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Innovations for safe and sustainable crop protection products
 9780841235380, 0841235384

Table of contents :
Safe and Sustainable Crop Protection......Page 2
Safe and Sustainable Crop Protection......Page 4
Library of Congress Cataloging-in-Publication Data......Page 5
Foreword......Page 6
Subject Index......Page 8
Preface......Page 10
The Microbiome of Fruit Flies as Novel Targets for Pest Management......Page 12
Invasive Fruit Flies: Small Insects of Big Economic Importance......Page 13
Current Status and Challenges of Fruit Fly Management......Page 20
The Fruit Flies’ Microbiome: Who Are the Players?......Page 21
Microbial Influence on Fruit Flies: Nutrition, Behavior, and Insecticide Resistance......Page 23
Leveraging Microbiomes to Develop Novel Management Strategies for Fruit Flies: Lessons from Different Insects......Page 25
Disruption of Microbiomes......Page 26
Paratransgenesis......Page 28
Attractants......Page 30
Optimization of SITs Using Probiotics......Page 31
References......Page 32
Modular Approach to Macrocyclic Picolinamides......Page 50
Figure 1. Structure of UK-2A.......Page 51
Figure 4. Macrocycle CAS-381 contains fewer esters and displays improved stability.......Page 52
Maximizing Synthetic Accessibility......Page 53
Synthesis of the Stereotriads......Page 54
Figure 10. Addition of an alcohol stereotriad into a Boc-aziridine carboxylate under Lewis acidic conditions.......Page 55
Modular Construction......Page 56
Figure 14. Scaffold markush for macrocycle derivatives with variations at the X,Y, and Z positions.......Page 57
References......Page 59
Background......Page 62
HRMS Analyzers......Page 63
Orbitrap......Page 64
Ionization Sources......Page 65
Other MS Technologies Applicable for Small-Molecule Structure Elucidation......Page 66
Typical Workflow for Impurity Identification......Page 67
GC/MS Study of Impurities in a Commercial Chlorpyrifos Sample......Page 68
Figure 4. The total ion chromatograms of (a) Chlorpyrifos and (b) Chlorpyrifos derivatized with BSTFA.......Page 69
Scheme 2. Haloxyfop-P Methyl Ester......Page 70
Figure 7. The positive ion ESI mass spectrum of Haloxyfop-P methyl ester showing [M + H]+, [M + Na]+, and [M + K]+.......Page 71
Figure 9. The negative ion ESI mass spectrum of Haloxyfop-P methyl ester showing [M − H]-, [M + O − H]+, and [M + O2 − H]+.......Page 72
Figure 11. The positive ion ESI MS/MS spectrum of [M + H]+ ions of Imp 1.......Page 73
Conclusion......Page 74
References......Page 75
Introduction......Page 78
Workflow for Development of Chiral Separation Methods......Page 79
Figure 1. Progression of chiral separation method development. (1) Receive racemic and enantioenriched sample. (2) Identify mobile phase composition. (3) Perform chiral stationary phase screening. (4) Optimize separation on best stationary phase. (5) Run both samples using optimized separation conditions and optimized sample prep. (6) Document method.......Page 80
Key Learnings......Page 81
Figure 3. Mixture of four isomers to be separated, containing two pairs of enantiomers.......Page 82
Figure 4. Progression of separation during method development using an OJ chiral stationary phase. (1) 15% IPA/hex at 40 °C. (2) 3% IPA/hex at 20 °C (first injection). (3) 1% IPA/hex at 20 °C. (4) 3% IPA/hex at 20 °C (second injection).......Page 83
Figure 6. Racemic molecule that needs to be separated into its enantiomers.......Page 84
Figure 8. Optimization of chiral separation conditions. (1) 2 mL/min, 5 μL injection of 1 mg/mL solution (0.005 mg), 214 nm. (2) 2 mL/min, 100 μL injection of 1 mg/mL solution (0.1 mg), 214 nm. (3) 2 mL/min, 100 μL injection of 10 mg/mL (1 mg), 214 nm. (4) 2 mL/min, 100 μL injection of 10 mg/mL (1 mg), 254 nm.......Page 85
Figure 10. Modified instrumentation configuration to allow analytical-scale isolation.......Page 86
Example 3: Application to Formulations......Page 87
Figure 15. Chiral separation developed for racemic material was used to analyze enantioenriched technical, which was then used to generate a formulation for field trials.......Page 88
Figure 17. Analysis of enantioenriched technical and formulation after 2 weeks at room temperature.......Page 89
Figure 20. Loss of enantiopurity observed upon addition of alkylamide solvent and dimethylamine to the formulation.......Page 90
Acknowledgments......Page 91
References......Page 92
Introduction......Page 94
Figure 1. Time difference of RP separation vs SFC separation of two chiral molecules.......Page 95
Figure 2. RPLC conditions and chromatograms.......Page 96
Figure 3. Conditions and representative chromatograms of proton donor evaluation.......Page 97
Figure 4. Calibration curves of prothioconazole from 50 to 6500 ppb with water added through the modifier and makeup flow and without water.......Page 98
Figure 5. RPLC and SFC ionization comparisons of prothioconazole.......Page 99
Figure 6. RPLC and SFC ionization comparisons of prothioconazole-desthio.......Page 100
Figure 7. Comparison of prothioconazole ionization efficiency using optimal mode and polarity in RPLC and SFC.......Page 101
Figure 8. Comparison of prothioconazole-desthio ionization efficiency using optimal mode and polarity in RPLC and SFC.......Page 102
Figure 10. SFC plot of S/N at different concentrations.......Page 103
References......Page 104
Introduction......Page 106
Methodology......Page 107
Results and Discussion......Page 108
Figure 3. Kinetics model for oxime degradation.......Page 109
Figure 5. Oxamyl degradation pathway.......Page 110
Figure 6. Oxamyl degradation and optimized data fit in soil.......Page 111
Figure 8. Optimized kinetics for conversion of cymoxanil to metabolites, NER, and CO2.......Page 112
Figure 9. Soil degradation pathway for cyhalofop butyl.......Page 113
Figure 10. Optimized kinetics for degradation of cyhalofop butyl.......Page 114
Figure 12. Optimized kinetics degradation of famoxadone and its metabolite H3310 in soil.......Page 115
References......Page 116
Creating Environmentally Resilient Agriculture Landscapes Using Precision Agriculture Technology: An Economic Perspective......Page 118
Introduction......Page 119
Incentive-Based Conservation......Page 120
Precision Agriculture Technology......Page 121
Precision Agriculture Application for Pesticide Mitigation......Page 122
Figure 2. Profit surface (dollar per acre) for a production soybean field in Lowndes County, Mississippi. Net Rev: net revenue.......Page 123
Figure 3. Total eligible area for CP-33, Filter Strips, on a grain farm in Tallahatchie, Mississippi. Reproduced with permission from reference 30. Copyright 2016 American Society of Agronomy, Crop Science Society of America, Soil Sciences Society of America.......Page 124
Figure 4. Total eligible area for CP-21, Habitat Buffers for Upland Birds, on a grain farm in Tallahatchie, Mississippi. Reproduced with permission from reference 30. Copyright 2016 American Society of Agronomy, Crop Science Society of America, Soil Sciences Society of America.......Page 125
Figure 6. Profit surface (dollar per acre) for a production soybean field with 120-foot CP-33, Habitat Buffers for Upland Birds, scenario in Lowndes County, Mississippi. Net Rev: net revenue.......Page 126
Discussion......Page 127
References......Page 128
The Evolution, History, and Market Demand for Contract Research Organizations......Page 134
The Regulatory Reality......Page 135
Managing Data......Page 137
The Role of the SD......Page 139
The Challenge of Flexibility......Page 140
The Challenge of New Technology......Page 141
Conclusions......Page 142
References......Page 143
Qi Yao......Page 144
Indexes......Page 146
Author Index......Page 148
I......Page 150
N......Page 151
P......Page 152

Citation preview

1334

innovate. This volume contains select innovations and advances in crop protection technology from the ACS Division of Agrochemicals. Chapters focus on biochemistry, like microbiomes and antibiotics, and workflows, like separation, characterization, and analysis. These contributions reflect the diverse program of the national meeting and the Agrochemicals division.

PUBLISHED BY THE

American Chemical Society SPONSORED BY THE

F O O D

As the world population grows, agrochemical researchers continue to

&

HIGHLIGHTS FROM THE ACS NATIONAL MEETING FALL 2018

A G R I C U L T U R E

VOLUME 1334

SAFE AND SUSTAINABLE CROP PROTECTION

ACS SYMPOSIUM SERIES

ACS SYMPOSIUM SERIES

SAFE AND SUSTAINABLE CROP PROTECTION

ACS Division of Agrochemicals

LYNN et al.

LYNN, MA, YANG & YAO

Safe and Sustainable Crop Protection

ACS SYMPOSIUM SERIES 1334

Safe and Sustainable Crop Protection Kari Lynn, Editor Corteva Agriscience Indianapolis, Indiana

Mingming Ma, Editor Corteva Agriscience Indianapolis, Indiana

Qiang Yang, Editor Corteva Agriscience Indianapolis, Indiana

Qi Yao, Editor University of Maryland College Park, Maryland

Sponsored by the ACS Division of Agrochemicals

American Chemical Society, Washington, DC

Library of Congress Cataloging-in-Publication Data Library of Congress Cataloging in Publication Control Number: 2019050262

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48n1984. Copyright © 2019 American Chemical Society All Rights Reserved. Reprographic copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Act is allowed for internal use only, provided that a per-chapter fee of $40.25 plus $0.75 per page is paid to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. Republication or reproduction for sale of pages in this book is permitted only under license from ACS. Direct these and other permission requests to ACS Copyright Office, Publications Division, 1155 16th Street, N.W., Washington, DC 20036. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

Foreword The purpose of the series is to publish timely, comprehensive books developed from the ACS sponsored symposia based on current scientific research. Occasionally, books are developed from symposia sponsored by other organizations when the topic is of keen interest to the chemistry audience. Before a book proposal is accepted, the proposed table of contents is reviewed for appropriate and comprehensive coverage and for interest to the audience. Some papers may be excluded to better focus the book; others may be added to provide comprehensiveness. When appropriate, overview or introductory chapters are added. Drafts of chapters are peer-reviewed prior to final acceptance or rejection. As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproductions of previous published papers are not accepted. ACS Books Department

Contents Preface .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

ix

1. The Microbiome of Fruit Flies as Novel Targets for Pest Management .  . . . . . . . . . . . . . . . . . . . . . . . . . . .  Adam Robert, Masroor Qadri, Jesse Blair, and Adam Chun-Nin Wong

1

2. Modular Approach to Macrocyclic Picolinamides .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  39 Kevin G. Meyer, Chenglin Yao, Ben Nugent, Karla Bravo-Altamirano, Jessica Herrick, William Dent, Fangzheng Li, Jeremy Wilmot, John F. Daeuble, Jonathan DeLorbe, Yu Lu, Rebecca LaLonde, Kyle DeKorver, and Timothy Boebel 3. Workflows for the Structure Elucidation of Impurities in Synthetic Agrochemicals Using Mass Spectrometry.  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  51 Chengli Zu, Daniel Knueppel, and Jeff Gilbert 4. Separation of Chiral Molecules in Support of Process Chemistry and Formulations Research .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  67 Daniel I. Knueppel, Jens T. Richards, and John M. Atkinson 5. Optimization of Critical Parameters in Chiral Analysis of Prothioconazole by Supercritical-Fluid Chromatography–Mass Spectrometry .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  83 Jens Richards, Daniel Knueppel, Jeff Godbey, and Chengli Zu 6. Characterization of Nonextractable Residues in Soil via Kinetics Modeling .  . . . . . . . . . . . . . . . . .  95 Ashok K. Sharma and Chengwei Fang 7. Creating Environmentally Resilient Agriculture Landscapes Using Precision Agriculture Technology: An Economic Perspective .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  107 Mark D. McConnell 8. Contract Research, Good Laboratory Practices, and Other Challenges for the Agrochemical Professional .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  123 Maria (Marian) Ponte and Megha Chandrashekhar Editors’ Biographies .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  133 Indexes Author Index .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  137 Subject Index .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  139

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Preface It is estimated that the world’s population will grow to 10 billion by the year 2050 and feeding this population will require that food production be increased by over 70%. To address the food security needs of the growing population, modern agriculture must continually develop technologies that increase production while ensuring human and environmental safety. Such technologies include developing innovative crop protection products to help fight crop damage by weeds, fungi, insects, fertilizers, and growth hormones that are widely used for agricultural purposes. To develop safe, sustainable, and cost-effective solutions for farmers and the environment, agriculture scientists are working diligently to understand how agrochemicals impact the environment, as well as devising protection strategies and potential cleaning approaches. The agrochemical (AGRO) division of the American Chemical Society (ACS) provides a key forum to help agrochemicals scientists work towards a common goal: Fostering Sustainable Agriculture and Protecting Public Health through Chemistry. Since 1974, the ACS symposium series has been publishing original research and review papers in book form. This mechanism provides a quick way of sharing scientific findings and information. This edition presents the highlights of the AGRO division symposia at the 256th fall ACS National Meeting in Boston, MA, USA in 2018. These symposia provided a great opportunity for agrochemicals scientists to discuss current emerging issues by gathering the leading experts within the agrochemical field. Fifty-four platform presentations and one poster section were presented within the AGRO division. The highlighted symposium speakers were invited to submit their research papers, and we are honored to have constructed their work into this book. This ACS ARGO highlight book consists of the innovative approaches of agrochemical design and characterization described in the chapters “Modular Approach to Macrocyclic Picolinamides,” “The Microbiomes of Fruit Flies as Novel Targets for Pest Management,” and “Characterization of Non-extractable Residues in Soil via Kinetics Modeling”; as well as usage of the state-of-art analytical technologies in agrochemical separation and identification, such as highlighted in the following chapters: “Optimization of Critical Parameters in Chiral Analysis of Prothioconazole by SFC-MS,” “Separation of Chiral Molecules in Support of Process Chemistry and Formulations Research,” and “Workflows for the Structure Elucidation of Impurities in Synthetic Agrochemicals Using Mass Spectrometry.” This book also includes an economic perspective of precision agriculture technology in the chapter “Creating Environmentally Resilient Agriculture Landscapes Using Precision Agriculture Technology: An Economic Perspective” and recommended laboratory practices in the chapter “Contract Research, Good Laboratory Practices, and Other Challenges for the Agrochemical Professional.” Overall this book has successfully captured the highlights of agrochemical research that directly relates to public welfare and environmental safety. We hope to share this book with students, chemists, engineers, regulators, and enterprisers to both promote a better understanding of agrochemicals and to work together to benefit public health and the environment. We are very grateful to the authors and peer-reviewers for contributing to the high quality of this edition. We are ix

also very thankful to the ACS Book Editorial office staff and for all technical support we received during the preparation of this book. Kari Lynn Predictive Safety Center Crop Protection Regulatory Sciences, R&D Corteva Agriscience Indianapolis, Indiana 46268, United States Mingming Ma Predictive Safety Center Crop Protection Regulatory Sciences, R&D Corteva Agriscience Indianapolis, Indiana 46268, United States Qiang Yang Product Design & Process R&D Corteva Agriscience 9330 Zionsville Road Indianapolis, Indiana 46268, United States Qi Yao Department of Chemical and Biomolecular Engineering University of Maryland College Park, Maryland 20902, United States

x

Chapter 1

The Microbiome of Fruit Flies as Novel Targets for Pest Management Adam Robert,#,1 Masroor Qadri,#,1 Jesse Blair,1 and Adam Chun-Nin Wong*,1,2 1Entomology and Nematology Department, University of Florida,

Gainesville, Florida 32611, United States 2Genetics Institute, University of Florida, Gainesville, Florida 32611, United States *E-mail: [email protected].

#Co-first authors.

Invasive fruit flies are global threats to food security and economy because they are destroying crops and inflicting multibillion-dollar losses annually. In the United States, regions with subtropical climates such as Florida, Georgia, and California with yearlong availability of cultivated crop and noncrop hosts, offer ongoing opportunities for fruit fly invasion. Incidences of tephritid invasion have resulted in unprecedented losses and eradication efforts. Drosophila suzukii (Matsumura) is another serious pest that has swept across 42 states since it first invaded California in 2008, causing millions of dollars in losses because of reduced crop yield and expenses associated with control measures and sorting processes for both fresh and processed fruits. Despite decades of research and pest management efforts on invasive fruit flies, several critical gaps of knowledge on their basic biology remain, including: How are they able to colonize and develop in vastly different host plants? To what extent are their life history traits (longevity, reproductive output, and dispersal) shaped by utilizing different food sources? And how do adults navigate the environment and make foraging or oviposition decisions? These important questions can be tied together by focusing on the extended phenotype of the flies’ microbiome in shaping the plant environment and feedback on pest fitness (such as larval development, starvation resistance, longevity, reproduction, and behavior such as mating, foraging, and oviposition). In this chapter, we review the latest developments in basic and applied microbiome research on invasive fruit flies, leading to a discussion of innovative microbialbased approaches that can be implemented into fruit fly management programs. Three different avenues will be discussed: suppression of fly performance by modifying the fly microbiome, development of microbial-based attractants and repellents, and optimization of sterile insect techniques using probiotics. The chapter uses the invasive D. suzukii as an example and outlines opportunities and obstacles for exploiting the microbiomes in D. suzukii management.

© 2019 American Chemical Society

Invasive Fruit Flies: Small Insects of Big Economic Importance The global fruits and vegetables processing market is estimated to be worth more than $240 billion USD and is set to grow favorably by 7% yearly (1, 2). Multiple socioeconomic factors have stimulated growth in this horticulture sector, including an increasing human population, particularly an upsurge in middle-class populations with improved income, changing consumer dietary lifestyles due to an awareness of healthy eating, and concerns with environmental impacts associated with livestock production (for example, antibiotic use (3) and greenhouse gas emissions (4, 5)). The rising demand for fruits and vegetables is beneficial in the context that it creates job opportunities and promotes economic growth. In the developing world, horticultural development also becomes a cornerstone in alleviating issues associated with poverty, hunger, and malnutrition (6, 7). However, such trends also present unique agricultural challenges. To keep pace with the fast-growing population and changing demographics, it was predicted that the global food supply will have to increase by up to 110% to meet the ongoing demand (8). One of the biggest hurdles to reach the goal of a prolific horticultural system is crop losses due to insect pests. Invasive fruit flies in the families Tephritidae and Drosophilidae are among the most destructive pests of fruits and vegetables. Tephritidae, also known as the true fruit flies, is a large family encompassing more than 4700 species from 500 genera (9, 10). Drosophilidae also consists of approximately 4000 species from 80 genera (11). At least 250 species of fruit flies are thought to be of economic significance (12). Among these, several species are some of the most significant crop pests in terms of invasiveness and the economic losses they induce. These include tephritid fruit flies such as the Mediterranean fruit fly (Ceratitis capitata), Anastrepha fruit flies such as the Mexican fruit fly (Anastrepha ludens), Bactrocera fruit flies such as the Oriental fruit fly (Bactrocera dorsalis), along with the drosophilid species Drosophila suzukii and the African fig fly (Zaprionus indianus) (13, 14). The economic burden caused by invasive fruit flies can be regional and worldwide. Taking D. suzukii in Florida as an example, it was first detected in a single county (Hillsborough) in 2009 (15). In just over four years, the pest has spread to 28 different Florida counties, resulting in $15 million in annual losses to the small fruit industry (16). On a global scale, D. suzukii has invaded South America (17, 18) and has been in most countries across Europe for at least 10 years (19). The economic loss caused by D. suzukii is on the scale of billions of dollars, based on figures from the United States (18, 20) and regional studies in Europe (21, 22). In the absence of interventions, fruit fly pests can cause crop damage leading to 80–100% losses (10). In terms of physiology, most fruit flies are characterized by a short generation time coupled with a high reproductive rate (23). D. suzukii takes between 7 and 12 days to develop from eggs to adults (23), allowing up to 13 generations per year (24). A single D. suzukii female can produce 200 to 600 eggs in her lifetime of 3 to 9 weeks (25). A strong flight and dispersal capacity may facilitate fruit fly invasion and spread. The flight capacity of the Oriental fruit fly, B. dorsalis, has been extensively studied (more than 20 studies) and was found to range from 0.13 to 3.1 km mean distance, with two independent studies showing that they could fly up to 22.7 km (26) and 94 km (27). (a summary of findings can be found in Table 1 of the review by Dominiak et al. (28)). Field recapture trials have shown C. capitata can disperse up to 9.5 km, although the majority of flies traveled a distance of 75%. Inhibition of the LEPTNO MET complex was measured for X-YZ scaffolds where R = H. LogD values for azoxystrobin and epoxiconazole were taken from the University of Hertfordshire Pesticide Properties Database: https://sitem.herts.ac.uk/aeru/ppdb/. a Biology

The macrocyclic analogs showed a broad range of lipophilicity with LogD values ranging from 4.3 (for the O-OO scaffold) to 5.7 (for the C-CC scaffold). Unsurprisingly, the LogD values correlate with the amount of ethereal oxygens present in the macrocycle ring, with LogD increasing as those oxygen atoms are replaced with more lipophilic methylenes. Lipophilicity appeared to have a negligible effect on the inhibition of LEPTNO MET, with all eight analogs showing subnanomolar IC50 values. Translation of the potent enzyme activity to whole-plant disease control gave mixed results. In general, the analogs with two or three ethereal oxygen atoms performed better than those with only one or no oxygen atoms. Control of wheat glume blotch, rice blast, and cucumber anthracnose across all analogs was very good, while control of sugar beet leaf spot and tomato early blight was poor. It should be noted that these tests merely show the efficacy of the molecules against these diseases averaged over three rates and may not accurately reflect the commercial broad-spectrum potential of these molecules because acceptable rates of application may be higher for some crops than others. Interestingly, for control of wheat leaf blotch and wheat leaf rust, the four analogs with oxygen at the Z position (O-OO, O-CO, C-OO, and C-CO) showed the most robust activity. Since the enzyme potency for these four molecules is similar to the other analogs in which Z = CH2, this 47

improved whole-plant control may suggest that oxygen at the Z position gives a slight plant mobility advantage in wheat.

Summary Eight macrocyclic fungicides similar in structure to the natural product UK-2A were investigated. Three positions in the macrocyclic framework were varied with either an oxygen atom or a methylene group. Determining the optimal number and positioning of oxygen atoms was best achieved using a modular synthetic strategy in which all targets could be derived from a small number of synthetic inputs. The study showed that a methylene linkage in the macrocycle backbone was easily accessible using a variety of chemical conditions and strategies and was thus preferred over an ether linkage. Biology tests revealed a preference for an oxygen atom to be in at least two of the three positions for lower lipophilicity and optimal whole-plant activity. Tests against wheat diseases also showed a preference for oxygen at the Z position (C8 of the macrocycle), which may suggest a plant mobility advantage. Taking into account both the optimal criteria for synthetic flexibility and optimal biological efficacy, the C-OO variant of the X-YZ scaffold emerged as the preferred macrocycle in this series.

Acknowledgments This research was performed in collaboration with Meiji Seika Pharma Co. Ltd. The authors wish to thank Meiji Seika Pharma Co. Ltd. for the generous supply of UK-2A to aid in SAR research. The authors would also like to thank the following Corteva Agriscience scientists for their suggestions and aid in collecting valuable data associated with this work: Tye Burgland, Scott Castetter, Paul Graupner, Stacy Meyer, Kyung Myung, W. John Owen, Jim Renga, Thomas Slanec, Nick Wang, Quanbo Xiong, and David Young.

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Dayan, F. E.; Cantrell, C. L.; Duke, S. O. Natural products in crop protection. Bioorg. Med. Chem. 2009, 17, 4022–4034. Cantrell, C. L.; Dayan, F. E.; Duke, S. O. Natural products as sources for new pesticides. J. Nat. Prod. 2012, 75, 1231–1242. Gerwick, B. C.; Sparks, T. C. Natural products for pest control: An analysis of their role, value and future. Pest Manag. Sci. 2014, 70, 1169–1185. Sparks, T. C.; Hahn, D. R.; Garizi, N. V. Natural products, their derivatives, mimics and synthetic equivalents: Role in agrochemical discovery. Pest Manag. Sci. 2017, 73, 700–715. Ueki, M.; Abe, K.; Hanafi, M.; Shibata, K.; Tanaka, T.; Taniguchi, M. UK-2A, B, C and D, Novel antibiotics from Streptomyces sp. 517-02. I. Fermentation, isolation and biological properties. J. Antibiotics 1996, 49, 639–643. Ueki, M.; Taniguchi, M. The mode of action of UK-2A and UK-3A, novel antifungal antibiotics from Streptomyces sp. 517-02. J. Antibiotics 1997, 50, 1052–1057. Torriani, S. F. F.; Melichar, J. P. E.; Mills, C.; Pain, N.; Sierotzki, H.; Courbot, M. Zymoseptoria tritici: A major threat to wheat production, integrated approaches to control. Fungal Genet. Biol. 2015, 79, 8–12.

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Owen, W. J.; Meyer, K. G.; Slanec, T. J.; Wang, N. X.; Meyer, S. T.; Niyaz, N. M.; Rogers, R. B.; Bravo-Altamirano, K.; Herrick, J. L.; Yao, C. Synthesis and biological activity of analogs of the antifungal antibiotic UK-2A. I. Impact of picolinamide ring replacement. Pest Manag. Sci. 2019, 75, 413–426. Owen, W. J.; Meyer, K. G.; Meyer, S. T.; Li, F.; Slanec, T. J.; Wang, N. X.; Yao, C. Synthesis and biological activity of analogs of the antifungal antibiotic UK-2A. II. Impact of modifications to the macrocycle benzyl position. Pest Manag. Sci. 2019, 75, 1831–1846. Owen, W. J.; Meyer, K. G.; Niyaz, N. N.; Fitzpatrick, G. M.; Meyer, S. T.; Slanec, T. J.; Wang, N. X.; Nugent, J.; Ricks, M. J.; Rogers, R. B.; Yao, C. Synthesis and biological activity of analogs of the antifungal antibiotic UK-2A. III. Impact of modifications to the macrocycle isobutyryl ester position. Pest Manag. Sci. DOI: 10.1002/ps.5511. Xiong, Q.; Myung, K.; Yao, C.; Graupner, P.; Adelfinskaya, Y. A.; Daeuble, J. F.; Meyer, S. T.; Buchan, Z.; Wang, N.; Meyer, K. G. Characterizing the surface abiotic degradation products of UK-2A. Abstracts of Papers, 254th ACS National Meeting & Exposition; Washington, DC, August 20–24, 2017; American Chemical Society: Washington, DC, 2017; AGRO 135. Owen, W. J.; Yao, C.; Myung, K.; Kemmitt, G.; Leader, A.; Meyer, K. G.; Bowling, A. J.; Slanec, T.; Kramer, V. Biological characterization of fenpicoxamid, a new fungicide with utility in cereals and other crops. Pest Manag Sci 2017, 73, 2005–2016. Nugent, B.; Meyer, K. G.; Yao, C.; Owen, W. J.; Renga, J.; Myung, K.; Daeuble, J.; Johnson, P. New macrocyclic compound for broad spectrum disease control. Abstracts of Papers, 254th ACS National Meeting & Exposition; Washington, DC, August 20–24, 2017; American Chemical Society: Washington, DC, 2017; AGRO 390. Whiteker, G. T.; Borromeo, P.; Li, F.; Roth, G. Process for the preparation of 4-alkoxy3-hydroxypicolinic acids via acetylation of 4-alkoxy-3-acetoxypicolinic acids under SchottenBaumann reaction conditions. World Patent Application WO 2017127791, 2017. Lalonde, R.; Meyer, K. G.; Li, F.; Wilmot, J.; Bravo-Altamirano, K.; Yao, C.; O’Callaghan, I.; Herrick, J.; DeKorver, K.; Lu, Y. Preparation and use of macrocyclic picolinamides as fungicides for control or prevention of fungal attack on plants. U.S. Patent 9,681,664, 2017. Boebel, T. A.; Lu, Y.; Meyer, K. G.; Yao, C.; Daeuble, J. F.; Bravo-Altamirano, K.; Nugent, B. M. Preparation of macrocyclic picolinamide derivatives as fungicides. U.S. Patent 9,955,691, 2018. Li, F.; Meyer, K. G.; Renga, J.; Yao, C.; Wilmot, J.; Herrick, J.; Bravo-Altamirano, K.; Boebel, T. Preparation of macrocyclic picolinamides as fungicides. U.S. Patent 9,629,365, 2017. DeKorver, K. A.; DeLorbe, J. E.; Meyer, K. G.; Yao, C.; Heemstra, R. J.; Dent, W. H., III. Preparation and use of macrocyclic picolinamides as fungicides. U.S. Patent 9,549,556, 2017. Wu, Y.-C.; Zhu, J. Asymmetric total synthesis of (-)-Renieramycin M and G and (-)Jorumycin using aziridine as a lynchpin. Org. Lett. 2009, 11, 5558–5561. Collier, P. N.; Patel, I.; Taylor, R. J. K. A concise, stereoselective synthesis of meso-2,6diaminopimelic acid (DAP). Tetrahedron Lett. 2001, 42, 5953–5954.

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Chapter 3

Workflows for the Structure Elucidation of Impurities in Synthetic Agrochemicals Using Mass Spectrometry Chengli Zu,* Daniel Knueppel, and Jeff Gilbert Crop Protection Product Design and Process R&D, Corteva AgriscienceTM, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States *E-mail: [email protected].

Identification of impurities in synthetic active ingredients is critical for the registration of products with regulatory agencies and to understand mechanisms of side reactions in order to eliminate or reduce the presence of impurities in the final product. Liquid chromatography or gas chromatography coupled with high-resolution mass spectrometry is an excellent tool for structure elucidation of impurities due to its combination of speed, sensitivity, and mass accuracy. The selection and application of appropriate data reduction tools are also important in this process. Efforts to improve the confidence of structural assignments and the overall throughput of impurity identification using commercial software and hardware tools are discussed. The characterization of the impurities present in two commercial agricultural chemicals is presented.

Background The world population is projected to exceed 9 billion by 2050. Consequently, significantly more food will be needed to feed the growing population of the planet. Additionally, dietary changes are occurring in emerging markets with rapidly growing populations, and their demand for grain is estimated to increase by more than 50% by 2050 (1). With limited arable lands, such a gain in yield will rely heavily on new technologies including novel crop protection agrochemicals and innovative seeds technologies. It is both desired by consumers and required by regulatory agencies that new agrochemicals and formulations be efficacious, ecofriendly, reasonably priced, and broad spectrum for sustainable market growth. Most active ingredients (AIs) are small-molecule organic compounds prepared by multistep organic synthesis. Side products (i.e., impurities) generated from these novel synthetic routes are usually new compounds that are often not registered in conventional databases. High-resolution mass spectrometry (HRMS) with accurate mass capabilities is critical for assigning the molecular formula of these unknowns, which is typically the first step in determining their structures (2). © 2019 American Chemical Society

Characterization of impurities in synthetic AIs is required for registration of products with regulatory agencies prior to their launch. Also, it is useful to understand the mechanisms of side reactions to eliminate or reduce the presence of impurities in the final product. HRMS coupled with separation technologies including liquid chromatography (LC) or gas chromatography (GC) is used in almost every stage of product development due to its speed, sensitivity, and selectivity. The development of a typical AI can be divided into several stages, as illustrated in Figure 1. In this discussion, we will describe several of the applications of HRMS in the development of new agricultural chemicals.

Figure 1. The role of mass spectrometry in the pipeline.

Mass Spectrometry Instruments Common analytical tools capable of fully or partially determining molecular structures include mass spectrometry (MS), NMR spectroscopy, X-ray crystallography, Fourier transform infrared spectroscopy, and UV spectroscopy. Each of these techniques has advantages and disadvantages, and as a result, researchers tend to use more than one approach to assign structures with high confidence. Often, it is necessary to synthesize the compound of the proposed structure, and then characterize it with the same analytical techniques to validate the structure assignment. Due to its speed, accuracy, selectivity, and sensitivity, HRMS in combination with a method of separation (e.g., LC or GC) is often the first choice for preliminary identification of unknown species. In the past, identification of structure and investigation of fragmentation pathways were accomplished mainly on low-resolution mass spectrometers. Thanks to recent advancements in HRMS instrumentation, several different types of high-resolution mass spectrometers are now commercially available. These mass spectrometers are typically categorized according to their inlet type, ionization sources, and mass analyzers. HRMS Analyzers Four types of mass analyzers are typically used for accurate mass measurement: magnetic sector mass analyzers, Fourier transform–ion cyclotron resonance, time of flight (TOF), and Orbitrap mass analyzers. The magnetic sector mass analyzer is less common due to its high cost, slow scan rates, and limited flexibility. Fourier transform–ion cyclotron resonance can provide ultra-high resolution; however, its use is also somewhat limited by its high cost and relatively slow scan rate, and thus is not well suited for LC or GC applications. Both TOF and Orbitrap mass analyzers provide medium to high resolution combined with fast scan rates and are the most commonly used instruments for LC/ MS and GC/MS workflows. 52

TOF and Quadrupole TOF The TOF analyzer is one of the most commonly used mass analyzers in the accurate mass analysis of both small organic molecules and biomolecules (3, 4). The basic principles of TOF are relatively straightforward. Ions generated in the ion source are pulsed into an evacuated tube under the influence of an electric field and travel a known distance to a detector. In practice, the length of the flight tube and the applied accelerating electric field are typically fixed; therefore, the time (t) of flight on an ion can be directly converted to measured m/z values. The general advantages and disadvantages of TOF analyzers are summarized in Table 1. Table 1. Advantages and Disadvantages of the TOF Analyzer Advantages

Disadvantages

Fast data acquisition rate

Narrow dynamic range

High sensitivity

Requires reference mass for optimal mass accuracy

Wide mass range

Difficult to resolve high-mass ions (>m/z 3000). However, this is not typically an issue for small molecules

Medium to high resolution Good mass accuracy Simple tuning and calibration Relatively low cost for a mass analyzer with accurate mass capability

The hybrid quadrupole TOF (QTOF) instrument combines a quadruple analyzer and a collision cell with a TOF analyzer. This configuration allows users to conduct tandem mass spectrometry (MS/MS) experiments with accurate mass measurement of product ions. Because the elemental compositions of both parent and fragment ions can be determined with high confidence with QTOF, it has become an attractive tool for de novo structure elucidation. Both TOF and QTOF analyzers are suitable for LC and GC applications due to their sensitivity and fast rates of data acquisition. Orbitrap The first Orbitrap MS system was brought to market by Thermo Fisher Scientific Inc. in 2005 (4, 5). Since then, its market share has grown significantly. In principle, the Orbitrap mass analyzer belongs to the Fourier transform ion trap family. It is basically composed of three electrodes. Two cuplike outer electrodes face each other and are electrically isolated by a central ring made of a dielectric material. The central electrode is a “spindle-shaped” rod positioned between the two outer electrodes and aligned via the dielectric end spacers. All the Orbitrap analyzers utilize a C-trap device to inject ions through a slot between the central and outer electrodes, using a compensation electrode relative to one of the outer electrodes. The ions then circulate in a radial electric field inside the trap created by the electrodes. An image current generated by harmonic axial oscillated ions is received by the outer electrodes; this is then Fourier-transformed from the frequency domain to produce a mass spectrum. Over the past decade, Thermo Fisher has introduced a series of Orbitrap systems. The Orbitrap instruments most often used for small-molecule identification are the Q Exactive Orbitrap 53

series. Currently, both LC and GC variants of the Q Exactive Orbitrap are commercially available. The general advantages and disadvantages of Orbitrap analyzers are summarized in Table 2. Table 2. Advantages and Disadvantages of the Orbitrap Analyzer High to ultra-high resolution

Slower scan rates than TOF analyzers

High sensitivity

Limited space charge

Broad dynamic range

Mass range limited by the “multiplication factor of 15”

High mass accuracy even without reference mass

Potential artifact peaks due to ion–water reaction in the C-trap

High stability

Ionization Sources Ionization technology is vital to the development of MS techniques, and rapid advances in this area have had far-reaching influence on other fields of science. For example, since the invention of electrospray ionization (ESI), the study of biomolecules has gone through a revolutionary change. Like most technologies, each ionization technology has its merits and limitations. The most common ionization tools used in LC/MS and GC/MS systems are discussed next. Electron ionization (EI) is one of the oldest ionization techniques that is still prevalent in many GC/MS systems (6). It is the only truly standardized ionization technique across all the commercial GC/MS systems. In EI, a molecule is introduced in the vapor state and bombarded with electrons, resulting in both ionization and, frequently, fragmentation. The type and intensity of the fragment ions produced under EI conditions are characteristic of the molecule, in many cases compounds produce a unique “fragmentation pattern” in their mass spectrum. The fragmentation pattern of a molecule in EI is impacted by the applied electron energy expressed in electron voltage (eV). and. Although this can be controlled, the default electron energy on most commercial GC/MS systems is 70 eV, which is the standard value for the generation of spectra that are searchable in National Institute of Standards and Technology and other commercial EI libraries. Compared with EI, lower energy is transferred in the chemical ionization (CI) process, resulting in less fragmentation and, typically, the observation of molecular adduct ions (7, 8). CI reactant ions are formed through the interaction of electrons with a reagent gas. Methane and ammonia are commonly used as reagent gases, which are ionized to produce reagent ions of different proton affinities. These reagent ions then react with the analytes to produce analyte adduct ions. For example, methane reagent gas generates CH5+, C2H5+, and C3H5+ reagent ions. These reagent ions react with the analyte (M) to produce [M + H]+, [M + C2H5]+, and [M + C3H5]+ adduct ions. CI can provide some selectivity in ionizing the material of interest through the selection of reagent gases that produce reagent ions with differing proton affinities. Low-energy reactant ions, like t-C4H9+, H3O+, or NH4+ from isobutane, water, or ammonia, respectively, frequently generate spectra that contain only [M + H]+. Although CI may provide less structural information than EI, the enhanced adduct ion species produced in CI is often a major advantage. Thus, CI is considered a complementary technique to EI. In addition, a CI source can often be operated in the negative ion polarity mode. Depending on the nature of the analyte and reagent gas selected, radical anions, deprotonated molecules, and anion adducts can be formed under these conditions (9, 10). Negative 54

chemical ionization (NCI)/MS has proven to be very useful for the analysis of many agricultural chemicals (11). In atmospheric pressure ionization (API), ions are generated at atmospheric pressure and transferred into the instrument for mass analysis and detection. There are three major API sources commonly used in LC/MS: ESI (12, 13), atmospheric pressure chemical ionization (APCI) (14), and atmospheric pressure photoionization (15). The related atmospheric pressure gas chromatography system is the result of an APCI device modified for GC application (16). It is generally accepted that ions generated with ESI are mostly preformed in solution. The transfer and separation of ions from solvent occur in a capillary tube with a high voltage applied, typically in the range of 2.5–6.0 kV, relative to other parts of the instrument. A nebulizer gas flow (e.g., nitrogen gas) surrounding the capillary tube aids the formation of a “Taylor Cone” of charged droplets consisting of ionic species and solvent after emission from the capillary. The charged droplets continuously shrink in size and eventually form ions in the gas phase due to Coulombic repulsion. This process is usually assisted by an elevated source temperature (e.g., 100–400 °C) and a concurrent stream of inert (sheath) gas flow. ESI is used in both small- and large-molecule analysis. In positive ESI, ions are typically formed as proton, ammonium, or sodium adducts, depending on the properties of the analytes and the presence of any added modifiers. Multiply charged ion species can also be formed if more than one ionizable functional group or site are present in a molecule. In APCI, the mobile-phase composition (i.e., solvents and modifiers) is critical in the formation of analyte ions. The solvent molecules, which are present in large excess relative to the analyte, are ionized to produce reagent ions through interactions with the electrons emitted from a corona discharge. Analyte molecules are then ionized by these solvent reagent ions via chemical ionization. Unlike in ESI, the solvent-evaporation and ion-formation processes are separate in APCI, thus allowing the use of low-polarity solvents that are unfavorable for ESI ion formation. This is especially important for normal phase LC/MS applications. Photoionization involves the absorption of a high-energy photons by a molecule and subsequent ejection of an electron. In direct atmospheric pressure photoionization, this analyte molecule is directly ionized, often forming the molecular radical cation M˙+. The analyte may be detected as M˙+, or it may react with surrounding molecules and be detected as an adduct ion. A common reaction is the abstraction of a hydrogen atom from the abundant solvent to form the stable [M + H]+ cation. If the LC eluents lack a photoionizable solvent, dopants such as acetone, hexane, or toluene may be introduced into the sample stream to create a source of reagent ions. Other MS Technologies Applicable for Small-Molecule Structure Elucidation At times, separation is unnecessary or impractical prior to MS analysis. An obvious example is when a sample only has a few analytes of interest, and these compounds are present at high concentrations. Under these circumstances, analysis may be quickly carried out by directly infusing the sample through a syringe pump or by performing flow injection of the sample solution directly into the mass spectrometer. Many novel ionization technologies have been designed for this purpose of direct MS analysis. These include direct analysis in real time (17), desorption electrospray ionization (18), and atmospheric solid analysis probe (19), all of which are commercially available. The benefits of employing these technologies are their fast analysis time and limited requirements for sample preparation.

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Conversely, the demands for increased sensitivity and comprehensive analysis may require the application of more sophisticated separations such as two-dimensional LC/MS (20, 21) and LC/ion mobility (IM)/MS (4, 22, 23). Ion mobility spectroscopy (IMS) is a technique that separates ionized species in the presence of inert gas under an applied electric field based on their collisional cross sections (CCS). IMS has been widely used in airport security to detect warfare reagents including explosives (24). IM–MS has been applied increasingly to the analysis of both small molecules and large molecules (biopolymers and industrial polymers). There are several types of IMS analyzers, including drift tube ion mobility spectrometry (22), traveling wave ion mobility spectrometry (25), field asymmetric ion mobility spectrometry (26), and trapped ion mobility spectrometry (27). In each of these devices, the IM separation is achieved based on the mass, charge, size, and shape of analytes, as well as the collision gas used and its temperature. With careful calibration, a drift tube ion mobility spectrometry DTIMS system can be used to measure drift times and CCS values with excellent reproducibility, often closely matching calculated CCS values. These data may provide additional structural information, which can be combined with molecular weight, retention time, and melting point, to characterize unknown compounds. CCS values can often be accurately predicted using quantum calculation methods, which can be used in the prioritization of proposed impurity structures. Efforts are underway to build databases that combine CCS values with MS and MSn spectra. These hold great promise for the rapid characterization of complex synthetic samples and formulation products.

Figure 2. A typical workflow for impurity identification in synthetic AI samples.

Typical Workflow for Impurity Identification Figure 2 outlines a general workflow for the characterization of process chemistry products. Typically, a sample is first analyzed by conventional chromatographic methods without MS detection. At this point, standards are rarely available, and the quantitation is reported as area % or by using a surrogate standard with a close response factor. As a rule of thumb, comprehensive MS analysis is then performed to identify the species close to or above 0.1 wt % following the same chromatographic conditions. It is worth noting that the mobile phase for the subsequent LC/ MS analysis should be compatible with mass spectrometry. For example, although phosphate is an excellent LC mobile-phase modifier for UV detection, it should be avoided for MS applications 56

due to its nonvolatile nature. Since many compounds produced in process chemistry are novel, elemental compositions are determined first using accurate mass data acquired on high-resolution mass spectrometers. Detailed structural information can then be deciphered by closely examining the fragment ions provided by high-energy ionization (e.g., EI/) or using MSn spectra of ionized molecules generated from one of the API sources. Although it is challenging to assign the structure of an unknown compound based solely on mass spectral data, informed proposals can often be made by combining expert knowledge with relevant chemistry information including synthetic routes, catalysts, reagents, starting materials, and solvents. Thereafter, if a given impurity is of interest, one of two actions are generally taken: (1) synthesizing the target molecule for definitive structure assignment, making it available as a standard for quantitation or (2) optimizing the synthesis process to reduce or eliminate the impurity. At times, it is possible that no reasonable structure can be proposed based solely on the information provided by MS. In those cases, it may be necessary to isolate the impurity with semipreparative LC (fractionation collection) for subsequent analysis by NMR or X-ray crystallography (if a single crystal can be obtained). Once a structure is proposed, it is still generally required to confirm the structure using total synthesis, which then provides reference material to use as a standard.

Derivatization Derivatization is an important methodology often used to convert an analyte into another molecule to obtain better chromatography and improved sensitivity and to enable identification. Researchers have developed numerous derivatization methods for both LC and GC applications (28–33). For example, alcohols can be converted to an ester sulfonate using 2-sulfobenzoic anhydride (2-SBA), which provides excellent sensitivity in the negative ion ESI/MS analysis (Figure 3) (34). For GC/MS, silylation reactions are frequently used to derivatize alcohols, amines, or acids for improved stability and volatility. In addition, the fact that a compound reacts with a derivatization reagent usually indicates the presence of certain functional groups. This will be demonstrated in several case studies in next section.

Figure 3. Derivatization of alcohols with 2-SBA for improved sensitivity in negative ion ESI/MS.

Case Studies GC/MS Study of Impurities in a Commercial Chlorpyrifos Sample A commercial chlorpyrifos sample (Scheme 1) was purchased from Sigma-Aldrich and analyzed on a GC/Orbitrap mass spectrometer for identification of impurities. Although none of the impurities were detected above 0.1 area %, the analysis was performed to demonstrate a typical identification workflow. Figure 4 shows the total ion chromatograms of the sample and the sample derivatized with N,O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA). Chlorpyrifos eluted at 14.2 min. The unknown compound observed at 8.17 min was present only in the derivatized sample, 57

indicating that it was the product of an impurity reacting with BSTFA. Figure 5 shows both EI and PCI-CH4 mass spectra of the derivatized impurity. The most intense observed ions in the EI mass spectrum were the [M − CH3]+ · isotopic ion series with m/z values of 253.94, 255.93, 257.93, and 259.93, indicating the presence of three chlorine atoms in the molecule. Molecular ions were observed at m/z values of 268.96, 270.96, 272.95, and 274.95, all at low abundances. The four isotopic ions of m/z 271.95, 273.94, 275.94, and 277.94 were assigned as [M − CH3 − H2O]+ ions. These were likely formed via reaction of the [M − CH3]+ ions with a trace amount of water in the C-trap of the Orbitrap instrument. This is a known phenomenon in ion traps, where certain types of ions can react with trace water due to longer residence times as compared to faster mass analyzers like TOF instruments (35, 36). The monoisotopic mass of the derivatized impurity was further confirmed by the m/z values observed in the PCI-CH4 spectrum. Three groups of adduct ions were detected, including the [M + H]+ ions at m/z 269.97, 271.96, 273.94, and 275.94; [M + C2H5]+ ions at m/z 297.99, 298.99, 301.99, and 303.99; and [M + C3H5]+ ions at m/z 309.99, 311.99, 313.99, and 315.99. Based on these mass spectral data, the most probable molecular formula was determined to be C8H10Cl3NOSi, which was consistent with the isotopic pattern indicating the presence of three chlorine atoms in the molecule. The observation of Si in the proposed molecular formula was consistent with the presence of trimethylsilyl (TMS) group from the derivatization. The elemental composition of the TMS group was then subtracted from the overall elemental composition to give the formula of the original compound as C5H2Cl3NO (eq 1). Since chlorpyrifos has a 2,5,6-trichloropyridine core structure, it is reasonable to assume that the unknown impurity may have a similar core to chlorpyrifos. The oxygen atom may be a hydroxyl group, as indicated by the formation of the TMS derivative. Therefore, the structure was tentatively proposed as 3,5,6trichloro-2-hydroxypyridine.

Figure 4. The total ion chromatograms of (a) Chlorpyrifos and (b) Chlorpyrifos derivatized with BSTFA.

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Figure 5. The EI and PCI-methane mass spectra of the impurity observed in chlorpyrifos derivatized with BSTFA.

Scheme 1. Chlorpyrifos LC/MS Study of Impurities in a Commercial Haloxyfop-P Methyl Ester Haloxyfop-P methyl ester (Scheme 2) is an herbicide used for selective control of grass weeds in crop and noncrop situations. A commercial sample was purchased from Sigma-Aldrich and analyzed by LC/MS in the positive ion ESI mode. Figure 6 displays the UV chromatogram of the sample, including an inset displaying an expanded chromatogram showing six impurity peaks. It is a recommended practice to examine the mass spectra of a known component (in this case, haloxyfopP methyl ester) to understand its ionization behavior and fragmentation pathway. These observations can then be used to aid in the interpretation of the mass spectra of unknowns in the same sample. It is also a good practice to ensure that the instrument is properly cleaned and calibrated for optimal resolution and mass accuracy. This can be confirmed by investigating the mass spectral data of the known compound.

Scheme 2. Haloxyfop-P Methyl Ester 59

Figure 6. The UV chromatogram of Haloxyfop-P methyl ester. As shown in Figure 7, positive ion ESI/MS analysis of Haloxyfop-P methyl ester produced [M +

H]+ at m/z 376.0574. In addition, both [M + Na]+ and [M + K]+ ions were detected as displayed in the inset. The observations of these adduct ions provided confirmation that the monoisotopic mass of Haloxyfop-P methyl ester was 375 Da. The ratio of ion peaks A:A+2 was approximately 3:1 which was also consistent with the proposal that the molecule contains one chlorine atom.

Figure 7. The positive ion ESI mass spectrum of Haloxyfop-P methyl ester showing [M + H]+, [M + Na]+, and [M + K]+. Figure 8 shows the MS/MS spectrum of protonated Haloxyfop-P methyl ester. The ion at m/z 316.0354 was likely the result of the loss of a methyl formate. The ion at m/z 288.0401 may have been formed upon methyl migration followed by the loss of a neutral C3H4O3. The ion at m/z 272.0086 was assigned as loss of a methyl 2-(l1-oxidanyl)propanoate moiety. The ion at m/z 119.0492 was likely a vinyloxybenzene cation. Interestingly, a tropylium ion of m/z 91.0544 was also observed, which may have been generated from the ion at m/z 288.0401 following the loss of the 3-chloro5-(trifluoromethyl)-2-pyridinol moiety. It is also worth noting that methyl transfer is a common rearrangement in the gas phase (37). In this case, our understanding of the fragmentation pathways of protonated Haloxyfop-P methyl ester provided important insight into the interpretation of the mass spectra of the other species in the sample. The major fragment ions generated from protonated Haloxyfop-P methyl ester are illustrated in Scheme 3.

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Scheme 3. Major Fragment Ions Generated from Protonated Haloxyfop-P Methyl Ester

Figure 8. The positive ion ESI MS/MS spectrum of [M + H]+ ions of Haloxyfop-P methyl ester. In some cases, there may be only a single adduct ion observed in the positive ion ESI mass spectrum, which makes the assignment of molecular mass difficult. In this situation, alternative ionization modes, such as using the negative ion mode, may prove helpful in confirming the molecular weight. Figure 9 shows the negative ion mass spectrum of the haloxyfop-P methyl ester. The deprotonated ion was observed at m/z 374.0391, confirming the molecular mass as 375 Da. The observed low-mass fragment ions, including m/z 195.9779, were likely formed via fragmentation in the ion source. It is also worth noting that the high-mass ions could be oxidized anions generated during the ionization process, which is not a common phenomenon.

Figure 9. The negative ion ESI mass spectrum of Haloxyfop-P methyl ester showing [M − H]-, [M + O − H]+, and [M + O2 − H]+.

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Figure 10. The positive ion ESI mass spectrum of Imp 1 showing both [M + H]+ and [M + Na]+. Figure 10 shows the full-scan mass spectrum of an impurity eluting at 15.4 min (Imp 1). This produced an [M + H]+ adduct at m/z 342.0944 and an [M + Na]+ adduct at m/z 364.0735. Therefore, the molecular mass was determined to be 341 Da, and the formula was then calculated to be C16H14F3NO4 with a mass error of 1.28 ppm. By comparing this formula with the formula for Haloxyfop-P methyl ester, it was observed that the elemental composition of Imp 1 lacked the chlorine atom of Haloxyfop-P methyl ester. The isotopic pattern also suggested that the molecule does not contain chlorine.

Figure 11. The positive ion ESI MS/MS spectrum of [M + H]+ ions of Imp 1. The MS/MS of the [M + H]+ adduct ion of Imp 1 (Figure 11) showed a similar fragmentation pattern to that of haloxyfop-P methyl ester (cf. Figure 8), which indicates that its structure is likely the des-chloro haloxyfop-P methyl ester. The identification of other impurities in the sample was accomplished using the same approach. It is worth mentioning that both Imp 2 and Imp 3 are isomers of haloxyfop-P methyl ester based on their mass spectral data (MS and MS/MS). However, it is not adequate to propose definitive isomer structures with only the mass spectral data. If it is considered necessary, alternative analytical tools like NMR should be used to assign the structures decisively, assuming that enough pure compounds can be isolated. Table 3 summarizes the proposed structures of the six impurities observed in the sample.

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Table 3. Summary of Tentative Assignment of the Trace Level Impurities Observed in Haloxyfop-P Methyl Ester

Future Improvements Software for processing complex MS data is a critical component of any modern MS system. Numerous software packages have been developed by instrument vendors, independent software vendors, and academic research groups. Many of these software tools were developed for proteomics or metabolomics research, driven by investment in biomedical research and the pharmaceutical industry. Although basic functions like elemental composition calculations, spectrum extraction, and chromatogram rebuilding are available from instrument vendors, many needs for advanced data reduction in impurity identification remain unmet. These include automated component detection and spectral extraction, improved assignments of formulas, tools for building and organizing compound databases, better prediction of fragmentation pathways, and so on. However, several recent software developments by both industrial and academic research groups indicate that the field is making progress toward the goal of automation and consistency in data interpretation (38–40).

Conclusion The use of HRMS in combination with LC or GC separations is an indispensable method for identification of impurities in agricultural chemicals. Definitive assignment of the structure of an unknown compound requires choosing appropriate instrumentation, methods, and operation parameters, as well as expertise in the interpretation of the resulting mass spectra. The two case studies described here illustrate the use of GC/MS and LC/MS in several different types of impurity analyses. Despite its wide use, MS has not yet reached its full potential, in part due to limitations in the available software tools specifically designed for small-molecule identification. It is our hope that both end users and instrument and software vendors will continue to work collaboratively in 63

the design and development of new software tools to improve both productivity and reliability of structure assignment.

Acknowledgments The author thanks Bruce Bell and Jennifer Jones for insightful discussion.

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30. Halket, J. M.; Zaikin, V. G. Derivatization in Mass Spectrometry-7. On-Line Derivatization/ Degradation. Eur. J. Mass Spectrom. 2006, 12 (1), 1–13. 31. Lin, D.-L.; Wang, S.-M.; Wu, C.-H.; Chen, B.-G.; Liu, R. H. Chemical Derivatization for the Analysis of Drugs by GC-MS - A Conceptual Review. Yaowu Shipin Fenxi 2008, 16 (1), 1–10. 32. Pedersen-Bjergaard, S. Gas Chromatography. In Bioanalysis of Pharmaceuticals; John Wiley & Sons Ltd.: New York, 2015; pp 173–206. 33. Thomas, B. F.; Daw, R. C.; Grabenauer, M. General Principles of Mass Spectrometry: GCMS, LC-MS, and LC-MS/MS. In Encyclopedia of Drug Metabolism and Interactions; Lyubimov, A. V., Ed.; John Wiley & Sons Ltd.: Hoboken, NY, 2012; Vol. 5, pp 21–46. 34. Zu, C.; Praay, H. N.; Bell, B. M.; Redwine, O. D. Derivatization of Fatty Alcohol Ethoxylate Non-Ionic Surfactants Using 2-sulfobenzoic Anhydride for Characterization by Liquid Chromatography/Mass Spectrometry. Rapid Commun. Mass Spectrom. 2010, 24 (1), 120–128. 35. Baumeister, T. U. H.; Ueberschaar, N.; Pohnert, G. Gas-Phase Chemistry in the GC Orbitrap Mass Spectrometer. J. Am. Soc. Mass Spectrom. 2019, 30 (4), 573–580. 36. Wood, K. V.; Bonham, C. C.; Jenks, M. A. The Effect of Water on the Ion Trap Analysis of Trimethylsilyl Derivatives of Long-Chain Fatty Acids and Alcohols. Rapid Commun. Mass Spectrom. 2001, 15 (11), 873–877. 37. Isbell, J. J.; Brodbelt, J. S. Effects of Functional Group Interactions on the Gas-Phase Methylation and Dissociation of Acids and Esters. J. Am. Soc. Mass Spectrom. 1996, 7 (6), 565–572. 38. Tsugawa, H.; Kind, T.; Nakabayashi, R.; Yukihira, D.; Tanaka, W.; Cajka, T.; Saito, K.; Fiehn, O.; Arita, M. Hydrogen Rearrangement Rules: Computational MS/MS Fragmentation and Structure Elucidation Using MS-FINDER Software. Anal. Chem. 2016, 88 (16), 7946–7958. 39. Li, Z.; Lu, Y.; Guo, Y.; Cao, H.; Wang, Q.; Shui, W. Comprehensive Evaluation of Untargeted Metabolomics Data Processing Software in Feature Detection, Quantification and Discriminating Marker Selection. Anal. Chim. Acta 2018, 1029, 50–57. 40. Božičević, A.; Dobrzyński, M.; De Bie, H.; Gafner, F.; Garo, E.; Hamburger, M. Automated Comparative Metabolite Profiling of Large LC-ESIMS Data Sets in an ACD/MS Workbook Suite Add-in, and Data Clustering on a New Open-Source Web Platform FreeClust. Anal. Chem. 2017, 89 (23), 12682–12689.

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Chapter 4

Separation of Chiral Molecules in Support of Process Chemistry and Formulations Research Daniel I. Knueppel,* Jens T. Richards, and John M. Atkinson Crop Protection Product Design and Process R&D, Corteva AgriscienceTM, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States *E-mail: [email protected].

Chiral molecules have been and continue to be of importance in crop protection research. The step-by-step work process employed for method development to separate enantiomers of chiral intermediates and active ingredients is discussed. Unusual behavior of a chiral stationary phase that was observed during analysis of chiral epoxides is highlighted. An analytical-scale isolation of enantiomers that required creative reconfiguration of the instrumentation to enable sufficient material to be isolated is showcased. Lastly, it is demonstrated that analyses of enantioenriched active ingredients may also be important in formulation samples in which undesired epimerization may occur.

Introduction Chiral molecules have been commercialized in the agrochemical market for many years. Close to 30% of agrochemicals possess chirality as outlined nicely in a review by Jeschke (1). Furthermore, with the steady rise of stereoselective chemical transformations, the opportunity to cost-effectively manufacture chiral agrochemicals has continued to increase. Corteva AgriscienceTM, Agriculture Division of DowDuPont, has a rich history of commercializing chiral agrochemicals. As a leader in developing natural products into crop protection products, the company has commercialized insecticidal spinosyn natural products including Spinosad and Spinetoram. These products were recognized by three Presidential Green Chemistry Awards. Spinetoram J, the major component of Spinetoram, possesses 17 chiral centers. Because the structural core is produced using a fermentation process, the enantiopure form of the molecule is generated by the bacterial species Saccharopolyspora spinosa.

© 2019 American Chemical Society

Another successful active ingredient with chirality that Dow AgroSciences has commercialized is the sap-feeding insecticide IsoclastTM active. This molecule possesses two stereocenters, but the active is produced as a racemic mixture of the four possible isomers (2) because rapid interconversion of both chiral centers occurs readily.

There has been a rise in the development of chiral agrochemicals that are intended to be commercialized in an enantiomerically pure form (1). In those instances, many analytical methods need to be developed that separate mixtures of enantiomers, not only for analysis of the chemical step in which chirality is introduced, but also for products of any subsequent reactions to ensure that enantiomeric purity has not diminished. More important, chiral technical grade active ingredients that are synthesized for toxicological and efficacy testing need to be characterized for their enantiopurity.

Workflow for Development of Chiral Separation Methods Development of analytical methods to determine enantiopurity of a material is generally welldescribed in the literature (3–5). Although the majority of literature on chiral separations involves pharmaceuticals, most of it applies equally well to agrochemicals because of their structural and physiochemical similarities (6). It was thought helpful to showcase the specific details of the analytical method development process for a chiral separation that has been found to work best in our hands. Every time a method is requested to determine enantiopurity, a racemic mixture needs to be supplied to ensure that the two antipodes are effectively resolved from each other. Our initial approach is usually to develop methods for this application using normal-phase liquid chromatography employing hexanes and isopropyl alcohol (IPA) as mobile phase with a chiral stationary phase. According to the literature, separation of enantiomers with reversed-phase conditions is equally as effective as with normal-phase conditions (7, 8); however, because most of our chiral stationary phases were historically dedicated to normal-phase chromatography, we opted to continue to predominantly develop chiral separations using normal-phase conditions. Supercritical fluid chromatography (SFC) is also employed when needed and has proven to be superior to normal-phase liquid chromatography in our experience because it allows employment of gradients, provides shorter methods, and is compatible with mass spectral detection (9). However, implementation of normal-phase chromatography methods in various internal and external

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laboratories is much easier than with SFC-based methods because liquid chromatography instrumentation is already widely available. For development of methods to separate enantiomers, a Thermo Fisher Dionex Ultimate 3000 UHPLC with a six-column compartment, two ternary mobile phase pumps, and a UV-Vis detector is employed. One of the pumps is configured for normal-phase and one for reversed-phase chromatography to allow ready toggling between phases based on method needs, but employing separate sets of chiral stationary phases dedicated to each phase per the manufacturer recommendations. This instrument also has fraction collection capabilities that can be employed for offline 2D-liquid chromatography when needed. Because most chiral separation methods are developed with normal-phase chromatography, our most common instrument setup is using isopropyl alcohol as the strong solvent and either hexanes, hexanes with 0.1% trifluoroacetic acid, or hexanes with 0.1% diethylamine as the weak solvent, depending on functional groups present in the molecule to be separated. We have historically used 250 × 4.6 mm columns with 5 micron particles and have continued to utilize columns with those dimensions for consistency. Smaller particle size packed columns have since been developed. In our work, we generally have not discovered any advantage in resolution compared with our current column format. The chiral stationary phases most frequently employed are from Chiral Technologies, including the AD, IA, IB, IC, ID, IE, OD, OJ, and OK phases. We also have found in select cases that TCI America’s Chiral MB-S or the Pirkle-type Whelk-O columns can be advantageous at resolving challenging-to-separate mixtures of enantiomers that were inseparable with any of the other chiral stationary phases. Step-by-Step Progression of Chiral Separation Method Development The step-by-step progression that is followed for a chiral method development project is highlighted in Figure 1.

Figure 1. Progression of chiral separation method development. (1) Receive racemic and enantioenriched sample. (2) Identify mobile phase composition. (3) Perform chiral stationary phase screening. (4) Optimize separation on best stationary phase. (5) Run both samples using optimized separation conditions and optimized sample prep. (6) Document method. The first step is to receive both a racemic and an entioenriched sample (Figure 1, Step 1). The racemic sample is crucial for identifying whether conditions are successfully resolving the two enantiomers, whereas the enantioenriched sample helps assign the correct peak with the representative enantiomer. 69

Prior to performing an extensive column screen, it is important to ensure that the analogues are adequately retained in order to provide optimal potential for success in performing a screen of potential stationary phases. Generally, because samples provided are fairly clean, retention times between ~2 and 8 minutes are targeted (Figure 1, Step 2). To begin, the racemic sample is prepared at 1 mg/mL with 30% isopropyl alcohol in hexanes, and a 5 μL injection using 30% isopropyl alcohol in hexanes as the mobile phase is run at 40 °C using a flow rate of 1 mL/min. The column temperature of 40 °C is selected because, except for a few outliers, peak shape and resolution are best at elevated temperatures. The mobile phase composition is then changed, if needed, to have the main peak(s) eluting in the desired range of ~2–8 minutes. With the appropriate mobile phase conditions identified, a stationary phase screen is conducted starting with the six phases that have historically demonstrated the best performance (discussed below). Screening data is usually easily interpreted with scrutiny of resolution and retention to establish the best stationary phase for chiral separation (Figure 1, Step 3). The chosen chromatography set is then further optimized for complete baseline resolution between the two peaks, if not already achieved during the initial stationary phase screen (Figure 1, Step 4). When resolution is similar between two stationary phases, preference is given to the chromatographic conditions that provide a shorter final method. Complete baseline resolution is crucial to enable accurate quantitation when mixtures are not 50:50 and smaller enantiomer peaks readily turn into difficult-to-integrate shoulders. With the ideal separation developed on a specific stationary phase and mobile phase setup, the sample prep is evaluated one last time. Usually an attempt is made to match the sample prep eluent with that of the LC mobile phase composition to avoid eroding a separation due to strength of the sample injection solvent. This is especially crucial when method conditions require only a small percentage of the strong solvent, such as 1% isopropyl alcohol in hexanes, but large injection volumes are required because of low UV absorbance of the analyte or poor solubility of the analyte in sample preparation solvents necessitating dilute sample preparation solutions. After the separation and sample solvent strength have been finalized, another set of fresh aliquots of the racemic and enantioenriched samples are prepared to confirm that the method is reproducible with duplicate injections (Figure 1, Step 5). The enantiomeric ratio from this analysis is also reported back to the requestor to complete the analysis of the enantioenriched sample of interest. Finally, a concise method summary is produced to document the results, share with the stakeholder, and archive for future analysis needs (Figure 1, Step 6). Key Learnings With a rise in the number of chiral agrochemicals under development in Corteva Agriscience’s pipeline, we have observed a steady increase in the number of chiral separation methods required. On an annual basis, over 30 separate methods are developed to support discovery, process chemistry research, tox lot and field sample characterization, and regulatory sciences studies. As previously mentioned, the vast majority of methods are developed using normal-phase conditions. A careful analysis of methods developed to this point revealed a clear trend in the best-performing chiral stationary phases. The four best stationary phases, starting with the most successful one, are the AD, OJ, OD, and ID chiral phases. Trailing those are the IA and IC stationary phases, which are about equally successful. Reversed-phase methods for chiral separations were predominantly developed in cases in which the polarity of a compound was so nonpolar (lipophilic) that even with 100% hexanes as the mobile phase, no retention and thus no resolution of peaks could be observed. Use of SFC has 70

increased because the advantages of this technology have outweighed the lack of broad availability of instruments. As our understanding and success of coupling SFC with mass spectrometry detectors has increased, this tool has found continually increasing use in development of chiral separation methods for characterization of active ingredients and metabolites, which have been successfully leveraged toward complex plant and animal matrices for regulatory studies. Use of chiral stationary phases has not only been very successful for the separation of enantiomers, but also when samples containing two or more chiral centers had to be analyzed. One such example that highlights the complexity of the separations required is shown below (Figure 2). Four isomers had to be resolved not only from each other but also from a chiral byproduct that existed as a mixture of enantiomers. With the successful separation of all six components, this method could be used to analyze a process intermediate for distribution of isomers, depending on how reaction conditions were modified.

Figure 2. Mixture of chiral diastereomers (four isomers) and chiral byproducts (two enantiomers).

Case Studies Although the overview of the general workflow can be helpful, the real opportunity for problem solving comes when method development for chiral separation methods does not proceed as expected or additional challenges are encountered along the way. This not only showcases useful learnings that might be of help to other scientists in similar situations, but also demonstrates the creativity and impact that chiral separation methods and analytical science can have throughout an R&D organization. Example 1: Interesting Findings—When Your Stationary Phase Behaves Unexpectedly Separation of a mixture of four chiral epoxides was required in order to determine the distribution of isomers. The racemic sample provided contained a mixture of two minor enantiomers and two major enantiomers (Figure 3).

Figure 3. Mixture of four isomers to be separated, containing two pairs of enantiomers.

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Execution of the typical method development screening process quickly confirmed acceptable resolution using the OJ chiral stationary phase with 15% isopropyl alcohol in hexanes at 40 °C (Figure 4, Chromatogram 1). This was one of the few cases in which, upon further optimization, it was found that lower temperatures improved the separation in combination with using 3% isopropyl alcohol in hexanes (Figure 4, Chromatogram 2). In order to save time during optimization of the mobile phase composition, an instrument sequence was run with subsequent injections at the same temperature using 1% isopropyl alcohol in hexanes (Figure 4, Chromatogram 3). Data showed that using 3% isopropyl alcohol had clearly provided superior chromatographic resolution and performance. As such, we switched back to those conditions to perform the final analysis and demonstrate robustness of the method. Yet, surprisingly, despite very sharp peaks, chromatographic resolution initially observed with 3% isopropyl alcohol was no longer achieved (Figure 4, Chromatogram 4).

Figure 4. Progression of separation during method development using an OJ chiral stationary phase. (1) 15% IPA/hex at 40 °C. (2) 3% IPA/hex at 20 °C (first injection). (3) 1% IPA/hex at 20 °C. (4) 3% IPA/hex at 20 °C (second injection). Because this result was puzzling, it was thought that maybe the column was not properly reequilibrated with 3% isopropyl alcohol in hexanes, despite having run two blank injections between the two runs. Yet even after equilibrating the instrument with 3% isopropyl alcohol in hexanes for close to an hour and doing another injection, the chromatogram again showed unresolved peaks. This prompted some additional investigation to understand what phenomenon might be at work. Because numerous attempts to reestablish resolution of all four isomers were unsuccessful, it was decided to return to conditions in the original sequence. Specifically, the mobile phase optimization started with 15% isopropyl alcohol in hexanes, followed by a blank injection and analysis with 3% isopropyl alcohol. When that sequence was followed, the successful separation of all four isomers was achieved once again. As a follow-up experiment, which was performed in triplicate to confirm this phenomenon, the system was equilibrated with 1% isopropyl alcohol in hexanes or straight hexanes for one hour, followed by a blank injection and analysis of the sample with 3% isopropyl alcohol to give coelution of two of the isomers (Figure 5, Chromatogram 1). Additionally, performing system equilibration at 15% or 40% isopropyl alcohol in hexanes for one hour followed by a blank injection and analysis of the sample with 3% isopropyl alcohol in hexanes gave complete resolution of all four isomer peaks (Figure 5, Chromatogram 2). Because of time constraints, this fascinating finding could not be investigated further. It would be interesting to understand how 72

long of an equilibration is actually needed. Yet all subsequent analyses of this epoxide mixture were performed by carrying out a 1-hour equilibration at 15% isopropyl alcohol in hexanes followed by a blank injection and sample analysis with 3% isopropyl alcohol in hexanes. A true explanation for the cause for this phenomenon has not been determined, yet the best hypothesis is that the stationary phase undergoes structural configurational changes in the presence of higher percentages of isopropyl alcohol that are crucial for the interactions with the epoxide isomers and subsequently their resolution. It is worth noting that this occurrence has never been observed with any other chemistry using the OJ or any of other chiral stationary phase during mobile phase optimization in this fashion.

Figure 5. Chromatograms of epoxide isomers at 20 °C after (1) equilibration at ≥15% IPA/hex for 1h followed by a blank injection at 3% IPA/hex and (2) equilibration at 90% is essential for a reliable analysis, and poor mass balance would increase the uncertainty of analysis. The model is optimized using the observed data. If a good visual fit for all transformations can be achieved without permitting any direct transformation of the parent compound into NER (unshaded arrow), then it is reasonable to conclude that the parent compound itself is not sequestered, and metabolites alone are responsible for the formation of the NER observed. In such cases, the risk associated with the uncharacterized NER can be evaluated based on degraded metabolites and not the parent compound. Several examples of such kinetics application are presented herein.

Results and Discussion Methomyl degrades rapidly in soil via the formation of one major metabolite, the oxime. The oxime metabolite does not accumulate and is seldom detected in amounts larger than 10%. Other minor metabolites are also found, but they individually account for a very small percentage of the total mass. Degradation in two representative soils was optimized to the observed data according to the model, which allows for the formation of NER and CO2 only from the oxime and other minor metabolites. The model shows good visual fit, and the kinetics parameters (rate constants for each transformation) appear reliable, as shown in Figure 2. Attempts to include the direct transformation of methomyl to NER did not improve the kinetics fit, and the rate constant for conversion of the parent compound to NER was negligible. Implication of such analysis is that there is very little incorporation of methomyl itself into NER via sequestration in soil. Instead, kinetics exercise 97

suggests that methomyl must degrade to oxime and perhaps further to even smaller degradates before NER and CO2 are produced. Because the amount of oxime and metabolites observed is small, one may argue that sequestration of methomyl into NER cannot be ruled out, despite a good data fit. In such a case, the conclusion derived from kinetics analysis for methomyl can be further supported by modeling the soil degradation of the oxime metabolite. A kinetics analysis conducted using data from studies in which the oxime was applied to the soil also showed substantial formation of NER and CO2 with no significant metabolites. Application of the kinetics model displayed in Figure 3 showed a good fit for the data observed (Figure 4).

Figure 2. Conversion of methomyl metabolites to NER in soil.

Figure 3. Kinetics model for oxime degradation. Results of the kinetics optimization analysis (Figure 4) confirm that the degradation of the oxime in soil is swift, and all degradation can be accounted for by the formation of NER and CO2 observed, with >90% degradation completed in just 7 days. 98

Figure 4. Conversion of oxime to NER in soil. In the kinetics analysis, transformation of oxime was permitted to only the NER and CO2, and the observed data fit for the degradation model verifies that the metabolite oxime is indeed the main source of NER. Rapid degradation, which is even faster than the rate of methomyl degradation, explains why the oxime is not observed in substantial amounts during the methomyl degradation study. A very consistent degradation model with parent and the main metabolite thus demonstrates that the NER found in soil from methomyl treatments should have no parent compound and only small fragments resulting from the oxime degradation. The oxime metabolite can be rationalized to be converting to smaller species such as acetic acid. Molecules such as acetic acid are known to mineralize as well as assimilate into natural constituents (11) at a rate similar to those we calculate in this kinetics analysis. Oxamyl is structurally similar to methomyl and readily degrades in soil with production of substantial amounts of CO2 and NER. In this case, degradation proceeds via initial conversion to a different oxime followed by the formation of N,N-dimethyloxamic acid (DMOA), as displayed in Figure 5. For oxamyl, the degradate DMOA degrades slowly enough that significant amounts are detected during the degradation study. In this case also, when the observed data were fitted to a kinetics model in which the NER originated only from the metabolites, a good fit for the observed data was found in all soils, two of which are illustrated in Figure 6.

Figure 5. Oxamyl degradation pathway.

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Even though the oxime is not observed in substantial quantities in the soil degradation, its transformation to DMOA followed by formation of oxalic acid is quite logical. A partial incorporation of species such as oxalic acid into natural organic matter, as well as mineralization, are likely and rationalized by the good data fit of the kinetics model. Because the formation and decline of DMOA fits well in the degradation model, a separate study with DMOA would be helpful for the conclusion. Nevertheless, based on the model presented, it would be justifiable to conclude that oxamyl itself plays no significant role in the formation of NER. The kinetics analysis of data supports the view that the NER is essentially all derived from small molecules such as DMOA, oxalic acid, or oxamic acid.

Figure 6. Oxamyl degradation and optimized data fit in soil. Cymoxanil is another molecule that degrades rapidly in soil via multiple metabolites (Figure 7). Many of the degradates displayed in the pathway have been identified in a variety of studies, but they never appear in significant amounts during soil degradation studies. Just like methomyl, cymoxanil metabolites collectively amount to no more than 10% in the soils studies. Presumably, the soil degradates degrade even more rapidly than the parent and fail to appear in substantial amounts. A kinetics model proposed in this chapter is optimized using the data set, and the resulting fit is visually quite good, as illustrated in Figure 8. Because no metabolite consistently exceeded 5% of the total composition, all minor metabolites were collectively represented as one compartment. Once again, the inference from such a fit is that the metabolites degrade into smaller molecules displayed near the end of the degradation scheme, and they become incorporated into natural organic matter to generate the observed NER. A significant portion of the applied material undergoes mineralization as well to produce a large amount of 14CO2. Based on the kinetics fit, it is concluded that cymoxanil itself contributes very little toward the NER formation.

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Figure 7. Degradation pathway of cymoxanil in soil.

Figure 8. Optimized kinetics for conversion of cymoxanil to metabolites, NER, and CO2. Although methomyl, oxamyl, and cymoxanil are easily visualized as degrading to two carbon acids that are easily assimilated into NER, that is not necessarily the case with larger molecules containing multiple rings. Cyhalofop butyl, which contains two rings (A and B), is an example, and its degradation was investigated in soil with two radiolabeled versions, viz., test substance that 101

contained the radiolabel in either ring A or ring B. Both test substances were examined in the same soil, and the two labeled versions served as replicates for the test substance. A proposed degradation pathway is presented in Figure 9, and two of the degradates (acid and diacid) were often observed in substantial amounts. In this case, if degraded metabolites are primarily responsible for the formation of NER, then the two rings would be expected to display substantially different amounts of NER as well as CO2 from the two radiolabels. Observation of such a difference between the labels is an initial indication that the degraded metabolites play a key role in the formation of NER. Such a difference in NER and CO2 for the two labels was highlighted previously for pyrithiobac sodium (9). In the case of cyhalofop butyl, degradation proceeding via hydroxyphenoxypropionic acid [HPPA, observed only in the degradation with the A label) and resulting in the formation of NER and CO2 would likely be in different amounts than that for the B label. That is exactly what we observed in the data, and for this reason, the kinetics analysis for the two labels also needed to be conducted separately. Optimization of the kinetics model (Figure 10) with each labeled version shows more mineralization from the B label and a larger proportion of NER from the A label, indicating that the two rings separated from each other during the degradation process before the rings or their fragments were incorporated into NER.

Figure 9. Soil degradation pathway for cyhalofop butyl. Famoxadone is another compound that contains multiple rings. Despite its complexity, degradation in soil is quite rapid. Several intervening metabolites have been identified (Figure 11) that also degrade readily and seldom individually exceed 3–5%. In addition, famoxadone tends to 102

display biphasic decline in many soils, and a portion of the parent compound is slow to degrade in those soils. Normally, such a behavior is associated with compounds that tend to be become sequestered in soil and themselves become a part of the bound residue. However, when we conducted kinetics analysis for the formation of NER and CO2, good fit for the data could be obtained only when the parent compound itself was not permitted to directly transform to NER. As illustrated in Figure 12, famoxadone degradation was indeed biphasic (double first order in parallel decline for parent compound); however, the metabolites did not appear in any significant quantity, and a large percentage of radioactivity was found in the NER and as mineralized CO2. Because the radiolabel is located in the middle phenyl ring, a significant production of CO2 suggested that the rings themselves were opening via oxidations and degrading into small molecules that eventually form CO2. Naturally, some of these smaller molecules also become incorporated into the natural organic matter. For a product that exhibits biphasic degradation and shows small quantities of metabolites, it is difficult to rationalize that such a molecule is not itself becoming nonextractable. However, if a terminal metabolite such as H3310 can be demonstrated to generate large amounts of NER and CO2, then an argument can be made that the metabolite, even if formed slowly, is degrading at a rate high enough to produce bound residue via degradation into small molecules. For famoxadone, degradation of H3310 in soil produced significant amounts of NER and CO2 (Figure 12, right panel) and further supported the view that famoxadone itself is not becoming part of the bound residue.

Figure 10. Optimized kinetics for degradation of cyhalofop butyl.

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Figure 11. Degradation pathway for famoxadone in soil.

Figure 12. Optimized kinetics degradation of famoxadone and its metabolite H3310 in soil. It is clear from the examples presented that in many compounds, NER formation can be at least partially characterized with the use of kinetics optimization and enable one to conclude that the metabolites and smaller degraded species are incorporated into the organic and hence become the nonextractable. In such cases, the toxicity of the NER should not be evaluated as equivalent to the parent compounds. There is some uncertainty in this analysis because the observed amounts of various compartments modeled have some experimental error, which manifests into the uncertainty of rate constants. Nevertheless, kinetics modeling does reliably help define the degradation pathway as well as the primary source of NER. We have seen many cases in which kinetics optimization analysis requires that a portion of the parent compound is itself sequestered and results in the incorporation into NER. We will present examples of those cases in a future publication. From the examples presented in this chapter, one can conclude that kinetics optimization is a useful tool in characterization of the observed NER. 104

References 1.

Brady, D. J. United States EPA, Guidance for Addressing Unextracted Residues in Laboratory Studies; 2014. https://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/ guidance-addressing-unextracted-pesticide-residues [accessed Mar 16, 2016]. 2. Kastner, M.; Trapp, S.; Schaffer, A. Consultancy Services for Supporting ECHA in Improving the Interpretation of Non-Extractable Residues [NERs] in Degradation Assessment; Discussion Paper draft, version IV; Apr 09, 2018. 3. Sandermann, H., Jr.; Hertkorn, N.; May, R.; Lange, B. Bound Pesticidal Residues in Crop Plants: Chemistry, Bioavailability, and Toxicology. In Pesticide Biotransformation in Plants and Microorganisms; Hall, J. C.; Hoagland, R. E.; Zablotowicz, R. M. , Eds; ACS Symposium Series; American Chemical Society; Washington, DC, 2001; Vol. 777, pp 119–128. 4. Richnow, H.; Eschenbach, A.; Mahro, B.; Kästner, M.; Annweiler, E.; Seifert, R.; Michaelis, W. Formation of Non-Extractable Soil Residues: A Stable Isotope Approach. Environ. Sci. Tech. 1999, 33, 3761–3767. 5. Li, J.; Dodgen, L.; Ye, Q.; Gan, J. Degradation Kinetics and Metabolites of Carbamazepine in Soil. Environ. Sci. Tech. 2013, 47, 3678–3684. 6. Loos, M.; Krauss, M.; Fenner, K. Pesticide Non-Extractable Residue Formation in Soil: Insights from Inverse Modeling of Degradation Time Series. Environ. Sci. Tech. 2012, 46, 9830–9837. 7. Trapp, S.; Brock, A. L.; Nowak, K. M.; Kästner, M. Prediction of the Formation of Biogenic Non-Extractable Residues During Degradation of Environmental Chemicals from Biomass Yields. Environ. Sci. Tech. 2018, 52, 663–672. 8. Nowak, K. M.; Miltner, A.; Gehre, M.; Schäffer, A.; Kästner, M. Formation and Fate of Bound Residues from Microbial Biomass During 2,4-D Degradation in Soil. Environ. Sci. Tech. 2011, 45, 999–1006. 9. Sharma, A. K.; Wen, L.; Hall, L. R.; Allan, J. G.; Clark, B. J. Metabolism of Pyrithiobac Sodium in Soil and Sediment, Addressing Bound Residue via Kinetics Modelling. J. Agric. Food Chem. 2016, 64, 5793–5802. 10. Guidance Document on Estimating Persistence and Degradation Kinetics from Environmental Fate Studies on Pesticides in EU Registration; The Final Report of the Workgroup on Degradation Kinetics of FOCUS; June 2006. 11. Ivarson, K. C.; Stevenson, I. L. The Decomposition of Radioactive Acetate in Soils. Can. J. Microbiol. 1964, 677–682.

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Chapter 7

Creating Environmentally Resilient Agriculture Landscapes Using Precision Agriculture Technology: An Economic Perspective Mark D. McConnell* Department of Wildlife, Fisheries, and Aquaculture, Mississippi State University, Mississippi State, Mississippi 39762, United States *E-mail: [email protected].

Agricultural landscapes face increased societal pressure to meet global food demands while simultaneously providing ecosystem services. Increased use of agricultural chemicals is expected in order to meet global yield objectives and reduce risk. However, increased use of agrochemicals poses significant risk to ecosystem function and sustainability. Natural plant communities, as a component of agricultural landscapes, can mitigate this risk and support ecosystem function by providing essential ecosystem services with broad societal value (i.e., pollination, beneficial insects, and wildlife populations). Sustainability of global agricultural systems will require greater focus on the strategic integration of conservation practices into production agricultural systems to protect and enhance ecosystem services and crop production. However, balancing these objectives creates challenges for producers as the allocation of land to noncrop uses (e.g., natural plant communities) entails economic opportunity costs for producers. Agricultural producers will implement conservation actions provided the economic incentives are equal to or greater than traditional farming. Therefore, it is essential for natural resource professionals to help producers identify and understand the economic opportunities of conservation implementation. Precision agriculture technology provides a unique framework for identifying economic and conservation opportunities in production agriculture. By using precision agriculture in a conservation framework, natural resource professionals can demonstrate the overlap between conservation eligibility and economic opportunity. I illustrate the application of this technology to create natural plant communities in production agricultural landscapes that increase field-level profitability and ecosystem services. This approach could easily be applied to increase the use, efficiency, and profitability of vegetative filter strips to reduce pesticide movement in the environment.

© 2019 American Chemical Society

Introduction Pesticides (e.g., herbicides, insecticides, fungicides, etc.) are necessary for meeting increasing global food demands (1). Although pesticide application has become widespread throughout the world (2), pesticides can pose significant risk to ecosystem services provided by agricultural landscapes (e.g., air quality, water quality, pollination, disease suppression, wildlife habitat, carbon storage, etc.) (3, 4). Prevailing environmental concerns from pesticide use include negative impacts of runoff into water bodies (5, 6) and drift into nontarget areas (7), each of which can have cascading environmental consequences (8). Pesticide runoff and drift have been attributed to numerous environmental impacts to soil, water, plants, insects, herpetofauna, and many other wildlife species (3). Whereas multiple efforts exist to minimize pesticide damage through more strategic application (9), other efforts exist to mitigate their impacts by strategically creating natural plant communities to intercept pesticide movement (10–16). Natural plant communities, as a component of agricultural landscapes, can mitigate pesticide risk and support ecosystem function by providing essential ecosystem services with broad societal value (i.e., pollination, beneficial insects, and wildlife populations) (16, 17). Mitigating environmental impacts of pesticides with natural plant communities is well developed (12). Whereas calls to increase pesticide retention effectiveness exist (2), opportunities to improve the prevalence and utility of strategic natural plant community implementation in agricultural landscapes remains a growing field of study. Multiple terms exists to describe strips or blocks of natural plant communities to slow runoff or drift, remove sediment, and maintain nutrients and pesticides in and around agricultural fields (e.g., vegetated filter strips, grass filters, grassed waterways, contour strips, wind breaks, riparian buffers, field borders, filter strips, buffer strips, etc.) (12, 18, 19). This chapter uses the term conservation buffer to consolidate all above-mentioned practices. Conservation buffers have been broadly defined as noncrop areas within and between agricultural fields (16). Agricultural landscapes have historically supported such natural plant communities in the form of riparian forests, wetlands, and hedges (20) to delineate property boundaries and contain livestock (21). The historic presence of fallow, natural plant communities was a by-product of limited farming capabilities, whereas the modern presence of such areas is a result of intentional decision making by agricultural producers. In other words, the landscape itself no longer produces conservation buffers on its own, rather the producer must now implement the practice deliberately. In recent decades, conservation buffers have reduced negative environmental impacts and improved ecosystem health (16, 19, 22). Documented benefits of conservation buffers include, but are not limited to, trapping sediment (23), removing fertilizers (24, 25) and herbicides (26), improving water quality (25), increasing wildlife populations (27–30), providing beneficial insect (31) and pollinator services (32, 33), improving crop yields (34, 35), and increasing land values (34). Using natural plant communities to mitigate pesticide risk often comes in the form of conservation buffers or wetlands. Both models for establishing natural plant communities have been deployed in agricultural landscapes with beneficial results (36, 37). Targeted use of conservation buffers for reducing pesticide runoff began as early as the 1970s (38) and continues today (39). However, the efficacy of conservation buffers at reducing pesticide movement is highly variable for numerous reasons (2, 9, 12, 16, 19, 39). Multiple targeted approaches exist for implementing conservation buffers to increase their pesticide reduction efficiency at the field (40), watershed (41), and landscape scales (42). Similarly, spatially optimizing conservation buffer placement to increase water quality performance using geospatial technology (43–47) creates tremendous opportunities for creating environmentally resilient landscapes. However, while the 108

multiple environmental benefits of conservation buffers are well established (10, 15, 48), buffer use has not increased with increasing agri-environmental needs. For example the U.S. Department of Agriculture’s (USDA’s) National Conservation Buffer Initiative sought to establish around 2 million miles of buffers by 2003 but fell short of that goal by 23% (22, 43), indicating challenges to conservation adoption. Hindrances to adopting conservation practices pose limitations regarding the long-term viability for enhancing ecosystem services and thus environmental resiliency in agricultural landscapes.

Hindrances to Conservation Adoption Hindrances to conservation adoption are numerous and largely understudied at large spatial scales. Addressing agricultural producers’ concerns and hesitations regarding conservation adoption is necessary to increasing environmental resiliency in agricultural landscapes. Gaines-day and Gratton identified a suite of hindrances to conservation program adoption including fear of attracting pests, lack of technical knowledge, and concerns about financial loss (49). Financial concerns drive most agricultural producer decisions (50); therefore, fear of economic loss from conservation implementation can hinder adoption of environmentally beneficial practices (30). Regardless of the environmental benefits of conservation enrollment, removing agricultural land from production poses an opportunity cost to producers from land that would have otherwise been in crop production (12, 51, 52). An abundance of research has indicated that financial considerations are major barriers to conservation adoption (53–57). Perhaps more importantly, research indicates that financial compensation for conservation implementation is necessary for agricultural producers to be able to afford reallocating land from production to conservation (58). Further research has shown that conservation payments are important incentives even if they do not cover the entire cost of establishing a conservation practice (55).

Incentive-Based Conservation The cost of societal benefits produced by conservation enrollment is disproportionately levied on the agricultural producers who provide the ecosystem service (59); therefore, incentive-based conservation has become the predominate mechanism for encouraging conservation enrollment. Incentive-based conservation reallocates the cost burden to society (60), who reap the benefits by subsidizing the cost of conservation establishment and management (61). Incentive-based conservation has proven effective around the world (62), but effectiveness varies (63). Federal conservation programs implemented by the USDA are vast and offer financial incentives and technical assistance to remove land from agricultural production and install conservation buffers (64, 65). This incentive-based approach has proven effective at increasing adoption (66), but it is also essential for producers to afford such changes in land use (58). To address economic loss from conservation enrollment (opportunity cost), it is essential to understand the economic tradeoffs of conservation incentives (i.e., payments) and the degree to which incentives offset loss from agricultural production. Contrary to many agricultural producers’ assumptions, research has shown that revenue from conservation enrollment can often exceed that of agricultural production when applied strategically (30, 52, 67). While financial incentives to reduce pesticide use are not mainstream in U.S. agriculture, there are incentives for conservation buffers to mitigate pesticide loss in addition to in-field practices such as no-till, conservation tillage, and cover cropping. Decreasing pesticide loss will undoubtedly 109

require increases in acreage devoted to natural plant communities to reduce runoff and drift. Unfortunately, addressing the economic outcome of establishing conservation buffers for pesticide mitigation has not been evaluated, which is surprising considering the magnitude of research dedicated to understanding and improving the structure, placement, configuration, and planting arrangement of conservation buffers to reduce pesticide loss. Discussions about decreasing pesticide loss at field or landscape scales are limited without considering the economic implications of reallocating agricultural land to conservation enrollment.

Mechanism for Conservation Delivery Agricultural policy in the United States offers numerous opportunities for creating natural plant communities to intercept pesticide movement (68). There are currently 36 separate conservation practices (CPs) in the Conservation Reserve Program (CRP), which is administered by the USDA Farm Service Agency. The CRP establishes contracts with agricultural producers to retire environmentally sensitive agricultural fields from production for 10 to 15 years through the conversion to a noncrop, natural plant community to provide environmental benefits in exchange for a rental fee (65). Within the CRP, there are multiple CPs that establish conservation buffers or conservation wetlands on a portion of agricultural fields. These practices vary in their objectives that range from improving water quality (CP-27, Farmable Wetlands) to creating wildlife habitat (CP-33, Habitat Buffers for Upland Birds). Each specific CRP practice comes with its own unique financial incentives to encourage enrollment. Financial incentives often include a soil rental rate equal to the land value, a sign-up incentive, and a practice incentive payment. Sign-up incentives are meant to encourage enrollment, and practice incentive payments are designed to offset the cost of establishing and maintaining conservation cover. However, practice-specific financial opportunities vary, making the economic outcomes of enrollment ambiguous among CPs in the CRP (30). Additionally, the spatial eligibility of some practices is very specific and tied to landscape features. For example, Filter Strips (CP-21) are only eligible immediately adjacent to seasonal or perennial streams, wetlands, or other permanent water bodies and can be no less than 20 feet and no more than 120 feet in width (69). The number and complexity of CP options within the CRP and their associated spatial and financial differences poses an unintentional challenge to conservation adoption. Therefore, advanced technology is required to better understand spatial eligibility and economic outcomes of conservation enrollment.

Precision Agriculture Technology Precision agriculture technology is “a philosophical shift in the management of variability within agricultural industries aimed at improving profitability and/or environmental impact (both short and long term)” (70). Precision agriculture technology represents a unique opportunity to integrate spatial eligibility and economic outcomes of conservation enrollment for agricultural producers seeking to reduce pesticide loss. Precision agriculture technology can be used to quantify direct economic cost of agricultural production versus conservation. Despite increasing adoption of precision agriculture technology across the United States (71), its application in conservation planning remains underutilized. When used in a conservation framework, McConnell and Burger illustrated that both profitability and environmental impact could be optimized through the strategic use of precision agriculture technology (30, 52). Their work represents the first application of precision agriculture technology to spatially quantify a per-acre economic outcome of conservation program enrollment. The application of precision agriculture technology to evaluate economic 110

opportunities in pesticide mitigation has not been evaluated but shares many similar components as McConnell and Burger and Capmourteres et al., who also quantified economic outcomes of conservation conversion (30, 67). The ultimate goal of this chapter is to illustrate the concept and application of using precision agriculture to mitigate the environmental effects of pesticides while considering the economic outcomes of those land use changes. Precision agriculture, when used in a precision conservation context, integrates geospatial technology, agricultural economics, agronomy, and conservation planning to identify the overlap between conservation and economic opportunities.

Figure 1. Yield map (bushels per acre) for a production soybean field in Lowndes County, Mississippi.

Precision Agriculture Application for Pesticide Mitigation Precision agriculture includes numerous tools and technologies used to improve farm efficiency (i.e., reduce inputs and increase yields). Yield monitors equipped with global positioning systems are the most widely adopted suite of precision agriculture technologies (71). Yield monitors produce yield maps that illustrate the spatial variability of yield (e.g., bushels per acre) across a field but provide no economic information (52) (Figure 1). When incorporated with crop input costs, commodity prices, and government payments, yield maps can be converted to profit maps that illustrate per-acre profitability of agricultural production (Figure 2). Profit maps can then be used to visually inspect unproductive field regions that could be reallocated to conservation use for pesticide mitigation. Low profitability areas are good candidates for conservation buffer implementation (72), but additional analysis is required to evaluate the economic outcome of conservation enrollment.

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Figure 2. Profit surface (dollar per acre) for a production soybean field in Lowndes County, Mississippi. Net Rev: net revenue. Precision agriculture can also be used to illustrate the spatial eligibility of conservation practices on a farm, incorporate practice-specific financial incentives, and calculate the profitability of conservation scenarios. McConnell and Burger used this approach to illustrate the spatial eligibility of CP-21, Filter Strips, and CP-33, Habitat Buffers for Upland Birds, on a working production farm in Tallahatchie County, Mississippi (30). Using a geospatial decision support tool that incorporates spatial and financial components of USDA conservation practices, they illustrated 257 acres of eligible area for CP-21 and 758 acres for CP-33 (Figures 3 and 4). These acres represented the eligible acres for implementing conservation buffers on the farm. These buffers could serve to reduce runoff and drift from on-farm pesticide applications (12); however, the economic outcomes of these potential conservation buffer enrollments could still prevent landowner adoption due to perceived economic loss. Economic evaluations require comparison of potential conservation acreage to that of agricultural production. Using conservation incentives and the profit maps depicting spatial variability in field-level profitability, McConnell and Burger’s approach can be used to create new profit surfaces for conservation buffer scenarios (30). Figures 5 and 6 illustrate a simulated profit surface with 30 feet and 120 feet CP-33 conservation buffer scenarios, respectively. Illustrating peracre profitability of multiple scenarios helps agricultural producers make informed decisions (Figure 7). Figure 7 illustrates the importance of buffer width on profitability when designing pesticide mitigation strategies using buffers. Buffer width is an important consideration when designing buffers to reduce pesticide loss (19, 40, 42), considering increasing buffer width does not always improve pesticide trapping (12). Therefore, the economic implications of buffer width should be balanced 112

with efficiency of buffer width for pesticide trapping. Precision agriculture technology provides the data necessary to enroll only where conservation is profitable. The decision to create conservation buffers beyond the profitable zone is up to the producer. Precision agriculture simply allows the producer to make an informed decision. However, it is important to remember that narrow buffers are better than no buffers at all (12). McConnell and Burger’s results illustrated that conservation buffers can increase field-level profitability across multiple buffer widths and commodity prices (30). Field margins are often the lowest yielding areas of a field (73) and, therefore, often the least profitable as well. By targeting low-yielding field margins with precision agriculture technology, agricultural producers can increase profitability by enrolling in incentive-based conservation programs while simultaneously reducing pesticide loss. This approach will increase environmental quality through the reduction in pesticide loss and increase profitability, making conservation an economically and environmentally viable farming practice.

Figure 3. Total eligible area for CP-33, Filter Strips, on a grain farm in Tallahatchie, Mississippi. Reproduced with permission from reference (30). Copyright 2016 American Society of Agronomy, Crop Science Society of America, Soil Sciences Society of America.

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Figure 4. Total eligible area for CP-21, Habitat Buffers for Upland Birds, on a grain farm in Tallahatchie, Mississippi. Reproduced with permission from reference (30). Copyright 2016 American Society of Agronomy, Crop Science Society of America, Soil Sciences Society of America.

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Figure 5. Profit surface (dollar per acre) for a production soybean field with 30-foot CP-33, Habitat Buffers for Upland Birds, scenario in Lowndes County, Mississippi. Net Rev: net revenue.

Figure 6. Profit surface (dollar per acre) for a production soybean field with 120-foot CP-33, Habitat Buffers for Upland Birds, scenario in Lowndes County, Mississippi. Net Rev: net revenue. 115

Figure 7. Bar graph illustrating the economic outcomes of two conservation profitability scenarios on a soybean field in Lowndes County, Mississippi.

Discussion As pesticide use continues to increase, greater effort to mitigate the associated negative environmental consequences will be required. Innovative conservation solutions that address both function and feasibility of conservation efforts must be addressed to provide agricultural producers and natural resource professionals with tools to minimize environmental damage without hindering necessary and responsible pesticide use. To date, no research on the use of precision agriculture technology to optimize pesticide loss and profitability through conservation buffers has been conducted. While this chapter does not specifically test this application, it illustrates the concept of using precision agriculture to balance economic and conservation needs in agriculture by framing previous work by McConnell and Burger in the context of creating conservation buffers to mitigate pesticide loss (30, 52). This chapter does not address the many facets of efficacy regarding conservation buffers and pesticide retention (vegetation type, density, width site conditions, pesticide being used, etc.). Rather, the goal is to illustrate a novel concept that could increase adoption by addressing financial concerns of agricultural producers. Conservation buffers are used globally to implement best management practices in agricultural landscapes (74). Conservation buffers generally have a universal goal—improving ecosystem health (16). Although conservation measures have historically been assumed to decrease agricultural revenue, this chapter illustrates that even efforts to reduce pesticide loss can have profitable outcomes on agricultural production. Research evaluating their effectiveness at pesticide retention is extensive in both breadth and geography. However, there is limited research quantifying the economic 116

outcome of implementing conservation buffers for pesticide retention. Future research should focus on using precision agriculture technology to identify the intersection of conservation and economic opportunities to increase pesticide mitigation and profitability. Specifically, research should integrate precision agriculture technology with other geospatial planning tools that identify the best location, width, vegetation type, and planting density to optimize conservation buffer placement. Conservation buffers are considered the “last line of defense” in pesticide mitigation (12). Conservation buffers are voluntary, incentive-based tools whose potential to achieve conservation goals is dependent on adoption by agricultural producers (64). By using precision agriculture technology to address economic opportunities of pesticide mitigation strategies, conservation buffers can play a greater part in conservation-focused agricultural production. Concomitantly achieving these goals should increase landowner satisfaction, farm-level profitability, and environmental resiliency in agricultural landscapes.

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49. Gaines-day, H. R.; Gratton, C. Understanding Barriers to Participation in Cost-Share Programs For Pollinator Conservation by Wisconsin (USA) Cranberry Growers. Insects 2017, 8, 79. 50. Kitchen, N. R.; Sudduth, K. A.; Myers, D. B.; Massey, R. E.; Sadler, E. J.; Lerch, R. N.; Hummel, J. W.; Palm, H. L. Development of a Conservation-Oriented Precision Agriculture System: Crop Production Assessment and Plant Implementation. J. Soil Water Conserv. 2005, 60, 421–430. 51. Jack, B. K.; Kousky, C.; Sims, K. R. E. Designing Payments for Ecosystem Services: Lessons from Previous Experience with Incentive-Based Mechanisms. PNAS. 2008, 105, 9465–9470. 52. McConnell, M. D. Using Precision Agriculture Technology to Evaluate the Environmental and Economic Tradeoffs of Alternative CP-33 Enrollments. Masters Thesis, Mississippi State University, 2011. 53. Lynne, G. D.; Shonkwiler, J. S.; Rola, L. R. Attitudes and Farmer Conservation Behavior. Am. J. Agric. Econ. 1988, 70, 12–19. 54. Macdonald, D. W.; Johnson, P. J. Farmers and the Custody of the Countryside: Trends in Loss and Conservaton of Non-Productive Habitats 1981–1998. Biol. Conserv. 2000, 94, 221–234. 55. Reimer, A. P.; Prokopy, L. S. Farmer Participation in U.S. Farm Bill Conservation Programs. Environ. Manage. 2014, 53, 318–332. 56. Sorice, M. G.; Haider, W.; Conner, J. R.; Ditton, R. B. Incentive Structure of and Private Landowner Participation in an Endangered Species Conservation Program. Conser. Biol. 2011, 25, 584–596. 57. Lute, M. I.; Gillespie, C. R.; Martin, D. R.; Fontaine, J. J. Landowner Practitioner Perspectives on Private Land Conservation Programs. Soc. Nat. Resour. 2017, 31, 1–14. DOI: 10.1080/ 08941920.2017.1376139. 58. Sweikert, L. A.; Gigliotti, L. M. Evaluating the Role of Farm Bill Conservation Program Participation in Conserving America’s Grasslands. Land Use Policy 2019, 81, 392–399. 59. Lynch, L.; Brown, C. Landowner Decision Making about Riparian Buffers. J. Agri. Appl. Econ. 2000, 32, 585–596. 60. Rolston, H., III. Life in Jeopardy on Private Property. In Balancing on the Brink of Extinction; Kohm, K. A., Ed.; Island Press: Washington, DC, 1991: pp 43–61. 61. Stubbs, M. Conservation Provisions in the 2014 Farm Bill; CRS Report R43504; 2014. 62. Ulber, L.; Klimek, S.; Steinmann, H-H.; Isselstein, J.; Groth, M. Implementing and Evaluating the Effectiveness of a Payment Scheme for Environmental Services from Agricultural Land. Environ. Conserv. 2011, 38, 464–472. 63. Nelson, E.; Polaksky, S.; Lewis, D. J.; Plantinga, A. J.; Lonsdorf, E.; White, D.; Bael, D.; Lawler, J. J. Efficiency of Incentives to Jointly Increase Carbon Sequestration and Species Conservation on a Landscape. PNAS. 2008, 105, 9471–9476. 64. Reimer, A. Ecological Modernization in U.S. Agri-Environmental Programs: Trends in the 2014 Farm Bill. Land Use Policy 2015, 47, 209–217. 65. Hellerstein, D. M. The U.S. Conservation Reserve Program: The Evolution of an Enrollment Mechanism. Land Use Policy 2017, 63, 601–610. 66. Lant, C.; Loftus, T.; Kraft, S. E.; Bennett, D. Land-Use Dynamics in a Southern Illinois (USA) Watershed. Environ. Manage. 2001, 28, 325–340.

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Chapter 8

Contract Research, Good Laboratory Practices, and Other Challenges for the Agrochemical Professional Maria (Marian) Ponte* and Megha Chandrashekhar Eurofins Agroscience Services, 625 Alfred Nobel Drive, Hercules, California 94547, United States *E-mail: [email protected].

Since their emergence, agrochemical Contract Research Organizations (CROs) have flourished as trusted advisers in a thorny world of tight regulations, complex science, and increasingly globalized business models. This success also brings many challenges for the modern CRO. Drawing on the research and insights presented by a panel of experts at the 2018 Annual Fall Meeting of the American Chemical Society, this chapter will examine many of these challenges and propose strategies for those determined to succeed. It will update readers on the latest Good Laboratory Practice guidelines and their implications, offering a perspective on how successful CROs think about compliance and data integrity in today’s intensely regulated industry. The Environmental Protection Agency’s 2017 inspection numbers will be discussed, revealing the risks levied by poor data management practices. To help CROs avoid these risks, this chapter argues for a way forward: strategic training and mentorship to incubate a new generation of scientists, a diversified service offering to build resilience into company objectives, and a grassroots approach to communication that will help all CROs contribute to sustained growth and prosperity.

The Evolution, History, and Market Demand for Contract Research Organizations Before there were agrochemical Contract Research Organizations (CROs) in the United States—indeed, before there were any CROs—there was a burgeoning and largely unregulated pharmaceutical industry. It may seem like a long time ago, but pharmaceutical manufacturers of the 1940s and 1950s were not altogether different from today. Like modern pioneers of genetic therapy and personalized medicine, researchers in America’s post-World War II labs were in close pursuit of new and life-changing interventions, including the polio vaccine (1954) and early oral contraceptives (1960). Like today, legislators then were playing catch-up, striving to modernize their regulations and guidelines to enable innovation while preserving safety and integrity.

© 2019 American Chemical Society

As these shifting factors conspired to make pharmaceutical development a more expensive and time-consuming enterprise (albeit with the chance for rich rewards), manufacturers began looking for cost-effective ways to outsource certain research functions on a contract-by-contract basis. This would free them to focus on core research activities while third-party experts ensured the ongoing quality and effectiveness of their products. Out of this need, the concept of the CRO was born. Since then, regulatory scrutiny has intensified and new testing requirements have emerged. Quality management guidelines, like the principles of Good Laboratory Practice (GLP), have introduced added complexities. The CRO has risen amid these changes as a trusted adviser, providing sponsors and clients with expertise and with the equipment and infrastructure needed to convert that expertise into production efficiently and inexpensively. With these skills, CROs flourished beyond their pharmaceutical roots, quickly expanding into the biotechnology and medical device sectors. In the 1990s and early 2000s, coincident with a surge in concern for the environmental fate of pesticides, another type of organization emerged: the agrochemical CRO. Like those in the pharmaceutical industry, companies involved in agrochemical production face compounding challenges. Market pressures continually drive up the cost of research and development as companies vie to offer crop protection solutions with lower application rates and higher efficiency. Regulatory complexity continually escalates as well. In North America, for example, agrochemical products undergo a risk-based assessment; in Europe, those same products are assessed not on risk, but on hazard. Emerging markets in places like Brazil, China, and India add further complexity to the mix with their own approach to regulatory requirements. These differentiated regulations pose a challenge for agrochemical companies who may find themselves with a product that is surging in one market and banned from another, making overall market growth a difficult and labyrinthian prospect. For many agrochemical firms, CROs diffuse this complexity. They have the expertise to navigate international regulations and the scientific sophistication to help agrochemical firms compete for market share in a challenging environment. Meanwhile, companies in other industries are seeing value in this expertise, too; as regulatory oversight of chemical production intensifies bilaterally across the pharmaceutical, veterinary, human health, and other industries, non-agrochemical clients are finding their way to the door of agrochemical CROs, seeking expert help and guidance. As a result, agrochemical CROs are busier than ever, with a client list that cuts across industries and that includes clients as large as the world’s top manufacturers and as small as independent domestic consultancies. It is a good, but challenging, time to be a CRO. This chapter will help CRO personnel understand these challenges in the context of our shared history and, for those able to adapt, our long and thriving future in the ever-changing agrochemical marketplace.

Trends in the Agrochemical Industry The Regulatory Reality Planning, performing, monitoring, recording, reporting, and archiving. To lab workers who test and assess agrochemicals for potential hazards and impurities, these 6 critical steps are like the 26 letters of the alphabet: elementary and familiar yet open to seemingly infinite expression. Depending on how they are applied, the result can spell success for a study, or it can spell noncompliance. The difference often comes down to another constellation of letters, GLP.

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When the GLP guidelines were introduced in the United States 40 years ago, they gave labs and the monitoring authorities who oversaw them a consistent framework for operationalizing these six steps in the testing process. Before the introduction of GLP guidelines, procedures varied from lab to lab, creating opportunities for CROs to backslide into negative outcomes. Post-GLP, those opportunities were largely extinguished through a shared understanding of the practices required to ensure a safer and more reliable agrochemical supply. At the heart of this surge toward standardized guidelines sits the central theme, and challenge, of modern labs everywhere: maintaining data integrity. In its forward to a series of documents called “The Principles of Good Laboratory Practice and Compliance Monitoring,” the Organisation for Economic Co-operation and Development (OECD), which maintains and distributes the principles internationally, writes that GLP is designed to “promote the quality and validity of test data used for determining the safety of chemicals and chemicals products (1).” The United States Environmental Protection Agency (EPA), which monitors the guidelines domestically under 40 CFR Part 160, puts an even finer point on it: “EPA’s Good Laboratory Practice Standards compliance monitoring program ensures the quality and integrity of test data submitted to the Agency in support of a pesticide product registration (2).” The principles of GLP provide structure to each of those six essential steps and help labs understand the criteria by which they will be assessed for compliance. But the principles of GLP are not the only guidance for labs, and herein lies a persistent gray zone. The United States Food and Drug Administration also oversees GLP compliance and subscribes to the premise that all data should be attributable, legible, contemporaneous, original, accurate, consistent, available, enduring, and complete. Meanwhile, other frameworks and principles further crowd this laundry list. For example, ISO 17025 was developed by the International Organization for Standardization to regulate and accredit the technical competence of testing labs. Good Manufacturing Process is an internationally recognized system developed to ensure products are consistently produced and controlled according to quality standards. The International Featured Standards, the Association for Assessment and Accreditation of Laboratory Animal Care International, and the Toxic Substances Control Act may also play a role in the regulatory life cycle of agrochemical CROs. As a result, there is a lot to track, understand, and apply to everyday activities in the lab. Additionally, increasing global awareness of environmental impacts and a growing demand for agrochemicals from emerging markets are imposing more complications on the regulatory landscape. The concept of a “typical” agrochemical CRO customer has all but expired; instead, the same CRO may find itself working with leading global giants one day and small consultancies or task forces the next, each seeking the regulatory expertise that is the CRO’s specialty. These clients are not limited to the agrochemical industry, either. In the past, the regulatory spotlight that searches for environmental risk was trained almost exclusively on crop protection companies and others in the traditional agricultural space. Today, its aperture has widened to include the pharmaceutical industry, animal health, industrial chemical production, health and personal care, and others. Essentially, any chemical which could potentially impact living organisms requires an environmental risk assessment, which in turn requires experienced and precise testing. As a result, agrochemical CROs, uniquely qualified for such testing, are seeing a surge in customers from a cross section of industries. Amid all this growth and change, agrochemical CROs must determine how to stay competitive and up to date.

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Surging toward Merging In the early 2000s, conflict and confusion abounded in this intricate regulatory world, particularly concerning international guidelines. CROs and other affected parties pushed for harmonization between American, Canadian, and European standards. The task was colossal, requiring in-depth revisions of almost every guideline. Under the principle of Mutual Acceptance of Data, the OECD updated its GLP documentation in 2017 (and again in 2018) to support the concept of “tested once, accepted for assessment everywhere.” The idea is to save the chemical industry the expense of duplicate testing for products marketed in multiple countries. Although this and other regulatory initiatives marked significant progress toward harmonized guidelines, the results are incomplete. Some individual regions persist in maintaining specific requirements that demand a customized approach. The gradual, imperfect emergence of harmonized guidelines created extensive work for CROs, which sought to preserve their reputations as centers of excellence while scaling up to meet globalized standards. This would require a painstaking reevaluation of every activity and a wholesale update to long-established reporting practices, ultimately, triggering a significant number of mergers and acquisitions among agrochemical CROs, particularly in the 2010s. These transactions were a strategic survival tactic designed to help CROs compete on a global scale while offering a wider breadth of services. The authors of this chapter know this story well. In 2017, their employer, EAG Laboratories, was acquired by Eurofins Scientific, whose CEO summarized the company’s motivations (and the motivations at the core of most CRO acquisitions of the time) perfectly: “EAG’s competencies, reputation for scientific excellence and footprint further strengthen Eurofins’ global offering in the highly attractive biopharmaceutical and agroscience testing markets (3).” Whether organically or through similar mergers and acquisitions, agrochemical CROs are offering more services than ever before to an increasingly global marketplace. Of course, this kind of rapid growth comes with an array of interesting and formidable challenges.

Challenges in the CRO Industry The Challenge of GLP Managing Data Mergers and acquisitions between agrochemical CROs have helped expand their global footprint and, in some ways, have aided in upholding complex global regulations; but challenges remain—some new, some old. CROs large and small remain beholden to the principles of GLP, which provide a necessary framework for quality assurance (QA) but contribute to an ongoing struggle to reconcile modern data collection activities with expectations of excellence. The bottom line is that while today’s lab management software makes it easier for labs to streamline and automate data collection, even the best technology cannot supplant the human lab worker, who bears responsibility for ensuring data integrity. Whenever that responsibility falters, vulnerabilities are introduced and violations can occur. These violations can levy significant consequences. There are hard costs like lost resources and time if a study must be repeated in order to achieve compliance. There are other, less immediate (but no less devastating) costs such as reputation damage by repeated violations. In a crowded, competitive market, CROs rely on credibility to find and retain new customers and sponsors. To 126

maintain that credibility, all lab workers need a thorough understanding of (and an unwavering commitment to) the practices that ensure data integrity inside the study site. Francisca E. Liem of the EPA has prepared a snapshot of how well this commitment to integrity is functioning in today’s agrochemical labs, as shown in Table 1 (4). Table 1. EPA Inspection Numbers: Compliance by Laboratory Discipline (FY 2017). Reproduced with permission from ref. (4). Copyright 2018 Environmental Protection Agency. Type of Laboratory

Number of Inspections

Inspections with NO Violations Found

Percent Compliance

Field Sites:

19

16

84

Product Chemistry:

15

8

53

Toxicology:

5

3

60

Analytical Chemistry:

11

10

91

Others:

14

5

36

Total:

64

42

66

Note. “Others” includes insecticide, rodenticide, antimicrobial efficacy, ecotoxicology, ecotox, archives, and processing labs (4).

The majority of violations concern insufficient record-keeping. Of the 19 inspections conducted at field sites, for example, 3 failed to comply with guidelines for correct data entry. Complete findings at the field sites included: • Lack of separate written records of routine and nonroutine repairs of equipment; • Protocol deviation: Nonionic surfactant was used in the application instead of crop oil concentrate; • Incorrect data entry; and • Data corrections that were noncompliant with GLP. In product chemistry labs, insufficient record-keeping is more prevalent. Of the seven inspections that uncovered violations, the majority revealed a notable lack of raw data. In two instances, noncompliant corrections to data were uncovered. Complete findings at the product chemistry labs included: • • • •

Lack of raw data; Data corrections that were noncompliant with GLP; Missing units; Instrument inspection recordings that were not differentiated between routine and nonroutine work; and • Oven temperature that was not monitored. To protect against these violations, CROs need two things. First, standardized operating procedures (SOPs) based on the principles of GLP are required to ensure a consistent and qualitydriven approach to data management. Second, and perhaps most important, CROs need a strategic plan for properly training lab workers on their roles and responsibilities. One without the other 127

(a rigorous set of SOPs without high-quality training or training based on individual preferences without the guidance of SOPs) is a formula for error. Compliance Starts with Training Training on the proper application of guidelines must cover a study’s entire life cycle—those six critical steps addressed by the principles of GLP and outlined previously. At one end sits the planning stage, which involves arranging equipment, methods, test items, and personnel into a functioning, harmonious unit. On the other end, archiving requires the equally colossal effort of consolidating raw data, test records, samples, record maintenance, and more (often across multiple locations and many years) into an accurate and comprehensive chronicle of a study’s life cycle. Between these poles, other study activities introduce their own unique and critical responsibilities. Clarifying these roles and ensuring that lab personnel have the time and resources to incubate needed skills and the expertise to fulfill expectations is the best assurance of compliance for a lab. An impurity profiling study is a good example. In order to register a new or generic product, the manufacturer must conduct a five-batch analysis to prove a chemical equivalence to the reference specifications issued by the Food and Agriculture Organization of the United Nations and the World Health Organization. Many manufacturers, realizing the scale of training and expertise required for such an analysis to succeed, rely on a CRO for these activities. This, of course, requires the CRO to maintain an updated training program. Depending on their role, lab workers conducting a five-batch analysis must be familiar with the latest certified reference standards; must know how to identify and verify impurities based on molecular weight or fragmentation pattern; must understand how to prepare and extract samples (5); and, above all, must be trained to meet regulatory and GLP requirements. While this means considerable training, it is necessary. It defends a CRO against the common pitfalls of complex analyses, including: • • • • •

Deleting or overwriting electronic raw data; Retesting out-of-specification results until acceptable standard is achieved; Not investigating out-of-specification results as required by the SOPs; Failing to provide an audit trail, data reviews, or both; and Performing questionable or incomplete documentation.

This list may be overwhelming to those concerned with data integrity. No CRO wants to tread anywhere near these violations, nor does any lab worker. Training programs help. Mentorship and experience help. Something else also helps: the omnipresent, exceptionally knowledgeable Study Director (SD). The Role of the SD The SD is the nerve center of GLP compliance during a study. In the planning stage, the SD works with the CRO’s client and sponsor to codevelop, endorse, and distribute the study plan to lab staff; in the archiving stage, the SD reviews all study data and provides a signed document included with the final report, stating that the study complied with standard GLP and QA guidelines and that the study data is, accordingly, valid. Between these first and last steps, the duties of the SD are varied and essential, from reviewing raw data and capturing any deviations in the SOPs (and the reasons for those deviations) to ensuring that test materials are available and that lab workers are trained and ready to perform their duties with 128

integrity. During client visits, the SD steps in as a knowledgeable host and a scientific interpreter. During the archiving stage, the SD oversees all documentation activities. This is an enormous responsibility. Like the ship captain who bears responsibility for navigating indifferent seas, the SD is chief navigator, ensuring that the journey from study plan to study compliance is conducted with confidence and integrity. The Challenge of an Aging Workforce As chief navigators, SDs are sometimes hard to come by, particularly at present. The scientists, managers, and consultants who began their careers 40 years ago are now retiring. These highly experienced professionals are leaving the workforce, taking with them valuable knowledge and expertise. To maintain stability and ensure ongoing GLP compliance, CROs need to plan for this drain in experienced personnel and, more than ever, develop a new generation of SDs. Nourishing the next wave of SDs is a threefold venture. First, CROs must attract new talent to the agrochemical industry by offering attractive compensation and benefits alongside a modernized, technologically sophisticated workplace where the best and most ambitious young scientists can envision a fulfilling career. Next, CROs must offer opportunities for new scientists to develop their skills and practice the principles of good data management. To do this, CROs must find ways to retain experienced and knowledgeable senior personnel, despite the draws of retirement or competing employment opportunities. Finally, successful CROs must develop a workplace culture of continuous improvement supported by formal and informal mentorships and feedback cycles. Megha Chandrashekhar has firsthand experience of the value that a mentoring culture imparts both to individual employees and to organizational health. Since joining EAG Laboratories as an Assistant Research Chemist in 2015, she has risen to the position of Staff Scientist I and is now SD on various studies. A strategic training program defined by close individual mentorship and weekly group meetings helped her follow this steeply ascending career path. The Challenge of Continuous Regulatory Change To remain competitive training initiatives must be up to date. This means that CROs have to ride the crest of the regulatory wave, continuously educating themselves about the guidelines and trends emerging from the established markets (United States, European Union, and Canada). Pure awareness is not enough; CROs must integrate these guidelines into their operations swiftly, seamlessly, and without losing quality while providing uninterrupted service to their clients. Regulatory change in developing markets are factors in this challenge, too. Brazil, Argentina, and China are emerging players in the international agrochemical industry, and each country presents its own nuanced set of regulatory guidelines that will become increasingly important as CROs continue to globalize. The OECD’s harmonization efforts, covered earlier in this chapter, will play a role here as well; in March of 2018, for example, Estonia achieved full compliance under the OECD’s Mutual Acceptance of Data principle (4). Other countries are quickly following suit. The Challenge of Flexibility By mastering the complex and ever-changing global regulatory landscape, a CRO has the expertise to design studies that fulfill the requirements of multiple geographic regions. This flexibility

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is a boon for clients pursuing studies in different markets and can truly differentiate CROs from their competition. However, offering multisite flexibility can quickly become very complicated. For one thing, the SD and the test sites may be in different parts of the world, triggering a landslide of challenges. Test sites may not be familiar with GLP, leading to a study plan that is noncompliant. Finally, even if the study plan does meet requirements, compliance may falter if the SD is not there to oversee it in action. Multisite studies may also introduce communication problems. Lead QA may not have been notified. Experiments may begin without the SD’s knowledge. Differences in time zones, languages, and cultural norms may hamper clear communication. Even differences in local laws and political agendas can complicate an ongoing study if the CRO and its study sites are geographically separated. For CROs who successfully overcome these challenges, offering multisite capabilities is a powerful market differentiator. Those who take flexibility further by accommodating tight schedules or increasingly detailed requests are even more ahead in the competitive race for new business. The Challenge of Diversification Adapting to shifting regulations and the pressures of greater flexibility is key. But for agrochemical CROs to continue flourishing, they must diversify their services beyond the agricultural industry. Like most other industries, agriculture can be unpredictable. Market demands soar one year and plummet the next, creating volatility that impacts all industry players, particularly CROs who are narrowly and vulnerably focused in a single service area. To insulate against a shifting, unpredictable market, visionary CROs are adding services adjacent to their agrochemical offering. Industrial and “down the drain” chemical testing is a good example, as is food chemistry investigation. However, for this diversification to scale across a CRO without impacting quality, a rigorous recruitment and training regimen is required to prepare lab workers to shift from one study type to another. The Challenge of New Technology Offering experienced staff who are prepared to work in diverse service areas helps CROs attract the interest of new clients. Converting that interest into a productive relationship often comes down to pricing. As clients begin expecting tailored study designs, and as chemistries become more complicated and regulations stricter, maintaining a competitive pricing model is increasingly difficult for many CROs. The answer is technology. By investing in the latest and best tools designed for labs in general and the agrochemical industry in particular, CROs can build efficiencies into each study’s life cycle. Some balk at the sticker price of initial purchase and installation, but a true cost-benefit analysis often reveals that, over time, the aggregate savings of a highly efficient, intuitive, digitally enabled lab far outweigh the upfront investment. Sometimes, investing in lab technology requires taking a risk; in order to outpace the competition, CROs need to anticipate what is next and prepare for it before a clear demand emerges in the market. The best way to stay current on the latest developments and predictions is to attend scientific conferences and to read texts like this one.

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The Holy Grail (Communication) The challenges highlighted in this chapter may appear numerous and formidable, but they have one weakness that CROs can exploit: consistent, timely, and clear communication. By fostering open and consistent lines of communication among different working groups, CROs can inoculate themselves against potential threats. An SD who communicates regularly with a sponsor helps to facilitate improved outcomes, particularly when multiple studies are underway for the same client. Communication between administrators for the CRO and for the client (including procurement, invoicing, and quoting units) helps to facilitate efficient business operations. Most of all, a culture of internal communication within the CRO can have a clear and measurable impact on the quality of ongoing studies. The authors of this chapter have seen these impacts firsthand. Eurofins Scientific has fostered a culture of communication through weekly meetings that bring management, operations, and QA together, inviting each aspect to share its opinion and making collective decisions about compliance activities. Figure 1 demonstrates that these meetings, along with other initiatives borne of regular communication (such as the advent of a centralized database of SOP deviations, which created opportunities for revising and clarifying SOPs), have had a direct and positive impact on our labs’ quality metrics (6).

Figure 1. The impact of communication initiatives at Eurofins on quality metrics. Major events in the second quarter of 2016 included establishing weekly Ops/QC/RW/QA meetings, new e-fate testing, and the established and management of a deviations tracking database (the second quarter of 2016) (6).

Conclusions Let us parlay 12 famous words into an observation on the modern agrochemical industry: “It was the best of times, it was the worst of times.” Though written long before anyone spoke in the alphabet of CROs, GLPs, or SOPs, these words epitomize the tension faced by an industry that is both surging in demand and flinching under increased regulatory pressure. Those who survive will learn to make this tension work for them, distinguishing themselves as scientific innovators who understand the principles of GLP better than anyone and who can offer a level of data integrity, technical sophistication, and bright, fresh scientific innovation that exceeds industry expectations. Only by learning this balance can agrochemical CROs 131

secure their future in a competitive world, ensuring that the worst of times are behind them and only the best of times remain.

References 1.

2.

3.

4. 5.

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OECD Environment Directorate. OECD Principles of Good Laboratory Practice; Series on Principles of Good Laboratory Practice and Compliance Monitoring No. 1; Environmental Health and Safety Publications, Organisation for Economic Co-operation and Development: Paris, 1998. http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote= env/mc/chem(98)17&doclanguage=en (accessed April 19, 2019). EPA. Good Laboratory Practices Standards Compliance Monitoring Program. https://www.epa. gov/compliance/good-laboratory-practices-standards-compliance-monitoring-program (accessed April 19, 2019). Eurofins Press Releases. Eurofins Announces the Successful Closing of the Acquisition of EAG Laboratories. https://www.eurofins.com/biopharma-services/product-testing/news-events/ press-releases/acquisition-of-eag-laboratories/ (accessed April 19, 2019). Liem, F. E. EPA Regulatory Update on Good Laboratory Practices. Presented at the American Chemical Society National Meeting, Boston, MA, August 19–23, 2018. Sanghani, L. U. Planning, Performing, Recording, Reporting and Archiving of Impurity Profiling Studies (Five Batch Analysis) in Compliance with GLP. Presented at the American Chemical Society National Meeting, Boston, MA, August 19–23, 2018. Dutton, J.; Hughes, C.; Sarff, P. Quality Metrics: The Use of Quality Metrics to Drive the Culture of Continual Improvements. Presented at the American Chemical Society National Meeting, Boston, MA, August 19–23, 2018.

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Editors’ Biographies Kari Lynn Kari Lynn is currently working at Corteva Agriscience in Indianapolis, Indiana, where she conducts and designs environmental fate studies to support discovery programs. She has been working in the Ag industry for 7 years. Prior to moving to Ag, Kari worked in Pharma for 20 years supporting drug metabolism, biopharmaceutics, and bioanalytical. Kari received her B.S. degree in Chemistry from Indiana University and her M.S. degree in Analytical Chemistry from Villanova University.

Mingming Ma Mingming Ma is currently a Regulatory Science Team Leader of Crop Protection Regulatory Sciences at Corteva Agriscience, where she leads the teams to support registrability assessment for new pesticide active discovery and development. She received a B.S. in Pharmacy from Zhejiang University and a Ph.D. in Pharmaceutical Sciences from the University of Wisconsin-Madison. Her Ph.D research focused on developing multi-faceted mass spectral technologies in crustacean neuropeptide discovery. She serves as an executive committee member in the ACS AGRO Division. Previously, she has been involved as a co-organizer for numerous symposia and an invited speaker for several AGRO symposia. She has authored or co-authored more than 20 papers in peer-reviewed journals.

Qiang Yang Qiang Yang is currently a Process Chemistry Team Leader in the Crop Protection Product Design & Process R&D at Corteva Agriscience in Indianapolis, Indiana, where he is designing and developing processes and leading process chemistry efforts to support the advancement of Crop Protection products. Prior to his employment at Corteva Agriscience, Qiang spent about 10 years in the Chemical Development group at Albany Molecular Research, Inc. (AMRI), focusing on route scouting, process development, and technology transfer of processes for pilot manufacture of Active Pharmaceutical Ingredients (APIs) intended for toxicological and clinical evaluations. Qiang received his B.S. in Chemistry from Beijing Normal University and M.S. in Organic Chemistry from Institute of Chemistry, the Chinese Academy of Sciences in Beijing. He completed his Ph.D. study under the direction of Prof. David C. Baker at University of Tennessee, Knoxville. His Ph.D. research focused on the total synthesis of C-/O- and S-/O- linked oligosaccharide mimetics of Hyaluronic Acid (HA). Qiang is a co-inventor for 50 issued patents and co-author for 25 peer-reviewed publications. Qiang has been a member of the Org. Process Res. & Dev. Editorial Advisory Board since January 2019.

Qi Yao Qi Yao (Ph.D., University of Maryland) is currently a postdoctoral researcher at University of Maryland, where she works on aerosol cloud condensation nuclei interaction and characterization. Prior to this employment, she was working on evaluating the effectiveness of vegetative environmental buffer (VEB) in reducing air pollutant emissions from poultry houses. She has earned a number of awards, including ACS AGRO Education Award (2016, 2017) and ACS IUPAC Student Award (2014). She is an ACS fellow and an AAAR fellow and has authored or co-authored 11 papers and 1 patent so far. © 2019 American Chemical Society

Indexes

Author Index Atkinson, J., 67 Blair, J., 1 Boebel, T., 39 Bravo-Altamirano, K., 39 Chandrashekhar, M., 123 Daeuble, J., 39 DeKorver, K., 39 DeLorbe, J., 39 Dent, W., 39 Fang, C., 95 Gilbert, J., 51 Godbey, J., 83 Herrick, J., 39 Knueppel, D., 51, 83 Knueppel, D., 67 LaLonde, R., 39 Li, F., 39 Lu, Y., 39

Lynn, K., x Ma, M., x McConnell, M., 107 Meyer, K., 39 Nugent, B., 39 Ponte, M., 123 Qadri, M., 1 Richards, J., 67 Richards, J., 83 Robert, A., 1 Sharma, A., 95 Wilmot, J., 39 Wong, A., 1 Yang, Q., x Yao, Q., x Yao, C., 39 Zu, C., 51, 83

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Subject Index prothioconazole, RPLC and SFC ionization comparisons, 88f prothioconazole-desthio, RPLC and SFC ionization comparisons, 89f prothioconazole with water and without water, calibration curves, 87f proton donor evaluation, conditions and representative chromatograms, 86f proton donor evaluation and corresponding S/Ns, parameters changed, 87t RPLC and SFC, comparison of prothioconazole-desthio ionization efficiency using optimal mode and polarity, 91f RPLC and SFC, comparison of prothioconazole ionization efficiency using optimal mode and polarity, 90f S/N, RPLC plot at different concentrations, 92f S/N, SFC plot at different concentrations, 92f

A Agrochemical professional, contract research, good laboratory practices, and challenges, 123 agrochemical industry, trends guidelines, GLP, 125 surging toward merging, 126 conclusions, 131 Contract Research Organizations, evolution, history, and market demand, 123 CRO industry, challenges aging workforce, challenge, 129 communication, holy grail, 131 compliance by laboratory discipline, EPA inspection numbers, 127t continuous regulatory change, challenge, 129 diversification, challenge, 130 Eurofins on quality metrics, impact of communication initiatives, 131f flexibility, challenge, 129 GLP, challenge, 126 managing data, 126 new technology, challenge, 130 SD, role, 128 training, compliance, 128

C Chiral analysis of prothioconazole by supercritical-fluid chromatography-mass spectrometry, optimization of critical parameters, 83 conclusion, 93 experimental section MS instrumentation, 86 RPLC conditions and chromatograms, 85f RPLC instrumentation, 85 sample preparation, 85 SFC instrumentation, 85 introduction, 83 RP separation vs SFC separation of two chiral molecules, time difference, 84f results and discussion, 86 linear regression, LOQ values, 92t

I Impurities in synthetic agrochemicals, structure elucidation using mass spectrometry, 51 background, 51 in the pipeline, role of mass spectrometry, 52f case studies chlorpyrifos, 59 chlorpyrifos and chlorpyrifos derivatized with BSTFA, total ion chromatograms, 58f commercial chlorpyrifos sample, GC/MS study of impurities, 57 haloxyfop-P methyl ester, 59 Haloxyfop-P methyl ester, negative ion ESI mass spectrum, 61f haloxyfop-P methyl ester, positive ion ESI mass spectrum, 60f Haloxyfop-P methyl ester, positive ion ESI MS/MS spectrum of [M + H]+ ions, 61f Haloxyfop-P methyl ester, tentative assignment of trace level impurities, 63t

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haloxyfop-P methyl ester, UV chromatogram, 60f Imp 1 showing [M + H]+ and [M + Na]+, positive ion ESI mass spectrum, 62f impurities in a commercial haloxyfop-P methyl ester, LC/MS study, 59 impurity in chlorpyrifos derivatized with BSTFA, EI and PCI-methane mass spectra, 59f [M + H]+ ions of Imp1, positive ion ESI MS/MS spectrum, 62f protonated Haloxyfop-P methyl ester, major fragment ions generated, 61 conclusion, 63 derivatization, 57 alcohols with 2-SBA, derivatization, 57f future improvements, 63 impurity identification, typical workflow, 56 instruments, mass spectrometry analyzers, HRMS, 52 ionization sources, 54 Orbitrap, 53 Orbitrap analyzer, advantages and disadvantages, 54t small-molecule structure elucidation, MS technologies, 55 synthetic AI samples, typical workflow for impurity identification, 56f TOF analyzer, advantages and disadvantages, 53t TOF and quadrupole TOF, 53

macrocyclization, 45 X-YZ macrocycles, macrocyclization methods, 45f molecule stability, improving, 41 CAS-424 and CAS-385, percent disease control against wheat pathogens, 42t esters and stability, macrocycle CAS-381, 41f UK-2A, hydrolyzable protecting group use to increase leaf surface stability, 41f UK-2A and CAS-381, acetoxymethyl ether derivatives, 42f natural product UK-2A, macrocyclic, 39 protectant application timing, UK-2A percent disease control, 40t UK-2A, structure, 40f UK-2A, structure-activity relationship, 41f retrosynthesis strategy, 43 modular construction of X-YZ macrocycles, retrosynthetic strategy, 43f stereotriads, synthesis, 43 olefinic and alcohol stereotriads, interconversion, 44f YZ substituents, early- and late-stage incorporation, 44f summary, 48 synthetic accessibility, maximizing, 42 CAS-385, scaffolds with atom variations at X, Y, and Z positions, 43f

N

M Macrocyclic picolinamides, modular approach, 39 comparisons, biology, 46 macrocycle derivatives, scaffold markush, 46f macrocycle scaffolds, physical attributes and biological activity, 47t construction, modular, 45 X-YZ macrocycle, matching the synthetic inputs, 46f fragments, stitching together, 44 Boc-aziridine carboxylate, addition of alcohol stereotriad under Lewis acidic conditions, 44f X = CH2 bond, formation, 45f

Nonextractable residues in soil, characterization via kinetics modeling, 95 introduction, 95 methodology, 96 nature of nonextractable residue, kinetics model, 97f results and discussion, 97 conversion of cymoxanil to metabolites, NER, and CO2, optimized kinetics, 101f cyhalofop butyl, soil degradation pathway, 102f cymoxanil in soil, degradation pathway, 101f degradation of cyhalofop butyl, optimized kinetics, 103f famoxadone and its metabolite H3310 in soil, optimized kinetics degradation, 104f

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famoxadone in soil, degradation pathway, 104f methomyl metabolites, conversion to NER in soil, 98f oxime, conversion to NER in soil, 99f oxime degradation, kinetics model, 98f pathway, oxamyl degradation, 99f soil, oxamyl degradation, 100f

P Pest management, microbiome of fruit flies as novel targets, 1 concluding remarks, 21 fruit fly management, current status and challenges, 9 insecticide resistance, microbial influence on fruit flies, 12 Drosophila development, microbial effects, 12 microbiome, fruit flies', 10 D. suzukii, microbiomes, 10 novel management strategies for fruit flies, leveraging microbiomes, 14 attractants and repellents, microbial-based, 19 CRISPR, gene editing, 16 insect technique, incompatible, 17 microbiomes, disruption, 15 modifying the insect microbiome, suppression of insect performance, 15 mosquitoes, paratransgenesis, 18 paratransgenesis, 17 pest management strategies, leveraging the microbiome of fruit flies, 15f probiotics, optimization of SITs, 20 small insects of big economic importance, invasive fruit flies, 2 fruit fly species, associated bacteria and fungi, 3t Precision agriculture technology, creating environmentally resilient agriculture landscapes, 107 conservation, incentive-based, 109 conservation adoption, hindrances, 109 conservation delivery, mechanism, 110 discussion, 116 introduction, 108

pesticide mitigation, precision agriculture application, 111 CP-21, Habitat Buffers for Upland Birds, on a grain farm, total eligible area, 114f CP-33, Filter Strips, on a grain farm, total eligible area, 113f production soybean field, profit surface, 112f production soybean field with 30-foot CP33, Habitat Buffers for Upland Birds, profit surface, 115f production soybean field with 120-foot CP33, Habitat Buffers for Upland Birds, profit surface, 115f soybean field, bar graph illustrating economic outcomes of two conservation profitability scenarios, 116f technology, precision agriculture, 110 production soybean field, yield map, 111f Process chemistry and formulations research, separation of chiral molecules, 67 case studies addition of acid to the formulation, racemization inhibited, 80f alkylamide solvent, structural motif, 79f alkylamide solvent and dimethylamine addition to the formulation, loss of enantiopurity, 79f analytical-scale isolation, creative, 73 analytical-scale isolation, modified instrumentation configuration, 75f chiral separation conditions, optimization, 74f Dionex Ultimate 3000 instrument, typical instrument configuration, 75f enantioenriched material, hypothesized racemization pathway, 79f enantioenriched technical and formulation, analysis, 78f enantiomer isolated in high enantiopurity, reanalysis of pooled fractions, 77f enantiomers that require isolation, chiral separation, 74f epoxide isomers after equilibration, chromatograms, 73f formulation components, interference screen, 78f formulation composition, 78t formulations, application, 76 141

how fractions were collected, example, 76f isolation run, minor peak shifting, 76f isomers to be separated, containing enantiomers, mixture, 71f method development using OJ chiral stationary phase, progression of separation, 72f racemic material used to analyze enantioenriched technical, chiral separation, 77f racemic molecule that requires chiral separation method, structural motif, 77f separated into enantiomers, racemic molecule, 73f

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when stationary phase behaves unexpectedly, interesting findings, 71 development of chiral separation methods, workflow, 68 chiral diastereomers and chiral byproducts, mixture, 71f chiral separation method development, progression, 69f chiral separation method development, step-by-step progression, 69 key learnings, 70 introduction, 67 summary, 80