 10 ml H 2 O + 10 ml ACN + I.S. Add 1.0 g NaCl and 4.0 g MgSO 4 Shake and centrifuge (4500 rpm x 5 min) Load 1.25mL extract on conditioned SPE cartridges GCB/PSA(0.25 g/0.5 g) Elute with 15 ml toluene (3:1 acetone:toluene) Reduce to ~100 µl, bring to 0.5 ml (toluene) + QC standards GC-MS/MS Analysis
Single Lab Validation Status 24 botanical materials have been studied All botanicals fortified at 10, 25, 100, 500 ppb in quadruplicates Incurred botanical analyzed in triplicates 14 point matrix matched standards constructed (0.5 ppb to 5000 ppb) LODs estimated statistically for all 24 botanicals using matrix matched standards Manuscript in preparation for publication
Collaborative Design for Proposed AOAC Study Two nested studies covering the same method and the same compounds Laboratories can sign up for one or both First study covers a limit number of pesticides (~30?) in all the matrices under study. Subset of the second study Second study which carry out in parallel will cover approximately 100-150 pesticides that are amenable to GC
Study Specifics Five botanicals will be tested by all laboratories under both study protocols Each matrix will be fortified at 50 and 250 ppb using a single standard mixture certified under ISO 17025 procedures Quadruplicate determinations will be performed and in addition 2-4 incurred matrices will be analyzed in triplicate by all laboratories
Acknowledgements U.S. Environmental Protection Agency National Pesticide Standard Repository U.S. Food and Drug Administration Field laboratories National Institutes of Health Office of Dietary Supplements IAG Y1-OD-6004-01