Journal of Analytical Toxicology, Vol. 24, May/June 2000
A New SpectrophotometricMethod for the
Toxicological Diagnosisof Cyanide Poisoning
A. Cruz-Landeira, M. L6pez-Rivadulla, L. Concheiro-Carro, P. Fern~indez-G6mez, and M.J. Tabernero-Duque
Forensic Toxicology Service, Legal Medicine Institute, University of Santiago of Compostela, San Francisco s/n. 15705,
Santiago of Compostela, Spain
Abstract
A spectrophotometric method for the determination of hydrogen
cyanide in biological fluids based on the release of cyanide ion
by the addition of a strong acid and its subsequent specific reaction
with hydroxocobalamin to give cyanocohalamin is proposed.
The release of cyanide ion is accelerated by aeration with a stream
of an inert gas (nitrogen) that carries it into the hydroxocobalamin
solution. Although the in vitro reaction develops to completion
within 20 rain, reproducible quantitation in biological media
takes 45 rain. The cyanocobalamin formed is quantitated by
second-derivative visible spectrophotometry from the absorbance
difference between 333 and 361 nm, the measured signal being
proportional to the cyanide ion concentration in the sample.
Introduction
Cyanide poisoning, which is clinically dramatic, has lost
much of the prominence it formerly had in forensic toxicology
as a homicidal or suicidal etiology, of which there are very few
instances today. However, the current massive use of cyanide
salts for industrial purposes and the release of hydrogen cyanide
through burning of plastics and other nitrogen-containing materials, results in frequent cases of accidental poisoning. In
fact, the two main toxic substances involved in fire casualties
are carbon monoxide (CO) and hydrogen cyanide (HCN) (1).
The toxicological diagnosis of cyanide poisoning usually has
little impact on clinical therapies because the analysis for
cyanide ion is usually performed with some delay; however, it
is always important with a view to the diagnostic confirmation
of clinical and forensic poisoning (2).
The greatest hurdles posed by cyanide ion analysis result
from the typically low concentrations involved in poisoning
cases and the intrinsic instability of cyanide ion, which is relatively easily volatilized (1). These two hindrances have raised the
need to develop a straightforward, expeditious method for the
quantitative determination of cyanide ion in a small volume of
sample and with minimal manipulation (to avoid losses).
266
Available analytical techniques for the toxicological diagnosis of cyanide poisoning include fluorimetry (3,4), electrochemical detection (5), gas chromatography (6--8), and visible
spectrophotometry (2,8-11). The latter provides some advantages over the others in terms of ease of operation and economy,
which makes it affordable to modest toxicological laboratories
(2). Many spectrophotometric methods rely on the formation of
a chromogen via the K~ning reaction as implemented in a
Conway cell or similar special device (9,10). The interference of
thiocyanate ion and plasma components is avoided by determining cyanide ion in erythrocytes (8).
The proposed method is based on releasing hydrogen cyanide
from a strongly acidic medium and sweeping it into a hydroxocobalamin solution with N2, forming cyanocobalamin (2,11) in
an amount proportional to the cyanide concentration in the
sample. The cyanocobalamin is quantitated by second-derivative
visible spectrophotometry. The procedure is applicable to whole
blood, plasma, urine, and in general, any biological fluid.
Materials and Methods
Reagents
All reagents used were analytical grade. The solutions employed included 0.5M H2SO4 (Merck), phosphate buffer of pH 7,
aqueous 25 mg/L KCN (equivalent to 10 mg/L CN- ion), 100
mg/L hydroxocobalamin in the phosphate buffer, and 100 mg/L
cyanocobalamin, also in the buffer. Antifoam B (Merck) was also
used. The pure N2 source was supplied by Air Liquide.
Apparatus
Measurements were made on a Perkin-Elmer Lambda 2 UVVis dual-beam spectrophotometer equipped with a derivative accessory and furnished with cuvettes of 1-cm light path and
1-mL capacity. The instrument's operation was governed via its
bundled software (PECSS).
The nitrogen aeration device (Figure 1) consisted of pure N2
(A); 10-mL Pyrex vials, which were made air-tight by means of
a rubber stopper and a metal O-ring (B and C); 3-mL cylindrical
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Journal of Analytical Toxicology, Vol. 24, May/June 2000
glass vials (D); flexible plastic tubing of 0.5-cm i.d., to which the
ends of intramuscular (IM) puncturing needles were connected
(E), that made the connection between all elements of the
device; and disposable plastic 5-mL syringes.
Nitrogen was driven from its source (A) to an air-tight vial
that was used as diverting repository (B). To this were connected the air-tight vials holding the samples (C), by fitting as
many plastic connectors as samples were to be analyzed (six in
our case). Finally, one cylindrical vial containing 1 mL of the
hydroxocobalamin solution (D) was connected to each vial
sample. As nitrogen was bubbled through the sample, the hydrogen cyanide was carried through the solution to form the
cyanocobalamin complex.
Procedure
The experimental procedure was conducted in the described
aeration chamber. For this purpose, 1 mL of sample (0.2 mL in
cases of severe poisoning) was placed in a 10-mL vial that was
tightly stoppered (C) and then immediately sealed with
Parafilm| to ensure that the vial was really air-tight. The cylindrical vial (D) was filled with 1 mL of 100-mg/L hydroxocobalamin solution. The sample (C) and reagent vials (D) were linked
via plastic connectors as shown in Figure 1. A syringe was then
used to aspirate 5 mL of 0.5M H2SO4 and a few drops of antifoam B, and the contents were deposited into vial C. The syringe was withdrawn and replaced with the N2 supply. After
checking that the N2 bubbled through the sample and the
reagent, the stream was allowed to pass through for 45 min at
room temperature.
Results and Discussion
The first step of the process involved examining the spectra
for hydroxocobalamin and cyanocobalamin in order to confirm the feasibility of simultaneously determining both in any
region of the visible spectrum. For this purpose, the absorbance
and the first-, second-, and third-derivative spectra for both
substances over the range 300-600 nm were obtained; the
spectra exhibited extensive band overlap except in the 320--400nm region, which was thus the most suitable for discrimination. However, the spectral profile was found to depend strongly
on pH and the hydroxocobalamin concentration.
Thus, spectra made at pH values ranged 5-9 showed that pH
exerted a pronounced effect. Experiments revealed the neutral
value necessary to provide the best conditions for determining
cyanocobalamin in the presence of hydroxocobalamin. The
second-derivative spectra obtained at pH 7 allowed cyanocobalamin to be quantitated from the absorbance difference between
0.0120
~p'
/
0.0660
D2
0.1440
A. 8ourc:e of Nz
II. DI,al~'tlng l eposIto0y of Nz
G, 8ample red (1 mL e4ood § $ mL
HzSO4+ antlfoom B)
O. Cyfll~r I~11Villi (1 mL hydroxocobeMmln eolutlon, 100 ~
In
phoephate buffer of pH 7)
IL Flexible plaMIr tubing of O.S-r Ld.,
with IM puncturing needle In the
erich.
Oev•umpkm
0.2220
I
0.3000
320.0 33@; 340.0 350.0 360.0 370.0 380.0 390.0 40@;
Figure 1. Sample preparation.
nm
Figure 3. Cyanocobalamin formed by addition of 1.25 pg of CN- ion to
1 mL of hydroxocobalamin solution. Scans performed at different times
(interval time between spectra = 100 s). Time to reach equilibrium, 20 rain.
0.1000
0.0200
Table I. Stabilityof the CyanocobalaminFormedby
Additionof CN- Ion to a HydroxocobalaminSolution
(25~ 24 h)
.0.1400
.0A~
CN- Conc.
.0.2200
IHI
.0.3000
320.0 ~.c;
~.,;
3~.i~ ~o
nm
37o.~ ~o.o 3,o.~
~.o
Figure 2. Second-derivative spectra of hydroxocobalamin (H) and
cyanocobalamin (C) in phosphate buffer at pH 7.
(mg/L)
1h
0
0.1
0.25
0.5
1
0.0048
0.0254
0.0551
0.1027
0.1919
2D333_361
24h
0.0055
0.0244
0.0542
0.1013
0.1916
267
Journal of Analytical Toxicology, Vol. 24, May/June 2000
concentration of 100 mg/L was found to provide the best reo
sults, both in the phosphate buffer and in biological fluids
(blood and urine).
After the most suitable conditions for the determination of
both compounds were established, the transformation of hydroxocobalamininto cyanocobalaminby addition of 1 mL of hydroxocobalaminto increasing concentrations (equivalent to 0,
0.1, 0.25, 0.5, and 1 rag/L) ofa 10-mg/Lcyanidesolution was examined. After a variable delay of 5-30 rain, a spectrophotometric scan in the second-derivative mode was done from 320
to 400 nm, which revealed that the absorbancesat 333 and 361
nm increased with an increase in the cyanide concentration
added because of the formation of increasing amounts of
cyanocobalamin.From scans performed at different times, the
reaction was found to reach equilibrium after 20 min at pH 7
(Figure 3); beyond that point, the cyanocobalamin concentration did not vary appreciably even after severalhours (TableI).
At other pH values, the reaction was slower,more unstable, or
did not even develop at all. The amount of cyanocobalamin
formed, quantitated by the difference between the secondderivative signals at 333 and 361 nm, was proportional to the
cyanide concentration added throughout the ranges studied
(Figure 4). The calibration curve provided by these experiments in phosphate buffer fitted the equation y = 0.1865x +
0.0069 with r = 0.9990 (Table II).
The followingexperimental stage involvedapplying the proposed procedure to biological samples; preliminarily,the release
of hydrogencyanide from them upon acidificationwas checked.
0.0000
The results were found to be affectedby the previous factorsand
several others. Thus, the acid concentration and volume, the N2
0.0180
flow-rate, the aeration time, and the sample and reagent volumes, were all found to markedly influence the results.
-0.0440
A volume of 5 mL of 0.5M H2SO 4 was thus chosen--more
D2
concentrated solutions acidified the hydroxocobalaminreagent
90.1060
and introduced substantial changes in its second-derivative
spectrum.
-0.1680
As regards the aeration time and N2 flow-rate,while the reaction in the phosphate buffer reached equilibrium within 20
-0.2300
rain, biological samples required a longer time. In fact, repro32o.o 330.o
o.o 3so.o
o.0
39o.o
ducibilitywas poor with an aeration time of 30 rain and became
nm
acceptable only after 45 rain.
Figure 4. Cyanocobalamin formed by addition of increasing concentraIdeally,the N2 flow-rate should have been adjusted by means
tions of CN- (0, 0.1,0.25, 0.5, and 1 mg/L)to I mL of hydroxocobalamin
of a fine regulating device that was unavailableto the authors.
solution.
In any case, the flow-rateprovidingthe best resuits was approximately 60 mL/min. Lower
Table II. Calibration Curves for Determination of Cyanide
flow-rates considerably slowed down the reWithin-assay
action. The lack of an N2 flow-rate control
precision
mechanism diminished the reproducibilityof
Conc. range
Yield rOD
(n = 6)
the method, as reflected in the high betweenCurve
(mg/L)
Equation
r
(%) (mg/L)
assay variability observed at an early stage of
1
the study. This problem was overcome by
(Phosph. buffer) 0-I
y = 0.1865x + 0.0069 0.9990 I00
0.0005
1.5
using the aeration device describedpreviously
2
(Figure 1), which allowed the simultaneous
(1 mL blood)
0-1
y = 0.1217x + 0.0084 0.9992
66
0.044
9
measurement of various samples under iden3
tical conditions. In this way,everypoint in the
(0.2 mL blood) 0-7.5 y = 0.0191x + 0.01492 0.9966 52
0.1
10
calibration curve was simultaneously pro4
8
cessed, so the N2 flow-rate and aeration time
(2 mL urine)
0-1
y : 0.1873x- 0.0034 0.9955
50
0.028
were the same for all. In analyzing unknown
333 and 361 nm, where the second-derivativespectrum for hydroxocobalamin was very close to the baseline (Figure 2). The
dramatic influence of pH on the spectra for cyanocobalamin
and hydroxocobalaminbecame apparent at an early stage of the
experiment, where the medium for the cyanide ion standard
was provided by a 1M KOH solution. The mere addition of 10 pL
of this solution to 1 mL of the hydroxocobalamin solution
seemingly caused its massive, instantaneous transformation
into cyanocobalamin. However,the transformation also took
place to a similar extent upon addition of 10 IJL of 1M KOH containing no cyanide,so it was undoubtedlydue to the pH change.
Even a 0.1M KOH solution was found to interfere, so the
cyanide standard solution was subsequently prepared in distilled water.
As stated, one other factor markedly influencing the shape of
the spectra was the hydroxocobalaminconcentration. For this
study, solutions (in phosphate buffer pH 7) of hydroxocobalamin and cyanocobalaminin concentrations ranged between 50
and 200 mg/L were made. At concentrations above 150 rag/L,
proportionality between the concentration and the spectral
signal vanished, and spectra began to be distorted. However,the
hydroxocobalaminconcentrations must lie within a range that
allows the quantitation of small amounts of cyanide (in order to
maximize sensitivity) while avoiding saturation at a high concentration of the analyte; hence the lack of linearity between it
and the cyanocobalamin concentration. A hydroxocobalarnin
268
Journal of Analytical Toxicology, Vol. 24, May/June 2000
samples, one should measure them against a calibration curve
run from standards processed under the same conditions as the
samples, which also, in our opinion, will permit the reduction
of the time of analysis in emergency situations.
As in the experiments in the phosphate buffer, the sample
volume used was initially 1 mL. However, the yield was found
to vary with the particular type of biological sample used and to
be higher for blood than for urine.
Blood samples were analyzed using two types of calibration
curve. For mild poisoning, a sample volume of 1 mL provided
a linear relationship between the cyanide concentration (0-1
rag/L) and the amount of cyanocobalamin formed fitting the
equationy = 0.1217x + 0.0084 with r = 0.9992. This is equivalent to a yield of 66% relative to the curve obtained in the
phosphate buffer. The limit of detection (LOD), calculated as
twice the standard deviation of the blank, was calculated to be
0.044 mg/L and the within-assay variability 9% (n = 6).
Although the concentration range from 0 to 1 mg/L may be acceptable for cases of mild poisoning, and for the determination of
cyanide in blood from smokers, the need to expand it (e.g., to
0-7.5 rag/L) in order to make the proposed method
applicable to cases of severe or even deadly poisoning was considered. With I mL of sample containing such high concentrations, the hydroxocobalamin reagent was saturated and the linear
relationship between the amount of cyanocobalamin formed and
the cyanide concentration vanished. The solution to this problem
was to use a larger volume of hydroxocobalamin solution or a
smaller volume of sample. In order to save sample and reagent,
we chose the latter and used 0.2 mL of blood for concentrations
of 0, 1, 2.5, 5, and 7.5 mg/L. The LOD thus obtained was 0.1
rag/L, the within-assay variability 10%, and the equation for the
linear regressiony = 0.0191x + 0.01492 with r = 0.9966.
Urine samples were initially analyzed by using volumes of 1
mL; however, the low yields thus obtained relative to the phosphate buffer led us to chose a sample volume of 2 mL. With
such a volume and 1 mL of the hydroxocobalamin reagent,
the linear concentration range spanned cyanide concentrations from 0 to 1 mg/L, with a yield of 50% relative to the
phosphate buffer, an LOD of 0.028 rag/L, and a within-assay
variability of 8%. The equation of the calibration curve was y =
0.1873x - 0.0034 with r = 0.9955.
We examined the influence of reported interferents potentially present in biological samples. Specifically, we investigated the effect of thiocyanate, cyanocobalamin, acetonitrile,
and thiosulfates. Thiocyanate is an inactive metabolite of
cyanide itself; theoretically, it can release cyanide ion upon
acidification and give grossly overestimated results. Some reported procedures avoid its interference by removing the
plasma and determining the cyanide in erythrocytes. In our experiments, thiocyanate concentrations below 20 mg/L posed no
interference, whereas concentrations above 30 mg/L gave a
spectrophotometric signal equivalent to 0.1 mg/L cyanide.
Vitamin B12 (cyanocobalamin) can also in theory release
cyanide by acid hydrolysis; however, concentrations below 400
mg/L exhibited no interference with our determinations as
they produced cyanide at concentrations below 0.04 mg/L,
which is lower than the LOD for the proposed method.
Acetonitrile is the origin of some cases of industrial poi-
soning as it is metabolized to cyanide. It undergoes acid hydrolysis to cyanide ion, which can thus interfere with the determination. Acetonitrile concentrations below 0.5 mg/L in
blood, however, were found not to interfere significantly with
the determination of cyanide.
Consistent with the phosphate buffer, the amount of
cyanocobalamin formed after aeration was stopped was found to
be negligible, both in urine and in blood. The solutions remained stable for several hours, even at room temperature.
Sodium thiosulfate is used as an antidote against cyanide
poisoning and is also known to interfere with the determination
of this ion. With I mL of whole blood, thiosulfate was found to
interfere only above a concentration of 0.5 g/L; with 0.2 mL of
blood (severe poisoning), the interference threshold rose to
2.5 g/L.
The mildness of the interferences observed allows the proposed method to be applied to whole blood--even hemolyzed
blood--without the need to isolate and clear erythrocytes,
which would make the procedure more labor-intensive and increase cyanide losses through manipulation of the sample.
Conclusions
A spectrophotometric method for the determination of
cyanide ion based on the formation of cyanocobalamin from hydroxocobalamin and on the quantitation of the former from its
second-derivative spectrum over the wavelength range 333-361
nm was developed. The method uses affordable equipment (i.e.,
a visible spectrophotometer furnished with a derivative device
and a straightforward, laboratory-made N2 aeration device to
aerate samples). The proposed method is applicable to blood,
urine, and other types of biological sample and is sensitive
enough for the rapid diagnosis of mild cyanide poisoning.
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Manuscript received June 15, 1999;
revision received September 9, 1999.