Synthesis and Storage Stability of Diisopropylfluorophosphate

Article abstract of DOI:10.1155/2016/3190891

Diisopropylfluorophosphate (DFP) is a potent acetylcholinesterase inhibitor commonly used in toxicological studies as an organophosphorus nerve agent surrogate. However, LD₅₀ values for DFP in the same species can differ widely even within the same laboratory, possibly due to the use of degraded DFP. The objectives here were to identify an efficient synthesis route for high purity DFP and assess the storage stability of both the in-house synthesized and commercial source of DFP at the manufacturer-recommended storage temperature of 4°C, as well as -10°C and -80°C. After 393 days, the commercial DFP stored at 4°C experienced significant degradation, while only minor degradation was observed at -10°C and none was observed at -80°C. DFP prepared using the newly identified synthesis route was significantly more stable, exhibiting only minor degradation at 4°C and none at -10°C or -80°C. The major degradation product was the monoacid derivative diisopropylphosphate, formed via hydrolysis of DFP. It was also found that storing DFP in glass containers may accelerate the degradation process by generating water in situ as hydrolytically generated hydrofluoric acid attacks the silica in the glass. Based on the results here, it is recommended that DFP be stored at or below -10°C, preferably in air-tight, nonglass containers.

Full text of DOI:10.1155/2016/3190891

Hindawi Publishing Corporation
Journal of Chemistry
Volume 2016, Article ID 3190891, 5 pages
http://dx.doi.org/10.1155/2016/3190891
Research Article
Synthesis and Storage Stability of Diisopropylfluorophosphate
Derik R. Heiss,1 Donald W. Zehnder II,1 David A. Jett,2 Gennady E. Platoff Jr.,3
David T. Yeung,2 and Bobby N. Brewer1
1Battelle Memorial Institute, 505 King Avenue, Columbus, OH 43201, USA
2National Institute of Neurological Disorders and Stroke, National Institutes of Health, 6001 Executive Boulevard,
Rockville, MD 20852, USA
3National Institute of Allergy and Infectious Diseases, National Institutes of Health, 5601 Fishers Lane, Rockville, MD 20892, USA
Correspondence should be addressed to Bobby N. Brewer; brewerb@battelle.org
Received 7 April 2016; Accepted 10 May 2016
Academic Editor: Teodorico C. Ramalho
Copyright © 2016 Derik R. Heiss et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Diisopropylfluorophosphate (DFP) is a potent acetylcholinesterase inhibitor commonly used in toxicological studies as an
organophosphorus nerve agent surrogate. However, LD values for DFP in the same species can differ widely even within the
50
same laboratory, possibly due to the use of degraded DFP. e objectives here were to identify an efficient synthesis route for high
purity DFP and assess the storage stability of both the in-house synthesized and commercial source of DFP at the manufacturer-
recommended storage temperature of 4∘C, as well as −10∘C and −80∘C. Afer 393 days, the commercial DFP stored at 4∘C
experienced significant degradation, while only minor degradation was observed at −10∘C and none was observed at −80∘C. DFP
prepared using the newly identified synthesis route was significantly more stable, exhibiting only minor degradation at 4∘C and
none at −10∘C or −80∘C. e major degradation product was the monoacid derivative diisopropylphosphate, formed via hydrolysis
of DFP. It was also found that storing DFP in glass containers may accelerate the degradation process by generating water in situ as
hydrolytically generated hydrofluoric acid attacks the silica in the glass. Based on the results here, it is recommended that DFP be
stored at or below −10∘C, preferably in air-tight, nonglass containers.
1. Introduction
e objectives of this study were to synthesize high
purity DFP and then evaluate the storage stability of both
the synthesized DFP and a commercial source of DFP [7].
e manufacturer-recommended storage temperature for
commercially available DFP is 4∘C [8]. erefore, this study
compared DFP stored at 4∘C against material stored at other
common laboratory storage temperatures, −10∘C and −80∘C.
Diisopropylfluorophosphate (DFP, see Figure 1) is a powerful
neurotoxin ofen used in research studies as a surrogate
for organophosphorus nerve agents such as sarin (GB) and
soman (GD) due to its ability to effectively inhibit the enzyme
acetylcholinesterase [1–3]. However, significant variability
has been observed in toxicological studies using commer-
cially available DFP. For example, published subcutaneous
2. Materials and Methods
LD values range from 0.0027 mg/kg to 6.4 mg/kg in the
50
mouse model [4–6].
2.1. Chemicals. Analytical grade DFP was procured from
Sigma-Aldrich. Diisopropylphosphate (DIPP) was obtained
from PolyOrg, Inc. All solvents and synthesis reagents were
purchased from Sigma-Aldrich and were of ACS grade or
better.
e storage stability of DFP, information especially useful
in support of long-term evaluations, has not been docu-
mented. However, anecdotal information suggests that DFP
degrades upon storage, and it is speculated that the wide
range in reported LD values may be caused by the use
of impure DFP. As such, proper storage of DFP for use in
analytical study is imperative.
50
2.2. Instrumentation. Nuclear magnetic resonance (NMR)
data were collected using a Bruker Advance 500 FT-NMR

2
Journal of Chemistry
chlorophosphate using a fluorinating agent [9–11]. In many
cases, the reaction is slow and ofen does not go to com-
pletion, leaving unreacted starting material remaining as an
impurity.
O
P
O
F
For this study, a previously reported one-pot synthesis
method [12] was modified to produce high purity DFP
from diisopropyl phosphite using a mixture of KF and 1,3-
dichloro-5,5-dimethylhydantoin (see Figure 2). e interme-
diate diisopropyl chlorophosphate formed in situ is rapidly
converted to the corresponding fluorophosphate. Vacuum
distillation of the resultant reaction mixture produced DFP
in 68% yield with a purity of 99%, as determined by 1H and
31P NMR.
O
DFP
Figure 1: Diisopropylfluorophosphate (DFP).
with an operating field of 11.75 Tesla. Fourier-transform
infrared (FT-IR) spectra were collected with a Digilab FTS-
7000 with UMA-600 microscope using NaCl plating tech-
nique. Gas chromatography-mass spectrometry (GC-MS)
data were obtained using an Agilent 6890 gas chromatograph
with Model 5973N mass spectrometer. X-ray diffraction
(XRD) data were recorded using a Rigaku Ultima IV diffrac-
tometer.
3.2. Storage Stability of DFP. Both the synthesized DFP
and the commercially acquired DFP were analyzed at the
beginning of the study (day 0) and found to have initial purity
values of >99% by 31P NMR. Purity assessments were then
conducted in duplicate at approximately 2-week intervals
over the course of 13 months (393 days) to evaluate the
stability of DFP under each of the three storage conditions.
Purity results for each replicate analysis are presented in
Figures 3, 4, and 5. DFP purity data are presented as outlined
and nonoutlined green triangles or red squares representing
individual synthesized and commercial samples, respectively.
As the purity results indicate, the commercial source
of DFP degraded significantly when stored at 4∘C, while
degradation was markedly slower when stored at −10∘C. In
this study, 88% of the vials of commercial DFP stored at
4∘C had degraded below 95% purity within 393 days, while
only 6% of the vials stored at −10∘C fell below 95% purity.
No degradation was observed in vials stored at −80∘C. By
contrast, the synthesized DFP was considerably more stable.
Only 21% of the vials of synthesized DFP stored at 4∘C and
none of the vials stored at −10∘C or −80∘C had degraded
to less than 95% purity within 393 days. e cause of the
disparity in degradation rates observed between the two
sources of DFP was not investigated for this study but can
likely be attributed to differences in the impurity profiles
introduced during synthesis.
As expected, the major degradation product of DFP
was found to be the hydrolysis product diisopropylphos-
phate (DIPP). is was confirmed upon comparison of
the degraded material to a known standard of DIPP using
31P NMR and FT-IR spectroscopy (see Figures 6 and 7).
In addition, a small amount of triisopropylphosphate had
formed, as indicated by GC-MS analysis.
A white solid began to form in the neat DFP as purity fell
to approximately 90% or below (see Figure 8). e presence
of the solid was somewhat confounding, as both DFP and its
hydrolysis products are liquids at room temperature.
2.3. Synthesis of DFP. Potassium fluoride (8.17 g, 140.9 mmol)
was added to 1,3-dichloro-5,5-dimethylhydantoin in acetoni-
trile (300 mL) and then stirred at room temperature for one
hour. Diisopropyl phosphite (18 g, 108 mmol) in acetonitrile
(100 mL) was added to the mixture all at once and then stirred
for 30 minutes. e resulting white precipitate was removed
by filtration over diatomaceous earth followed by a 0.45 ꢀm
PTFE membrane filter. e concentrated crude product was
purified by distillation (bp. 63∘C, 8 mmHg) affording 13.7 g
(68% yield) of a clear, colorless liquid with a purity of 99%, as
determined by 1H and 31P NMR.
2.4. DFP Stability Study. Two sources of DFP, one procured
from Sigma-Aldrich and one synthesized as described above,
were each divided into approximately 30 mg, single-use
aliquots in clear glass vials with PTFE-lined screw caps.
e vials were stored in the dark at either 4∘C, −10∘C, or
−80∘C ( 2∘C) surrounded with cold packs inside coolers
to prevent unwanted thermal cycling. At approximately 2-
week intervals, duplicate sacrificial vials of each material at
each storage temperature were removed and warmed to room
temperature in a desiccator. e samples were then dissolved
in acetonitrile-ꢁ3 or other appropriate solvents and analyzed
by 31P NMR to determine purity.
Degradation products were determined using 31P NMR
by dissolving an aliquot of DFP in acetonitrile-ꢁ3. In addition,
a second sample of DFP was analyzed in neat form by FT-IR
while a third portion of DFP was extracted with methylene
chloride, centrifuged to remove undissolved solids, and
analyzed by GC-MS. e solid remaining from this aliquot
was rinsed with methylene chloride, dried, and analyzed by
FT-IR and XRD.
e solid was identified using FT-IR and XRD as a
hexafluorosilicate salt. Its presence in the degraded DFP
can likely be explained by a secondary reaction between
hydrofluoric acid (HF), formed as DFP hydrolyzes, and silica
from the glass storage vials. HF is known to react with silicate
glass and is commonly used as a wet chemical etchant in
industrial processes [13].
3. Results and Discussion
3.1. Synthesis of DFP. Dialkylfluorophosphates, including
DFP, are traditionally synthesized from the corresponding

Journal of Chemistry
3
O
O
P
O
P
O
CH3CN
H
O
F
Cl
N
+
+
KF
O
O
N
O
Cl
DFP
Figure 2: One-pot synthesis of DFP.
4∘C storage
−80∘C storage
100
98
96
94
92
90
100
90
80
70
60
50
40
30
0
50
100
150
200
250
300
350
400
0
50
100
150
200
250
300
350
400
Time (days)
Time (days)
Synthesized DFP
Commercial DFP
Synthesized DFP
Commercial DFP
Figure 5: Purity results for synthesized DFP and commercial DFP
Figure 3: Purity results for synthesized DFP and commercial DFP
when stored at −80∘C.
when stored at 4∘C.
−10∘C storage
O
100
O
F
P
O
40
30
20
10
0
95
90
85
80
75
DFP
O
P
O
OH
0
50
100
150
200
250
300
350
400
O
Time (days)
DIPP
Synthesized DFP
Commercial DFP
0
−2
−4
−6
−8 −10 −12 −14
(ppm)
Figure 4: Purity results for synthesized DFP and commercial DFP
when stored at −10∘C.
Figure 6: 31P NMR spectra of pure DFP (top), partially degraded
commercial DFP (middle), and a pure DIPP standard (bottom).
Interestingly, this side reaction may be responsible for
accelerating the degradation of DFP in two ways: (1) by
consuming HF, thus driving the equilibrium toward the
products side of the hydrolysis reaction (i.e., to the right), and
(2) by generating water in situ, resulting in a self-sustaining
hydrolysis cycle (Figure 9).
solid) and water. e water generated by the reaction can
then initiate further hydrolysis, resulting in an autocatalytic
reaction cycle that ultimately accelerates the degradation of
DFP.
Because DFP degrades rapidly once hydrolysis becomes
self-sustaining, small differences in surface reactivity of the
glass vials or the amount of surface area exposed to the
degraded DFP can likely lead to large discrepancies in purity
values among replicates of the same material. is might
explain the variability observed in some of the purity results
for the same material, most notably the commercial DFP
In the initial stages of DFP degradation, hydrolysis is the
primary reaction mechanism and appears to proceed accord-
ing to standard first- or second-order kinetics. However, afer
a certain induction period elapses and the material degrades
further, the HF produced may begin to react with silica in
the glass forming the insoluble hexafluorosilicate salt (white

4
Journal of Chemistry
100
50
O
P
O
O
P
O
P
Hydrolysis
O
O
F
F
OH
O
H2O
+
+
HF
O
O
DFP
0
−50
DFP
DIPP
+
SiO2
+
2H2O
H2SiF6
6HF
Figure 9: Autocatalytic hydrolysis of DFP when stored in glass vials.
−100
−150
O
O
OH
P
O
DIPP
important to ensure appropriate storage of DFP in order to
retain potency so that toxicity values are accurate and there is
consistency among toxicology studies across laboratories.
For this study, high purity DFP was synthesized
using a one-pot approach by fluorinating the associated
phosphite with a mixture of KF and 1,3-dichloro-5,5-
dimethylhydantoin. e synthesized material showed
4000 3500 3000 2500 2000 1500 1000
500
Wavenumber (cm−1
)
Figure 7: FT-IR spectra of pure DFP (top), partially degraded
commercial DFP (middle), and a pure DIPP standard (bottom).
minimal (≈4%) degradation when stored at 4∘C and no
degradation when stored at −10∘C or below through 393
days. Conversely, significant degradation was observed
in commercially acquired DFP when stored at the
manufacturer-recommended storage temperature of 4∘C and
minor degradation (≈5%) when stored at −10∘C within the
same time period.
Based on these results, DFP should be stored at −10∘C or
below to ensure long-term chemical stability. Storage above
this temperature would likely result in premature degradation
and surreptitiously impact results generated from the use of
the material. Additionally, alternatives to glass storage con-
tainers and incorporation of stabilizers should be considered.
Figure 8: White solid observed in partially degraded DFP.
stored at 4∘C (see Figure 3). For this study, it was assumed
that all vials of the same material stored under the same
conditions would behave similarly. If this assumption does
not hold, which appears to be the case here, variability can
be introduced even if all other parameters are held constant.
As such, it is recommended that DFP be stored in nonglass
containers.
Similar storage issues resulting from the formation of HF
upon degradation have been observed for other fluorinated
alkylphosphates. Most notably, some stockpiles of sarin in
the US arsenal require stabilizers such as tributylamine
or diisopropylcarbodiimide to mitigate the corrosion of
metal storage containers and munitions by scavenging the
acid generated when the parent compound hydrolyzes [14].
Similarly, these types of compounds might act as stabilizers
of DFP when stored in glass containers by neutralizing
the HF generated as the material degrades, thus preventing
autocatalytic degradation (Figure 9) from occurring.
Disclosure
e views expressed in this paper are those of the authors and
do not reflect the official policy of the NIH, HHS, DoD, or
the US Government. No official support or endorsement of
this paper by the DoD, NIAID, NINDS, or NIH is intended
or should be inferred. e sponsor developed the concept of
the study and contributed to its design and the interpretation
of the data as well as the preparation of the paper and the
decision to submit it for publication. e sponsor also made
similar contributions to other studies occurring at Battelle
during the same time frame.
Competing Interests
e authors have no known competing interests.
Acknowledgments
4. Conclusions
Funding for this work was provided by the National Institutes
of Health Office of the Director through an interagency
agreement (Y1-OD-0387-01) between the National Institute of
Allergy and Infectious Diseases (NIAID) and Department of
Defense (DoD) and prepared under the auspices of the NIH,
NIAID, NINDS, and the DoD Defense Technical Information
Proper storage of DFP for use in toxicological evaluations is
critical. Degradation of DFP is likely to elicit a concomitant
reduction in overall toxicity since the primary degradation
product identified in this study, DIPP, has previously been
shown not to inhibit cholinesterase activity [15]. It is therefore

Journal of Chemistry
5
Center (DTIC) via the Chemical, Biological, Radiological &
Nuclear Defense Information Analysis Center (CBRNIAC)
program, Contract no. SP0700-00-D-3180, Delivery Order
no. 0794, CBRNIAC Task 689/CB-13-0689.
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Chemical Weapons, Army Chemical Review, 2006.
[15] H. Imaizumi, K. Nagamatsu, A. Hasegawa, Y. Ohno, and A.
Takanaka, “Metabolism and toxicity of acid phosphate esters,
metabolites of organophosphorous insecticides, in rat,” Japanese
Journal of Toxicology and Environmental Health, vol. 39, no. 6,
pp. 566–571, 1993.
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