Preventing nitrite contamination in tetramethylammonium peroxynitrite solutions

Article abstract of DOI:10.1021/ic049161k

Peroxynitrite prepared from superoxide and nitric oxide in liquid ammonia does not contain detectable levels of nitrite. However, the dissolution of nitrite salts can lead to variable levels of peroxynitrite depending on the conditions used to disolve the salt. Low levels of nitrite result when frozen peroxynitrite solutions are first brought to +1 °C and then to room temperature. These undergo only 2-3% decomposition after 1 h, in contrast with the findings of a recent report (Lymar, S. V.; Khairutdinov, R. F.; Hurst, J. K. Inorg. Chem. 2003, 42, 5259-5266), where high levels of nitrite (～20%) result from rapid thawing of these solutions to room temperature. Warming the frozen peroxynitrite solution directly to room temperature in 30 min leads to a nitrite level of 28%.

Full text of DOI:10.1021/ic049161k

Inorg. Chem. 2004, 43, 6519−6521
Preventing Nitrite Contamination in Tetramethylammonium Peroxynitrite
Solutions
Petr Latal,† Reinhard Kissner,† D. Scott Bohle,‡ and Willem H. Koppenol*,†
Laboratorium fu¨r Anorganische Chemie, Departement Chemie und Angewandte Biowissenschaften,
ETH-Ho¨nggerberg, Zu¨rich, Switzerland, and Department of Chemistry, McGill UniVersity,
Montreal, Canada
Received June 28, 2004
Peroxynitrite prepared from superoxide and nitric oxide in liquid
ammonia does not contain detectable levels of nitrite. However,
the dissolution of nitrite salts can lead to variable levels of
peroxynitrite depending on the conditions used to disolve the salt.
Low levels of nitrite result when frozen peroxynitrite solutions are
Peroxynitrite decomposition yields nitrite and dioxygen
in a 2:1 ratio,4 and the yields of these products, relative to
peroxynitrite, can provide information about the mechanism
of decomposition. Generally, frozen stock solutions are
melted and warmed to 0,5 25,6,7 or 37 °C4 and then mixed
with varying concentrations of phosphoric acid at the
appropriate temperature to obtain different pH values at 25
or 37 °C, followed by a nitrite determination. If nitrite is
already generated by the warming process and if the
concentration of nitrite is a function of the time that the stock
solution is kept at a particular temperature, then this would
complicate the analysis, even if a determination is made at
low pH some time after the stock solution has melted. This
issue of nitrite generation in the alkaline solution needs to
be resolved for progress to be made on the mechanistic front.
Herein, we demonstrate how, through warming above 0
°C, alkaline (0.01 M potassium hydroxide) stock solutions
can become heavily contaminated with nitrite, and we
describe the steps that should be followed to avoid this
problem.
The tetramethylammonium peroxynitrite used in these
studies was prepared by treating tetramethylammonium
superoxide with nitrogen monoxide at -77 °C in liquid
ammonia, followed by isolation as a crystalline solid via
removal of the ammonia. From combustion analysis, this
solid corresponds to (NMe4)(O3N); contamination with as
little as 6.6% (NMe4)(NO2) would significantly alter these
figures.8-10 Clearly, the C, H, and N analyses alone are not
consistent with 20% contamination with nitrite. Greater
precision might be possible when oxygen analysis is included
first brought to
undergo only 2
findings of a recent report (Lymar, S. V.; Khairutdinov, R. F.; Hurst,
J. K. Inorg. Chem. 2003, 42, 5259 5266), where high levels of
nitrite ( 20%) result from rapid thawing of these solutions to room
+1
°
C and then to room temperature. These
−3% decomposition after 1 h, in contrast with the
−
∼
temperature. Warming the frozen peroxynitrite solution directly to
room temperature in 30 min leads to a nitrite level of 28%.
The mechanism of peroxynitrite [systematic name: oxo-
peroxonitrate(1-)] decomposition has been intensively stud-
ied and vigorously debated in the past decade. Among the
critical mechanistic issues are the reaction products, which
include nitrite and dioxygen when the initial concentration
of peroxynitrite is high.
ONOOH + ONOO- f 2NO2- + O2 + H+
(1)
In a recent paper on the thermodynamics of peroxynitrous
acid, Hurst and co-workers1 criticized our model to describe
the formation of nitrite and dioxygen and claimed that their
high-pressure studies support homolysis. Furthermore, these
authors mention that the peroxynitrite supplied by us
contained 20% nitrite. Whereas the first two points have
already been addressed,2,3 the origin of the nitrite contamina-
tion needs to be scrutinized carefully.
(4) Pfeiffer, S.; Gorren, A. C. F.; Schmidt, K.; Werner, E. R.; Hansert,
B.; Bohle, D. S.; Mayer, B. J. Biol. Chem. 1997, 272, 3465-3470.
(5) Kissner, R.; Koppenol, W. H. J. Am. Chem. Soc. 2002, 124, 234-
239.
* To whom correspondence should be addressed. E-mail: koppenol@
inorg.chem.ethz.ch.
(6) Coddington, J. W.; Hurst, J. K.; Lymar, S. V. J. Am. Chem. Soc. 1999,
121, 2438-2443.
† ETH-Ho¨nggerberg.
‡ McGill University.
(7) Kirsch, M.; Korth, H.-G.; Wensing, A.; Sustmann, R.; De Groot, H.
Arch. Biochem. Biophys. 2003, 418, 133-150.
(1) Lymar, S. V.; Khairutdinov, R. F.; Hurst, J. K. Inorg. Chem. 2003,
42, 5259-5266.
(8) Bohle, D. S.; Hansert, B.; Paulson, S. C.; Smith, B. D. J. Am. Chem.
Soc. 1994, 116, 7423-7424.
(2) Kissner, R.; Nauser, T.; Kurz, C.; Koppenol, W. H. IUBMB Life 2003,
55, 567-572.
(9) Bohle, D. S.; Glassbrenner, P. A.; Hansert, B. Methods Enzymol. 1996,
269, 302-311.
(3) Kissner, R.; Thomas, C.; Hamsa, M. S. A.; van Eldik, R.; Koppenol,
W. H. J. Phys. Chem. A 2003, 107, 11261-11263.
(10) Bohle, D. S.; Sagan, E. S. Inorg. Synth. 2004, 34, 36-41.
10.1021/ic049161k CCC: $27.50
Published on Web 09/17/2004
© 2004 American Chemical Society
Inorganic Chemistry, Vol. 43, No. 21, 2004 6519

COMMUNICATION
in this determination. Furthermore, given the absence of the
strong ν(NO)sym band at 1040 cm-1 in the Raman spectrum,
the salt is not contaminated with nitrate. This band and other
characteristic signals for nitrite and nitrate are also not
observed by IR spectrum. Finally, the 15N NMR spectra of
these salts in both liquid ammonia at -40 °C and D2O at
pH 14 are consistent with the presence of tetramethylam-
monium and peroxynitrite nitrogens alone. Because peroxy-
nitrite and nitrite both have geometries bent at the nitrogen
atom, we expect similar relaxation times for these species.
The 15N NMR results are consistent with less than 1%
contamination with nitrite. Given that nitrate exhibits D3h
geometry and longer relaxation times, we also used longer
relaxation delays in measurements in an attempt to detect
its presence in these experiments as well. None was observed,
and the clear conclusion from all of these experiments is
that nitrite is not formed during synthesis.8-10 Thus, if it is
present, it must result from either the dissolution or the
subsequent treatment. Finally, we note that only one con-
formation of peroxynitrite is observed in these spectroscopic
methods, and this is consistent with the cis geometry
determined by X-ray diffraction.11
Figure 1. Decomposition of [(CH3)4N]+[ONOO]- as a function of
temperature and time. One-milliliter samples of 20.1 mM [(CH3)4N]+-
[ONOO]-, pH ) 12, were thawed and warmed as indicated: b, from -80
°C directly to room temperature; 2, from -80 °C to +1 °C in ice-cold
water and then to room temperature; and 9, from -80 °C to +1 °C in
ice-cold water and kept at that temperature. The samples were immediately
analyzed for nitrite by the Griess method and by ion chromatography. No
significant differences were found between these two analytical methods.
To prepare aqueous solutions of peroxynitrite, it is best
to dissolve the tetramethylammonium salt in cold (0-2 °C)
0.01 M potassium hydroxide that has been freshly prepared
to avoid carbonate contamination. Either this alkaline per-
oxynitrite solution can be used immediately, or it can be
stored, e.g., as 1-mL aliquots, ca. 20 mM in peroxynitrite,
in small Eppendorf tubes and frozen by dropping the tubes
into liquid dinitrogen. Peroxynitrite so prepared was stored
at -80 °C. The sample of peroxynitrite used by Prof. Hurst
and co-workers was prepared in this way and was sent on
dry ice by courier.
We analyze stored peroxynitrite by ion chromatography
as previously described5 from time to time for nitrite, and
values of ca. 1% or less are always found. The claim that
the material contains 25% nitrite is, thus, misleading. That
analyses by Hurst and co-workers of peroxynitrite decom-
position products1,6 generally show higher nitrite values and
more scatter than ours2;57 under comparable conditions does
not account for the difference between 1% and 20%. Also,
the addition of hydroxide1 is not expected to increase the
nitrite level.
Figure 2. Temperature vs time profiles of the samples during warming.
Symbols as described in the caption of Figure 1.
analyses, by way of the Griess method and anion chroma-
tography, are shown in Figure 1. The temperature-time
profiles were obtained with frozen 0.01 M potassium
hydroxide samples and are shown in Figure 2. The samples
“warmed up” in ice water reached 0 °C within 100 s; those
in air took ca. 250 s and stayed near 0 °C for ca. 12 min,
after which their temperature increased again.
The sample allowed to warm to room temperature directly
had a nitrite level comparable to that reported by Hurst and
co-workers in less than 30 min.1 The other samples had nitrite
levels near 1% after 1 h that increased only slowly with time.
This applied also to the sample that was diluted while
melting. We are of the opinion that the 1% nitrite is also
caused by decomposition. Clearly, temperatures above 0 °C
We suggest that the nitrite might have originated through
decomposition (reaction 1), which is much enhanced at
higher concentrations of peroxynitrite.5 To investigate this
possibility, we took several frozen 1-mL samples, warmed
them in three different ways, and analyzed them for nitrite.
One sample was brought directly from -80 °C to room
temperature (heat exchange by air), and others were allowed
to melt in an ice-bath at or near 0 °C first or to remain at
that temperature. None of the samples were stirred. One
sample was warmed by hand until the frozen pellet fell into
ice-cold potassium hydroxide. The results of the nitrite
(11) Wo¨rle, M.; Latal, P.; Kissner, R.; Nesper, R.; Koppenol, W. H. Chem.
Res. Toxicol. 1999, 12, 305-307.
6520 Inorganic Chemistry, Vol. 43, No. 21, 2004

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lead to a significant conversion of peroxynitrite to nitrite and
dioxygen, in agreement with earlier observations.5
shipment or storage, a temperature of at least -20 °C was
not maintained. In either case, the high nitrite levels reported
by these workers could result.
We conclude that handling and storage procedures are
critical for the quality of tetramethylammonium peroxynitrite
solutions, and we speculate that accelerated decomposition
might be due to high heats of dissolution and solvation. It is
quite plausible that the high levels of nitrite contamination
reported1 were caused by keeping the peroxynitrite solution
at room temperature. Alternatively, it is possible that, during
Acknowledgment. We thank Dr. P. L. Bounds for
discussion. Our studies on peroxynitrite were supported by
the Swiss NF and the ETH (W.H.K.) and by NSERC through
a discovery grant (D.S.B.).
IC049161K
Inorganic Chemistry, Vol. 43, No. 21, 2004 6521