Journal of Analytical Toxicology, Vol. 27, May/June 2003
Acute NitrobenzenePoisoningwith SevereAssociated
Methemoglobinemia:Identificationin Whole Bloodby
GC-FID and GC-MS
M.A. Martinez 1,*, S. Ballesteros 2, E. Almarza 1, C. S;inchez de la Torre 1, and S. B•a 3
1Chemistry Department, National Institute of Toxicology, Ministry of Justice, C/Luis Cabrera 9, 28002 Madrid, Spain;
2Spanish Poison Control Center, National Institute of Toxicology, Madrid, Spain; and 3Intensive Care Unit, Hospital de M6stoles,
C./RfoJ6car, s/n, 28935 M6stoles, Madrid, Spain
Abstract[
A rare fatal case of self-poisoningwith nitrobenzene following
oral ingestionis reported. On presentation to the hospital,
severe methemoglobinemia (70%) was observed in an 82-yearold malewho had ingested 250 mL of an unknown substance
in the previous24 h. Methylene blue and exchangetransfusion
were the therapeutic methods applied in the treatment
of the methemoglohinemia. Forty-eight hours after ingestion,
a blood sample was collected in ICU and sent to our laboratory.
We detected that the blood contained 3.2 pg/mL of
nitrobenzene. The determination of nitrobenzene was
performed usingthe combination of GC-FID for screening
analysisand quantitation and GC-MS for confirmation of
the obtained results.
Introduction
Nitrobenzene (NB), also known as nitrobenzol, mirbane oil,
or essence of mirbane, is a pale yellow oily liquid with an odor
of bitter almonds. NB has a melting point of 5.85~ and a
boiling point of 210.9~ It is slightly soluble in water, readily
soluble in organic solvents such as alcohol, ether, and benzene, and very soluble in lipids. Itwas first synthesized in 1834
by reacting benzene and fuming nitric acid and was made commercially in England by 1856. It serves as an intermediate in
the synthesis of aniline dyes, a solvent for the manufacture of
cellulose ethers and acetate, a flavoring agent, a perfume for
soap, and in the rubber industry (1).
NB can be absorbed by humans followingoral, inhalation, or
dermal exposure.When introduced into the blood, it is metabolized by reduction to aniline, which induces methemoglobinemia (MetHb). NB oxidizesthe iron in hemoglobin to
9Author to whom correspondenceshouldbe [email protected].
form methemoglobin, this reduces the oxygen carrying capacity of the blood and also impairs oxygen delivery to the tissues. Acute oral exposure of NB has resulted in MetHb,
cyanosis, anemia, and neurological effects including headache,
nausea, vertigo, confusion, unconsciousness, apnea, coma, and
death (2). Signs and symptoms may be delayed several hours
because some chemicals do not directly produce MetHb,but require biochemical transformation to toxic metabolites.
MetI-Ib imparts a chocolate hue to the blood. The diagnosis
should be suspected when a blood sample is brown colored
and does not redden on exposure to air. The aetiology of MetHb
may be congenital because of deficiency in cythocrome b5 reductase and structural abnormalities in the hemoglobin
molecule or could be acquired as the result of the oxidant
stress from various drugs or chemicals, most commonly nitrites
or nitrates (3). Concentrations of methemoglobin up to 20%
are generally well tolerated, although values above 40% are
associated with cardiorespiratory symptoms. Life threatening
MetHb is rare and is usually the result of acute poisoning by oxidizing compounds (4). Lethal concentrations of methemoglobin are probably greater than 70%.
Numerous cases of acute nonfatal and fatal poisoning because of the ingestion or dermal absorption of NB have been reported in the scientific literature of the 1970s, or earlier (5-9)
at a time when the chemical was a common constituent of
soaps, shoe dyes, inks, and other household products (10).
More recently, one case has been reported in Germany (11)
and three in India (12-14). However,NB poisoning is an uncommon cause of death nowadays in occidental countries. We
report a severe MetHb case after an acute ingestion of NB. The
substance was analyzed in the blood sample by gas chromatography with flame ionization detector (GC--FID)and gas
chromatography-mass spectrometry (GC-MS), following a
toxicological screening procedure used routinely in our laboratory. The analytical method was validated and described in
this paper.
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221
Journal of Analytical Toxicology,Vol, 27, May/June2003
Case History
pound and the samples were kept frozen at -25~ until used.
A telephone consult was made to our Poison Control Center
(PCC) from the Intensive Care Unit (ICU) of M6stoles Hospital
(Madrid, Spain) concerning an 82-year-old male who had intentionally ingested, in the previous 24 h, approximately 250
mL of a substance that the family alleged to be 'INhite Spirit".
The ingestion occurred in a mechanical garage owned by the
patient's son, and the container was unlabeled. At admission
into the hospital, the patient had, as the main clinical features,
a metabolic coma of toxic origin (possibly autolytic), a MetHb
of 70%, and anuric renal failure. He also suffered severe
cyanosis mainly in acra areas, mild hypotension, and Glasgow
coma score 6. He was admitted into the ICU and was intubated
under mechanical ventilation. Computed Axial Tomography
was consistent with hypoxic isquemic encephalopathy. The
chest radiograph showed a pattern consistent with bronchoaspiration. He was treated with oxygen 100%, activated
charcoal, and methylene blue, as well as exchange transfusion.
Significant blood parameters revealed: pH, 7.36; pCOz. 27
mmHg; pO~, 138 mmHg; bicarbonate, 15.7 mEq/L; leukocytes,
12,920 cells/mL; hemoglobin, 12 g/dL; hematocfit, 39%; glucose, 174 mg/dL; urea, 49 mg/dL; creatinine, 13.8 mg/dL;
sodium, 136 mEq/L; potassium, 4.2 mEq/L; and CPK, 83
lag/mL.
Because the clinical features did not correspond to a case of
poisoning with '~/hite Spirit", a differential diagnosis was made
in our PCC with acquired causes of MetHb, and a toxicological
screening was suggested. In ICU, a nasogastric tube was introduced and a thick liquid with solvent odour was extracted. A
blood sample was taken and sent for analysis to the National Institute of Toxicologywith instructions to investigate the presence of solvents.
The patient had hemodynamic instability refractory to intravenous liquids and vasopressor agents (dopamine and noradrena[ine). Shock and renal failure persisted, so continuous
venovenous hemofiltration was initiated. In spite of methylene
blue administration, MetHb persisted at 30%. Therefore, exchange transfusion was made with reduction to 18%. However, refractory shock with asystolia and death occurred three
days after admission.
Instrumentation
Experimental
Materials
Sodium sulfate, diethyl ether, and methanol of analytical
grade were obtained from Scharlau (Barcelona, Spain). The
NB and N-octylbenzene (internal standard, IS) were purchased
from Fluka-Sigma Aldrich (Buch, Switzerland). Stock solutions (1 mg/mL) were prepared by dissolving the appropriate
amount of each substance in methanol. These stock solutions
were stored in glass tubes at -25~
In order to perform the validation of the method, a pool of citrated human whole blood samples provided by Hospital 12 de
Octubre (Madrid, Spain), were tested to verify the possibility for
use as blanks. No interferences were found for the studied corn222
A P-Selecta Centronic S centrifuge was obtained from Selecta
(Barcelona, Spain). GC-flame ionization detection (PtD) analysis of the extracts was performed on a model 5890 Series II gas
chromatograph equipped with a flame ionization detector, an
autosampler 7673A, and linked to a 3396A integrator (all from
Hewlett-Packard, Avondale, PA).
A 25-m x 0.20-ram i.d. fused-silica capillary column coated
with cross-linked methylsilicone (0.1-1Jm film thickness)
(Hewlett-Packard) was employed. The carrier gas was helium
(Air Liquid, Madrid, Spain) delivered at a column head pressure
of 22 psi. The injection port and detector temperatures were
280~ and 300~ respectively. The split ratio was 1:24. The
column temperature was initially held at 40~ for 3 rain and
then increased to 280~ at 10~
The total chromatographic time, including 2 rain of equilibration time, was 29
rain. The detector gases were hydrogen and air (Air Liquid), delivered at flow rates of 40 and 400 mUmin, respectively.Insert
liners silanized with dimethyldichlorosilane:toluene (5:100)
and packed with Supelco silanized glass wool (Bellefonte, PA)
were used.
GC-MS analysis was performed on a model 5791 mass-selective detector linked to an MS Chemstation Series II (all from
Hewlett-Packard). The autosampler, gas chromatograph, and
column were as mentioned above. The MS was operated in the
electron impact mode using an electron energy of 70 eV and a
full scan mass-to-charge (re~z) ratio range of 35-650 ainu.
Transfer line and ion-source temperatures were both maintained at 280~ The presence of NB was confirmed using identical chromatographic conditions.
Sample preparation and extraction
Liquid-liquid extraction was performed in a 10-mL screwcapped glass tube by adding 1 mL of diethy[ ether (cold at 4~
and 15 mg of anhydrous sodium sulfate to 3 mL of whole blood
added with 100 laL IS solution (N-octylbenzenemethanolic solution of 100 lag/mL), vortex mixed for 3 rain, centrifuged at
4000 rpm for 10 rain, and cold after being centrifuged at 4~
The organic layer was then collected and transferred to a gas
vial and 3 laLwere injected first for GC-FID screening analysis
and later for GC-MS confirmation of the obtained results.
Validation of the method
The method described here was validated by adding NB to
human whole blood (100 mL) to achieve 0.1, 0.3, 1, 3, and 5
lag/mL.The blood was sonicated for 5 min at room temperature
then submitted to liquid-liquid extraction using the extraction
procedure described previously and quantitated by GC-FID.
Calibration curves were prepared with standard solutions of
NB in diethyl ether. The concentrations were 0.3, 1, 5, 10, and
20 lag/mL and the concentration of the IS was fixed at 10
pg/mL. NB to IS area ratios were measured, and the calibration
curves were generated from least-squares linear regression.
The regression lines were used to calculate the absolute recoveries (n = 6) of NB from spiked blood at five concentration
levels.
Journal of Analytical Toxicology, Vol. 27, May/June 2003
The intra-assay precision was assessed at five concentration
levels by the extraction and analysis on the same day of six
spiked blood samples for each level. The interassay precision
was assessed by analyzing, on two different days, a set of ten
spiked blood samples at each concentration.
The limit of detection (LOD) and limit of quantitation (LOQ)
were determined as the lowest concentration, giving a response
of three- and ten-times, respectively, the average of the base line
noise defined from six control samples.
The linearity of the method for NB was checked by preparing
six replicates of the calibration curves at five different concentrations ranging from 100-5000 ng/mL, by the addition of
known amounts of NB to human whole blood.
The sensitivity of GC-MS as a confirmation technique was
also assessed by analyzing (n = 3) the lowest NB blood spiked
levels (0.1, 0.3, and 1 IJg/mL), in the total ion chromatogram
(TIC) and selected-ion monitoring (SIM) modes.
found, and the time for the chromatographic analysis was 27
rain for one sample. Recoveries in the studied range were more
than 93.0%, with intra- and interassay precisions of less than
8.4% and 12.0%, respectively. The LOD and LOQ were 79
ng/mL and 263 ng/mL, respectively. An excellent lineafity (r2 =
TIC: 1 ~ 1 ~ 0 1 0 ~ . D
L4.4,4
3]
(50000
100000
SSO000
2
500000
12.110
4S0000
1
&00000.
8.21
)50000.
JO0000.
Results and Discussion
ISO000~
Assay characteristics
A comprehensive toxicological screening for solvents was
performed in the blood sample. This included ethanol and
volatile analysis by headspace GC-FID. The blood GC-FID
screening analysis using the described method revealed the
presence of 3.2 IJg/mL of NB. This quantitation was performed
using a blood calibration curve in the range of 0.1-5.0 IJg/mL.
During the development of the GC-FID analysis, propofol, an
anesthetic and hypnotic agent administrated in the hospital,
was also detected. The presence of other hydrocarbons and
volatile solvents were discarded. The obtained results were confirmed by GC-MS analysis in TIC mode. Figures I and 2 show
the GC-FID chromatogram and a zoom of the TIC obtained in
the (]C-MS analysis of the patient's blood, respectively. Figures
3 and 4 show the GC-MS mass-spectrum of analyte peaks
eluting at 8.21 and 12.80 rain and the resulting match of the
computerized library searches, respectively.
The results of the validation of the GC--FIDanalytical method
for NB detection are shown in Table I. No interferences were
100000~
.s0ooo4
,ooooo4
soooo4
or.-_.
,.'oo
' " ~.o'.0o " " "~='.ao"
T i m e (mln)
"z,'.oo" " " "
Figure 2. Zoom of the TIC in G C - ~ 5 analysis of the patient's blood. 1 =
NB, 2 = propofol, 3 = 5, 4 = ionol.
Scan 460 (8.207 m l n ) : M Z I 6 0 1 0 6 . D
A
(*)
B000
i 6000
SI
123
4000
2000
!
"
3
5
0
6
i ....
30
?,'
i ....
40
A/....
i ....
50,
I
i ............
60
70
m/z
16104: Benzene, nitro-
I
80
1.1,.i
....
i ....
i.l,,i
,,
90
100
110
120
130
(CAS) $$ Ni~robenzene $$ M~rbane oi
B
:1
1
2
E
E
L.
0000
!
6000
51
123
4000
b
Figure1. GCJID chromatogramof the patienfsbloodobtainedat the
hospital48 h afterthe ingestionof a commercialproduct.Detectedare:
1 = NS, 2 -- propofol,3 -- IS,4 -- iono[,and 5 and 6 = fattyacids.
2000
0
30
40
50
60
70
a0
9Q
I00
110
120
~30
m/z
Figure 3. GC-MS mass spectrum of analyte eluting at 8.21 min (A) and
nitrobenzene from data system reference library (B).
223
Journal of Analytical Toxicology, Vol. 27, May/June 2003
0.999) was observed from LOQ up to 5.0 pg/mL. The sensitivity study of GC-MS as confirmation technique should be
confirmed in GC-MS, using SIM mode. The remainder concentrations studied could be confirmed using TIC mode.
For toxicological screening in our method, N-octylbenzene
was chosen as the IS because it is not present in commercial
products, as far as we know. Its chemical structure makes it
suitable for use as an IS of a wide variety of hydrocarbon compounds (Figure 5). According to our toxicological analytical
experience, it has a very good extraction behavior [the recovery
obtained for this standard was 103% with a relative standard deScan
?94
mLn): Hg~6010$.D
(12,794
;3
(~)
A
800C
00(]
~
4000
,~
178
2000
43
77
9 i . . . .
SO
1.].'/
I
i . . . .
100
t . . . .
150
91
i
2bO
#46:
. . . .
nVz
i
250
. . . .
PROIPOFOL
i
300
. . . .
r , - ,
350
400 . . . .
m
(*)
P
800(
O
600C
~ 400C
178
2000
7791
0o '
l
2az
31z
~29
m/z
Figure4. GC-MS mass spectrum of an analyte eluting at 12.80 min (A)
and propofol from data system reference library (B).
'
50
100
190
.r
. . . .
200
i . . . .
250
J ,
300
..
, i . . . .
350
i , .~
400__
'
~)TCH3
[•2
Nitrobenzene
N-Octylbenzene
Figure5. Chemical structures of nitrobenzene and IS.
Table I. Validation Data For Nitrobenzene in Whole Blood
Liquid-Liquid Extraction and Analyzed by GC-FID
Nitrobenzene
concentration
(pg/mL)
0.1
0.3
1.0
3.0
5.0
224
Intra-assay
precision
%Recovery %RSD
(n = 6)
(n = 6)
109.0
104.0
93.0
95.2
98.0
4.2
2.2
3.0
2.2
8.4
Interassay
precision
%RSD
Linearity
(n = 10) r 2 (n = 6)
12.0
6.6
4.6
6.0
10.2
0.999
viation (RSD) of 5%].
As far as we know, there are very few reports describing analytical methods for NB detection in biological specimens
(11,23). The methods that have been developed to test NB in biological samples such as blood or gastric content are just briefly
described, and no validation data has been reported; Ewert et al.
(11) used dichloromethane and Shormakov et al. (23) used
toluene as the extraction solvent.
Poisoning characteristics
This is an unusual case of voluntary ingestion of a substance
that had MetHb as the main clinical manifestation. Relevant features of the case are the uncertainty of the etiologic agent and
the erroneous information given by the patient's relatives. The
product mentioned as the cause of the poisoning was '~Vhite
Spirit", which is actually a mixture of aromatic hydrocarbons
with boiling points in the range of 150-200~ and sold as a substitute of turpentine. They have not been described as a cause of
MetHb, therefore nitrated organic solvents were suspected by
our PCC.
From 1991 to 2000, Spanish PCC reported 5000 exposures to
products able to produce MetHb. According to our experience,
when this sign was already present, causes of poisoning were
aniline dye derivatives (present in shoe or clothes dyes and
inks), phenols, creosotes, nitrates (such as fertilizers and nitrates), and mothballs. The main routes of exposure were respiratory and cutaneous, and intoxication affected mainly
workers. Other less common etiology of MetHb, although accidents are frequently reported, include an artificial fingernail
remover (15) and herbicides (monolinuron, metobromuron)
(16,17), even diesel fuel additives. MetHb has also been described after dermal contact with an octane booster containing
80% aniline and 20% toluene (18). Etiology of poisoning with
all these products is mainly accidental, but suicidal cases are
also described (9,11,17).
Nitro and amino derivatives of aromatic hydrocarbons are
used in the chemical industry in synthetic dyes, but significant
acute damage because of them is rare in Spain. In our country,
NB or its derivatives are present at low concentration in household products (metal and furniture polish) and at higher concentrations in industrial products. NB is also used illegally to
improve combustion in car races; this could be the reason for
its presence in a mechanical garage. Only two other cases of NB
poisoning in adults had been reported to our PCC in the past 10
years. Routes of exposures were inhalation of
vapor and dermal and ocular contact with
using
vapor and liquid. They occurred in the workplace (in a printmaking industry) and in the
University (a laboratory in the chemistry department).
LOD
LOQ
The metabolism route of NB is oxidation to
(ng/mt) (ng/mt)
p-nitrophenol and reduction to aniline, which
is further oxidized to p-aminophenol. Nitrosobenzene and phenylhydroxylamine, highly
79
263
toxic compounds, are believed to be produced
as intermediates in the reduction of nitroben~
zene to aniline, which oxidizes hemoglobin to
methemoglobin. NB and its intermediary prod-
Journal of AnalyticalToxicology,Vol. 27, May/June2003
ucts are eliminatedvia the lungs and kidneys partly unchanged
and partlyafter reduction and conjugation (19). Only 13-16%
of a dose is excreted in the urine as p-nitrophenol, and probably
less than 10% as p-aminophenol. Both substances are eliminated in the form of sulfate or glucuronide conjugates. The
half-lifeof these substances is between 2 and 20 days, which explains the duration of MetHb (20) and the possibility of analytical detection after 48 h of ingestion. Unfortunately, no other
samples such as urine or gastric contents were submitted for
toxicological analysis, so a distribution study was not possible.
Our patient suffered a severe poisoning with progressive deterioration leading to fatality. Contributing factors could be the
delay in hospital arrival and the old age, because elders are at
greater risk of developing MetHb. On the other hand, NB is
slowly eliminated in the presence of renal failure causing prolonged MetHb. There could also be a delayed release of NB
from stores in the adipose tissue and gastrointestinal tract, because of its high liposolubility. Formation of MetHb by NB occurs more slowly than with aniline, but cyanosis is more
persistent. Howeverthis analytical method was also validated for
aniline (LOD=107 ng/mL), this compound was not detected in
the blood sample. Methylene blue is recommended for reversal
of the often severe MetHb that results from nitrobenzene exposure (5,21,22). There is some evidence to suggest that exchange transfusion may be beneficial when methylene blue is
ineffective. However, in spite of all therapeutic interventions,
the patient died four days after exposure.
Conclusions
A rare and fatal case of self-poisoning with methemoglobinemia after the ingestion of NB is reported. Clues for diagnosis were the history of chemical ingestion at a garage and
the clinical characteristics. NB is a substance rarely present in
commercial products in our country. Therefore, a general toxicology screening for substances causing MetI-Ib was undertaken. The toxin was rapidly identified and quantitated by a
combination of GC-FID/GC-MSscreening method in daily use
in our laboratory.To date, this is the first case of NB poisoning
for which the analytical data are described and the analytical
method was also described and validated. These results complete the previous data on NB in humans and highlight the importance of an accurate analytical identification for a more
comprehensive understanding of unusual compounds involved
in poisonings in occidental countries.
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Manuscript received June 7, 2002;
Revision received November 14, 2002.
225