Journal of Analytical Toxicology,Vol. 29, October 2005
Spectrophotometric Detection of Iodide and
Chromic (111)in Urine after Oxidation to Iodine and
Chromate (VI)*
Buddha D. Pault and Aaron lacobs
Division of Forensic Toxicology, Office of the Armed ForcesMedical Examiner,Armed Forces Institute of Pathology,
Rockville, Maryland 20850
[ Abstract
[
Introduction
Tests for oxidizing adulterants in urine are a continuing challenge to
the drug-testing program. Iodine was found to destroy morphine and
6-acetylmorphine almost immediately. The effects were less evident
on 11-nor-Ag-tetrahydrocannabinol-9-carboxylic acid (THC-acid).
When the urine solution was tested for iodine by a chromogenic
substrate, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS), no iodine was detected. Masking drug and adulterant
simultaneously made iodine a preferred oxidizing adulterant for
drug abusers. In this study, the reduced iodide was oxidized by
sodium nitrite to iodine. The excess nitrite was decomposed by
sulfamic acid and the iodine was detected by ABTS. Linearity was
12.7 to 635 mg/L (0.1 to 5 retool/L, y= 0.9966x § 0.0016,
R2 = 1.0000). Precisions (coefficient of variation) were within
• 4.1% and quantitative accuracies were within 97% of expected
values (n = 5). Chromate, iodate, periodate, and persulfate
interfered with the method. To alleviate the problem, the positive
specimens were tested again by an iodine-specific method. After
oxidation, the samples were treated with sodium azide and
ammonium thiocyanate. In presence of thiocyanate, the azide
reduced iodine to iodide almost immediately, and the solutions
showed negative response to ABTS. The results were compared with
that of a control group tested without thiocyanate. When iodine was
present, the ratios of thiocyanate to control were less than 6%.
Chromate was also found to destroy THC-acid in urine, and during
storage most of the chromate changed to chromic (111).In this study,
chromic was oxidized to chromate by hydrogen peroxide and
sodium hydroxide and detected by 1,5-diphenylcarbazide. Linearity
was 5.2 to 156 mg/L (0.1 to 3.0 retool/L, y = 1.0285x - 0.0034,
R2 = 0.9998). Precisions were within • 8.5% and quantitative
accuracies were within 92% of expected values (n = 5). The test was
not interfered by other oxidizing agents. Both iodide and chromic
oxidation methods showed urine backgrounds less than 1.27 and
0.52 rag/L, respectively (< 0.01 retool/L). It indicated that a response
more than 10 times of the background could be considered as
oxidant contamination or adulteration of urine specimens.
* The opinion expressed herein are those of the authors and are not to be construed as official
or as reflecting the views of ~he Department of the Army or the Department (if Defense
t Author to whom correspondence should be addressed. E-mail: paul@afip osd.mil.
658
Urine adulteration to conceal drug-positive results is an ongoing challenge to the drug-testing program. Some oxidizing
agents are effective in destroying 11-nor-Ag-tetrahydrocannabinol-9-carboxylic acid (THC-acid),a THC metabolite in
urine (1-5). The effects are less evident on morphine. Chromate oxidized THC-acid completely within 24 h, whereas most
of the morphine remained unaffected (4). Moreover, some of
these agents interfere with the detection procedures (6-12). In
presence of chromate, acid hydrolysis destroyed morphine
completely. To avoid interferences, reduction of oxidizing
agents before analysis was recommended (2,5). When drug-detection procedures demonstrate interference, tests for the adulterants are recommended (13). Chromate can be detected by a
color reaction with 1,5-diphenylcarbazide (DPC) and measuring the color intensities by a spectroscopic method (4,11).
Diazo-color test is suitable for detecting nitrite in urine (14).
Methods for other oxidizing agents are difficult to establish. To
alleviate the problem, attempts were made to characterize
oxidizing properties of urine by several spectroscopic methods
(15). Unusual oxidizing properties could be attributed to adulteration of urine by oxidizing agents. However, these methods
have limited application when the oxidizing agents are lost
due to reduction. The extent of reduction may depend on the
urine constituents, storage conditions, and the type and concentrations of oxidants. Almost 45% of the chromate was lost
after addition to urine. The loss is likely due to reduction to
chromic (Cr UI). Iodine (I~ is also a potential oxidizing agent,
and like chromate, it may oxidize a drug and then be reduced
to iodide. Therefore, a specimen could be reported as negative
for a drug and oxidizing agent when both were present in the
urine. The current study is designed to investigate the effects
of iodine on THC-acid and opiates and develop a method
for detection of iodine in urine. Procedure for detecting
chromium will also be discussed.
Reproduction(photocopyinglof editorialcontentof this journal is prohibitedwithoutpuNisher'spermission.
Journal of Analytical Toxicology, Vol. 29, October 2005
Materials and Methods
Chemicals, reagents, and supplies
All oxidizing agents and chromogenic compoundswere purchased from Sigma-Aldrich Chemicals (Milwaukee,WI or St.
Louis, MO).A mixed solution of iodine (1M) and potassium iodide (2.18M)was used as an iodine stock solution. Solvents and
reagents are of analytical or high-performance liquid chromatography grade. Negative urine was collected from volunteers and used without preservatives. Phosphate buffer (0.5M,
pH 5.3) was prepared by adding a solution of Na2HPO4 (0.5M,
pH 8.95) to a solution of NaH2PO4 (0.5M, pH 4.40).
Equipment
A Spectronic | spectrophotometer (model GenesisTM 2) with
a tungsten lamp was used. The cuvettes were made of glass
suitable for absorption spectra in the range of 334 to 2500
nm. The optical path and the cell volume were 10 mm and 1.4
mL, respectively.
Preparation of 2,2'-azino-bis(3-ethylbenzthiazoline-6sulfonic acid) diammonium salt (ABTS) solution
ABTS (68.6 mg, MW 549) was dissolved in approximately
5 mL of water and then diluted to 100 mL using phosphate
buffer (0.5M, pH 5.3). The final ABTS concentration was 1.25
mmol/L (0.686 g/L). The solution was stable for at least
3 months at 3-5~
Detection of iodine in urine by color reaction with ABTS
Solutions of chromogenic substrate, ABTS (2 mL) and HCI
(50 IJL, 0.3M) were added to 50 IJL of water blank, water solutions of a mixture of iodine/potassium iodide standard (I0/I-,
1.0/2.18 mmol/L, 127/277 rag/L), controls (I0/I-, 0.5/1.09 and
2.0/4.36 mmol/L, 63.5/138 and 254/554 mg/L), and dilution
control (I~ -, 3.90/8.51 mmol/L, 495/1081 mg/L), and urine
specimens in separate tubes. All solutions with iodine turned
green. After 3 rain at room temperature, phosphate buffer (6
mL) was added to the dilution control. The absorptions were
recorded at 415 nm. Water blank was set to zero before sample
readings.
Detection of iodide in urine by nitrite oxidation and color
reaction with ABTS
The reaction involved oxidation of iodide to iodine and detection of the iodine by ABTS. Solutions of sodium nitrite (50
IJL, 50 retool/L, 3.45 g/L) and HCl (50 IJL,0.3M) were added to
50 IJL of water blank, water solutions of iodide standard (1.0
mmol/L, 127 rag/L), controls (0.5 and 2.0 mmol/L, 63.5 and
254 mg/L), and oxidation/dilution control (iodine/iodide,
3.90/8.51 retool/L, 495/1081 rag/L), and urine specimens in
separate tubes. The iodide was oxidized to iodine almost immediately. The excess nitrite was decomposed by adding a
water solution of sulfamic acid (50 IJL, 0.1M, 9.7 g/L). Immediate effervescence of nitrogen was observed. The solutions
were left at room temperature for 5 min for complete decomposition of nitrite. When a solution of ABTS (2 mL) was added
to the tubes, all solutions with iodine turned green. After 3 min
at room temperature, phosphate buffer (6 mL) was added to the
oxidation/dilution control. The absorptions were recorded at
415 nm. Water blank was set to zero before sample readings.
The absorptions were stable for at least 60 min.
Iodine-specific iodine-azide reaction in
presence of thiocyanate
In the test, two sets of standard, controls, and specimens
were used. After oxidation of iodide to iodine and decomposition
of excess nitrite by sulfamic acid, a solution of sodium azide (50
pL, 50 mmol/L, 3.25 g/L in water) was added to the tubes. To
stimulate the azide reduction of iodine, a small amount of ammonium thiocyanate (50 laL,2 mmol/L, 152 mg/L in water) was
added to one set of tubes (thiocyanate group). To the other set,
50 laL of water was added (control group). A solution of ABTS (2
mL) was added to all tubes. After 3 rain of reaction at room temperature, the absorptions were recorded at 415 nm. Water blank
from the control group was set to zero before sample readings.
Standard (1.0 mmol/L, 127 mg/L) from the control group was
used as a calibrator. Responses of standard and controls from
the thiocyanate group were less than 6% of that of standard and
controls from the control group without thiocyanate. A specimen that shows positive response without thiocyanate and
negative response with thiocyanate (< 10% of positive) indicated
the presence of iodine.
Preparation of DPC solution
DPC (151 mg, MW 242) was dissolved in approximately 15
mL of acetone in a 25-mL volumetric flask. The solution was
mixed with 5 mL of 2.5M hydrochloric acid and finally diluted
to 25 mL with acetone. The final concentration of DPC was
6.04 g/L (25 mmol/L). The reagent was used for 30 days when
stored at 3-5~ and then discarded.
Detection of chromate (Cr VI) in urine by color reaction
with DPC
A solution of DPC (100 IJL) was added to 50 pL of water
blank, water solutions of potassium chromate standard (Cr
VI, 1.0 retool/L, 52 mg/L), controls (Cr VI, 0.5 and 2.0 retool/L,
26 and 104 mg/L), and dilution control (Cr VI, 5 retool/L, 260
mg/L), and urine samples. The solutions were diluted with
3 mL of water. After 3 rain of reaction at room temperature,
3.5 mL of water was added to the dilution control and the
absorptions of all samples were taken at 541 nm. Water blank
was set to zero before sample readings.
Detection of chromic (Cr III) in urine by hydrogen peroxide
oxidation and color reaction with DPC
Solutions of sodium hydroxide (50 IJL, 0.5M) and hydrogen
peroxide (50 IJL, 0.5 tool/L) were added to 50 IJL of water
blank, water solutions of chromic chloride standard (Cr III, 1.0
mmol/L, 52 mg/L), controls (Cr III, 0.5 and 2.0 mmol/L, 26 and
104 mg/L), dilution control (Cr III, 5.0 mmol/L, 260 rag/L),
potassium chromate oxidation control (Cr VI, 1.0 mmol/L, 52
mg/L), and urine samples in separate tubes. The tubes were
closed and heated at 70~ for 15 min. The solutions were
cooled to room temperature. A solution of DPC (100 IJL) was
added to the tubes followed by dilution with 1 mL of water.
After 3 min of reaction time at room temperature, 1.25 mL of
659
Journal of Analytical Toxicology, Vol. 29, October 2005
water was added to the dilution control. The absorptions were
taken at 541 nm. Water blank was set to zero before sample
readings. The absorptions were stable for at least 60 min.
Resultsand Discussion
addition of a chromogenic compound (ABTS) and measuring
the color intensities at 415 nm. The absorption spectra with the
maxima at 415 nm are shown in Figure 1. But when iodine was
added to urine and tested immediately, no response was observed. It appeared that the iodine was reduced to iodide immediately after addition to urine. In a modified test procedure, iodide was oxidized to iodine by excess sodium nitrite and
then detected by the ABTS (Figure 2, Reactions A and D). Nitrite is known to interfere with the ABTS procedure. Therefore,
sulfamic acid was used to decompose the excess nitrite. The
evolution of nitrogen was immediate but took 5 rain at room
temperature for complete decomposition (Reaction B). Urea
could also be used to remove nitrite, but required heating at
50~ for 5 rain (Reaction C).
The quantitation of iodide in water was linear over concentration range of 12.7 to 635 mg/L (0.1 to 5.0 retool/L, y =
0.9966x + 0.0016, R 2 = 1.0000). Slope and intercept indicated
excellent linearity in this method. Because of optical saturation, the responses above the range of linearity were less than
theory. Precision and accuracy at concentrations 12.7, 127,
and 635 mg/L (0.1, 1.0, and 5.0 retool/L) were determined in
five replicates. The precisions [coefficients of variation (CV%)]
were 4.1, 1.5, and 1.8%, respectively, and the quantitative accuracies were within 97% of the expected values. In this
method, the limit of detection is considered as the lowest limit
of quantitation (0.1 mmol/L, 12.7 rag/L).
To ensure complete oxidation in a batch analysis, a solution
of iodine/iodide in water (495/1081 rag/L, 3.9/8.51 retool/L) was
used as a control. After the color reaction with ABTS, the solution was diluted with 6 mL of phosphate buffer (0.5M, pH
5.3). The final concentration was within _+87% of the expected
value (1576 rag/L). Dilution of color instead of dilution of
specimen is the advantage of this method. When iodide concentrations in specimens were above linearity, the colored solutions were diluted and the absorptions were recorded again.
Stable absorption for at least 60 rain minimized drift in results
in a batch analysis of more than 20 samples. For an iodide solution of 127 mg/L (1.0 mmol/L), the absorption at 60 min was
within 98% of that at 3 min.
Twelve urine specimens found negative for iodine were
spiked with iodine/iodide to a concentration of 495/1081
Iodine effect on drugs and its stability in urine
Many oxidizing adulterants are known to destroy THC-acid
and morphine in urine (1-6). Most of the oxidants that contained oxygen in the molecule require hydrogen ion to facilitate oxidation. When both acid and oxidizing agent are added
to the urine, the pH may drop below the normal pH of urine
(normal, pH 4.5-8.0) (16). To investigate adulteration, the
drug-testing program requires all specimens to be tested for pH
(13). Iodine is also a potential oxidizing adulterant for drugs of
abuse but requires no acid for oxidation. Moreover, iodine
could easily be reduced to iodide, and there is no suitable procedure available for detection of iodide in urine. Iodine is
poorly soluble in water, but dissolves in a solution of KI. In an
experiment, 97.4 IJL of iodine/iodide (1.0/2.18N) was added to
25 mL of urine preheated to body temperature (97~ After addition, the final temperature dropped to 92~ and the color was
slightly iodine-type (tint red) but within the realm of normal
urine (straw color). On standing for 24 h at room temperature,
the color gradually reverted to the original color of the urine,
indicating possible reduction of iodine to iodide. Effects of iodine/iodide on 6-acetylmorphine (50 ng/mL) and morphine
(2000 ng/mL) in urine at 97~ were evaluated. After addition to
the drug solutions, the final concentrations of iodine/iodide
were 495/1081 mg/L (3.9/8.51 retool/L). The drugs were extracted immediately using a solid-phase extraction procedure
previously described (17). In the extraction, acid hydrolysis
was avoided and to save the internal standards from oxidation, 6-acetylmorphine-d6 and morphine-d3 were added after
the solid-phase extraction. Both 6-acetylmorphine and morphine could not be detected in urine, indicating total oxidation
of the compounds by iodine. Iodide showed no effect on the
drugs even when acid hydrolysis was performed on morphine
glucuronide.
Iodine effects on oxidation of THC, 11hydroxy-THC, and THC-acid were also
0.900 ]
evaluated. The oxidation reactions were
0.800
similar to that used for morphine. The
samples were tested immediately and after
0.700
24 h at room temperature using a proce060O
Iodine oxidized ABTS
dure previously described (5). Recoveries
0.5oo
of THC, 11-hydroxy-THC, and THC-acid
=~
0.400
were 92%, 62%, and 66%, respectively.
~
0.300 I
No further losses were observed when
tested after 24 h at room temperature. It
appeared that the iodine oxidized the
drugs immediately and then reduced to
45O
inactive iodide.
~
Chromate-DPC Complex
541 nm
4r~"~Z,
0.20O
0.100
Test for iodide/iodine
Iodine in water can be tested by simple
660
5OO
55O
6O0
65O
Wavelength(nm)
Figure 1. Absorption spectra of iodine-oxidized ABTS and chromate-DPC complex.
70o
Journal of Analytical Toxicology, Vol. 29, October 2005
mg/L and tested for free iodine. No iodine was detected. The
specimens were stored frozen at -18~ for 15 days and tested
for total iodide using nitrite oxidation method. In 11 specimens, the concentrations were 1154 • 105 mg/L (recoveries
86 + 7.8%), compared to 1340 mg/L for the iodine/iodide
dilution control. In the test, the colored solutions were diluted to get concentrations within the limit of linearity. In
one specimen (Specimen 9) the recovery was only 32%. To
examine the storage effect at 2--4~ the specimens were
stored again in a refrigerator for 7 days. The recoveries after
the oxidation reaction were 69.9 • 9.6% (58 to 85%, n = 11)
of dilution control (1381 rag/L) (Table I). At this time, iodide
was not detected in specimen 9 (< 2 mg/mL). The total loss
may be due to substitution of iodide on some urine constituents that could not be oxidized by nitrite. However, in
most cases (11/12) the iodide can be detected when frozen for
at least 15 days.
ABTS reaction is not specific for iodine. Other oxidizing
agents react with this reagent (15). After initial screening by
the nitrite oxidation method, the specimens were tested again
A.
41"+ 2NO2" + 6H +
9
410 + N 2 0 + 3H20
B.
NO2- (excess)+ H + + "OSO2NH2
9
N2 + HSO4- + H 2 0
C.
2N02" (excess) + NI'I2CON'I-12+ 21-I+
D.
I~ + ABTS
m, 2Nz + CO2 + 3H20
9
ABTS-chromogen
(C_n-een, kin,= 415 rim)
Figure2. Oxidation of iodide to iodine and detection of iodine by ABTS.
by an iodine-specific detection procedure. The reactions and
test procedure are outlined in Figure 3. In the experiment, two
sets of standards, controls, and specimens were used. After
the nitrite oxidation of iodide to iodine, sodium azide was
added to the samples. Iodine and azide reaction is very slow, but
in presence of ammonium thiocyanate, the reaction was almost
instantaneous. In this process, iodine was reduced to iodide.
After adding sodium azide in all tubes, a small amount of ammonium thiocyanate (50 IlL, 152 mg/L, 2 mmol/L) was added
to one set of samples [ammonium thiocyanate (SCN) group].
Water (50 pL) was added to the other set to compensate the
volume (control group). When ABTSwas added to the samples,
the control group without ammonium thiocyanate showed
positive color response while the other group with thiocyanate
showed no response (Table I). Samples with iodide showed response with thiocyanate less than 6% of that without thiocyanate (ratio of SCN:control < 6%).
To determine the specificity of the thiocyanate-induced reaction, several oxidizing agents at concentration 20 mmol/L
were tested (Table II). In the oxidation reaction, hydrogen peroxide and sodium oxychloride (bleach) showed no response.
Chromate, iodate, periodate, and persulfate responded to ABTS,
but after the sodium azide-thiocyanate reaction the SCN:control response ratios were 87% or more, compared to less than
6% for the iodide standard and controls. None of the oxidizing
agents interfered with the assay.
To determine urine adulteration by iodine, a background
response was established. The same 12 urine specimens as
blanks were tested by the nitrite oxidation procedure. The iodide concentrations were less than 1.27 mg/L (0.01 retool/L) in
Table I. Nitrite Oxidation of Iodide to Iodine Followed by Sodium Azide Reduction in Presence of Ammonium Thiocyanate
(SCN)
Concentration
Control (No SCN)
Sample*
I~ + I(mg/L)
Absorption
Standard, IControl, IControl, IControl, I~
127
63.5
254
1576
0.340
0.169
0.691
1.024
1576
1576
1576
1576
1576
1576
1576
1576
1576
1576
1576
1576
0.805
0.648
0.635
0.620
0.787
0.872
0.752
0.691
0.004
0.856
0.598
0.646
Conc.found
(mg/L)
127
63.1
258
1381
With SCN
Conc.found
(mg/L)
SCN/ControlRatio
(%)
0.006
-0.021
0.037
0.03
2.2
nd*
13.8
11.2
1.8
<I
5.4
0.8
0.014
-0.004
0.009
0
0.033
0.041
0.01
0.024
-0.001
0.013
0.015
-0.008
5.2
nd
3.4
nd
12.3
15.3
3.7
9.0
nd
4.9
5.6
nd
0.5
<I
0.4
<1
1.2
1.3
0.4
1.0
nd
0.4
0.7
<1
Absorption
Specimens
I
2
3
4
5
6
7
8
9
10
11
12
1085
874
856
836
1061
1176
1014
932
nd (< 2)
1154
806
871
* Specimens were spiked with iodine/iodide, 495/I 081 mglL (total 1576 mg/L) and stored at -I 8~ for I S days and again at 2-4~ for 7 days before analysis.
t Absorption readings of iodine/iodide control and spiked specimens were after 3.61 times dilution.
* n d = not detected.
661
Journal of Analytical Toxicology, Vol. 29, October 2005
11 specimens. In specimen-9,the concentration was 58.4 mg/L.
When examined under the SCANmode (400 to 700 nm), the
spectral characteristic of specimen-9 was completely different
from that of the standard. Moreover, in the iodine-specific
thiocyanate procedure, the SCN/control ratio of the specimen9 was 96%. It clearly indicated that the interfering response
from specimen-9 was not from iodide or iodine. From the
urine background it appeared that a specimen could be considered as contaminated or adulterated when the iodine concentration is more than 10-fold of the background (> 12.7
mg/L, > 0.1 mmol/L).
Chromate effect on drugs and its stability in urine
Like iodine, chromate is also an effectiveadulterant in concealing drug-positive results (4--6). Loss of THC-acidwas more
than 94% when exposedto chromate in urine. The oxidant also
interferes with the detection of morphine. Potassium chromate
in urine was tested by a chrornogenic reagent DPC. Solutions of
chromate in four urine specimens (156 rag/L, 3 retool/L) were
left at room temperature for 18 h and tested by DPC. The reReactions:
2I~ + 2NAN3
2I~ + 2NaNa
~
SCN"
2NaI + 3Nx (Slow re,action)
9
2Nal + 3N2 (Fast reaction)
Procedure:
I~ + N3Test[ Control
scNz_...j L__~SCNi- + N2 4------'---"
~
l~
ABTS
I
-
ABTS;
No or weak absorption (T)
Positive absorption (C)
Requirement: To be positive for iodine the percent of T/C has be less than 10%,
Figure 3. Iodine specific
iodine-azide reaction and detection procedure.
coverieswere 75 + 9% (63-85%). On standing for additional 22
days at 3-5~ the recoveries decreasedto 55 _+9% (42-64%). It
appeared that the recoveries depend on storage conditions and
constituents in urine. The loss of chromate could be attributed
to reduction chromate to chromic (Cr III or Cr3§
Test for chromic/chromate
Chromic in water was oxidized to chromate by hydrogen
peroxide and sodium hydroxide. The oxidation was complete
within 15 min at room temperature. But in urine the oxidation required heating at 70~ for 15 rain. The chromate was
detected by a solution of DPC in acetone and hydrochloric
acid (Figure 4). The absorption spectra of chromate-DPC
complex with the maxima at 541 nm are shown in Figure 1.
The quantitation of chromic in water was linear over the
concentration range of 5.2 to 156 mg/L (0.1 to 3.0 mmol/L,
y = 1.0285x - 0.0034, R2 = 0.9998). Slope and intercept indicated excellent linearity. Optical saturation was observed
above the linearity. Precisions (CV%,n = 5) at concentrations
5.2, 52, and 156 mg/L were 4.4, 6.3, and 8.5%, respectively.
The accuracies at the same concentrations were within 92~
of the expected values. To ensure complete oxidation, a solution of chromate in water (52 rag/L) was used as control.
The average chromate concentration in seven different batch
analyses was 52 + 1.7 mg/L, indicating complete oxidation of
chromic to chromate. The batch also contained a dilution
control with chromic concentration at 260 mg/L (5.0
mmol/L). After the color reaction, the solution was diluted
with equal amount of water. The final concentration was
within 95% of the expected value. In specimen analysis, when
the concentrations were outside the range of linearity, the
colored solutions were diluted and the absorptions were
recorded again. This avoided specimen reanalysis when the
concentrations were more than 156 mg/L. Stable absorption
for at least 60 rain minimized drift in results in a batch analysis of more than 20 samples.
Table II. Detection of Urinary Iodide and Interfering Oxidants by Nitrite Oxidation Followed by Sodium Azide Reduction in
Presence of Ammonium Thiocyanate (SCN)
Concentration
Control(No SCN)
I~ + I-
Sample
Iodide standard
Iodide control
(mg/L)
127
63.5
Absorption
Conc.found
(rag/L)
With SCN
Absorption
Conc.found
(mg/t)
SCN/ControlRatio*
(%)
0.330
0.168
127
64.8
0.001
0
0
0
< 1
< 1
0.042
0.185
0.325'
-0.001
-0.005
0.992
6.8
98
189*
nd*
nd
578
0.044
0.163
0.486t
0.004
-0.004
1.464
6.8
85.8
281'
nd
nd
852
100
87
> 100
nd
nd
> 100
Interfering oxidants
Chromate
Iodate
Periodate
Hydrogenperoxide
Sodiumoxychloride
Persulfate
1040
3500
3820
680
1030
3940
* Interfering oxidants either not detected or showed ratios > 87% compared to < 1% for standard and control.
t Values were increasing over time.
* nd = not detected.
662
Journal of Analytical Toxicology, Vol. 29, October 2005
References
A.
2Cr~++ 3H2Oz + 8OH-
I~ 2CRO42"+ 2H + + 6H20
B.
Cr042"+ DPC
9
Chromium-DPC
(Red-violet, ~
complex
541 run)
Figure 4. Hydrogen peroxide oxidation of chromic to chromate and detection of chromateby DPC.
Chromate recoveries in four urine specimens were evaluated
at concentration of 156 mg/L. After 18 h at room temperature
and 22 daysat 3-5~ the recoveries were 54%, 64%, 42%, and
59%, respectively.But after oxidation, the recoveries were 93,
94, 94, and 92%, respectively,for these specimens. It indicated
that the hydrogen peroxide oxidation of chromic to chromate
was almost quantitative for these specimens. Urine background
from 12 specimens was evaluated and found to be less than
0.52 mg/L (0.01 retool/L, 0-0.0093 retool/L). Therefore, a specimen that shows chromic oxidation response more than 10-fold
of background may be considered as contaminated or adulterated by chromium compounds. The procedure was tested for
interferences from iodine, iodate, periodate, nitrite, sodium
oxychloride,and persulfate at 20 mmol/L in urine. Iodate and
periodate responses were 0.61 and 0.63 retool/L, respectively,
but in SCAN(400-700 nm) the absorption spectra were completely different from that of the standard. Cross-reactions
from all other oxidants were almost none. Final verification
under the SCANmode is important to eliminate false-positive
result from interferences.
Conclusions
Capillary electrophoresis with indirect UV detection and
high-performance ion chromatography with conductivity
detector are viable alternatives for detection of iodide, chromate, and chromic ions. But these separation technologies require processing each sample separately and may take at least
3 h for a batch analysis of 20 samples. In the chemical oxidation and chromogenic detection procedures, the reactions are
specific to the target compounds and required only 20-30 rain
for analysis of the same number of samples. Moreover, the
chemical procedure may be automated to test a large number
of samples in a relatively short time. In the chemical procedure, it is recommended that the specimens initially be
screened by taking readings at the absorption maxima (km~),
and if positive, the same solutions may be tested again under
SCANmode in 400-700 nm and the results compared with that
of a standard.
1. C. Baiker, L. Serrano, and B. Lindner. Hypochlorite adulteration
of urine causing decreased concentration of delta-9-THC by
GC/MS. J. Anal. Toxicol. 18:101-103 (1994).
2. M.A. ELSohly, S. Feng, W.J. Kopycki, T.R Murphy, A.B. Jones,
A. Davis, and D. Carr. A procedure to overcome interferences
caused by adulterant "Klear" in the GC-MS analysis of 11-nor-AgTHC-9-COOH. J. Anal. Toxicol. 21" 240-242 (1997).
3. S.-C.J. Tsai, M.A. EISohly, T. Dubrovsky, B. Twarowska, J. Towt,
and S.J.Salamone. Determination of five abused drugs in nitriteadulterated urine by immunoassays and gas chromatography-mass spectrometry. J. Anal. Toxicol. 22:474-480 (1998).
4. B.D. Paul, K.K. Martin, J. Maguilo, and M.L. Smith. Effects of
pyridinium chlorochromate adulterant (Urine Luck) on testing
drugs of abuse and a method for quantitative detection of
chromium (VI) in urine. J. Anal. Toxicol. 24:233-237 (2000).
5. B.D. Paul and A. Jacobs. Effects of oxidizing adulterants on detection of 11-nor-A9-THC-9-carboxylic acid in urine. J. Anal. Toxicol. 26:460-463 (2002).
6. J.T. Cody, S. Valtier, and J. Kuhlman. Analysis of morphine and
codeine in samples adulterated with StealthTM. J. Anal. Toxicol.
25:572-575 (2001).
7. J.T. Cody and R.H. Schwarzhoff. Impact of adulterants on RIA
analysis of urine for drugs of abuse. J. Anal. Toxicol. 13:277-284
(1989).
8. A. Warner. Interference of common household chemicals in immunoassay methods for drugs of abuse. Clin. Chem. 35:648-651
(1989).
9. W. Bronner, P. Nyman, and D. yon Minden. Detectability of
phencyclidine and 11-nor-A9-tetrahydrocannabinol-9-carboxylic
acid in adulterated urine by radioimmunoassay and fluorescence
polarization immunoassay. J. Anal. Toxicol. 14:368-371 (1990).
10. R. Schwarzhoff and J.T. Cody. The effects of adulterating agents on
FPIA analysis of urine for drugs of abuse. J. Anal. Toxicol. 17:
14-17 (1993).
11. A.H.B. Wu, B. Bristol, K. Sexton, G. Cassella-McLane,
V. Holtman, and D.W. Hill. Adulteration of urine by "Urine
Luck". Clin. Chem. 45:1051-1057 (1999).
12. J.T. Cody and S. Valtier. Effects of StealthTM adulterant on immunoassay testing for drugs of abuse. J. Anal. Toxicol. 25:
466-470 (2001).
13. Department of Health Human Services. Mandatory guidelines
for federal workplace drug testing programs. Fed. Regist. Part III.
69:19644-19673 (2004).
14. P. Griess. Remarks on the paper of H.H. Wesley and Benedikt
Uber on some azocompounds (in German). Ber. Deutsch Chem.
Gen. 12:426 (1879).
15. B.D. Paul. Six spectroscopic methods for detection of oxidants in
urine: Implication in differentiation of normal and adulterated
urine. ]. Anal. Toxicol. 28:599-608 (2004).
16. E. Jungreis. Spot Test Analysis. John Wiley & Sons, New York, NY,
1985, pp 29-30.
17. B.D. Paul, E.T.Shimomura, and M.L. Smith. A practical approach
to determine cutoff concentrations for opiate testing with simultaneous detection of codeine, morphine, and 6-acetylmorphine in
urine. Clin. Chem. 45:510-519 (1999).
663