Alkyl nitrite esters have been abused, particularly among male
homosexuals,10 and high doses result in toxic11,12 and fatal13-15
effects, such as methemoglobinemia.13 Immunosuppressive10 and
blood cell destructive16 effects have been also reported. These
esters are unstable in blood and water and readily undergo
hydrolysis to nitrite and alcohol.6,17,18 In this report, we introduce
briefly a case of IBN poisoning, report the experimental results
concerning the behavior of IBN in a canned coffee drink solution,
and report important information for toxicologists concerning the
decomposition of toxic substances and related formation on other
toxic substances with regard to artifacts.
tions were determined by the headspace gas chromatography (HS-
GC) method. A total of 500 µL of the above-mentioned gas phase
was injected into the following GC. The GC instrument used was
a model HP 6890 gas chromatograph (Yokogawa Analytical
Systems, Tokyo, Japan) equipped with a flame ionization detector
and split injector. A capillary column HP-WAX (30 m × 0.25 mm
i.d., 0.25-µm thickness, Yokogawa Analytical Systems) was used
as the stationary phase. The column head pressure of carrier gas
(helium) flow was adjusted to 7.67 psi, and the splitter ratio was
adjusted to 30. The oven temperature was controlled by a
temperature program (initially at 40 °C (3-min hold), then to 115
°C at 15 °C/ min). The injection port and detector were maintained
at 200 and 220 °C, respectively.
EXPERIMENTAL PROCEDURES
i
The presence of IBN and BuOH were confirmed by GC/ mass
Reagent. IBN was purchased from Tokyo Chemical Industry
(Tokyo, Japan). All other chemicals used were of analytical grade.
All aqueous solutions were prepared with deionized, distilled
water.
spectrometry (MS), which consisted of an HP 5890 series II gas
chromatograph combined with an HP 5989B quadrupole mass
spectrometer (Yokogawa Analytical Systems). The stationary
phase was DB-WAX (30 m × 0.25 mm i.d., 0.25-µm thickness,
J&W Scientific, Folson, CA). The carrier gas (helium) flow rate
was 0.67 mL/ min. The injection port, transfer line, and ion source
were maintained at 200, 280, and 200 °C, respectively. The splitter
ratio was adjusted to 47. Electron impact ionization (EI, ionization
energy 70 eV, ionization current 60 µA) was used as the ionization
mode. The oven temperature was controlled by a temperature
program (initially at 40 °C (3-min hold), then to 200 °C at 10 °C/
min (3-min hold)). The acquisition mass range was 33-150, and
the sampling was 0.8 scan/ s. Acquisition was started 4 min after
sample injection.
Cyanide Assay by Headspace Gas Chromatography. Cya-
nide concentrations were determined by the HS-GC method, as
described previously.19,20 A total of 500 µL of the above-mentioned
gas phase was injected into an HP 5890A gas chromatograph
(Yokogawa Analytical Systems), which was equipped with a
nitrogen-phosphorus detector and a GS-Q column (30 m × 0.53
mm i.d., J&W Scientific). The carrier gas (helium) flow rate was
4.7 mL/ min. The injection port, detector, and column oven were
maintained at 200, 250, and 140 °C, respectively. The splitter ratio
was adjusted to 3.5. The quantification limit was roughly 0.05 nmol
(S/ N ) 3.5) of cyanide in the HS vial.
Capillary Electrophoretic Determination of Nitrite and
Nitrate Anions. Nitrite and nitrate anions were determined using
a Quanta 4000E CE system (Waters, Milford, MA). The capillary
column used was a fused-silica column (75 µm i.d. × 60 cm), and
the electrophoresis buffer was 5 mM sodium chromate (pH 8.0)
containing 0.5 mM CIA-Pak anion-BT (Waters). The voltage was
set at 20 kV with a negative power supply. The detection was by
indirect ultraviolet absorption at 254 nm. The column temperature
was maintained at 25 °C. Samples were applied hydrodynamically
for 30 s. Nitrite and nitrate migrated at 3.5 and 3.6 min and were
well separated from chloride (3.3 min) and sulfate (3.4 min).
Headspace Incubation of Isobutyl Nitrite in Various Types
of Solutions. In a screw cap septum vial (7 mL, GL Science,
Tokyo, Japan), 0.05 mL of acetone solution containing IBN or
isobutyl alcohol (iBuOH) and 0.95 mL of an aqueous solution
which contained various additives (buffer or coffee solution) were
added and the vial was then sealed with a Tuf-Bond disk. For the
i
IBN and BuOH assay, the mixture was allowed to stand at 30 °C
for an appropriate time interval, and 0.5 mL of the gas phase in
the headspace was injected into a GC using a glass syringe. For
the cyanide assay, the mixture was allowed to stand at 50 °C for
30 min, and 0.5 mL of the gas phase was injected into a GC.
Storage Experiment for Isobutyl Nitrite in Water and a
Coffee Solution. Twenty milliliters of water or a commercial
canned coffee drink (Gorgia, Japan Coca Cola Co., Tokyo, Japan)
and 140 mg of IBN were added to a glass-stoppered conical beaker
and stored at 4 °C. Two hundred microliters of the mixed solution
was periodically sampled into an HS vial containing 0.5 mL of 1
M phosphate buffer (pH 7.0), 0.25 mL of water, and 50 µL of
acetone and incubated at 30 °C for 10 min, and 0.5 mL of the gas
i
phase was injected into GC for IBN and BuOH analysis. For the
cyanide analysis, 0.2 mL of the storage solution was sampled into
an HS vial containing 0.5 mL of 1 M phosphate buffer (pH 6.0)
and 0.3 mL of water and incubated at 50 °C for 30 min, and 0.5
mL of the gas phase was injected into GC. For the nitrite and
nitrate assay, an aliquot of the storage sample was diluted 100-
fold with water, filtered through a 0.45-µm cellulose acetate
membrane, and applied to capillary electrophoresis (CE).
Headspace Gas Chromatographic Determination of Isobu-
tyl Nitrite and the Isobutyl Alcohol. IBN and iBuOH concentra-
(10) Haverkos, H. W.; Kopstein, A. N.; Wilson, H.; Drotman, P. Environ. Health
Perspect. 1 9 9 4 , 102, 858-861.
(11) Sharp, C. W.; Brehm, M. L. Ann. Int. Med. 1 9 8 0 , 92, 700-701.
(12) Osterloh, J.; Olson, K. Ann. Int. Med. 1 9 8 6 , 104, 727.
(13) Dixon, D. S.; Reisch, R. F.; Santinga, P. H. J. Forensic Sci. 1 9 8 1 , 26, 587-
593.
RESULTS AND DISCUSSION
Report of the P oisoning Case. In the autumn of 1998 at a
(14) O’Toole, J. B.; Robbins, G. B.; Dixon, D. S. J. Forensic Sci. 1 9 8 7 , 32, 1811-
1812.
(15) Bradberry, S. M.; Whittington, R. M.; Parry, D. A.; Vale, J. J. Toxicol. Clin.
local motor hotel, one of a young couple drank one portion of a
canned coffee drink taken from a refrigerator. She immediately
noticed a strange taste and spit most of the liquid from her mouth.
Toxicol. 1 9 9 4 , 32, 179-184.
(16) Soderberg, L. S. F.; Flick, J. T.; Barnett, J. B. Exp. Hematol. 1 9 9 6 , 24, 592-
596.
(17) Osterloh, J.; Goldfield, D. J. Anal. Toxicol. 1 9 8 4 , 8, 164-169.
(18) Osterloh, J. D.; Goldfield, D. J. Pharm. Sci. 1 9 8 5 , 74, 780-782.
(19) Seto, Y.; Tsunoda, N.; Ohta, H.; Shinohara, T. Anal. Chim. Acta 1 9 9 3 , 276,
247-259.
(20) Seto, Y. Jpn. J. Toxicol. Environ. Health 1 9 9 6 , 42, 319-325.
5188 Analytical Chemistry, Vol. 72, No. 21, November 1, 2000