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HOW DO YOU COMPLY WITH <USP 232/233>? HOW DO YOU ADDRESS AN FDA RESPONSE LETTER ASAP? HOW DO YOU MEASURE PESTICIDE DEGRADATION RATES? HOW DO YOU QUANTIFY ADDITIVES IN PLASTIC? HOW DO YOU PREDICT THE EFFECTS OF HEAT, COLD AND HUMIDITY? HOW DO YOU KNOW IT WILL WITHSTAND A BLAST? HOW DO YOU MAKE A BETTER BATTERY? HOW DO YOU KNOW IF A PATENT HAS BEEN INFRINGED? HOW DO YOU KNOW WHEN TO CONDUCT E/L STUDIES? HOW DO YOU PREVENT ELECTRONICS FAIL-URE? HOW DO YOU PERFORM A SUCCESSFUL ILV? HOW DO YOU UNDERSTAND THE CAUSE OF A PAINT FAILURE? HOW DO YOU MEET GLOBAL REGULATORY REQUIREMENTS? HOW DO YOU DETERMINE THE CAUSE OF LANDING GEAR FAILURE? HOW DO YOU KNOW WHY A HARD DRIVE CRASHED? HOW DO YOU ESTABLISH LIABILITY THROUGH ENGINEERING ANALYSIS? HOW DO YOU FULLY CHARACTERIZE AN ANTIBODY DRUG CONJUGATE? HOW DO YOU TEST FOR BIOCOMPATIBILITY? HOW DO YOU RESPOND TO A DATA CALL-IN? HOW DO YOU IDENTIFY CONTAMINANTS? HOW DO YOU PREVENT PACKAGING ISSUES? HOW DO YOU FIND CONTAMINATION ON SATELLITE PARTS? HOW DO YOU VALIDATE A CIRCUIT MODEL? HOW DO YOU DETERMINE THE ROOT CAUSE OF AN INDUSTRIAL FIRE? HOW DO YOU MONITOR CHARGE VARIANTS AND DEGRADATIONS? HOW DO YOU MEASURE PARTI-CLE SIZE? HOW DO YOU TACKLE A GLOBAL REGISTRATION? HOW DO YOU ENSURE CONSISTENT CRYSTAL FORM ACROSS BATCHES? HOW DO YOU PREVENT CONTAMINATION? HOW DO YOU TEST RELIABILITY OF NIGHT VISION SENSORS? HOW DO YOU MAKE AN LED BRIGHTER OR MORE AFFORDABLE? HOW DO YOU KNOW IF SOMEONE IS USING YOUR TRADE SECRETS? HOW DO YOU DETECT POST-TRANSLATIONAL MODIFICATIONS? HOW DO YOU DETERMINE THE CAUSE OF FAILURE? HOW DO YOU MEASURE CHEMICAL RESIDUE IN MILK? HOW DO YOU KNOW WHAT ANALYTICAL TECHNIQUE TO USE? HOW DO YOU IMPROVE BIODEGRADABILITY? HOW DO YOU IDENTIFY METAL FATIGUE? HOW DO YOU VALI-DATE SUPPLY CHAIN QUALITY? HOW DO YOU KNOW IF THERE IS A LEGAL CONFLICT? HOW DO YOU RE-OPTIMIZE AN ELISA METHOD WHEN A REAGENT LOT CHANGES? HOW DO YOU KNOW WHETHER THE LINER IS THE PROBLEM? HOW DO YOU EVALUATE HYDROPHOBIC COMPOUNDS BY SPME? HOW DO YOU COMPARE FEEDSTOCK SUPPLIERS? HOW DO

YOU COMPARE FEEDSTOCK SUPPLIERS? HOW DO YOU TEST MEDICAL DEVICES IN A MALPRACTICE CASE? HOW DO YOU DETERMINE WHETHER A COMPOSITE IS SAFE? HOW DO YOU MEASURE THE ROUGHNESS OF A COATING? HOW DO YOU COMPLY WITH <USP 232/233>? HOW DO YOU ADDRESS AN FDA RESPONSE LETTER ASAP? HOW DO YOU MEAS-URE PESTICIDE DEGRADATION RATES? HOW DO YOU QUANTIFY ADDITIVES IN PLASTIC? HOW DO YOU PREDICT THE EFFECTS OF HEAT, COLD AND HUMIDITY? HOW DO YOU KNOW IT WILL WITHSTAND A BLAST? HOW DO YOU MAKE A BETTER BATTERY? HOW DO YOU KNOW IF A PATENT HAS BEEN INFRINGED? HOW DO YOU KNOW WHEN TO CONDUCT E/L STUDIES? HOW DO YOU PREVENT ELECTRONICS FAILURE? HOW DO YOU PERFORM A SUCCESSFUL ILV? HOW DO YOU UNDERSTAND THE CAUSE OF A PAINT FAILURE? HOW DO YOU MEET GLOBAL REGULATORY REQUIREMENTS? HOW DO YOU DETERMINE THE CAUSE OF LANDING GEAR FAILURE? HOW DO YOU KNOW WHY A HARD DRIVE CRASHED? HOW DO YOU ESTABLISH LIABILITY THROUGH ENGINEERING ANALYSIS? HOW DO YOU FULLY CHARACTERIZE AN ANTIBODY DRUG CONJUGATE? HOW DO YOU TEST FOR BIOCOMPATI-BILITY? HOW DO YOU RESPOND TO A DATA CALL-IN? HOW DO YOU IDENTIFY CONTAMINANTS? HOW DO YOU PREVENT PACKAGING ISSUES? HOW DO YOU FIND CONTAMINATION ON SATELLITE PARTS? HOW DO YOU VALIDATE A CIRCUIT MODEL? HOW DO YOU DETERMINE THE ROOT CAUSE OF AN INDUSTRIAL FIRE? HOW DO YOU MONITOR CHARGE VARI-ANTS AND DEGRADATIONS? HOW DO YOU MEASURE PARTICLE SIZE? HOW DO YOU TACKLE A GLOBAL REGISTRATION? HOW DO YOU ENSURE CONSISTENT CRYSTAL FORM ACROSS BATCHES? HOW DO YOU PREVENT CONTAMINATION? HOW DO YOU TEST RELIABILITY OF NIGHT VISION SENSORS? HOW DO YOU MAKE AN LED BRIGHTER OR MORE AF-FORDABLE? HOW DO YOU KNOW IF SOMEONE IS USING YOUR TRADE SECRETS? HOW DO YOU DETECT POST-TRANSLATIONAL MODIFICATIONS? HOW DO YOU DETERMINE THE CAUSE OF FAILURE? HOW DO YOU MEASURE CHEMICAL RESIDUE IN MILK? HOW DO YOU KNOW WHAT ANALYTICAL TECHNIQUE TO USE? HOW DO YOU IMPROVE BIODEGRADABILI-TY? HOW DO YOU IDENTIFY METAL FATIGUE? HOW DO YOU VALIDATE SUPPLY CHAIN QUALITY? HOW DO YOU KNOW IF THERE IS A LEGAL CONFLICT? HOW DO YOU RE-OPTIMIZE AN

HOW DO YOU COMPLY WITH <USP 232/233>? HOW DO YOU ADDRESS AN FDA RESPONSE LETTER ASAP? HOW DO YOU MEASURE PESTICIDE DEGRADATION RATES? HOW DO YOU QUANTIFY ADDITIVES IN PLASTIC? HOW DO YOU PREDICT THE EFFECTS OF HEAT, COLD AND HUMIDITY? HOW DO YOU KNOW IT WILL WITHSTAND A BLAST? HOW DO YOU MAKE A BETTER BATTERY? HOW DO YOU KNOW IF A PATENT HAS BEEN INFRINGED? HOW DO YOU KNOW WHEN TO CONDUCT E/L STUDIES? HOW DO YOU PREVENT ELECTRONICS FAIL-URE? HOW DO YOU PERFORM A SUCCESSFUL ILV? HOW DO YOU UNDERSTAND THE CAUSE OF A PAINT FAILURE? HOW DO YOU MEET GLOBAL REGULATORY REQUIREMENTS? HOW DO YOU DETERMINE THE CAUSE OF LANDING GEAR FAILURE? HOW DO YOU KNOW WHY A HARD DRIVE CRASHED? HOW DO YOU ESTABLISH LIABILITY THROUGH ENGINEERING ANALYSIS? HOW DO YOU FULLY CHARACTERIZE AN ANTIBODY DRUG CONJUGATE? HOW DO YOU TEST FOR BIOCOMPATIBILITY? HOW DO YOU RESPOND TO A DATA CALL-IN? HOW DO YOU IDENTIFY CONTAMINANTS? HOW DO YOU PREVENT PACKAGING ISSUES? HOW DO YOU FIND CONTAMINATION ON SATELLITE PARTS? HOW DO YOU VALIDATE A CIRCUIT MODEL? HOW DO YOU DETERMINE THE ROOT CAUSE OF AN INDUSTRIAL FIRE? HOW DO YOU MONITOR CHARGE VARIANTS AND DEGRADATIONS? HOW DO YOU MEASURE PARTI-CLE SIZE? HOW DO YOU TACKLE A GLOBAL REGISTRATION? HOW DO YOU ENSURE CONSISTENT CRYSTAL FORM ACROSS BATCHES? HOW DO YOU PREVENT CONTAMINATION? HOW DO YOU TEST RELIABILITY OF NIGHT VISION SENSORS? HOW DO YOU MAKE AN LED BRIGHTER OR MORE AFFORDABLE? HOW DO YOU KNOW IF SOMEONE IS USING YOUR TRADE SECRETS? HOW DO YOU DETECT POST-TRANSLATIONAL MODIFICATIONS? HOW DO YOU DETERMINE THE CAUSE OF FAILURE? HOW DO YOU MEASURE CHEMICAL RESIDUE IN MILK? HOW DO YOU KNOW WHAT ANALYTICAL TECHNIQUE TO USE? HOW DO YOU IMPROVE BIODEGRADABILITY? HOW DO YOU IDENTIFY METAL FATIGUE? HOW DO YOU VALI-DATE SUPPLY CHAIN QUALITY? HOW DO YOU KNOW IF THERE IS A LEGAL CONFLICT? HOW DO YOU RE-OPTIMIZE AN ELISA METHOD WHEN A REAGENT LOT CHANGES? HOW DO YOU KNOW WHETHER THE LINER IS THE PROBLEM? HOW DO YOU EVALUATE HYDROPHOBIC COMPOUNDS BY SPME? HOW DO YOU COMPARE FEEDSTOCK SUPPLIERS? HOW DO YOU COMPARE FEEDSTOCK SUPPLIERS? HOW DO YOU TEST MEDICAL DEVICES IN A MALPRACTICE CASE? HOW DO YOU DETERMINE WHETHER A COMPOSITE IS SAFE? HOW DO YOU MEASURE THE ROUGHNESS OF A COATING? HOW DO YOU COMPLY WITH <USP 232/233>? HOW DO YOU ADDRESS AN FDA RESPONSE LETTER ASAP? HOW DO YOU MEAS-URE PESTICIDE DEGRADATION RATES? HOW DO YOU QUANTIFY ADDITIVES IN PLASTIC? HOW DO YOU PREDICT THE EFFECTS OF HEAT, COLD AND HUMIDITY? HOW DO YOU KNOW IT WILL WITHSTAND A BLAST? HOW DO YOU MAKE A BETTER BATTERY? HOW DO YOU KNOW IF A PATENT HAS BEEN INFRINGED? HOW DO YOU KNOW WHEN TO CONDUCT E/L STUDIES? HOW DO YOU PREVENT ELECTRONICS FAILURE? HOW DO YOU PERFORM A SUCCESSFUL ILV? HOW DO YOU UNDERSTAND THE CAUSE OF A PAINT FAILURE? HOW DO YOU MEET GLOBAL REGULATORY REQUIREMENTS? HOW DO YOU DETERMINE THE CAUSE OF LANDING GEAR FAILURE? HOW DO YOU KNOW WHY A HARD DRIVE CRASHED? HOW DO YOU ESTABLISH LIABILITY THROUGH ENGINEERING ANALYSIS? HOW DO YOU FULLY CHARACTERIZE AN ANTIBODY DRUG CONJUGATE? HOW DO YOU TEST FOR BIOCOMPATI-BILITY? HOW DO YOU RESPOND TO A DATA CALL-IN? HOW DO YOU IDENTIFY CONTAMINANTS? HOW DO YOU PREVENT PACKAGING ISSUES? HOW DO YOU FIND CONTAMINATION

HOW DO YOU DETERMINE THE ADVANTAGES OF USING 14C MATERIALS IN YOUR TESTING?

M-032717COPYRIGHT © 2017 EAG, INC. | REv. 01.03.18

INTRODUCTIONThe trifluoromethyl (CF3) group on small molecules (alkyl, aryl, and 5-/6-membered heterocyclic compounds) is a common component of agricultural chemicals such as insecticides, herbicides and fungicides. Approximately 8.5% (77 among 908 agrochemicals) of the pesticide entries in The Pesticide Manual (1) contained a CF3 group.

EXAMPLE OF PESTICIDES CONTAINING A CF3 GROUP:• Herbicides: Beflubutamid, Benfluralin, Cyflufenamid,

Diflufenican, Flazasulfuron, Fluazifop-P, Flufenacet, Fluometuron, Flupyrsulfuronmethyl, Flurochloridone, Flurtamone, Haloxyfop, Isoxaflutole, Oxyfluorfen, Penoxsulam, Picolinafen, Prosulfuron, Pyroxsulam, Tembotrione, Triflusulfuron, Tritosulfuron, etc.

• Fungicides: Flonicamid, Fluazinam, Fluopicolide, Fluopyram, Flutonanil, Penthiopyrad, Picoxystrobin, Trifloxystrobin, Triflumizole, Flutianil, etc.

• Insecticides/Acaricides: Acrinathrin,Bifenthrin, Cyflumetofen, Cyhalothrin, Fipronil, Fluvalinate, Indoxacarb, Lufenuron, Tefluthrin, Metaflumizone, Pyridalyl, Saflufenacil, Sulfoxaflor, etc.

[Phenyl-U-14C] or [5-membered heterocyclic-14C] pesticide containing a CF3 group was applied onto soil and various crops (wheat, lettuce, radish, carrot, and spinach) were grown in the soil for crop metabolism studies.

A 14C unknown polar metabolite in extracts of various crop and

soil samples was found as a major degradation product by > 10% of the total radioactive residues and > 0.01 mg/kg which triggers attempts to identify the metabolite.

The unknown polar metabolite in the crop and soil extracts was identified as trifluoroacetic acid (TFA) by high resolution LC/MS/MS, and confirmed by cochromatography using an authentic reference standard [14C]TFA.

Here, EAG Laboratories present various analytical techniques including sample preparation using HPLC, TLC, and state-of-the-art mass spectrometry using 14C tracer technology for identification of TFA in extracts of plant matrices.

METHODSGENERAL PROCEDURE FOR IDENTIFICATION/CONFIRMATION OF [14C]TFA AS AN UNKNOWN:

• [Phenyl-U-14C] or [5-membered heterocyclic-14C]pesticides containing a CF3 group was applied onto soil. various crops (wheat, lettuce, radish, carrot, and spinach) were planted in treated soil for crop metabolism studies.

• The raw agricultural commodities (immature/mature, root top, and forage, hay, straw and grain of crops) and soils were collected.

• Plant samples collected from treated soil were extracted with an acetonitrile/water mixture and acetonitrile. Also in a few studies the 14C-treated soil was also sampled and extracted with acetonitrile/1N HCl and acetonitrile/1N NaOH mixture after harvest. Extracts were combined and concentrated prior to HPLC analysis.

• Determined a 14C unknown polar metabolite (RT4) in extracts of the test systems as a major degradation product by > 10% of the total radioactive residues and > 0.01 mg/kg.

• Isolated the unknown polar metabolite using an analytical (C18) column.

• Purified the isolate using a mixed mode WAX-1 column.

• The isolate was identified using a high-resolution LC/MS.

• The isolate was confirmed by HPLC and TLC, cochromatographed with an authentic 14C-TFA.

APPLICATION NOTE

Identification of Trifluoroacetic Acid as Polar Metabolite from Pesticides Containing a Trifluoroacetic (CF3) Moiety Using 14C Tracer TechnologyBy Ki Chang An, Ying Yng Choy, Thomas Fleischmann, and David Dohn

Figure 1. An example of chemical structures including a trifluoromethyl group and 14C-test substances (* indicates position of 14C label)

EAG.COMCOPYRIGHT © 2017 EAG, INC. | REv. 01.03.18 M-032317

INSTRUMENTS FOR [14C]RADIOACTIVE DETECTION/IDENTIFICATION:

• HPLC with radioisotope detections was performed using Beta-RAM detector equipped with 500 μL liquid cell (LabLogic Systems, Ltd.) or collection of 15-20 sec fractions with subsequent liquid scintillation counting (MicroBeta2 Microplate Counter, PerkinElmer).

• Fractionation to vials and microplates: Gilson FC204 Fraction Collector.

• Radioisotope detection for TLC used phosphor imaging plate with the radioactivity visualized using a Molecular Dynamics Storm 820 Phosphorimager.

• LC-MS high resolution mass spectrometry was conducted with Thermo Scientific Q Exactive mass spectrometer interfaced with a Thermo Scientific Ultimate 3000 UHPLC system.

RESULTS1. A 14C-unknown polar metabolite eluting close to the HPLC

solvent front was found as a major degradation product (>0.01 mg/kg and >10% of total radioactive residues).

2. The 14C-unknown polar metabolite was purified on a mixed mode HPLC column with reversed-phase and weak anionic exchange chromatography after the isolation on the above C18 column.

3. Identification of the unknown polar metabolite using a high-resolution LC/MS

WHITE PAPER

Identification of Trifluoroacetic Acid as Polar Metabolite from Pesticides Containing a Trifluoroacetic (CF3) Moiety Using 14C Tracer Technology

Figure 1.1: C18 reverse phase HPLC from a wheat sample grown in soil treated with [phenyl-U-14C] pesticide

Figure 1.2: C18 reverse phase HPLC HPLC from a spinach sample grown in soil treated with [5-membered heterocyclic-14C] pesticide In a spinach sample grown in soil treated with [5-membered heterocyclic-14C]pesticide

Figure 1.3: Normal phase silica TLC

Figure 1.4: C18 reverse phase HPLC from a soil sample treated with [phenyl-U-14C] pesticide

Figure 2.1: C18 reverse phase HPLC from a wheat sample grown in soil treated with [phenyl-U-14C] pesticide

Figure 2.2: In a soil sample treated with [phenyl-U-14C]pesticide after harvest

Figure 3.1 (ESI-) LC-MS Analysis of [14C]Unknown Polar Metabolite as TFA

A. Extracted ion chromatogramB. MS spectrum showing molecular ion isotope pattern

m/z 112-114C. MS2 fragmentation pattern for m/z 112D. List of isotopic ratios of m/z 112

EAG.COMCOPYRIGHT © 2017 EAG, INC. | REv. 01.03.18 M-032717

APPLICATION NOTE

Identification of Trifluoroacetic Acid as Polar Metabolite from Pesticides Containing a Trifluoroacetic (CF3) Moiety Using 14C Tracer Technology

4. Confirmation of the unknown polar metabolite using an authentic 14C-TFA standard

Figure 3.2: Compared with commercial TFA Standard (ESI-) LC-MS Analysis of commercial TFA Standard

A. Extracted ion chromatogramB. MS spectrum showing molecular ion isotope pattern m/z

112-114C. MS2 fragmentation pattern for m/z 112D. List of isotopic ratios of m/z 112

Figure 3.3: Proposed chemical structure of the 14C-unknown polar metabolite

Figure 4.1: Spinach extract

Figure 4.2: Authentic 14C-TFA standard

Figure 4.3: Spinach extract and authentic 14C-TFA

Figure 4.4: In a wheat sample grown in soil treated with [phenyl-U-14C]pesticide [Mixed Mode HPLC]

Figure 4.5: In a soil sample treated with [phenyl-U-14C]

Figure 4.6: In a wheat sample grown in soil treated with [phenyl-U-14C]pesticide [Normal phase silica TLC]

Figure 4.7: In a spinach sample grown in soil treated with [5-membered heterocyclic-14C]pesticide

EAG.COMCOPYRIGHT © 2017 EAG, INC. | REv. 01.03.18 M-032717

APPLICATION NOTE

Identification of Trifluoroacetic Acid as Polar Metabolite from Pesticides Containing a Trifluoroacetic (CF3) Moiety Using 14C Tracer TechnologyCONCLUSIONS/DISCUSSION• When 14C-test pesticides containing a CF3 group were applied

to soil, a significant amount of an unknown polar metabolite (close to solvent front) in all crops grown in soil was determined a major product by > 0.01 mg/kg and >10% TRR.

• The unknown polar metabolite isolated by a reversed C18 column and a mixed mode WAX column was identified as TFA by a high-resolution LC/MS. The unknown metabolite was confirmed with an authentic 14C-TFA.

• TFA was observed in various crops (root, leafy vegetable, and small grain) via soil uptake, and soil.

• TFA was not found in target crops after foliar application, It is likely that TFA or its precursor is formed in soil by soil microorganism.

• A literature2 described agrochemicals of the formation of TFA in metabolism studies: Benfluralin, Fluazinam, Flufenacet, Fluormeturon, Flurtamone, Halozyfop-R, Oxyfluorfen, Saflufenacil, Trifloxystrobin, and Tritosulfuron

• The toxicological reference values derived for the metabolite TFA via the diet will not result in a consumer exposure. Therefore, TFA is unlikely to pose a public health concern.2

• In the analytical consideration for the metabolism studies, the major unknown polar metabolite was determined/identified as TFA.

ACKNOWLEDGEMENTS: We thank Julie Huang and Matthew Wilson for their kind contributions to this study.

REFERENCES1. The Pesticide Manual, 15th Edition-British Crop Production Council (2009). There are a total of 908 pesticide entries in this edition. Seventy-seven pesticides contained a CF3 moiety.

2. European Food Safety Authority, 2014, Reasoned opinion on the setting of MRLs for saflufenacil in various crops, considering the risk related to the metabolite trifluoroacetic acid (TFA), EFSA Journal, 2014;12(2):3585