Cyanide and Thiocyanate in Human Saliva by Gas - Journal of ...

Journal of Analytical Toxicology, Vol. 30, October 2006
Cyanide and Thiocyanate in Human Saliva by
Gas Chromatography-Mass Spectrometry
Buddha D. Paul and Michael t. Smith
Division of Forensic Toxicology, Office of the Armed Forces Medical Examiner, Armed Forces Institute of Pathology,
Rockville, Maryland 20850
r Abstract ]
A method is described for simultaneous determination of cyanide
(CN) and thiocyanate (SCN) in human saliva, or oral fluid. SCN
concentrations in body fluids appeared to be important in
classifying patients as smokers or nonsmokers, in determining
some clinical conditions, and in specimen validity testing in
forensic drug testing. The human saliva samples were diluted and
the anions were separated by an extractive alkylation technique.
Tetrabutylammonium sulfate was used as phase-transfer catalyst
and pentafluorobenzyl bromide as the derivatizing agent. The
products were analyzed by a gas chromatography-mass
spectrometry (GC-MS) with selected ion monitoring method.
2,5-Dibromotoluene was used as internal standard for quantitation
of CN and SCN in saliva. The calibration plot was linear over the
concentration range from 1 to 100 pmol/t (0.026-2.60 pg/mL) for
CN (R = 0.9978) and 5 to 200 pmol/L (0.29-11.6 pg/mL) for SCN
(R = 0.9996). The method was used to examine 10 saliva
specimens. The concentration ranged from 4.8 to 29 pmol//
(0.13-0.75 pg/mL) for CN and 293 to 1029 pmol/L (17-59.7
pg/mL) for SCN. The SCN results were similar to those obtained
from a method using oxidation of SCN to CN with colorimetric
detection (R = 0.9882). The proposed GC-MS confirmatory
method was found useful when the concentrations of CN and SCN
in saliva needed to be accurately determined.
Introduction
Thiocyanate (SCN) is usually present in serum, saliva, and
urine in low concentration (1--4). It is the principal metabolic
product of cyanide (CN) metabolism. High concentration may
be due to HCN ingestion from tobacco smoke and metabolic
conversion to SCN (5,6). When sodium nitroprusside is used to
treat a variety of cardiovascular conditions, SCN in plasma
may increase to a potentially toxic level (7). In contrast, a low
concentration of 8CN in plasma of a tobacco smoker may con-
firm a toxic amblyopia (8,9). Other clinical disorders like ataxic
neuropathy (10), tropical diabetes (11), and endemic goiter and
cretinism (12) may be due to elevated level of SCN in body
fluids from ingestion of cyanogenic glycosides in cassava. Mea-
surements of CN and SCN are of growing importance in mon-
itoring some clinical conditions and distinguishing tobacco
smokers from nonsmokers.
Many colorimetric methods have been reported for deter-
mination of SCN (1,3,6,13-16) and CN in blood and plasma
(17,18). The colorimetric methods for detecting SCN are based
on reaction between SCN and ferric nitrate as a chromogenic
reagent (16) and the Konig reaction (19). Ion chromatography
(IC) (20,21), gas chromatography (GC) (2,22-25), and high-
performance liquid chromatography (HPLC) (26) were also
used to detect SCN and CN in blood. SCN in saliva has been in-
vestigated by color reaction (4), HPLC (27), flow injection
(28), IC (29,30), fourier transform infrared spectrometry (31),
and capillary zone electrophoresis (32,33). In one procedure,
CN and SCN were separated by a gas-phase diffusion tech-
nique, transforming the ions to the corresponding chlorides by
chloramine-T, and detecting the products by a GC-MS method
(34). The procedure required careful handling of volatile com-
ponents and had several reaction steps (CN-HCN-CNC1). We re-
port here a GC-MS procedure for simultaneous determination
of CN and SCN in saliva, or oral fluid, after an extractive alky-
lation using a phase-transfer catalyst. We also present the ef-
fect of the phase-transfer catalysts on conversion of SCN to CN.
Materials and Methods
Chemical, reagents, and supplies
Pentafluorobenzyl bromide (PFB-Br), 2,5-dibromotoIuene
(2,5-DBT, internal standard), tetrabutylammonium sulfate
(TBAS), sodium cyanide, and ammonium thiocyanate were
purchased from Sigma-Aldrich (Milwaukee, WI). All solvents
and reagents were of analytical or HPLC grade.
Equipment
A GC-MS system, consisting of a 6890N GC and 5975 mass
selective detector (MSD) from Agilent Technologies (Palo Alto,
CA) was used. Helium was used as the carrier gas. The head
pressure on the capillary column (15 m x 0.25-ram i.d. x 0.20
]Jm, 5% phenyl polysiloxane, J&W Scientific, Rancho Cordova,
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. 5'[ I

CA) was 8-10 psi. The instrument was operated in splitless and
temperature program modes. To avoid peak tailing, the purge
valve was turned on at 0.50 rain after sample injection. The
MSD was operated in electron impact mode at 70 eV. The de-
tector was initially off and was turned on after a strong reagent
peak of PFB-Br (approximately 3.0 rain).
Preparation of standards and reagents
Stock solutions of CN and SCN were prepared at a concen-
tration of 10 mmol/L in water. Working solutions of CN/SCN
at concentrations of 0, 25, 50, and 100 IJmol/L in water were
used for specimen analysis. An SCN control (200 IJmol/L in
water) was used to monitor artifact formation of CN. The
phase-transfer catalyst (TBAS, 10 mmol/L) was prepared in a
saturated solution of sodium borate (pH 9.2). Derivatizing
agent (PFB-Br) and internal standard (2,5-DBT) were prepared
in ethyl acetate at concentrations of 20 mmol/L and 50 ]Jmol/L,
respectively. All solutions were stored at 2-5~ except the
TBAS solution, which was left at room temperature. The
reagents were stable for at least six months. For K6nig color
reaction, 0.6 g of barbituric acid was dissolved in 10 mL of a
mixture of pyridine and 1M hydrochloric acid (3:7, v/v). The
solution had a short shelf-life and was prepared before the
analysis.
Preparation of saliva samples
Ten unstimulated saliva (oral fluid) specimens were col-
lected from three male (M1, M2, M3) and two female (F1, F2)
nonsmoking subjects who expectorated directly into
polypropylene collection tubes. The waiting time was at least
a week when collected from the same subject. Approximately
3 mL of saliva was centrifuged at RCF 7300 xg for 30 rain. For
analysis, 0.5 mL of the clear solution was diluted to 5 mL with
water for a 10-fold dilution.
Extractive alkylation procedure
Solutions of 0.3 mL of PFB-Br (20 mmol/L), 0.2 mL of 2,5-
DBT (50 lJmol/L), and 0.8 mL of TBAS (10 mmol/L) were
added to 0.2 mL of standards (CN/SCN 0, 25, 50, 100 ]Jmol/L),
control (SCN 200 IJmol/L), and saliva specimens in 5-mL glass
centrifuge tubes. The tubes were capped, vortex mixed for 1
min, and heated at 55~ for 30 rain in a water bath. The solu-
tions were centrifuged at RCF 1700 x g for 5 rain, and ap-
proximately 100 IJL of the clear ethyl acetate solutions was
transferred into screw-capped injection vials.
GC-MS analysis
The oven temperature was increased from 60~ (held for 1.0
rain) to 90~ at 10~ and then to 150~ at 30~
Both injector and detector were set at 200~ Ions monitored
were as follows: m/z 207, 188, and 157 for PFB-CN; m/z 239,
181, and 161 for PFB-SCN; m/z 250 and 169 for 2,5-DBT (IS).
Electron multiplier was set 200 V above autotune. To enhance
sensitivity, PFB-CN in one group and PFB-SCN and IS in an-
other group were monitored. Approximately 1-3 IJL of the
ethyl acetate solution was injected onto the instrument. The
retention times for PFB-CN, PFB-SCN, and 2,5-DBT were 3.37,
5.16, and 5.41 rain, respectively.
512
Journal of Analytical Toxicology, Vol. 30, October 2006
K~nig color test for saliva specimens
The procedure was the same as that used to test CN and SCN
in blood with minor modifications (18). A solution of 0.8 mL
of perchloric acid (0.33 tool/L) was added to 0.2 mL of stan-
dards (SCN 0, 25, 50, 100, 150 tJmol/L) and saliva specimens.
After 2 rain of oxidation of SCN to CN, 0.2 mL of sodium ac-
etate (1M) and 60 IJL of sodium hypochlorite (50 mmol/L)
were added to the solutions. The so]ution was mixed, and
within a minute, 0.2 mL of barbituric acid-pyridine reagent
was added. After 5 rain, the absorbance was recorded at 583
nm. The instrument was set to zero using a water blank prior
to analysis of specimens.
Results and Discussion
In extractive alkylation, CN and SCN in an aqueous solution
reacted poorly with PFB-Br in ethyl acetate. But with the use
of TBAS as a phase-transfer catalyst the reaction began im-
mediately (CN ~ PFB-CN, SCN ~ PFB-SCN). After 30 min of
reaction, the total ion chromatogram (TIC) areas were greater
than 177,000 and 25,000 after an on-column injection of 5 pi-
comol (0.29 ng) of SCN and 12.5 picomol (0.33 ng) of CN, re-
spectively. In both cases the background was less than 1%. The
reactions were studied over a period of 120 rain. When moni-
tored by ion/IS ratios, the reactions were almost complete
after 90 rain for both CN and SCN. Because of the excellent on-
column sensitivity, we ran the reaction for only 30 rain. At this
reaction time, the yield was 55-65% for both CN and SCN. In
GC-MS analysis, three ions from PFB-CN in one group and
Table I. Cyanide and Thiocyanate (pmol/L) in Ten Saliva
Specimens Collected from Three Male (M) and Two
Female (F) Nonsmoking Subjects*
Colorimetric
GC-MS Method Method
(CN + SCN) CN/SCN (CN + SCN)
Saliva CN SCN pmol/L % pmol/L
M1-1 29 794 823 3.7 929
M1-2 11 442 453 2.5 555
M1-3 12 741 753 1.6 825
M1-4 8.2 1029 1037 0.8 1056
M1-5 5.7 595 601 1.0 634
M2-1 4.8 293 298 i .6 323
M3-1 8.8 463 472 1.9 494
F1-1 6.9 726 733 1.0 797
F1-2 15 771 786 1.9 815
F2-1 17 573 590 3.0 615
Min 4.8 293 298 0.8 323
Max 29 1029 1037 3.7 1056
Median 9.9 661 667 1.8 716
Average 11.8 643 655 1.9 704
Std 7.2 213 215 0.9 220
* Method comparison for (CN + SCN), intercept = -26.7, slope = 0.9673,
r = 0.9882.

Journal of Analytical Toxicology, Vol. 30, October 2006
three ions from PFB-SCN and two ions from IS in another
group were monitored. The identification of the compounds
was based on comparing retention times ( 2%) and relative
ion abundances ( 20%) with the reference compounds. Ex-
cellent linearities were observed over the concentration range
of i to 100 lamol/L (0.026-2.60 pg/mL) for CN and 5 to 200
lamol/L (0.29-11.6 lag/mL) for SCN. The intercept, slope, and
correlation coefficient were -0.0073, 0.9933, and 0.9978 for CN
and 0.0695, 0.0.9989, and 0.9996 for SCN, respectively. In-
terrun variations of ion/IS ratios were less than 11% (N = 5).
The procedure was used to examine 10 saliva specimens
collected from 3 male (M) and 2 female (F) subjects. Saliva in
our study is used synonymously with oral fluid to remain con-
sistent with terminology in past publications. The specimens
were initially centrifuged, and the clear solutions diluted 10-
Table II. Literature Reported Thiocyanate Concentrations
in Saliva from Nonsmokers
SCN mmol/L
Reference Methods Average -+ SD Range N
4 Colorimetric* 0.54 + 0.41 20
30 IC 0.51 + 0.31 10
31 FT-IR 0.83 + 0.42 0.41-1.25 25
32 CZE 0,23-1.20 48
Present method GC-MS 0.66 + 0.22 0,30-1.04 10
Present method Colorimetric 0.70 + 0.22 0.32-1,06 10
* For colorimetric method, the amounts represent (CN + SCN).
l,ge+0
1.6e+0
1.4r
1.2e+0
lc+07
~c+06
6c+06
4e+06
2e+05
! 3+§ to.~9.~ /
l+Fo
4,+ 1o. tlt~
+i .......... I .......
9 . .~. +, ,.+..*++ .
.... t t +
. . . . . 72 ......
~lxii t 1+4 n.,,r
%
3.1s i';-/ '~s ' '3':C L~i
5.16 PFB-SCN
5.41 I5
3.37 PFB-CN
+ A
3.20 3.40 3.60 3.80 4.00 4.20 4,40 4.60 4.80 5.00 5.20 5.40 5.60 5,80
Time (min)
Figure 1. Total ion chromatogram (TIC) and selected ion monitoring (SIM) of CN as PFB-CN (RT
3.374 rain, 5.7 +stool/L) and SCN as PFB-SCN (RT 5.16 rain, 595 tJmol/L) from a saliva specimen
(M1-5). 2,5-Dibromotoluene was used as internal standard (IS, RT 5.41 rain).
fold before testing. The results are recorded in Table I. The
method was verified by testing the same specimens by a col-
orimetric method used to test SCN and CN in blood (18). In
the procedure the SCN was oxidized to CN and tested by the
Kgnig procedure. Therefore, the result indicated total of CN
and SCN. The total CN and SCN values between the two
methods compared favorably (r = 0.9882). In all cases, the
values in GC-MS were less than those of the colorimetric
method (intercept = -26.7 pmol/L). This difference may be due
to an increase in background absorbance from the saliva ma-
trix. The SCN concentrations found in our experiments are
similar to those published in the literature (Table II). Consid-
erable variation was observed in specimens collected from the
same subject (M1, 442-1029 pmol/L). Generally, the amount
of CN in all specimens was low (average 11.8 pmol/L) and
varied in the range of 0.8-3.7% of SCN. The ion chromatogram
of specimen M1-5 is presented in Figure 1.
Linearity and precision of CN and SCN were verified in
saliva. A sample (F2-1) was tested at 5, 10, 15, 20-fold dilutions.
The corresponding final concentrations were 20.0, 16.5, 15.4,
and 15 t~mol/L (average 16.7 _+ 14%) for CN and 600, 573,
597, and 607 lJmol/L (average 594 2.5%) for SCN. Two more
specimens also gave similar results (M3-1, CN 7.9 + 11%, SCN
477 + 3.9%, and F1-2, CN 11.4 + 1.5%, SCN 827 ___ 9.2%). For
precision, M1-5 was tested in five replicates. The average +
CV% were 5.7 11.6% for CN and 595 4.3% for SCN. It ap-
peared that salivary concentrations of CN and SCN determined
by this method were linear and reproducible.
Cyanide was reported as an artifact when SCN was deriva-
tized under a basic condition (35). A basic condition was also
used to determine CN and SCN in blood (25).
In this method, SCN was derivatized in pres-
ence of benzyldimethyltetradecylammonium
chloride (BDM-TDA) as a phase-transfer cata-
lyst. We studied the BDM-TDA method and
monitored the artifact formation of CN from
SCN. Solutions of SCN (0.8 mL, 25-200
pmol/L) were mixed with 0.3 mL of PFB-Br
(20 retool/L), 0.2 mL of 2,5-DBT (IS, 50
1Jmol/L), and 0.8 mL of BDM-TDA (5 mmol/L
in saturate sodium borate in water, pH 9.2)
and heated at 55~ for 30 min. In the GC-MS
chromatogram, a peak of CN was observed.
The artifact CN concentrations were 7.2-9.2%
of SCN (average 8.5 _+ 9%), compared to only
0-0.34% by the TBAS method. Moreover,
when tested by the BDM-TDA method, the
amount of CN increased with increasing time
of reaction. No significant change was ob-
served with the TBAS method. It indicated
that the small amount of CN detected by the
TBAS was not an artifact but an impurity pre-
sent in the commercial SeN. Artifact forma-
tion of CN from SeN was also monitored in
specimen analysis. When specimen F2-1 was
tested by the BMD-TDA method, the CN and
SeN concentrations were 86 and 514 pmol/L,
respectively, compared to 17 and 573 pmol/L,
513

espectively, by the TBAS method (Table I). Standard solutions
of CN and SCN were stable in water for at least 2 months. In
different batch analysis, the ratios of CN/IS or SCN/IS were
within + 20%. Both CN and SCN were stable in saliva stored at
2--4~ for 7 days. When six specimens were tested again, the re-
sults were within _+ 20% of the original values.
Three previously published studies found concentrations of
SCN in saliva of smokers to be 1655 +_ 841 IJmol/L (N = 20),
3620 _+ 1720 tJmol/L (N = 5), and 2050 _+ 450 lJmol/L (N = 3)
(4,28,33). These concentrations are much higher than those
found for nonsmokers in this study, 655 _+ 215 IJmol/L. In a
study of smokers with toxic amblyopia, the investigators found
that plasma SCN concentrations returned to the nonsmoker
range due to an inability to convert CN to SCN (8,9,15). Other
clinical abnormalities have also been reported in individuals
who ingest cassava and have elevated plasma levels of SCN
(10-12). It has also been proposed that identifying endoge-
nous components of saliva, such as CN and SCN, could be
used to prove that specimens collected in drug testing pro-
grams are valid (36). One would expect that these various
groups could be distinguished based on CN and SCN concen-
trations in their saliva.
Conclusions
Both CN and SCN are present in body fluids in different
amounts. The validated GC-MS procedure presented here de-
tects CN and SCN in saliva as low as 1.0 and 5.0 IJmol/L, re-
spectively. In 10 specimens, the CN and SCN concentrations
ranged from 4.8 to 29 lJmol/L and 293 to 1029 IJmol/L, re-
spectively. The CN concentration was 0.8-3.7% of the SCN
concentration. The saliva SCN concentrations found in this ex-
periment were comparable with other literature procedures,
supporting validity of the GC-MS method. The procedure may
be useful in forensic drug testing when specimen validity
testing is required and also in classifying patients as smokers
or non-smokers.
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