Nitrite Adulteration of Workplace Urine Drug-Testing Specimens I ...

Journal of Analytical Toxicology, Vol. 22, March/April I998
Nitrite Adulteration of Workplace Urine Drug-Testing
Specimens I. Sources and Associated Concentrations
of Nitrite in Urine and Distinction Between Natural
Sources and Adulteration
Francis M. Urry 1,2, Gabor Komaromy-Hiller 2, Brian Staley 2, David K. Crockett 1, Mark Kushnir 1, Gordon Nelson 1,
and Richard E. Struempler 1
1ARUP Laboratories, Inc., 500 Chipeta Way, Salt Lake City, Utah 84108 and 2Department of Pathology, University of Utah
School of Medicine, Salt Lake City, Utah 84112
Abstract
The active ingredient in the commercial workplace urine
drug-testing adulterant, Klear, was previously determined to be
nitrite ion. Nitrite adulteration compromises the confirmation of
some drugs, notably the marijuana metabolite. A previously
reported bisulfite step overcomes some nitrite adulteration, but it
cannot do so in every case, which leaves the laboratory to report
the specimen as not suitable for testing. Unlike many other
adulterants, nitrite is found in normal urine at low concentrations.
In order to defend a report of nitrite adulteration, it is necessary
to provide evidence that the amount of nitrite in a workplace
urine specimen could not arise by normal means. The objectives
of this study were to identify all sources of nitrite in urine and the
range of concentrations associated with these sources and to
determine if nitrite adulteration can be supported based upon a
quantitative result. The scientific literature was reviewed for
internal and external sources of nitrite and their concentration
ranges and are reported. The following specimens were obtained
and nitrite concentrations measured by a spectrophotometric
method: clinical specimens nitrite positive by test strip
(< 15 pg/mL); specimens culture positive for nitrate-reducing
microorganisms (< 36 pg/mL); specimens from patients on
medications that may metabolize to nitrite (< 6 pg/mt); and
drug-test specimens, both negative (< 130 pg/mL) and others that
appeared to be adulterated with nitrite (range 1910-12,2])0
pg/mL, mean 5910). The literature and the nitrite measurements
of this study indicate a substantial difference between
concentrations from natural sources compared with adulteration.
A quantitative measurement of nitrite by a well-structured assay
can provide scientifically valid and forensically defensible proof
of adulteration with a nitrite-containing substance.
Introduction
The growth of employment drug testing has been accompa-
nied an increase in commercial products to help the donor avoid
being detected as a drug user. A current commercial product,
Klear, is widely available and is represented as being able to
clear all positive drug-test results. The Klear product consists of
two rnicrotubes of a white crystalline material, and each tube
contains approximately 500 rag. When added to urine after
voiding, the material readily dissolves without altering the tem-
perature, and no visual change occurs in the specimen. At the
laboratory, there is no obvious sign of adulteration, and there are
no changes in normal values for creatinine, specific gravity, or
pH. The laboratory first becomes aware of an adulterant when
the specimens' initial test is positive (usually for cannabinoids)
but fails to confirm by gas chromatography-mass spectrometry
(GC-MS) because of a substantial reduction in the recovery of
the monitored ions of 11-nor-9-carboxy-A"-tetrahydrocannabinol
(9-THCA) and its internal standard. Informal communications
among laboratorians suggested that the active ingredient may be
a form of nitrite. E1Sohly et al. (1) determined the substance to
be potassium nitrite and provided evidence that nitrite leads to
decomposition of the ions of 9-THCA and its internal standard.
They indicated that the interference could be overcome through
the use ofa bisulfite step at the beginning of sample preparation
for GC-MS analysis. The bisulfite step is helpful, but, in our
experience, it reverses the nitrite pattern only about half the
time, thus preventing the confirmation of some specimens
whose initial test is positive for cannabinoids.
If any specimen is determined to be adulterated by the lab-
oratory, and the result can be defended scientifically and foren-
sically, the Department of Transportation has authorized the
laboratory to report the specimen as adulterated and has given
the medical review officer guidance on how to proceed (2).
However, unlike other adulterants such as glutaraldehyde (e.g.,
UrinAid), soap (e.g., Mary Jane Super Clean 13), and bleach,
nitrite at low concentrations is a constituent of normal human
urine. The purposes of this study were to identify the various
sources of nitrite in urine and their associated concentrations
from the scientific literature and direct testing and to deter-
mine if a clear distinction between adulteration and other
explanations for nitrite presence can be made based on con-
centration.
Reproduction (photocopying) of editorial content of Ihis journal is F, rohibited without publisher's pemfission. 89

Experimental
The commercial product Klear was purchased from a local
merchant. The package insert directs the donor to "Add the
contents of one tube of Klear to each urine sample...For large
samples of more than 4 oz. use two tubes". The urine test strip
Chemstrip TM was used to screen for nitrite and was obtained
from Boehringer Mannheim (Indianapolis, IN).
Specimens for determination of nitrite concentrations
In order to determine the concentration of nitrite in urine, the
following specimen types were employed: clean-catch specimens
for medical urinalysis from patients who had tested positive for
nitrite with the Chemstrip and other specimens that were positive
by culture for nitrate-reducing pathogens; specimens from
patients taking medications that contained nitrate, nitro, or azo
groups, nitroglycerin, isosorbide dinitrate, a combination of nitro-
glycerin and isosorbide dinitrate, isosorbide-5-mononitrate, nitro-
furantoin, nifedipine, phenazopyridine, nitroprusside, ranitidine,
and metronidazole; specimens submitted for drug testing that
demonstrated that they may have been adulterated with nitrite by
a GC-MS assay and the Chemstrip nitrite test; and specimens
submitted for employment drug testing that were certified as
negative. In order to determine if nitrite concentration increased
with time from bacterial metabolism at room temperature, some
medical urinalysis and negative drug-test specimens were analyzed
after receipt, then held at room temperature 6-8 days and retested.
Identification and quantitation of nitrite in urine
Nitrite analysis was performed on a Lachat QuikChem AT TM
automated continuous flow analyzer (Lachat Instruments,
Milwaukee, WI) using U.S. Environmental Protection Agency
(EPA) method 353.2, Determination of Nitrate-Nitrite Nitrogen
by Automated Colorimetry, wavelength, 543 nm (3). The method
is authorized for nitrite and nitrate testing in drinking water.
When operated without the cadmium sulfate reduction step,
the method is specific for nitrite; any nitrate present does not
cross-react. The detection limit of nitrite in urine is 0.612g/mL,
which is expressed in nitrogen from nitrite ion. Because nitrite
measurements in biological matrices and in the medical litera-
ture are reported primarily in micrograms-per-milliliter nitrite
ion, the concentrations obtained by the EPA method were mul-
tiplied by the factor 3.284 to convert to micrograms-per-milliliter
nitrite ion. Negative interference occurred from residual chlo-
rine, iron, copper, and other metals. According to the EPA pro-
cedure, there are no known substances in drinking water that
give a positive interference. The testing was performed by the
Utah State Public Health Laboratory (Salt Lake City, UT), which
is certified by the EPA. The statistical significance of nitrite con-
centration changes over time was evaluated by the method of
randomized, paired comparison.
Results and Discussion
Nitrite in urine may arise from internal sources (i.e., for-
mation in tile body) or from external sources.
90
Journal of Analytical Toxicology, Vol. 22, March/April 1998
Nitrite in urine from internal sources
Mammalian biochemistry. In 1981, it was determined that
rodents and humans excrete larger amounts of nitrate than
could be accounted for from the diet, suggesting that mam-
malian cells have the biochemical processes necessary to syn-
thesize oxides of nitrogen (4,5). Studies from many disciplines
converged to identify nitric oxide radical (NO.) as the precursor
of endogenously formed nitrite and nitrate, l-Arginine is metab-
olized to citrulline and NO., which requires molecular oxygen
and reduced NADP, and is catalyzed by the enzyme nitric oxide
synthase (NOS). NO. is oxidized further to nitrite and nitrate,
which are eventually excreted primarily in the urine. NO. is an
important secretory chemical involved in the regulation of the
cardiovascular, nervous, and immune systems and among the
most vigorously researched entities in biochemistry and the
medical sciences. NOS occurs in three isoforms (6). In neu-
ronal tissues (Type I, nNOS) and in endothelium (Type III, eNOS)
the enzyme is constitutive; they are normal tissue components.
Type II (iNOS) is nonconstitutive and is discussed under Patho-
logical Conditions. The NO. produced from nNOS activity func-
tions in neurotransmission. NO. from eNOS functions as the
primary regulator of blood vessel dilation, and also has throm-
bolytic activity, as part of the body's physiological processes.
The amount of nitrite/nitrate formed from NO. by nNOS and
eNOS activities is very low (picomoles). The nitrite formed by
actions of all three NOS enzymes is taken up by red blood cells
and interacts with oxygenated hemoglobin to produce nitrate ion
and methemoglobin (7). Therefore, nitrate concentration in
urine from NOS activity is much greater than the nitrite con-
centration. With regard to the ratio of nitrate to nitrite in urine,
Moshage et al. (8) reported a study of 13 healthy volunteers
who consumed diets not regulated for nitrate content. The data
indicated average concentrations of 61 IJg/mL nitrate and 0.2
!ag/mL nitrite. The nitrite concentration was only 0.4% of the
total nitrate plus nitrite. Hovenga et al. (9) reported a normal
range of nitrate plus nitrite in urine of 0-124 1Jg/mL, which is
consistent with the data of Moshage et al. (8) cited here previ-
ously.
Pathological conditions. During inflammatory and infec-
tious processes, cytokines and endotoxins stimulate several cell
types to synthesize a third form of NOS, inducible NOS (Type II,
iNOS), iNOS activity is implicated in a wide range of patholog-
ical conditions and produces NO. under these listed condi-
tions: sepsis, asthma, rheumatoid arthritis, atherosclerotic
lesions, tuberculosis, inflammatory bowel disease, surgical
diversions of the urinary tract, allograft rejection, Alzheimer's
disease, and multiple sclerosis (6). Although nitrite concentra-
tions from iNOS are greater than that from normal physiolog-
ical processes involving NO., they are relatively modest.
Moncada et al. (10) in their literature review found that for
nNOS and eNOS, concentrations of NO. released are in pico-
moles, whereas concentrations of NO, released from iNOS are
1000-fold greater, in nanomoles. Sasajima et al. (11) reported an
increase in urinary nitrite excretion of up to 48 IJg/mL in the
healing phase of ulcerative colitis. Wagner and Tannenbaum
(12), while doing nitrate balance studies in humans on low
nitrate diets (120 pmoles per day), observed that one person in
the study became ill with nonspecific intestinal diarrhea. The

Journal of Analytical Toxicology, Vol. 22, March/April 1998
subject's average daily output of nitrate was approximately 750
lJmoles, but the nitrate output went to 4500 pmoles, a sixfold
increase, on the third day of illness. By the fifth day of illness,
nitrate output began to approach pre-illness levels. They repli-
cated this pattern in the rat by injecting a pyrogenic factor
from Escherichia coli, and obtained a ninefold increase in
nitrate output over baseline. The highest level obtained, 4500
pmoles per day, assuming average daily urinary output of 1500
mL, would produce a nitrate concentration of about 186 IJg/mL.
Nitrite was not independently measured, but assuming that
nitrite is less than 1% of the total as indicated in the Moshage
et al. (8) study, it would be less than 2 pg/mL.
Urinary tract infection. Such enterobacteriaceae as
Escherichia coli, Klebsiella, Proteus, Staphylococcus, and Pseu-
domona are frequent causes of urinary tract infections. When
numbers of these organisms are present in bladder urine in the
range of ix 10S/mL, nitrate is reduced to nitrite by the microbial
enzyme nitrate reductase. A semiquantitative test for nitrite was
recommended as early as 1922 as an indicator of a significant
urinary tract infection (13) and is still used extensively,
employing commercially available urine test strips. These strips
are based upon the Griess test for nitrite (14). The Chemstrip
indicates a limit of detection of 0.5 pg/mL. Ten clean-catch spec-
imens were obtained from a clinical urinalysis laboratory and the
nitrite quantitated by the EPA method (Table I) to assess the con-
centration of nitrite associated with medical specimens which
are test-strip nitrite positive. In order to determine if an increase
in nitrite may occur upon incubation, the specimens were stored
at room temperature for 8 days and retested. There was no sig-
nificant change (p = 0.40) due to the extended holding; six of the
specimens decreased and four increased in nitrite concentration.
The data from 18 clean-catch specimens for which culturing
indicated an enterobacteriaceae infection of approximately
100,000 colony-forming units per milliliter of urine are also
listed in Table I. Averages and ranges for specimens 1-8 (initial
and 7-day incubations) and all 18 specimens indicated no appre-
ciable difference in the average or the ranges (p = 0.93). With this
group, two specimens decreased in nitrite concentration while
six specimens increased. The highest nitrite level in any of the 28
specimens in these two categories was 35.5 IJg/mL. These data
are consistent with the Chemstrip product literature, which
indicated that urine specimens infected with nitrate reducing
organisms do not usually produce more than 100 IJg/mL nitrite
(15). As far as is presently known, nitrate reductase enzymes
capable of reducing nitrate to nitrite have not been identified in
humans or other mammals (16). In addition to the nitrite
formed from microbial nitro reductase activity, some portion of
the nitrite measured in urinary tract infection is contributed by
stimulation of iNOS activity by endotoxins from the enterobac-
teriaceae organisms (17).
Nitrite in urine from external sources
In the environment, nitrogen in the combined state occurs
mostly as potassium and sodium nitrates. From the environment,
Table I. Nitrite Concentrations in Urine Specimens that Were Nitrite Positive by Test Strip and in Specimens Positive for
Enterobacteriaceae by Culture
Nitrite (pg/mL) Nitrite (pg/mL)
Test strip nitrite positive Day 0 Day 8 Enterobacteriaceae positive Day 0 Day 7
Average
Range
1 18.4
1 5.9 4.6 2 33.5
2 7.2

industry, through biological processes of nature and agricultural
practices, nitrate and nitrite occur in food, water, and air. \~en
these items are consumed, nitrite may appear in the urine.
Food. Nitrate and nitrite salts are used as a preservative of
meats to delay decomposition and maintain color for consumer
appeal. Their concentration as a food additive is low, at 50-200
parts per million. Some vegetables, such as spinach, lettuce,
celery, and potatoes, are particularly nitrate-rich. Consumption
of vegetables and nitrite-preserved foods result in nitrite inges-
tion of 0.8-8.4 m~day (18). Under normal gastrointestinal con-
ditions, most of the ingested nitrite is destroyed by food and
gastric juices (19) and by oxygenated hemoglobin in the red
blood cells as described. Vegetables and preserved meats would
contribute less than 1 IJg/mL nitrite to the urine.
Water Nitrate in drinking water, which is largely florn runoff
flom agricultural use of nitrate-containing fertilizers, is a recog-
nized public health issue. Nitrate in drinking water can produce
methemoglobinemia (through formation of nitrite), which is a
serious condition for newborn and very young infants. In the
United States, the EPA enforces a maximum contaminant level
(MCL) of 10.2 m~L of nitrogen as nitrate plus nitrite, and an MCL
of 1 m~L of nitrite nitrogen (20). Assuming a daily consumption
of 3 L of drinking water, and a reduction of nitrite in the stomach,
the portion of nitrite in urine originating from drinking water
92
Table II. Nitrite Concentrations in Urine Specimens from Patients Receiving
Organic Nitrate/Nitro/Azo Medications*
Medication Nitrite (pg/mL) Medication Nitrite (pg/mL)
Nitroglycerin

Jourr, al of Analytical Toxicology, Vol. 22, March/April 1998
Metronidazole was of interest because it can be given in doses as
high as 4 g/day. Of the two patients on metronidazole, one was
receiving 1.5 g/day, which produced 0.8 IJg/mL nitrite. Ranitidine
resulted in a similar level of nitrite in the urine. The urinary tract
analgesic phenazopyridine was included. Although not con-
taining a nitro group, it was tested because the Chemstrip
package insert indicated that it might give a false-positive
reading for nitrite because of the orange-red color it imparts to
the urine (15). The modestly increased apparent nitrite con-
centration with phenazopyridine listed in Table II may be due to
color from phenazopyridine. Specimens from patients taking
antifungal and antineoplastic drugs were not tested. The anti-
fungal drugs are applied topically, and relatively little would
reach the urine. With antineoplastic agents, the daily dosage
does not exceed 300 mg of parent drug, and it would not increase
urinary nitrite beyond a few nanograms per milliliter.
Medical care: Medications which stimulate the in vivo for-
mation of nitrite~nitrate through induction of NOS.
]ndomethacin transfusion for premature infants lead to a 25-
fold increase in urine nitrite concentration through a currently
unknown mechanism (26). The increase returned to baseline
after 3 days, indicating that this was a transitory effect not
related to chronic administration. In order to assess the possible
effect of indomethacin in a workplace setting, two male subjecL~
of working age were given a standard dose of indomethacin
(25 rng three times per day) with specimens taken at 2, 8, 12, 24,
and 48 h after the initial dose. All specimens from both subjects
had a nitrite concentration below the limit of detection of the
method, 0.6 lJg/mL. No effect was observed.
lnterleukin-2 in cancer treatment can increase urinary
Table III. Nitrite Concentrations in Urine Specimens Received for
Employment Drug Testing which Appeared to Have Been Adulterated
With Excess Nitrite
Specimen No. Nitrite (pg/mL) Specimen No. Nitrite (pg/mL)
A* 3640 + 17 5490
1 1910 18 6300
2 4400 19 4200
3 7390 20 7030
4 10,700 21 4140
5 6770 22 4530
6 4400 23 2500
7 11,500 24 11,600
8 5420 25 7780
9 12,200 26 6730
10 8340 27 7750
11 6930 28 4040
12 4630 29 4990
13 3810 30 4140
14 3740 31 6540
15 4070 32 2920
16 4560
Average 59 I0
Range 1910-12,200
* Certified drug-negative urir+e i)(x)l, negative by lest strip for nitrite, fotlified with one vial of Klear in 50 mL urine.
r Results rounded to three significant figures because of 1000+f(~kl dilution of specimet~s.
nitrate and nitrite through the induction of cytokines and
NOS (27). This study produced a peak urine nitrate concen-
tration of 183 tJg/mL.
Intentional addition to the urine after voiding
"Fable III lists the concentrations of nitrite found in ~outine
workplace drug-testing specimens which presented to the labo-
ratory with a pattern consistent with adulteration: an initial
test positive (for cannabinoids), a failure to confirm by GC-MS
due to little or no recovery of the ions of 9-THCA and its deuter-
ated internal standard, and a strongly positive reaction for nitrite
on the Chemstrip. A clinical specimen being screened for a uri-
nary tract infection produces a light pink color on the Chem-
strip. A specimen to which Klear has been added (or crystalline
potassium nitrite for experimental purposes) produces a vivid
red-violet color that persists for approximately 20 s and then
fades to a pale orange. Each microtube of Klear contains about
500 mg of potassium nitrite. With a minimal acceptable urine
volume of 30 mL, an estimated maximum volume of 120 mL (as
may occur in a split specimen collection using a collection con-
tainer), a variable percent purity of potassium nitrite (85-96%+),
and the potential for addition of either one or two microtubes of
Klear, a urine specimen to which Klear was added would be
expected to contain between 1900 and 15,000 IJg/mL nitrite.
The 32 specimens suspected of nitrite adulteration ranged from
a low of 1910 to a high of 12,200 1Jg/mL with an average of
5910. None of the 32 specimens had a concentration less than
1500 IJg/mL. Specimen A was prepared in the laboratory by
adding one microtube of Klear to 50 mL of certified drug-free
urine that was negative for nitrite with the Chemstrip. It pro-
duced a nitrite concentration of 3640 IJg/mL.
Background nitrite concentration
in drug-negative specimens
In order to assess background levels of nitrite
in urine specimens for workplace drug testing,
specimens which were initial test negative were
evaluated. From among 200 specimens which
were tested by the Chemstrip, 15 were selected
as producing the most color on the Chemstrip
(Note: none of the 200 produced the typical
intense pattern of nitrite adulteration). These
specimens were stored at room temperature
for 2 weeks, and then tested for nitrite concen-
tration. They were held an additional 6 days
and retested. This process, although uncharac-
teristic of holding times and conditions for
employment testing specimens, was used to
represent an extreme circumstance in the
amount of background nitrite that might be
found in a negative workplace drug-testing
specimen. The data are displayed in Table IV. On
the first test (2 weeks from collection), the
group averaged 50.2 IJg/mL with a range of
6.2-128.1. The data on the retest were very
similar. The maximum nitrite concentration
obtained was 129.1 IJg/mL, which was the
highest concentration obtained in the entire
93

study, including urinary tract infections and medications. It is
probable that the four specimens with nitrite concentrations in
excess of 100 tJg/mL were contaminated by enterobacteriaceae and
had sufficient nitrate present to achieve these levels. In contrast to
these specimens, which were allowed to incubate at room tem-
perature, 15 other drug-negative specimens were selected at
random from 60 in an initial test batch and tested for nitrite con-
centration. The data are not contained in the table because all 15
were below the limit of detection of the method, 0.6 IJg/mL. These
data illustrate that specimens more typical of the age of workplace
drug-testing specimens, 12-24 h, tend to be negative or have low
concentrations of nitrite, whereas those that are allowed to incu-
bate can reach concentrations relatively high for nonadulterated
specimens.
94
Table IV. Nitrite Concentrations in Urine Specimens
Received for Employment Drug Testing which were
Initial Test Negative and Held at Room Temperature for
2-3 Weeks
Nitrite (pg/mL)
Specimen No. 2weeks 3weeks
1 19.7 33.8
2 121.2 121.2
3 37.1 36.8
4 43.0 37.4
5 6.9 7.2
6 34.8 33.8
7 122.2 118.6
8 43.0 39.7
9 6.9 7.2
10 34,2 36.1
11 128.1 129.1
12 42.7 37.8
13 6.2 7.2
14 43.7 36.4
15 63.7 116.9
Average 50.2 53.3
Range 6.2-128.1 7.2-129.I
Table V. Sources of Nitrite in Urine and Approximate
Concentrations Achievable From Them*
Source Approximate nitrite concentration (pg/mL)
Internal sources
Urinary tract infection 150
Palhological conditions 100
Normal biochemistry Trace
External source.g
Medications 25
All other natural sources
Food, water, air,
occupational exposure,
induction by medications

Journal of Analytical Toxicology, Vot. 22, March/April 1998
in the range of that obtained by adulteration were to arise in the
urine from filtration of the blood in the kidneys, the donor would
be in a toxic, life-threatening circumstance at the time of voiding.
Symptoms of nitrite poisoning include headache, nausea, vom-
iting, diarrhea, convulsions, and coma. This would be obvious to
the collector and anyone else in the donor's presence. The posi-
tive initial test result and the typical pattern in the confirmatory
test are evidences of motive on the part of the donor to adulterate.
A concentration of 1000 IJg/mL or greater of nitrite ion, deter-
mined with a well-structured and documented analysis, is scien-
tifically valid and forensically defensible proof of adulteration of
the specimen with a nitrite-containing substance. A cutoff con-
centration between 500 and 1000 pg/mL may also be supportable.
Part II of this series will present a quantitative method for analysis
of nitrite in urine, and will suggest a test algorithm for detecting
and reporting nitrite adulteration.
Acknowledgment
The authors acknowledge the excellent, professional assis-
tance of the Utah State Health Laboratory in performing
instrumental analysis for nitrite as used in public health envi-
ronmental monitoring programs.
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Manuscript received August 28, 1997;
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