The threats of chemical and biological attack have lead to remediation
efforts using various chemical oxidants (Raber and McGuire 2002).
Among the new reagents being tested is Oxone, potassium peroxymonosulfate,
a product of Dupont, Wilmington, Delaware. This compound has been
tested on contaminated substrates with encouraging results, but
its effects on subsequent tests of forensic interest still need
to be determined (Tumosa et al. 2002).
The initial examination of bloodstains usually includes at least one test based
on the catalytic nature of hemoglobin (Gaensslen 1983). In particular, a test
based on the oxidation of benzidine or more recently tetramethylbenzidine in the
presence of peroxide is often used. The similar phenolphthalin (reduced phenolphthalein)
test is also common. Because these tests are based on the oxidation of the reagent,
interferences that yield false-positive reactions are often oxidizers. Iron and
copper oxides as well as permanganates, sodium hypochlorite, nitric acid, and
ferrocyanides have been reported to interfere with the test (Garner et al. 1976;
Hunt et al. 1960). Other interferences have been addressed as well (Cox 1991;
The active ingredient of Oxone is potassium peroxymonosulfate.
The salt is present as a component of a triple salt, 2KHSO5
KHSO4 K2SO4, potassium hydrogen
peroxymonosulfate [CAS-RN 70693-62-8]. Oxone is a mild oxidizing
agent and has been evaluated for the mitigation of the effects of
biological and chemical warfare agents, as well as some viral agents
(Raber and McGuire 2002). In addition, other oxidizing compounds,
such as sodium perborate and sodium percarbonate, are present in
commonly available cleaning and disinfecting products. These compounds
were tested for reactions with the 3,3',5,5'-tetramethylbenzidine
(TMB) and phenolphthalin reagents as well.
Materials and Methods
The tests were performed in a two-step process. The test reagent was added
first, the color of the solution was noted, and then the oxidizing reagent was
added. The TMB reagent was prepared as a two-percent solution in glacial acetic
acid, and a three-percent hydrogen peroxide solution was used in the second step
of the test (Lee 1982). The phenolphthalin reagent (Kastle-Meyer reagent) was
prepared by reducing phenolphthalein with zinc metal in basic solution and an
approximately 20-percent sodium perborate solution was used in the second step
of the test (Culliford 1971). The tests were performed by adding two drops of
TMB or phenolphthalin reagents to similar volumes of test material (oxidizing
agent), and the color of each was noted after one minute. The three-percent hydrogen
peroxide or 20-percent (weight/volume) sodium perborate was then added and the
color and time-to-color development was noted.
Solutions of sodium percarbonate, sodium perborate, and Oxone were
made to one percent (weight/volume) in deionized water.
The influence of blood in contact with the oxidizing agents was
also investigated. The same procedure as above was followed but
with human or pig blood on solid substrates, such as filter paper
or cotton swabs. Dried samples of human and pig blood of known age,
from one week to three years old, on filter paper, cotton swabs,
or as crusts were used to test the reagents and to act as controls.
Negative controls (both test reagents only) and positive controls
(both test reagents with blood) reacted as expected.
All chemicals were of analytical grade, and test reagents were
prepared fresh before use.
The one-percent Oxone solution reacted with the tetramethylbenzidine
reagent, first to give a purple precipitate, then a green color,
and finally a yellow solution. This color change also occurred when
the one-percent Oxone was dried on cotton swabs and the test reagent
added to the swabs. In neither case did the developed color mimic
that formed when the TMB reagent and hydrogen peroxide reacted with
human or pig blood. This was true whether or not the blood was tested
on a cotton or filter paper substrate, as crusts, or dissolved in
saline solution. Because the TMB test reagent alone gave color changes
with the Oxone, further tests with blood were not performed.
The one-percent sodium percarbonate solution did not react with
the tetramethylbenzidine reagent, as might be expected. In the presence
of blood with no other peroxide source added, the characteristic
color of the test developed quickly. The percarbonate acted as a
source of peroxide and functioned as a substitute for the commonly
used hydrogen peroxide in the tests. Solutions of sodium percarbonate
in contact with visible blood stains evolved bubbles of oxygen gas,
as do three-percent solutions of hydrogen peroxide. The sodium percarbonate
solution when air dried on swabs failed to react to give the characteristic
color (or any color) in the presence of blood and the test reagent
alone. The addition of peroxide gave the characteristic color.
Phenolphthalin reagent (Kastle-Meyer reagent)
The one-percent Oxone solution reacted very slowly with the phenolphthalin
reagent alone to give a faint pink color after about one minute.
This color did not intensify even after five minutes. The color
was neither as intense nor as immediate as it would be with an authentic
blood sample. In the presence of blood, the color was intense and
immediate without the further addition of perborate or hydrogen
Sodium percarbonate solutions did not react with the phenolphthalin
reagent alone but gave the characteristic red color if blood were
present whether in solution or on cotton substrates.
Discussion and Conclusions
Oxone in aqueous concentrations typically used for the decontamination
of surfaces will react with the tetramethylbenzidine reagent as
used in common forensic practice. The initial colored compound and
the color of the ultimate reaction solution are unlike those encountered
with authentic blood samples. The phenolphthalin reagent reacted
slowly but not in the manner of an actual blood stain, and so the
test results should not be mistaken for blood.
Sodium percarbonate in the presence of blood could give a positive-color
test for blood without the addition of the usual developer peroxide.
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for blood, Journal of Forensic Sciences (1991) 36:1503-1511.
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U.S. Department of Justice, Washington, DC, 1983, pp. 101-116.
Garner, D. D., Cano, K. M., Peimer, R. S., and Yeshion, T. E. Evaluation of
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blood stains: A critical survey, Journal of Forensic Medicine (1960)
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