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A Gas Chromatography-Mass Spectrometry (GC-MS) Method for the Detection and Quantitation of Monofluoroacetate in Plants Toxic to Livestock Joyce Santos-Barbosa, Stephen T. Lee, Daniel Cook, Dale R. Gardner, Luis Henrique Viana, and Nilva Ré J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00294 • Publication Date (Web): 28 Jan 2017 Downloaded from http://pubs.acs.org on January 29, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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A Gas Chromatography-Mass Spectrometry (GC-MS) Method for the Detection and Quantitation of Monofluoroacetate in Plants Toxic to Livestock

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Joyce M. Santos-Barbosa†, Stephen T. Lee‡, Daniel Cook‡, Dale R. Gardner‡, Luis Henrique Viana† and Nilva Ré †

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Instituto de Química, Universidade Federal de Mato Grosso do Sul, Avenida Senador Filinto Muller, 1555, 79074-460, Campo Grande, MS, Brazil ‡

Poisonous Plant Research Laboratory, Agricultural Research Service, United States Department of Agriculture, 1150 E. 1400 N., Logan, UT 84341, USA

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

*Corresponding author Tel: (435) 752-2941; Fax: (435) 797-5681; E-mail: [email protected] 1

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ABSTRACT

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Monofluoroacetate (MFA) is a potent toxin that occurs in over 50 plant species in Africa,

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Australia, and South America and is responsible for significant livestock deaths in these regions.

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A gas chromatography–mass spectrometry (GC-MS) method for the analysis of MFA in plants

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based on the derivatization of MFA with n-propanol in the presence of sulfuric acid to form

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propyl fluoroacetate was developed. This method compared favorably to a currently employed

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high performance liquid chromatography-mass spectrometry (HPLC-MS) method for the

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analysis of MFA in plants. The GC-MS method was applied to the analysis of MFA in

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herbarium specimens of Fridericia elegans, Niedenzuella stannea, N. multiglandulosa, N.

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acutifolia, and Aenigmatanthera lasiandra. This is the first report of MFA being detected in F.

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elegans, N. multiglandulosa, N. acutifolia, and A. lasiandra, some of which have been reported

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to cause sudden death or that are toxic to livestock.

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Keywords: monofluoroacetate (MFA); Friderica elegans; Niedenzuella multiglandulosa;

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Niedenzuella acutifolia; Aenigmatanthera lasiandra; gas chromatography-mass spectrometry

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(GC-MS)

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INTRODUCTION

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Monofluoroacetate (MFA) is a naturally occurring toxin found in over 50 plant species

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distributed through the Fabaceae, Rubiaceae, Bignoniaceae, Malpighiaceae and Dichapetalaceae

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families in Africa, Australia, and South America. Several Gastrolobium and Dichapetalum spp.

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within the Fabaceae and Dichapetalaceae families, respectively, have been documented to

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contain MFA.1 The MFA containing Gastrolobium species in Australia and the Dichapetalum

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species in Africa have been responsible for significant livestock deaths in these regions with D.

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cymosum reported as the third most important poisonous plant in South Africa.2,3

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Plants that contain MFA in South America belong to the Rubiaceae, Bignoniaceae, and

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Malpighiaceae families.1 These plants poison livestock causing a disease called “sudden death

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syndrome” which is responsible for half of all cattle deaths in Brazil.4 Livestock poisoned by

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MFA containing plants exhibit loss of balance, ataxia, tachycardia and labored breathing,

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ultimately leading to death shortly after initial clinical signs.5 Palicourea marcgravii, a member

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of the Rubiaceae family, is the most significant poisonous plant in Brazil because of its

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palatability, acute toxicity to livestock, and its wide geographical distribution.4 In addition to P.

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marcgravii, several other Palicourea spp., P. aeneofusca, P. barraensis, P grandiflora, P. aff.

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juruana, and P. longiflora contain MFA,1,5-7 likewise, in the Malpighiaceae family several

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Amorimia spp., A. amazonica, A. exotropica, A. publiflora, A. rigidia, and A. septentrionalis,

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contain MFA and cause “sudden death”.1,4,8 In both these genera other species have been

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reported to contain MFA; however, they have yet to be associated with this syndrome.6,8

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Recently, another species within the Malpighiaceae, N. stannea, was reported to cause “sudden

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death” and contain MFA.9 In the Bignoniaceae, two species (Tanaecium bilabiatum and F. 3

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elegans) are reported to cause the syndrome,1,4 where T. bilabiatum has been reported to contain

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MFA.10 Another Bignoniaceae species, F. japurensis was previously thought to cause “sudden

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death”; however a recent paper demonstrated that the original specimens were misidentified and

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are correctly classified as the MFA-containing plant T. bilabiatum.11

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Several analytical methods have been developed for the analysis of monofluoroacetate in plants

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including: colorimetry,12 19F NMR spectroscopy,10,13 ion chromatography with conductivity

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detection,14 HPLC with UV detection,15 gas chromatography–mass spectrometry (GC-MS) with

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derivatization of MFA with pentafluorobenzyl bromide,16 and, high performance liquid

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chromatography-mass spectrometry (HPLC-MS).8

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Monofluoroacetate in the form as the sodium salt, also known as “Compound 1080” is extremely

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toxic and has been used in many countries as a rodenticide and for the control of predators. The

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wide historical and more regulated current use of sodium monofluoroacetate for the control of

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pests has resulted in the development of various analytical methods for analysis of sodium

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monofluoroacetate in baits, serum, urine, animal tissues and foods. Most recently, HPLC-MS

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methods have been developed in response to threats by environmental activists to contaminate

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milk and infant formulas with sodium monofluoroacetate.17-19 Several gas chromatography (GC)

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methods have been developed for the analysis of sodium monofluoroacetate in these matrices.

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One report describes the GC-MS analysis of residues of sodium monofluoroacetate on sheep

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wool/skin as the free acid, however, the authors describe the persistence of “ghost peaks” in

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blank samples after analysis of MFA containing samples due to adsorption of MFA in the GC

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injection port and at the head of the GC column.20 Because MFA is highly polar and relatively

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non-volatile, successful GC analysis has, in general, required derivatization prior to analysis. 4

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Derivatization prior to final analysis can also add selectivity in that many of the compounds

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extracted from a matrix will not react with the derivatizing agent and thus, will not contribute to

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interferences in the final analysis. Monofluoroacetate has been analyzed in water, beverages,

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baits, animal stomach contents, blood, plasma, serum, urine, animal and human tissues by GC

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after derivatization with a variety of dervatizing agents including: pentafluorobenzyl bromide;21-

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25

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hydroxide;28 diazomethane;29 ethanol;30,31 and ethanol and n-propanol.32

2,4-dichloroaniline;26 1-pyrenyldiazomethane;27 benzyldimethylphenylammonium

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Due to the significance of livestock losses from MFA containing plants around the world and

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because of the availability of GC-MS in many laboratories an uncomplicated, inexpensive, GC-

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MS method for the analysis of MFA in plant samples was developed. Only one report of the

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analysis of plant material for MFA with GC-MS has been reported.16 The method described

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herein is based on the extraction of MFA from the plant matrix, reaction of n-propanol with the

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extracted MFA, and final GC-MS analysis of the propyl fluoroacetate (PFA). Using this method

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herbarium voucher specimens of F. elegans, a plant reported to cause sudden death, were

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analyzed for MFA. In addition, several taxa morphologically and/or phylogenetically related to

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N. stannea,33,34 a species recently reported to contain MFA,9 were analyzed for MFA. We

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suspected that they may contain MFA as some of these taxa cause clinical signs in livestock

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similar to those observed in sub-lethal doses of MFA-containing taxa that cause sudden death.4

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MATERIALS AND METHODS

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Chemical and Reagents. Sodium monofluoroacetate (SMFA) and sodium monochloroacetate

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(SMCA) were purchased from Sigma-Aldrich (St. Louis, MO). N-propanol and anhydrous 5

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sodium sulfate were purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ), sulfuric acid

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was obtained from (Pharmacy Products (Brookfield, CT) and chloroform was purchased from

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Fisher Scientific (Pittsburg, PA). Pure water (18.2 MΩ/cm) was obtained from a WaterPro PS

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Station (Labconco, Kansas City, MO).

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Standard solutions. Calibration standards were prepared in water from SMFA and SMCA.

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Stock solutions of 100 µg/mL of SMFA and SMCA were prepared. Each stock solution was

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diluted resulting in working solutions of 2 µg/mL. All of the stock and working solutions were

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stored at 4 °C. Aliquots of the working solutions were used to prepare the calibration curve for

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SMFA.

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Plant material. Collections of A. septentrionalis were made in the state of Paraíba, Brazil at

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two locations (S7° 09.47 W37° 19.06; elevation 305 m; S7° 12.24 W37° 15.11; elevation 749

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m). Voucher specimens were deposited at the USDA-ARS Poisonous Plant Research Laboratory

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Herbarium.7 Individual A. septentrionalis plants were separated into leaves, stems, and floral

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parts, air dried and ground to pass through a 2 mm screen. After grinding the individual plants

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and plant parts, a portion of the ground samples were combined and mixed thoroughly by

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repeatedly inverting the container for 24 h to create a composite A. septentrionalis sample. P.

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marcgravii leaves collected in Brazil were air dried, ground, and combined as described to create

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a composite P. marcgravii sample. Likewise, G. bilobum was collected near Albany, Western

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Australia, Australia and subsequently air dried, ground, and combined to form a “MFA QC

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Pool” sample. Vegetative and/or floral material was sampled from herbarium specimens from

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the Missouri Botanical Garden of select Bignoniaceae and Malpighiaceae taxa: F. elegans, N.

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stannea, N. multiglandulosa (Tetrapterys multiglandulosa), N. acutifolia (T. acutiflolia), and A. 6

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lasiandra (Mascagnia lasiandra). The Tetrapterys and Mascagnia species sampled have

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undergone a recent taxonomic revision accounting for the new scientific names.33

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Extraction and derivatization reaction. Extraction of plant material for monofluoroacetate

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(MFA) was accomplished by the method of Lee et al.8 Briefly, dry ground plant material (100

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mg) was weighed into a 13 mL screw top test tube equipped with Teflon lined caps. Water

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(deionized, 5 mL) was added to each test tube and placed on a mechanical shaker for 30 min,

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then centrifuged (2570 x g; room temperature) for 5 min to separate the plant residue and water

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extract. The plant residue was extracted once more with water (5 mL) for 30 min, centrifuged,

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and the water extracts combined for a total of 10 mL. For herbarium specimens, ground plant

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material (25 mg) was weighed into a 2 mL screw-cap micro-centrifuge tube. Water (deionized,

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1.5 mL) was added to each test tube and placed in a mechanical shaker for 18 h, then centrifuged

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to separate the plant residue and water extract. A water aliquot (1 mL) was filtered through a

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filter made from a Kimwipe (Kimberly-Clark Corp. Irving, TX) and a disposable pipette and

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subsequently transferred to a clean vial.

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An aliquot (500 µL) of the water extract was transferred to a 5 mL screw top vial with a Teflon

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lined cap. The internal standard SMCA (50 µL of a 2.08 µg/mL solution) was added and the

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extract was then evaporated to dryness under a stream of N2 at 40 °C. Concentrated sulphuric

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acid (30 µL) and n-propanol (70 µL) were added to the 5 mL screw top vial containing the

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evaporated extract reacted for 10 min. As the reaction was taking place, the vial was periodically

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stirred such that the sulfuric acid/n-propanol mixture came in contact with the surface of the vials

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where the residue of the evaporated water extract and SMCA solution remained. After the 10

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min reaction time, water (2 mL) and chloroform (1 mL) were added to the 5 mL vial. The vial 7

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was shaken vigorously and centrifuged (2570 x g; RT) for 5 min. The chloroform was removed

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and filtered, through a filter made by inserting a section of Kimwipe (1 mm x 1 mm) into the top

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of a disposable Pasteur pipette and pushing it to the top of the narrow portion of the pipette and

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adding 0.35 g of anhydrous sodium sulfate, into a clean 5 mL screw top vial. The Kimwipe-

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sodium sulfate filter was washed with chloroform (1 mL) into the 5 mL vial. The filtrate was

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evaporated almost to dryness under stream of N2 at room temperature, and reconstituted with

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chloroform (200 µL).

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To quantitate samples, a calibration curve with a range of 0.024 – 1.0 µg/mL (0.024, 0.039,

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0.079, 0.16, 0.31, 0.47, 0.71 and 1.0 µg/mL) of MFA in water were prepared using SMFA and

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SMCA prepared in the same manner as the samples. The concentrations of MFA in the

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composite P. marcgravii extract were more concentrated than the other samples analyzed and

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was diluted (625X) in water by two serial 1:25 dilutions. The standards/samples and the internal

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standard were analyzed as propyl fluoroacetate (PFA) and the propyl chloroacetate (PCA),

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respectively.

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Spiked samples for recovery analysis were performed at two different levels in both the A.

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septentrionalis composite and the P. marcgravii composite extracts. Ten µL and 20 µL of 2.04

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µg/mL SMFA in water was spiked into four aliquots (0.5 mL) from extracts of 100 mg

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septentrionalis composite and P. marcgravii composite extracts. This resulted in spikes at two

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levels in A. septentrionalis composite (0.0157 µg MFA, n=4 and 0.0314 µg MFA, n=4) and P.

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marcgravii composite (0.0157 µg MFA, n=4 and 0.0314 µg MFA n=3) extracts.

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Instrumental conditions. GC-MS analyses were carried out using a 7890B gas chromatograph

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equipped with a split/splitless injector coupled to a 5977A MSD mass spectrometer and a 7693

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autosampler (Agilent Technologies, Santa Clara, CA) which injected 1 µL of sample.

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Separations were performed using a 30 m x 0.250 mm i.d., DB-5MS column (Agilent

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Technologies, Santa Clara, CA). The carrier gas was helium at a flow of 1 mL/min. The oven

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temperature was 40 °C for 1 min; 40 - 60 °C at 3 °C/min; 60 °C - 250 °C at 30 °C/min; and at

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held 250 °C for 1 min for a total run time of 15 min. Split injection was performed with a 1:1

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split ratio. The transfer line, ion source, and quadrupole temperatures were 275 °C, 230 °C, and

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150 °C, respectively, the electron impact ionization voltage (EI) was 70 eV. The mass

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spectrometer was operated in both full scan and SIM modes with data acquired after a 4 min

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solvent delay. In full scan mode the mass range m/z 30-650 was scanned. In SIM mode m/z 43,

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61, 79, and 91 were monitored from 4.0 – 6.4 min and m/z 49, 77, 79, 95, 97, 107 were

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monitored from 6.4 to 15.0 min. For quantitative analysis, ions with m/z 61, 79 and ions with

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m/z 49, 79, 97, 107 for PFA and PCA, respectively, were extracted and peak areas integrated.

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Under these conditions PFA eluted at 5.76 min and the PCA internal standard eluted at 9.34 min

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(Figures 1 and 2).

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RESULTS AND DISCUSSION

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Experimental rationale. Several analytical methods have been developed for the quantitation

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MFA in plants. Only one GC and/or GC-MS method has been reported for the quantitation of

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MFA in plant tissues.16 This method required esterification of MFA with pentafluorobenzyl

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bromide and final analysis by GC-MS, but only after extensive sample extraction and preparation

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procedures including two, 72 h, pH-dependent liquid-liquid extraction steps to sufficiently 9

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remove contaminants so that no interfering peaks were observed. The pH of the aqueous phase

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used in the liquid-liquid extractions had to be carefully monitored to prevent the sulfuric acid

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used in the extraction procedure from consuming the reaction reagent. Other reports have

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described the analysis of SMFA (1080) in water, blood, plasma, animal stomach contents, animal

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and human tissues by gas chromatography after esterification with methanol using

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diazomethane;29 and by esterification with ethanol30-32 and n-propanol32 in the presence of

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concentrated sulfuric acid. The semi-quantitative method with the esterification of MFA by

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methanol and n-propanol reported by Peterson32 required extensive sample preparation including

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the use of cation and anion exchange resins and distillation of fluoroacetate in the presence of

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concentrated sulfuric acid.32 The methods described by Mori et al.30 and Koryagina et al.31 were

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simple in that the aqueous extract was evaporated to dryness and reacted with ethanol in the

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presence of sulfuric acid.30, 31 However, in both these methods, the ethyl fluoroacetate was

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sampled using head space procedures for final GC analysis. In this study we investigated the

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reaction of short chained alcohols with MFA in the presence of sulfuric acid and were interested

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in developing a method in which an auto-sampler compatible with liquid sample solvents could

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be used.

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Method development. We evaluated esterification of MFA and GC-MS analysis of the

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resulting MFA-esters with ethanol, n-propanol, and n-butanol. We were able to extract the

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resulting alkyl fluoroacetates from the aqueous reaction mixtures into chloroform for final GC-

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MS analysis. Detection of the butyl fluoroacetate derivative was problematic, with interfering

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peaks even after employing different oven temperature hold times and ramp rates. We also

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found that the ethyl fluoroacetate eluted on the tail of the solvent peak. However, the propyl 10

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fluoroacetate (PFA) was resolved from interfering peaks and was retained sufficiently that it was

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well resolved from the solvent peak (Figures 1 and 3). This derivatization scheme was then

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directly applied to evaporated water extracts from MFA containing plants (Figures 1 and 5). The

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EI mass spectra of the PFA and the propyl chloroacetate (PCA) internal standard are shown in

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Figure 2.

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Method validation. For quantitative purposes, ions at m/z 61, 79 for PFA and ions at m/z 49,

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79, 97, 107 for PCA were extracted and peak areas integrated. A linear calibration curve

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(R2=0.9944) over the range of 0.024 – 1.0 µg/mL µg/mL MFA in water with SMCA as an

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internal standard was used for quantitation. It should be noted that sodium fluoroacetate 13C2

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isotope is a superior choice for an internal standard and is commercially available, however, it

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was cost prohibitive for this application. The limit of detection (LOD, 3X S/N), and limit of

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quantitation (LOQ, 10X S/N) of MFA were determined to be 0.0079 µg/mL and 0.024 µg/mL,

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respectively (Figure 3). Additionally, recoveries of monofluoroacetate were determined at two

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different levels of MFA in both the A. septentrionalis composite and P. marcgravii composite

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samples. Recoveries of MFA ranged from 89.8-113% at both levels and in both of the different

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plants (Table 1). Intra-day and inter-day variation were also determined by the analysis the A.

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septentrionalis composite over 1 day (n=8) and seven days (n=7), respectively. The intra-day

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variation was determined to be 2.2% and the inter-day variation over 7 days was 4.6%.

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Samples of Gastrolobium and Amorimia spp. were compared using different methods within and

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between laboratories. A Gastrolobium bilobum composite sample labeled “MFA QC Pool” was

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prepared and analyzed by scientists at the Department of Agriculture and Food, Western 11

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Australia, South Perth, Western Australia (DAFWA) using the HPLC-MS method of Lee et al.7

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and by S. T. Lee at the Poisonous Plant Laboratory in Logan, Utah by the same method. (Table

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2). This G. bilobum “MFA QC Pool” sample was analyzed by the GC-MS method described in

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this report. Comparison of the results in Table 2 between the HPLC-MS method run at

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laboratories in Australia and in the U.S.A. and with the GC-MS method reported herein are

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acceptable. Additionally, thirteen individual samples of A. septentrionalis plant parts consisting

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of stem, leaves, and floral plant parts previously analyzed using the HPLC-MS method described

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by Lee et al.7 were also analyzed using the GC-MS method described. A good correlation

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described by the equation y = 0.86x -2.6 (R2 = 0.993) was observed between results using the

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HPLC-MS and GC-MS methods. This comparison supports the specificity and robustness of the

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developed method.

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Detection of MFA herbarium samples. Herbarium specimens have been used previously to

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detect MFA in Palicourea and Amorimia species.6,8 Using this method we analyzed herbarium

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specimens of several taxa reported or suspected to contain MFA. MFA was detected in all six of

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the F. elegans specimens analyzed using the GC-MS method in SIM mode. MFA was also

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detected in 8-25% of the N. stannea, N. multiglandulosa, N. acutifolia, and A. lasiandra

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specimens analyzed (Table 3). The detection frequency of MFA in these specimens was similar

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to those reported for Amorimia spp.8 MFA was detected in F. elegans and N. stannea, consistent

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with the reports that these species cause sudden death.1,4,9 In addition, MFA was detected in N.

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multiglandulosa and N. acutifolia, two species that are reported to cause sub-acute clinical signs

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similar to other MFA containing plants that cause sudden death.4 Lastly, MFA was detected in

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A. lasiandra, a plant phylogenetically related to the Niedenzuella species investigated and not yet 12

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reported to be toxic.34 Caution should be taken with respect to the MFA concentrations reported

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from herbarium specimens. The herbarium concentration values reported may be biased due to a

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number of factors such as limited sample size, variability in the age of the herbarium specimens,

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and possible differences in the storage conditions etc.

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In conclusion, an inexpensive, straight forward extraction, derivatization, sample preparation,

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GC-MS method for the analysis of MFA in plants was developed. The method compared

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favorably to a currently employed HPLC-MS method for the analysis of MFA in plants. This

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method was applied to the analysis of MFA in herbarium specimens and MFA was detected in F.

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elegans, N. stannea, N. multiglandulosa, N. acutifolia, and Aenigmatanthera lasiandra. This is

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the first report of MFA being detected in F. elegans, N. multiglandulosa, N. acutifolia, and

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Aenigmatanthera lasiandra. It is anticipated that the method is sufficiently simple for routine

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screening of suspected MFA-containing plants in Brazil and in other parts of the world where

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this toxin is a significant cause of livestock deaths.

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SUPPORTING INFORMATION

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List of plant taxa, voucher specimen numbers, and associated herbaria. This material is available

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free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGEMENTS

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We thank FUNDECT and CAPES for financial support of J. M. Santos-Barbosa. We thank

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Gerard Smith, Department of Agriculture and Food, Western Australia, South Perth, Western

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Australia, Australia (DAFWA) for the HPLC-MS analysis of the G. bilobum “MFA QC Pool”

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sample. We thank the Herbarium at the Missouri Botanical Gardens for providing material from

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select taxa for analysis.

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REFERENCES

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1. Lee, S. T.; Cook. D.; Pfister, J. A.; Allen, J. G.; Colegate, S. M.; Riet-Correa, F.; Taylor, C.

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M. Monofluoroacetate-containing plants that are potentially toxic to livestock. J. Agric.

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Food Chem. 2014, 62, 7345-7354.

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2. Aplin, T. H. E. Poison plants of Western Australia: the toxic species of Gastrolobium and Oxylobium. Western Australian Department of Agriculture Bulletin 3772; 1971.

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3. Kellerman, T. S.; Coetzer, J. A.W.; Naude, T. W.; Botha, C. J. Cardiovascular system. In

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Plant Poisonings and Mycotoxicoses of Livestock in Southern Africa, 2nd ed.; Oxford

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University Press, Cape Town, South Africa, 2005; pp. 146-153.

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4. Tokarnia, C. H.; Döbereiner, J.; Peixoto, P. V. Poisonous plants affecting livestock in Brazil. Toxicon 2002, 40, 1635-1660. 5. Riet-Correa, F.; Medeiros, R. M. T.; Pfister, J.; Schild, A. L.; Dantas, A. F. M.. Poisonings

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by Plants, Mycotoxins and Related Substances in Brazilian Livestock; Sociedade Vicente

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Pallotti: Patos, Brazil, 2009; pp 15–24.

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6. Cook, D.; Lee, S. T.; Taylor, C. M.; Bassuner, B.; Riet-Correa, F.; Pfister, Gardner, D. R.

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Detection of toxic monofluoroacetate in Palicourea species. Toxicon 2014, 80, 9-16.

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7. Carvalho, F. K. L.; Cook, D.; Lee, S. T.; Taylor, C. M.; Oliveira, J. B. S.; Riet-Correa, F.

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Determination of toxicity in rabbits and corresponding detection of monofluoroacetate in

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four Palicourea (Rubiaceae) species from the Amazonas State, Brazil. Toxicon 2016, 109,

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42-44.

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8. Lee, S. T.; Cook, D.; Riet-Correa, F.; Pfister, J.; Anderson, W. R.; Lima, F. G.; Gardner, D.

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R. Detection of monofluoroacetate in Palicourea and Amorimia species. Toxicon 2012, 60,

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791-796. 15

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9. Arruda, F. P.; Caldeira, F. H. P.; Ducatti, K. R.; Bezerra, K. S.; Marcolongo-Pereira, C.; Lee,

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S. T.; Cook, D.; Riet-Correa, F.; Colodel, E. M. Experimental poisoning in Niedenzuella

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stannea (Malpighiaceae) in cattle and corresponding detection of monofluoroacetate. Cienc.

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Rural 2017, 47, 1-6.

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10. Krebs, H. C.; Kemmerling, W.; Habermehl, G. Qualitative and quantitative determination of

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fluoroacetic acid in Arrabidea bilabiata and Palicourea marcgravii by 19F-NMR

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spectroscopy. Toxicon 1994, 32, 909-913.

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11. Lima, E. F.; Medeiros, R. M. T.; Cook, D.; Lee, S. T.; Kaehler, M.; Barbosa, J.; Riet-Correa,

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F. Studies in regard to the classification and putative toxicity of Fridericia japurensis

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(Arrabidaea japurensis) in Brazil. Toxicon 2016, 115, 22-27.

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12. Vickery, B.; Vickery, M. L.; Ashu, J. T. Analysis of plant for fluoroacetic acids. Phytochemistry 1973, 12, 145-147. 13. Baron, M. L.; Bothroyd, C. M.; Rogers, G. I.; Staffa, A.; Rae, I. D. Detection and

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measurement of fluoroacetate in plant extracts by 19F NMR. Phytochemistry 1987, 26, 2293-

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2295.

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14. O’Hagan, D. O.; Perry, R.; Lock, J. M.; Meyer, J. J. M.; Dasaradi, L.; Hamilton, J. T. G.;

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Harper, D. B. High levels of monofluoroacetate in Dichapetalum braunii. Phytochemistry

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1993, 33, 1043-1045.

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15. Minnaar, P. P.; Swan, G. E.; McCrindle, R. I.; de Beer, W. H. J.; Naude, T. W. A high-

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performance liquid chromatographic method for the determination of monofluoroacetate. J.

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Chromatogr. Sci. 2000, 38, 16-20.

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16. Vartiainen, T.; Kauranen, P. The determination of traces of fluoroacetic acid by extractive

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alkylation, pentafluorobenzylation and capillary gas chromatography-mass spectrometry.

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Anal. Chim. Acta 1984, 157, 91-97.

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17. Joseph, G. Determination of sodium monofluoroacetate in dairy powders by liquid

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chromatography tandem mass spectrometry (LC-MS/MS): First Action 2015.02. J. AOAC

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Int. 2015, 98, 1121-1126.

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18. Bessaire, T.; Tarres, A.; Goyon, A.; Mottier, P.; Dubois, M.; Tan, W. P.; Delatour, T.

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Quantitative determination of sodium monofluoroacetate “1080” in infant formulas and dairy

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products by isotope dilution LC-MS/MS. Food Addit. Contam., Part A. 2015, 32, 1815-

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1892.

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19. Cooney, T. P.; Varelis, P.; Bendall, J. G. High-throughput quantification of

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monofluoroacetate (1080) in milk as a response to extortion threat. J. Food Prot. 2016, 79,

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273-281.

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20. Kimball, B. A.; Mishalanie, E. A. Gas chromatographic determination of sodium

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monofluoroacetate as the free acid in an aqueous solvent. J. Chromatogr. 1993, 634, 289-

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21. Okuno, I.; Meeker, D. L.; Felton, R. R. Modified gas-liquid chromatographic method for the

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determination of Compound 1080 (sodium fluoroacetate). J. Assoc. Off. Anal. Chem. 1982,

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65, 1102-1105.

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22. Okuno, I.; Connolly, G. E.; Savarie, P. J.; Breidenstein, C. P. Gas chromatographic analysis

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of coyote and magpie tissue for residues of Compound 1080 (sodium fluoroacetate). J. -

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Assoc. Off. Anal. Chem. 1984, 67, 549-553.

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23. Casper, H. H.; McMahon, T. L.; Paulson, G. D. Capillary gas chromatographic-mass

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spectrometric determination of fluoroacetate residues in animal tissues. J. - Assoc. Off. Anal.

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Chem. 1985, 68, 722-725.

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24. Allender, W. J. Determination of sodium fluoroacetate (compound 1080) in biological tissues. J. Anal. Toxicol. 1990, 14, 45-49.

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25. Miki, A.; Tsuchihashi, H.; Yamashita, M. GC and GC-MS determination of fluoroacetic acid

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and phenoxy acid herbicides via triphasal extractive pentafluorobenzylation using a polymer-

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bound phase-transfer catalyst. J. Anal. Toxicol. 1998, 22, 237-245.

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26. Demarchi, A.C.; Menezer, M. L.; Mercadant, E, A.; Vassillief, I. Determination of the

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sodium monofluoroacetate in serum by gas chromatography. Chromatographia 2001, 27,

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402-404.

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27. Sporkert, F.; Pragst, F.; Hubner, S.; Mills, G. Headspace solid-phase microextration with 1-

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pyrenyldiazomethane on-fibre derivatisation for analysis of fluoroacetic acid in biological

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samples. J. Chromatogr. B: Biomed. Sci. Appl. 2002, 772, 45-51.

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28. Hoogenboom J. L.; Rammell, C. G. Determination of determination of sodium

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monofluoroacetate (Compound 1080) in tissues and baits as its benzyl ester by reaction-

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capillary gas chromatography. J. Anal. Toxicol. 1987, 11, 140-143.

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29. Stevens, H. M.; Moffat, A.C.; Drayton, J.V. The recovery and identification of

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fluoroacetamide and fluoroacetic acid from tissues. Forensic Sci. 1976, 8, 131-137.

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30. Mori, M.; Kakajima, H.; Seto, Y. Determination of fluoroacetate in aqueous samples by

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headspace gas chromatography. J. Chromatogr. A 1996, 736, 229-234. 31. Koryagina, N. L.; Savelieva, E. I.; Khlebnikova, N. S.; Goncharov, N. V.; Jenkins, R. O.; Radilov, E. S. Determination of fluoroacetic acid in water and biological samples by GC-FID 18

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and GC-MS in combination with solid-phase microextration. Anal. Bioanal. Chem. 2006,

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386, 1395-1400.

383 384 385 386 387 388

32. Peterson, J. E. A gas chromatographic method for sodium fluoroacetate (Compound 1080) in biological materials. Bull. Environ. Contam. Toxicol. 1975, 15, 751-757. 33. Anderson, W.R. Eight segregates from the neotropical genus Mascagnia (Malpighiaceae). Novon 2006, 16, 168-204. 34. Davis, C.C., Anderson, W. R. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. Am. J. Bot. 2010, 97, 2031-2048.

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FIGURE CAPTIONS

391 392

Figure 1. Reconstructed GC-MS ion chromatograms of (m/z 61.0, 79.0) from (A) water extract of

393

A. septentrionalis composite sample and (B) water extract of P. marcgravii composite

394

sample diluted 1:625 v:v. * denotes MFA peak (detected as propyl fluoroacetate). IS

395

denotes internal standard peak.

396

Figure 2. Full scan mass spectrum of (A) propyl fluoracetate and (B) propyl chloroacetate. *

397

denotes m/z used for qualitative determination of peak identity. ** denotes m/z used

398

for quantitative analysis.

399

Figure 3. Reconstructed GC-MS ion chromatograms of (m/z 61.0, 79.0) from (A) a blank

400

reaction (B) a 0.0079 µg/mL MFA standard (LOD) and (C) a 0.024 µg/mL MFA

401

standard (LOQ). * denotes MFA peak as the propyl fluoroacetate derivative.

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Table 1. Recoveries Obtained for Extracts of A. septentrionalis and P. marcgravii Spiked at Two Levels of MFA. MFA Spike (µg) Samples Mean Recovery (µg) RSD (%) Recovery (%)

0.0157 A. septentrionalis P. marcgravii n=4 n=4 0.0141 ± 0.0005 0.0168 ± 0.0019 3.54 11.3 89.8 107

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0.0314 A. septentrionalis P. marcgravii n=4 n=3 0.0324 ± 0.0023 0.0354 ± 0.0020 7.10 5.65 103 113

Journal of Agricultural and Food Chemistry

Table 2. Comparison of Analysis of G. bilobum Composite MFA QC Pool Sample Between Different Laboratories and Different Analysis Methods. Laboratory DAFWA n = 10 PPRL n = 4 PPRL n = 4

Method HPLC-MS HPLC-MS GC/MS

MFA ± STDV (µg/g) 50 ± 5 60 ± 3 47 ± 2

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Table 3. GC-MS Analysis of Herbarium Specimens Representing Different Plant Taxa Reported or Suspected of Containing MFA. Number of Specimens

Specimens Containing MFA

MFA Conc. Range (µg/g)

Fridericia elegans

6

6

0.31 – 1.3

Malpighiaceae

Aenigmatanthera lasiandra

8

2

nd – 5.2

Malpighiaceae

Niedenzuella acutifolia

8

1

nd – 0.21

Malpighiaceae

Niedenzuella multiglandulosa

8

1

nd – 1.7

Malpighiaceae

Niedenzuella stannea

13

1

nd – 1.4

Family

Scientific Name

Bignoniaceae

23

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Figure 1

A

6

IS

5 4 3

*

Counts x104

2 1 0 6

B

IS

5 4 3 2

*

1 0

4

5

6

7

8

9

Time (min)

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11

12

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Figure 2

1.1 1.0

** 61.0

A

* 43.0

31.0

0.8

32.0

42.1 41.1

** 79.0

0.6

Counts x104

0.4

33.0 36.0

59.1 57.0

83.0

0.2 0 2.5

*

91.0

*

B

77.0 43.1

2.0 1.5

0

57.1

**

32.0

71.1 75.0

49.0

0.5 30

34

38

42

46

50

*

95.0

** 79.0

42.1

1.0

54

58

62

66

70

74

** 97.0

89.1

78

82

86

(m/z)

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94

98

**

107.0

117.1 119.0

102 106 110 114 118

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Figure 3

4 3

A

2

Counts x103

1 0 4 3

B

2 1 0 4 3

* C

2

*

1 0

5.0

5.5

6.0

6.5

Time (min)

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7.0

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O

4

*

2 0

Counts x104

O

6 F

Counts x104

TOC Graphic

4

1.2

5

6

7 61.0

43.0

0.8

8 Time (min)

9

10

Time (min) 79.0

91.0

0.4 0

30

40

50

60

70 80 (m/z)

90

100

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