A Simple Spectrophotometric Method for the Determination of Cyanide in Blood
Jerome Smith, PhD, Deborah Sammons, BS, Christine Toennis, BS, Barbara MacKenzie, BS,
Cynthia Striley, PhD, John Snawder, PhD, Marissa Alexander-Scott, DVM, Shirley Robertson
Introduction
Cyanide (CN-) is a very short-acting and powerful toxic agent. Effects of
inhalation of hydrogen cyanide (HCN) or ingestion of CN- salts are
encountered in clinical and forensic science practice. CN- is also widely
used in industry (electroplating, metal refining, fumigation, soil
sterilization). Other sources of exposure to CN- include cyanogenic
glycosides occurring in digestible plants, motor vehicle exhaust fumes,
tobacco smoke, and therapeutic treatment with sodium nitroprusside.
Blood CN- concentrations are also raised in fire victims (both survivors
and fatalities) after inhaling smoke containing HCN due to pyrolysis of
nitrogen-containing polymers. When absorbed, CN- is rapidly distributed
to all parts of the body, especially the lungs, heart, kidneys and brain. The
main toxic effects of CN- are due to its high affinity with the iron atom of
cytochrome oxidase in mitochondria which results in cytotoxic hypoxia.
Measurement of cyanide in blood is performed with a wide variety of
electrochemical and colorimetric techniques but more recent techniques
often use GC and GC-MS (1). These techniques are sensitive and specific
but involve sophisticated expensive equipment. Our goal was to develop
a simple technique that could be performed with an inexpensive ELISA
plate reader.
The
assay is an adaptation of a method developed for
in equine
blood (2). In the method, blood is added to a H2SO4 solution in a larger
cup which also contains a NaOH solution in a separate smaller cup and
the HCN generated from the blood is collected overnight in a NaOH
solution in a smaller cup. The collected CN- is then analyzed by a
colorimetric reaction. The equine blood method uses HCN generation
with 10 ml 1 N H2SO4 in a 120 ml plastic cup and collection of
generated HCN in 2.5 ml 0.25 M NaOH in a 10 ml cup. The collected
cyanide ion is reacted with chloramine-T to convert it to cyanogen
chloride which is then reacted with pyridine-barbituric acid reagent to
form a red-blue complex, whose intensity is measured
spectrophotometrically at 570 nm (Figure 1). The equine blood
procedure employs an autoanalyzer to perform the colorimetric analysis
of the collected CN-. Since the development of color by the reagents
does not have a definite endpoint but reaches a maximum intensity and
then fades, both the accuracy and the precision of the method depend on
being able to add sample and reagents at reproducible intervals which
use of the autoanalyzer allows. The autoanalyzer requires preparation of
relatively large volumes of reagents but the samples can be analyzed
automatically over a period of time
D
B
CN-
E
C
A
Figure 2: Steps in Performing Method A. Sample added to H2SO4
solution in 10 ml glass tube. B. Rubber cap with cup with NaOH solution
put on tube. C. Capped tube incubated overnight. D. Sample solution and
reagents added to 96 well plate with 8 channel electronic pipettes. E. Plate
read with ELISA reader in kinetic mode for 20 min.
The response of the method was determined for two types of samples: 1.
prepared solutions 2. spiked water. The prepared solutions were made by
diluting a standard CN- solution to the proper concentration. The spiked water
solutions were made by spiking water with standard solutions. The spiked
water solutions were then added to acid solutions in the 10 ml tubes (Figure 2
A) and the generated HCN was collected overnight in NaOH solution (Figure 2
B and 2C). Recovery was calculated by comparing the concentration of the
recovered CN to that predicted from the spiked value.
Results
pyridine
Response for Spiked Water
2500
2000
y = 2239x - 10.058
R² = 1
1500
1000
500
0
0
0.2
0.4
0.6
0.8
1
Absorbance
Figure 4: Response of method for spiked water. Water was spiked
with CN- over a range of concentrations and the CN- was recovered by
addition to H2SO4 solution in glass tube and collection of HCN
in cup with NaOH solution
Recovery from spiked water
1800
y = 0.846x - 8.6213
R² = 1
1600
1400
1200
1000
800
600
400
200
0
0
500
1000
1500
2000
2500
Spike Concentration (ng/ml)
Response for Prepared Solutions
Figure 5: CN- recovered from spiked water. Concentration recovered
versus expected concentration if 100% recovery
2500
Conclusions
2000
barbituric acid
Figure 1: Color Development Reaction
In our adaptation of the method, we added 350 µl water samples to 1 ml
1 N H2SO4 in a 10 ml glass tube which was quickly sealed with a rubber
cap which had a small cup with 350 µl 0.25 M NaOH for overnight
collection of HCN generated from the samples. The smaller tubes were
easier to handle than the larger cups and the caps were easy to install.
The same colorimetric reaction used with the equine samples was
employed but the reagent volumes were scaled to use a 96 well ELISA
plate to perform the colorimetric reactions (Figure 2). Samples and
reagents were added rapidly and accurately with 8 channel electronic
pipettes (Matrix, Thermo Fisher, Inc). The plate was incubated for 5 min
on a shaker after addition of the chloramine-T reagent to allow
conversion of the CN- to cyanogen chloride. The reading of the
absorbance in the wells of the plate was started with an ELISA plate
reader immediately after addition of the pyridine-barbituric acid reagent.
Since color development doesn’t reach a stable endpoint, the plate was
read in a kinetic mode for 20 min which records the absorbance at fixed
intervals during the time period and the maximum value of absorbance is
used as the end point in the assay and is plotted as a function of
concentration.
Concentration (ng/ml)
Cyanide poisoning occurs with exposure to cyanide compounds, such as
hydrogen and potassium cyanide, present in insecticides, smoke, car
exhaust, and some industrial processes, resulting in weakness, paralysis,
hypothyroidism, miscarriages or even death. Cyanide blood levels are a
marker of exposure often measured by gas chromatography-mass
spectrometry, which is accurate, but expensive. We have adapted an
autoanalyzer technique for cyanide measurement in equine blood, using
human blood and low reagent volumes by using an Enzyme Linked
Immunosorbant Assay (ELISA) plate reader. Cyanide in a 350 µl water
or blood sample is converted to hydrogen cyanide by adding the sample
to 1 ml of 1 M H2SO4 in a 10 ml test tube, which is rapidly capped with
a rubber cap containing a small collection cup with 350 µl of 0.25 N
NaOH which captures the generated hydrogen cyanide as cyanide ion.
After collecting overnight, the cyanide ion is reacted with chloramine-T,
in a 96-well plate, to convert it to cyanogen chloride which is then
reacted with pyridine-barbituric acid to form a red-blue complex, whose
intensity is measured spectrophotometrically at 570 nm using an ELISA
plate reader. Since color development by these reagents is without a
definite endpoint, but reaches a maximum intensity and then fades, the
absorbance measurement is started immediately after addition of
pyridine-barbituric acid reagent and the plate is read kinetically for 20
minutes. Maximum absorbance in each well is determined. Preliminary
data indicates the method has linear response from 31.2 to 2000 ng/ml
for prepared solutions and spiked water samples and the data from the
spiked samples is well correlated with prepared solutions. Further study
will better define the range and precision of the method as well as study
the recovery from spiked blood samples and determine if this method
may be used for application in biomonitoring.
CN-
Recovered Concentration (ng/ml)
Methods
Abstract
Spiked Concentration (ng/ml)
Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Cincinnati, OH
y = 1894.3x - 17.149
R² = 0.9991
•
The method is capable of measuring from 31.2 to 2000
ng/ml in water and will be tested with blood
•
The use of the ELISA plate reader makes the method
usable by more labs since the plate reader is a common
laboratory instrument
•
Use of 10 ml tubes with rubber caps is more convenient
than larger 120 ml plastic cups
•
Method could be automated using robotic ELISA sample
preparation and would have high throughput if this were
done
References
1500
1000
500
0
0
0.2
0.4
0.6
0.8
1
1.2
Absorbance
Figure 3: Response of method for prepared solutions.
Solutions were prepared by dilution of a standard solution
and run with the method
1. G Frison et. al., Rapid Commun. Mass Spectrom. 2006; 20: 2932–2938
2. C Hughes et. al., Toxicology Mechanisms and Methods, 13:129–138, 2003
The findings and conclusions in this abstract have not been formally disseminated by the National Institute for
Occupational Safety and Health (NIOSH) and should not be construed to represent any agency determination or
policy. Mention of company names and/or products does not constitute endorsement by NIOSH.