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

Article abstract of DOI:10.1021/acs.jafc.7b00294

Monofluoroacetate (MFA) is a potent toxin that occurs in over 50 plant species in Africa, Australia, and South America and is responsible for significant livestock deaths in these regions. A gas chromatography-mass spectrometry (GC-MS) method for the analysis of MFA in plants based on the derivatization of MFA with n-propanol in the presence of sulfuric acid to form propyl fluoroacetate was developed. This method compared favorably to a currently employed high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for the analysis of MFA in plants. The GC-MS method was applied to the analysis of MFA in herbarium specimens of Fridericia elegans, Niedenzuella stannea, N. multiglandulosa, N. acutifolia, and Aenigmatanthera lasiandra. This is the first report of MFA being detected in F. elegans, N. multiglandulosa, N. acutifolia, and A. lasiandra, some of which have been reported to cause sudden death or that are toxic to livestock.

Full text of DOI:10.1021/acs.jafc.7b00294

Subscriber access provided by University of Newcastle, Australia
Article
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
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
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.

Page 1 of 27
Journal of Agricultural and Food Chemistry
1
2
A Gas Chromatography-Mass Spectrometry (GC-MS) Method for the Detection and
Quantitation of Monofluoroacetate in Plants Toxic to Livestock
3
4
Joyce M. SantosꢀBarbosa†, Stephen T. Lee‡, Daniel Cook‡ Dale R. Gardner‡, Luis Henrique
,
Viana† and Nilva Ré †
5
6
7
8
†Instituto de Química, Universidade Federal de Mato Grosso do Sul, Avenida Senador Filinto
Muller, 1555, 79074ꢀ460, Campo Grande, MS, Brazil
9
10
11
‡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) 752ꢀ2941; Fax: (435) 797ꢀ5681; Eꢀmail:
stephen.lee@ars.usda.gov
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 27
30
ABSTRACT
31
32
33
34
35
36
37
38
39
40
41
Monofluoroacetate (MFA) is a potent toxin that occurs in over 50 plant species in Africa,
Australia, and South America and is responsible for significant livestock deaths in these regions.
A gas chromatography–mass spectrometry (GCꢀMS) method for the analysis of MFA in plants
based on the derivatization of MFA with nꢀpropanol in the presence of sulfuric acid to form
propyl fluoroacetate was developed. This method compared favorably to a currently employed
high performance liquid chromatographyꢀmass spectrometry (HPLCꢀMS) method for the
analysis of MFA in plants. The GCꢀMS method was applied to the analysis of MFA in
herbarium specimens of Fridericia elegans, Niedenzuella stannea, N. multiglandulosa, N.
acutifolia, and Aenigmatanthera lasiandra. This is the first report of MFA being detected in F.
elegans, N. multiglandulosa, N. acutifolia, and A. lasiandra, some of which have been reported
to cause sudden death or that are toxic to livestock.
42
43
44
45
46
Keywords: monofluoroacetate (MFA); Friderica elegans; Niedenzuella multiglandulosa;
Niedenzuella acutifolia; Aenigmatanthera lasiandra; gas chromatographyꢀmass spectrometry
(GCꢀMS)
47
48
2
ACS Paragon Plus Environment

Page 3 of 27
Journal of Agricultural and Food Chemistry
49
INTRODUCTION
50
51
52
53
54
55
56
Monofluoroacetate (MFA) is a naturally occurring toxin found in over 50 plant species
distributed through the Fabaceae, Rubiaceae, Bignoniaceae, Malpighiaceae and Dichapetalaceae
families in Africa, Australia, and South America. Several Gastrolobium and Dichapetalum spp.
within the Fabaceae and Dichapetalaceae families, respectively, have been documented to
contain MFA.1 The MFA containing Gastrolobium species in Australia and the Dichapetalum
species in Africa have been responsible for significant livestock deaths in these regions with D.
cymosum reported as the third most important poisonous plant in South Africa.2,3
57
58
59
60
61
62
63
64
65
66
67
68
69
70
Plants that contain MFA in South America belong to the Rubiaceae, Bignoniaceae, and
Malpighiaceae families.1 These plants poison livestock causing a disease called “sudden death
syndrome” which is responsible for half of all cattle deaths in Brazil.4 Livestock poisoned by
MFA containing plants exhibit loss of balance, ataxia, tachycardia and labored breathing,
ultimately leading to death shortly after initial clinical signs.5 Palicourea marcgravii, a member
of the Rubiaceae family, is the most significant poisonous plant in Brazil because of its
palatability, acute toxicity to livestock, and its wide geographical distribution.4 In addition to P.
marcgravii, several other Palicourea spp., P. aeneofusca, P. barraensis, P grandiflora, P. aff.
juruana, and P. longiflora contain MFA,1,5ꢀ7 likewise, in the Malpighiaceae family several
Amorimia spp., A. amazonica, A. exotropica, A. publiflora, A. rigidia, and A. septentrionalis,
contain MFA and cause “sudden death”.1,4,8 In both these genera other species have been
reported to contain MFA; however, they have yet to be associated with this syndrome.6,8
Recently, another species within the Malpighiaceae, N. stannea, was reported to cause “sudden
death” and contain MFA.9 In the Bignoniaceae, two species (Tanaecium bilabiatum and F.
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 27
71
72
73
74
elegans) are reported to cause the syndrome,1,4 where T. bilabiatum has been reported to contain
MFA.10 Another Bignoniaceae species, F. japurensis was previously thought to cause “sudden
death”; however a recent paper demonstrated that the original specimens were misidentified and
are correctly classified as the MFAꢀcontaining plant T. bilabiatum.11
75
76
77
78
79
Several analytical methods have been developed for the analysis of monofluoroacetate in plants
including: colorimetry,12 19F NMR spectroscopy,10,13 ion chromatography with conductivity
detection,14 HPLC with UV detection,15 gas chromatography–mass spectrometry (GCꢀMS) with
derivatization of MFA with pentafluorobenzyl bromide,16 and, high performance liquid
chromatographyꢀmass spectrometry (HPLCꢀMS).8
80
81
82
83
84
85
86
87
88
89
90
91
92
Monofluoroacetate in the form as the sodium salt, also known as “Compound 1080” is extremely
toxic and has been used in many countries as a rodenticide and for the control of predators. The
wide historical and more regulated current use of sodium monofluoroacetate for the control of
pests has resulted in the development of various analytical methods for analysis of sodium
monofluoroacetate in baits, serum, urine, animal tissues and foods. Most recently, HPLCꢀMS
methods have been developed in response to threats by environmental activists to contaminate
milk and infant formulas with sodium monofluoroacetate.17ꢀ19 Several gas chromatography (GC)
methods have been developed for the analysis of sodium monofluoroacetate in these matrices.
One report describes the GCꢀMS analysis of residues of sodium monofluoroacetate on sheep
wool/skin as the free acid, however, the authors describe the persistence of “ghost peaks” in
blank samples after analysis of MFA containing samples due to adsorption of MFA in the GC
injection port and at the head of the GC column.20 Because MFA is highly polar and relatively
nonꢀvolatile, successful GC analysis has, in general, required derivatization prior to analysis.
4
ACS Paragon Plus Environment

Page 5 of 27
Journal of Agricultural and Food Chemistry
93
94
95
96
97
98
99
Derivatization prior to final analysis can also add selectivity in that many of the compounds
extracted from a matrix will not react with the derivatizing agent and thus, will not contribute to
interferences in the final analysis. Monofluoroacetate has been analyzed in water, beverages,
baits, animal stomach contents, blood, plasma, serum, urine, animal and human tissues by GC
after derivatization with a variety of dervatizing agents including: pentafluorobenzyl bromide;21ꢀ
25 2,4ꢀdichloroaniline;26 1ꢀpyrenyldiazomethane;27 benzyldimethylphenylammonium
hydroxide;28 diazomethane;29 ethanol;30,31 and ethanol and nꢀpropanol.32
100
101
102
103
104
105
106
107
108
109
110
Due to the significance of livestock losses from MFA containing plants around the world and
because of the availability of GCꢀMS in many laboratories an uncomplicated, inexpensive, GCꢀ
MS method for the analysis of MFA in plant samples was developed. Only one report of the
analysis of plant material for MFA with GCꢀMS has been reported.16 The method described
herein is based on the extraction of MFA from the plant matrix, reaction of nꢀpropanol with the
extracted MFA, and final GCꢀMS analysis of the propyl fluoroacetate (PFA). Using this method
herbarium voucher specimens of F. elegans, a plant reported to cause sudden death, were
analyzed for MFA. In addition, several taxa morphologically and/or phylogenetically related to
N. stannea,33,34 a species recently reported to contain MFA,9 were analyzed for MFA. We
suspected that they may contain MFA as some of these taxa cause clinical signs in livestock
similar to those observed in subꢀlethal doses of MFAꢀcontaining taxa that cause sudden death.4
111
MATERIALS AND METHODS
112
113
Chemical and Reagents. Sodium monofluoroacetate (SMFA) and sodium monochloroacetate
(SMCA) were purchased from SigmaꢀAldrich (St. Louis, MO). Nꢀpropanol and anhydrous
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 27
114
115
116
117
sodium sulfate were purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ), sulfuric acid
was obtained from (Pharmacy Products (Brookfield, CT) and chloroform was purchased from
Fisher Scientific (Pittsburg, PA). Pure water (18.2 MΩ/cm) was obtained from a WaterPro PS
Station (Labconco, Kansas City, MO).
118
119
120
121
122
Standard solutions. Calibration standards were prepared in water from SMFA and SMCA.
Stock solutions of 100 µg/mL of SMFA and SMCA were prepared. Each stock solution was
diluted resulting in working solutions of 2 µg/mL. All of the stock and working solutions were
stored at 4 °C. Aliquots of the working solutions were used to prepare the calibration curve for
SMFA.
123
124
125
126
127
128
129
130
131
132
133
134
135
Plant material. Collections of A. septentrionalis were made in the state of Paraíba, Brazil at
two locations (S7° 09.47 W37° 19.06; elevation 305 m; S7° 12.24 W37° 15.11; elevation 749
m). Voucher specimens were deposited at the USDAꢀARS Poisonous Plant Research Laboratory
Herbarium.7 Individual A. septentrionalis plants were separated into leaves, stems, and floral
parts, air dried and ground to pass through a 2 mm screen. After grinding the individual plants
and plant parts, a portion of the ground samples were combined and mixed thoroughly by
repeatedly inverting the container for 24 h to create a composite A. septentrionalis sample. P.
marcgravii leaves collected in Brazil were air dried, ground, and combined as described to create
a composite P. marcgravii sample. Likewise, G. bilobum was collected near Albany, Western
Australia, Australia and subsequently air dried, ground, and combined to form a “MFA QC
Pool” sample. Vegetative and/or floral material was sampled from herbarium specimens from
the Missouri Botanical Garden of select Bignoniaceae and Malpighiaceae taxa: F. elegans, N.
stannea, N. multiglandulosa (Tetrapterys multiglandulosa), N. acutifolia (T. acutiflolia), and A.
6
ACS Paragon Plus Environment

Page 7 of 27
Journal of Agricultural and Food Chemistry
136
137
lasiandra (Mascagnia lasiandra). The Tetrapterys and Mascagnia species sampled have
undergone a recent taxonomic revision accounting for the new scientific names.33
138
139
140
141
142
143
144
145
146
147
148
149
Extraction and derivatization reaction. Extraction of plant material for monofluoroacetate
(MFA) was accomplished by the method of Lee et al.8 Briefly, dry ground plant material (100
mg) was weighed into a 13 mL screw top test tube equipped with Teflon lined caps. Water
(deionized, 5 mL) was added to each test tube and placed on a mechanical shaker for 30 min,
then centrifuged (2570 x g; room temperature) for 5 min to separate the plant residue and water
extract. The plant residue was extracted once more with water (5 mL) for 30 min, centrifuged,
and the water extracts combined for a total of 10 mL. For herbarium specimens, ground plant
material (25 mg) was weighed into a 2 mL screwꢀcap microꢀcentrifuge tube. Water (deionized,
1.5 mL) was added to each test tube and placed in a mechanical shaker for 18 h, then centrifuged
to separate the plant residue and water extract. A water aliquot (1 mL) was filtered through a
filter made from a Kimwipe (KimberlyꢀClark Corp. Irving, TX) and a disposable pipette and
subsequently transferred to a clean vial.
150
151
152
153
154
155
156
157
An aliquot (500 µL) of the water extract was transferred to a 5 mL screw top vial with a Teflon
lined cap. The internal standard SMCA (50 µL of a 2.08 µg/mL solution) was added and the
extract was then evaporated to dryness under a stream of N2 at 40 °C. Concentrated sulphuric
acid (30 µL) and nꢀpropanol (70 µL) were added to the 5 mL screw top vial containing the
evaporated extract reacted for 10 min. As the reaction was taking place, the vial was periodically
stirred such that the sulfuric acid/nꢀpropanol mixture came in contact with the surface of the vials
where the residue of the evaporated water extract and SMCA solution remained. After the 10
min reaction time, water (2 mL) and chloroform (1 mL) were added to the 5 mL vial. The vial
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 27
158
159
160
161
162
163
164
was shaken vigorously and centrifuged (2570 x g; RT) for 5 min. The chloroform was removed
and filtered, through a filter made by inserting a section of Kimwipe (1 mm x 1 mm) into the top
of a disposable Pasteur pipette and pushing it to the top of the narrow portion of the pipette and
adding 0.35 g of anhydrous sodium sulfate, into a clean 5 mL screw top vial. The Kimwipeꢀ
sodium sulfate filter was washed with chloroform (1 mL) into the 5 mL vial. The filtrate was
evaporated almost to dryness under stream of N2 at room temperature, and reconstituted with
chloroform (200 µL).
165
166
167
168
169
170
171
To quantitate samples, a calibration curve with a range of 0.024 – 1.0 µg/mL (0.024, 0.039,
0.079, 0.16, 0.31, 0.47, 0.71 and 1.0 µg/mL) of MFA in water were prepared using SMFA and
SMCA prepared in the same manner as the samples. The concentrations of MFA in the
composite P. marcgravii extract were more concentrated than the other samples analyzed and
was diluted (625X) in water by two serial 1:25 dilutions. The standards/samples and the internal
standard were analyzed as propyl fluoroacetate (PFA) and the propyl chloroacetate (PCA),
respectively.
172
173
174
175
176
177
Spiked samples for recovery analysis were performed at two different levels in both the A.
septentrionalis composite and the P. marcgravii composite extracts. Ten µL and 20 µL of 2.04
µg/mL SMFA in water was spiked into four aliquots (0.5 mL) from extracts of 100 mg
septentrionalis composite and P. marcgravii composite extracts. This resulted in spikes at two
levels in A. septentrionalis composite (0.0157 µg MFA, n=4 and 0.0314 µg MFA, n=4) and P.
marcgravii composite (0.0157 µg MFA, n=4 and 0.0314 µg MFA n=3) extracts.
8
ACS Paragon Plus Environment

Page 9 of 27
Journal of Agricultural and Food Chemistry
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
Instrumental conditions. GCꢀMS analyses were carried out using a 7890B gas chromatograph
equipped with a split/splitless injector coupled to a 5977A MSD mass spectrometer and a 7693
autosampler (Agilent Technologies, Santa Clara, CA) which injected 1 µL of sample.
Separations were performed using a 30 m x 0.250 mm i.d., DBꢀ5MS column (Agilent
Technologies, Santa Clara, CA). The carrier gas was helium at a flow of 1 mL/min. The oven
temperature was 40 °C for 1 min; 40 ꢀ 60 °C at 3 °C/min; 60 °C ꢀ 250 °C at 30 °C/min; and at
held 250 °C for 1 min for a total run time of 15 min. Split injection was performed with a 1:1
split ratio. The transfer line, ion source, and quadrupole temperatures were 275 °C, 230 °C, and
150 °C, respectively, the electron impact ionization voltage (EI) was 70 eV. The mass
spectrometer was operated in both full scan and SIM modes with data acquired after a 4 min
solvent delay. In full scan mode the mass range m/z 30ꢀ650 was scanned. In SIM mode m/z 43,
61, 79, and 91 were monitored from 4.0 – 6.4 min and m/z 49, 77, 79, 95, 97, 107 were
monitored from 6.4 to 15.0 min. For quantitative analysis, ions with m/z 61, 79 and ions with
m/z 49, 79, 97, 107 for PFA and PCA, respectively, were extracted and peak areas integrated.
Under these conditions PFA eluted at 5.76 min and the PCA internal standard eluted at 9.34 min
(Figures 1 and 2).
194
RESULTS AND DISCUSSION
195
196
197
198
199
Experimental rationale. Several analytical methods have been developed for the quantitation
MFA in plants. Only one GC and/or GCꢀMS method has been reported for the quantitation of
MFA in plant tissues.16 This method required esterification of MFA with pentafluorobenzyl
bromide and final analysis by GCꢀMS, but only after extensive sample extraction and preparation
procedures including two, 72 h, pHꢀdependent liquidꢀliquid extraction steps to sufficiently
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 27
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
remove contaminants so that no interfering peaks were observed. The pH of the aqueous phase
used in the liquidꢀliquid extractions had to be carefully monitored to prevent the sulfuric acid
used in the extraction procedure from consuming the reaction reagent. Other reports have
described the analysis of SMFA (1080) in water, blood, plasma, animal stomach contents, animal
and human tissues by gas chromatography after esterification with methanol using
diazomethane;29 and by esterification with ethanol30ꢀ32 and nꢀpropanol32 in the presence of
concentrated sulfuric acid. The semiꢀquantitative method with the esterification of MFA by
methanol and nꢀpropanol reported by Peterson32 required extensive sample preparation including
the use of cation and anion exchange resins and distillation of fluoroacetate in the presence of
concentrated sulfuric acid.32 The methods described by Mori et al.30 and Koryagina et al.31 were
simple in that the aqueous extract was evaporated to dryness and reacted with ethanol in the
presence of sulfuric acid.30, 31 However, in both these methods, the ethyl fluoroacetate was
sampled using head space procedures for final GC analysis. In this study we investigated the
reaction of short chained alcohols with MFA in the presence of sulfuric acid and were interested
in developing a method in which an autoꢀsampler compatible with liquid sample solvents could
be used.
Method development. We evaluated esterification of MFA and GCꢀMS analysis of the
resulting MFAꢀesters with ethanol, nꢀpropanol, and nꢀbutanol. We were able to extract the
resulting alkyl fluoroacetates from the aqueous reaction mixtures into chloroform for final GCꢀ
MS analysis. Detection of the butyl fluoroacetate derivative was problematic, with interfering
peaks even after employing different oven temperature hold times and ramp rates. We also
found that the ethyl fluoroacetate eluted on the tail of the solvent peak. However, the propyl
10
ACS Paragon Plus Environment

Page 11 of 27
Journal of Agricultural and Food Chemistry
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
fluoroacetate (PFA) was resolved from interfering peaks and was retained sufficiently that it was
well resolved from the solvent peak (Figures 1 and 3). This derivatization scheme was then
directly applied to evaporated water extracts from MFA containing plants (Figures 1 and 5). The
EI mass spectra of the PFA and the propyl chloroacetate (PCA) internal standard are shown in
Figure 2.
Method validation. For quantitative purposes, ions at m/z 61, 79 for PFA and ions at m/z 49,
79, 97, 107 for PCA were extracted and peak areas integrated. A linear calibration curve
(R2=0.9944) over the range of 0.024 – 1.0 µg/mL µg/mL MFA in water with SMCA as an
internal standard was used for quantitation. It should be noted that sodium fluoroacetate 13C2
isotope is a superior choice for an internal standard and is commercially available, however, it
was cost prohibitive for this application. The limit of detection (LOD, 3X S/N), and limit of
quantitation (LOQ, 10X S/N) of MFA were determined to be 0.0079 µg/mL and 0.024 µg/mL,
respectively (Figure 3). Additionally, recoveries of monofluoroacetate were determined at two
different levels of MFA in both the A. septentrionalis composite and P. marcgravii composite
samples. Recoveries of MFA ranged from 89.8ꢀ113% at both levels and in both of the different
plants (Table 1). Intraꢀday and interꢀday variation were also determined by the analysis the A.
septentrionalis composite over 1 day (n=8) and seven days (n=7), respectively. The intraꢀday
variation was determined to be 2.2% and the interꢀday variation over 7 days was 4.6%.
242
243
244
Samples of Gastrolobium and Amorimia spp. were compared using different methods within and
between laboratories. A Gastrolobium bilobum composite sample labeled “MFA QC Pool” was
prepared and analyzed by scientists at the Department of Agriculture and Food, Western
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 27
245
246
247
248
249
250
251
252
253
254
255
Australia, South Perth, Western Australia (DAFWA) using the HPLCꢀMS method of Lee et al.7
and by S. T. Lee at the Poisonous Plant Laboratory in Logan, Utah by the same method. (Table
2). This G. bilobum “MFA QC Pool” sample was analyzed by the GCꢀMS method described in
this report. Comparison of the results in Table 2 between the HPLCꢀMS method run at
laboratories in Australia and in the U.S.A. and with the GCꢀMS method reported herein are
acceptable. Additionally, thirteen individual samples of A. septentrionalis plant parts consisting
of stem, leaves, and floral plant parts previously analyzed using the HPLCꢀMS method described
by Lee et al.7 were also analyzed using the GCꢀMS method described. A good correlation
described by the equation y = 0.86x ꢀ2.6 (R2 = 0.993) was observed between results using the
HPLCꢀMS and GCꢀMS methods. This comparison supports the specificity and robustness of the
developed method.
256
257
258
259
260
261
262
263
264
265
266
Detection of MFA herbarium samples. Herbarium specimens have been used previously to
detect MFA in Palicourea and Amorimia species.6,8 Using this method we analyzed herbarium
specimens of several taxa reported or suspected to contain MFA. MFA was detected in all six of
the F. elegans specimens analyzed using the GCꢀMS method in SIM mode. MFA was also
detected in 8ꢀ25% of the N. stannea, N. multiglandulosa, N. acutifolia, and A. lasiandra
specimens analyzed (Table 3). The detection frequency of MFA in these specimens was similar
to those reported for Amorimia spp.8 MFA was detected in F. elegans and N. stannea, consistent
with the reports that these species cause sudden death.1,4,9 In addition, MFA was detected in N.
multiglandulosa and N. acutifolia, two species that are reported to cause subꢀacute clinical signs
similar to other MFA containing plants that cause sudden death.4 Lastly, MFA was detected in
A. lasiandra, a plant phylogenetically related to the Niedenzuella species investigated and not yet
12
ACS Paragon Plus Environment

Page 13 of 27
Journal of Agricultural and Food Chemistry
267
268
269
270
reported to be toxic.34 Caution should be taken with respect to the MFA concentrations reported
from herbarium specimens. The herbarium concentration values reported may be biased due to a
number of factors such as limited sample size, variability in the age of the herbarium specimens,
and possible differences in the storage conditions etc.
271
272
273
274
275
276
277
278
279
In conclusion, an inexpensive, straight forward extraction, derivatization, sample preparation,
GCꢀMS method for the analysis of MFA in plants was developed. The method compared
favorably to a currently employed HPLCꢀMS method for the analysis of MFA in plants. This
method was applied to the analysis of MFA in herbarium specimens and MFA was detected in F.
elegans, N. stannea, N. multiglandulosa, N. acutifolia, and Aenigmatanthera lasiandra. This is
the first report of MFA being detected in F. elegans, N. multiglandulosa, N. acutifolia, and
Aenigmatanthera lasiandra. It is anticipated that the method is sufficiently simple for routine
screening of suspected MFAꢀcontaining plants in Brazil and in other parts of the world where
this toxin is a significant cause of livestock deaths.
280
281
SUPPORTING INFORMATION
282
283
List of plant taxa, voucher specimen numbers, and associated herbaria. This material is available
free of charge via the Internet at http://pubs.acs.org.
284
285
ACKNOWLEDGEMENTS
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 27
286
287
288
289
290
We thank FUNDECT and CAPES for financial support of J. M. SantosꢀBarbosa. We thank
Gerard Smith, Department of Agriculture and Food, Western Australia, South Perth, Western
Australia, Australia (DAFWA) for the HPLCꢀMS analysis of the G. bilobum “MFA QC Pool”
sample. We thank the Herbarium at the Missouri Botanical Gardens for providing material from
select taxa for analysis.
291
14
ACS Paragon Plus Environment

Page 15 of 27
Journal of Agricultural and Food Chemistry
292
REFERENCES
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
1. Lee, S. T.; Cook. D.; Pfister, J. A.; Allen, J. G.; Colegate, S. M.; RietꢀCorrea, F.; Taylor, C.
M. Monofluoroacetateꢀcontaining plants that are potentially toxic to livestock. J. Agric.
Food Chem. 2014, 62, 7345ꢀ7354.
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.
3. Kellerman, T. S.; Coetzer, J. A.W.; Naude, T. W.; Botha, C. J. Cardiovascular system. In
Plant Poisonings and Mycotoxicoses of Livestock in Southern Africa, 2nd ed.; Oxford
University Press, Cape Town, South Africa, 2005; pp. 146ꢀ153.
4. Tokarnia, C. H.; Döbereiner, J.; Peixoto, P. V. Poisonous plants affecting livestock in Brazil.
Toxicon 2002, 40, 1635ꢀ1660.
5. RietꢀCorrea, F.; Medeiros, R. M. T.; Pfister, J.; Schild, A. L.; Dantas, A. F. M.. Poisonings
by Plants, Mycotoxins and Related Substances in Brazilian Livestock; Sociedade Vicente
Pallotti: Patos, Brazil, 2009; pp 15–24.
6. Cook, D.; Lee, S. T.; Taylor, C. M.; Bassuner, B.; RietꢀCorrea, F.; Pfister, Gardner, D. R.
Detection of toxic monofluoroacetate in Palicourea species. Toxicon 2014, 80, 9ꢀ16.
7. Carvalho, F. K. L.; Cook, D.; Lee, S. T.; Taylor, C. M.; Oliveira, J. B. S.; RietꢀCorrea, F.
Determination of toxicity in rabbits and corresponding detection of monofluoroacetate in
four Palicourea (Rubiaceae) species from the Amazonas State, Brazil. Toxicon 2016, 109,
42ꢀ44.
8. Lee, S. T.; Cook, D.; RietꢀCorrea, F.; Pfister, J.; Anderson, W. R.; Lima, F. G.; Gardner, D.
R. Detection of monofluoroacetate in Palicourea and Amorimia species. Toxicon 2012, 60,
791ꢀ796.
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 27
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
9. Arruda, F. P.; Caldeira, F. H. P.; Ducatti, K. R.; Bezerra, K. S.; MarcolongoꢀPereira, C.; Lee,
S. T.; Cook, D.; RietꢀCorrea, F.; Colodel, E. M. Experimental poisoning in Niedenzuella
stannea (Malpighiaceae) in cattle and corresponding detection of monofluoroacetate. Cienc.
Rural 2017, 47, 1ꢀ6.
10. Krebs, H. C.; Kemmerling, W.; Habermehl, G. Qualitative and quantitative determination of
fluoroacetic acid in Arrabidea bilabiata and Palicourea marcgravii by 19FꢀNMR
spectroscopy. Toxicon 1994, 32, 909ꢀ913.
11. Lima, E. F.; Medeiros, R. M. T.; Cook, D.; Lee, S. T.; Kaehler, M.; Barbosa, J.; RietꢀCorrea,
F. Studies in regard to the classification and putative toxicity of Fridericia japurensis
(Arrabidaea japurensis) in Brazil. Toxicon 2016, 115, 22ꢀ27.
12. Vickery, B.; Vickery, M. L.; Ashu, J. T. Analysis of plant for fluoroacetic acids.
Phytochemistry 1973, 12, 145ꢀ147.
13. Baron, M. L.; Bothroyd, C. M.; Rogers, G. I.; Staffa, A.; Rae, I. D. Detection and
measurement of fluoroacetate in plant extracts by 19F NMR. Phytochemistry 1987, 26, 2293ꢀ
2295.
14. O’Hagan, D. O.; Perry, R.; Lock, J. M.; Meyer, J. J. M.; Dasaradi, L.; Hamilton, J. T. G.;
Harper, D. B. High levels of monofluoroacetate in Dichapetalum braunii. Phytochemistry
1993, 33, 1043ꢀ1045.
333
334
335
15. Minnaar, P. P.; Swan, G. E.; McCrindle, R. I.; de Beer, W. H. J.; Naude, T. W. A highꢀ
performance liquid chromatographic method for the determination of monofluoroacetate. J.
Chromatogr. Sci. 2000, 38, 16ꢀ20.
16
ACS Paragon Plus Environment

Page 17 of 27
Journal of Agricultural and Food Chemistry
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
16. Vartiainen, T.; Kauranen, P. The determination of traces of fluoroacetic acid by extractive
alkylation, pentafluorobenzylation and capillary gas chromatographyꢀmass spectrometry.
Anal. Chim. Acta 1984, 157, 91ꢀ97.
17. Joseph, G. Determination of sodium monofluoroacetate in dairy powders by liquid
chromatography tandem mass spectrometry (LCꢀMS/MS): First Action 2015.02. J. AOAC
Int. 2015, 98, 1121ꢀ1126.
18. Bessaire, T.; Tarres, A.; Goyon, A.; Mottier, P.; Dubois, M.; Tan, W. P.; Delatour, T.
Quantitative determination of sodium monofluoroacetate “1080” in infant formulas and dairy
products by isotope dilution LCꢀMS/MS. Food Addit. Contam., Part A. 2015, 32, 1815ꢀ
1892.
19. Cooney, T. P.; Varelis, P.; Bendall, J. G. Highꢀthroughput quantification of
monofluoroacetate (1080) in milk as a response to extortion threat. J. Food Prot. 2016, 79,
273ꢀ281.
20. Kimball, B. A.; Mishalanie, E. A. Gas chromatographic determination of sodium
monofluoroacetate as the free acid in an aqueous solvent. J. Chromatogr. 1993, 634, 289ꢀ
296.
21. Okuno, I.; Meeker, D. L.; Felton, R. R. Modified gasꢀliquid chromatographic method for the
determination of Compound 1080 (sodium fluoroacetate). J. Assoc. Off. Anal. Chem. 1982,
65, 1102ꢀ1105.
22. Okuno, I.; Connolly, G. E.; Savarie, P. J.; Breidenstein, C. P. Gas chromatographic analysis
of coyote and magpie tissue for residues of Compound 1080 (sodium fluoroacetate). J. -
Assoc. Off. Anal. Chem. 1984, 67, 549ꢀ553.
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 27
358
359
360
23. Casper, H. H.; McMahon, T. L.; Paulson, G. D. Capillary gas chromatographicꢀmass
spectrometric determination of fluoroacetate residues in animal tissues. J. - Assoc. Off. Anal.
Chem. 1985, 68, 722ꢀ725.
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
24. Allender, W. J. Determination of sodium fluoroacetate (compound 1080) in biological
tissues. J. Anal. Toxicol. 1990, 14, 45ꢀ49.
25. Miki, A.; Tsuchihashi, H.; Yamashita, M. GC and GCꢀMS determination of fluoroacetic acid
and phenoxy acid herbicides via triphasal extractive pentafluorobenzylation using a polymerꢀ
bound phaseꢀtransfer catalyst. J. Anal. Toxicol. 1998, 22, 237ꢀ245.
26. Demarchi, A.C.; Menezer, M. L.; Mercadant, E, A.; Vassillief, I. Determination of the
sodium monofluoroacetate in serum by gas chromatography. Chromatographia 2001, 27,
402ꢀ404.
27. Sporkert, F.; Pragst, F.; Hubner, S.; Mills, G. Headspace solidꢀphase microextration with 1ꢀ
pyrenyldiazomethane onꢀfibre derivatisation for analysis of fluoroacetic acid in biological
samples. J. Chromatogr. B: Biomed. Sci. Appl. 2002, 772, 45ꢀ51.
28. Hoogenboom J. L.; Rammell, C. G. Determination of determination of sodium
monofluoroacetate (Compound 1080) in tissues and baits as its benzyl ester by reactionꢀ
capillary gas chromatography. J. Anal. Toxicol. 1987, 11, 140ꢀ143.
29. Stevens, H. M.; Moffat, A.C.; Drayton, J.V. The recovery and identification of
fluoroacetamide and fluoroacetic acid from tissues. Forensic Sci. 1976, 8, 131ꢀ137.
30. Mori, M.; Kakajima, H.; Seto, Y. Determination of fluoroacetate in aqueous samples by
headspace gas chromatography. J. Chromatogr. A 1996, 736, 229ꢀ234.
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 GCꢀFID
18
ACS Paragon Plus Environment

Page 19 of 27
Journal of Agricultural and Food Chemistry
381
382
383
384
385
386
387
388
and GCꢀMS in combination with solidꢀphase microextration. Anal. Bioanal. Chem. 2006,
386, 1395ꢀ1400.
32. Peterson, J. E. A gas chromatographic method for sodium fluoroacetate (Compound 1080) in
biological materials. Bull. Environ. Contam. Toxicol. 1975, 15, 751ꢀ757.
33. Anderson, W.R. Eight segregates from the neotropical genus Mascagnia (Malpighiaceae).
Novon 2006, 16, 168ꢀ204.
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, 2031ꢀ2048.
389
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 27
390
391
392
FIGURE CAPTIONS
Figure 1. Reconstructed GCꢀMS ion chromatograms of (m/z 61.0, 79.0) from (A) water extract of
A. septentrionalis composite sample and (B) water extract of P. marcgravii composite
sample diluted 1:625 v:v. * denotes MFA peak (detected as propyl fluoroacetate). IS
denotes internal standard peak.
393
394
395
396
397
398
Figure 2. Full scan mass spectrum of (A) propyl fluoracetate and (B) propyl chloroacetate. *
denotes m/z used for qualitative determination of peak identity. ** denotes m/z used
for quantitative analysis.
399
400
401
Figure 3. Reconstructed GCꢀMS ion chromatograms of (m/z 61.0, 79.0) from (A) a blank
reaction (B) a 0.0079 µg/mL MFA standard (LOD) and (C) a 0.024 µg/mL MFA
standard (LOQ). * denotes MFA peak as the propyl fluoroacetate derivative.
20
ACS Paragon Plus Environment

Page 21 of 27
Journal of Agricultural and Food Chemistry
Table 1. Recoveries Obtained for Extracts of A. septentrionalis and P. marcgravii Spiked at
Two Levels of MFA.
MFA Spike (µg)
0.0157
A. septentrionalis
0.0314
P. marcgravii
n = 4
A. septentrionalis P. marcgravii
Samples
Mean Recovery (µg)
RSD (%)
n = 4
0.0141 ± 0.0005
3.54
n = 4
n = 3
0.0168 ± 0.0019
11.3
0.0324 ± 0.0023 0.0354 ± 0.0020
7.10
103
5.65
113
Recovery (%)
89.8
107
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 27
Table 2. Comparison of Analysis of G. bilobum Composite MFA QC Pool Sample Between
Different Laboratories and Different Analysis Methods.
MFA ± STDV
Laboratory
Method
(µg/g)
50 ± 5
60 ± 3
47 ± 2
DAFWA n = 10
PPRL n = 4
PPRL n = 4
HPLCꢀMS
HPLCꢀMS
GC/MS
22
ACS Paragon Plus Environment

Page 23 of 27
Journal of Agricultural and Food Chemistry
Table 3. GCꢀMS 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)
Family
Scientific Name
Bignoniaceae
Fridericia elegans
6
8
6
2
0.31 – 1.3
nd – 5.2
Malpighiaceae
Aenigmatanthera
lasiandra
Malpighiaceae
Malpighiaceae
Niedenzuella acutifolia
8
8
1
1
nd – 0.21
nd – 1.7
Niedenzuella
multiglandulosa
Malpighiaceae
Niedenzuella stannea
13
1
nd – 1.4
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 27
Figure 1
IS
6
A
5
4
3
2
1
*
0
6
B
IS
5
4
3
2
1
0
*
4
5
6
7
8
9
10
11
12
Time (min)
24
ACS Paragon Plus Environment

Page 25 of 27
Journal of Agricultural and Food Chemistry
Figure 2
1.1
1.0
**
A
61.0
*
31.0
43.0
42.1
41.1
0.8
0.6
0.4
0.2
32.0
**
79.0
*
91.0
33.0
36.0
59.1
57.0
83.0
0
2.5
*
77.0
B
43.1
2.0
1.5
1.0
0.5
0
*
95.0
42.1
**
79.0
57.1
32.0
117.1
119.0
**
107.0
**
75.0
74
**
49.0
71.1
70
89.1
97.0
30
34
38
42
46
50
54
58
62
66
78
82
86
90
94
98 102 106 110 114 118
(m/z)
25
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 27
Figure 3
4
A
3
2
1
0
4
B
3
2
1
0
4
*
*
C
3
2
1
0
5.0
5.5
6.0
6.5
7.0
Time (min)
26
ACS Paragon Plus Environment

Page 27 of 27
Journal of Agricultural and Food Chemistry
TOC Graphic
6
4
2
0
F
O
*
4
5
6
7
8
9
10
Time (min)
1.2
61.0
43.0
Time (min)
0.8
0.4
0
79.0
91.0
90
30
40
50
60
70
(m/z)
80
100
27
ACS Paragon Plus Environment