Clinical Chemistry 45: 1051-1057, 1999;
(Clinical Chemistry. 1999;45:1051-1057.)
© 1999 American Association for Clinical Chemistry, Inc.
Adulteration of Urine by "Urine Luck"
Alan H.B. Wu1,a,
Ben Bristol1,
Karen Sexton1,
Gina Cassella-McLane1,
Verena Holtman1 and
Dennis W. Hill2
1
Department of Pathology and Laboratory Medicine, Toxicology Laboratory, Hartford Hospital, 80 Seymour St., Hartford, CT 06102.
2
Microchemistry Laboratory, U-193, University of
Connecticut, Storrs, CT 06269.
a Author for correspondence. Fax 860-545-3733; e-mail awu{at}harthosp.org
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Abstract
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Background: In vitro adulterants are used to invalidate assays for
urine drugs of abuse. The present study examined the effect of
pyridinium chlorochromate (PCC) found in the product "Urine Luck".
Methods: PCC was prepared and added to positive urine controls at
concentrations of 0, 10, 50, and 100 g/L. The controls were assayed for
methamphetamine, benzoylecgonine (BE), codeine and morphine,
tetrahydrocannabinol (THC), and phencyclidine (PCP) with the Emit II
(Syva) and Abuscreen Online (Roche) immunoassays, and by gas
chromatography/mass spectrometry (GC/MS). Two tests were also developed
to detect PCC in urine: a spot test to detect chromate ions using 10
g/L 1,5-diphenylcarbazide as the indicator, and a GC/MS assay for
pyridine. We tested 150 samples submitted for routine urinalysis,
compliance, and workplace drug testing for PCC, using these assays.
Results: Response rates decreased at 100 g/L PCC for all Emit II
drug assays and for the Abuscreen morphine and THC assays. In contrast,
the Abuscreen amphetamine assay produced apparently higher results, and
no effect was seen on the results for BE or PCP. The PCC did not affect
the GC/MS recovery of methamphetamine, BE, PCP, or their deuterated
internal standards, but decreased GC/MS recovery of the opiates at both
intermediate (50 g/L) and high (100 g/L) PCC concentrations and
apparent concentrations of THC and THC-d3 at
all PCC concentrations. Two of 50 samples submitted for workplace drug
testing under chain-of-custody conditions were positive for PCC,
whereas none of the remaining 100 specimens submitted for routine
urinalysis or compliance drug testing were positive.
Conclusions: PCC is an effective adulterant for urine drug
testing of THC and opiates. Identification of PCC use can be
accomplished with use of a spot test for the oxidant.© 1999
American Association for Clinical Chemistry
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Introduction
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Workplace urine drug testing continues to be an important program
for employers in the private and public sectors. Under the guidelines
established by the Substance Abuse and Mental Health Services
Administration, testing involves a two-step process of screening by
immunoassay, and confirmation by gas chromatography/mass spectrometry
(GC/MS)1
(1). Employees who test positive for a drug class
may suffer untoward consequences, ranging from denial of employment to
suspension from work and dismissal. As a result, some individuals
attempt to conceal their drug abuse by drinking large volumes of
liquids to dilute urine drug concentrations (in vivo adulteration) or
by adding foreign substances to urine after collection to invalidate
testing procedures (in vitro adulteration) (2). Some in
vitro adulterants act by interfering with the immunoassay detection
scheme. Others convert the target drug to compounds that do not bind to
the antibodies used in the immunoassays or that produce negative
results in subsequent confirmation testing (2). Some states
have outlawed the use of urine drug testing adulterants. Commercial
products are available to assist an individual in "passing a drug
test". One of the earliest products was "UrinAid". The active
ingredient was glutaraldehyde, which interfered with screening
immunoassays by producing final absorbance rate readings that were
lower than those of true negative urine samples (3). The
presence of glutaraldehyde can be detected by direct colorimetric
analysis for aldehydes. "Klear" or "Whizzies" are commercial
adulterants that contain potassium nitrate and cause interferences in
the GC/MS analysis of marijuana [tetrahydrocannabinol (THC)]
metabolites (4). Their presence can be suspected by a low or
absent recovery of added deuterated THC internal standard (IS) and
confirmed by direct measurement using colorimetric procedures
(5). The latest product is "Urine Luck". The active
ingredient of this adulterant is 200 mmol/L pyridinium chlorochromate
(PCC). The present studies were conducted to determine the conditions
under which Urine Luck interferes with screening and confirmation
analyses for drugs of abuse.
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Materials and Methods
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materials
Urine Luck was purchased through the Internet from Spectrum
Laboratories. Commercial urine quality-control materials were from
Bio-Rad Laboratories (Lyphochek) and Euro/DPC Ltd. (CONDOA Level II).
Two different controls were used because they exhibited different pH-
buffering capacities. The pH of the Bio-Rad control was adjusted to 6.0
and 8.0 before the addition of PCC. The pH of the CONDOA was adjusted
to 6.0 before adulteration. All pH measurements were performed on a
digital meter. For the reaction-vs-time study, the CONDOA control
was used without prior pH adjustment (pH 5.9). Adulterated
urine controls were stored at room temperature before analysis. The
controls nominally contained 1200 µg/L methamphetamine; 360 µg/L
total morphine; 360 µg/L benzoylecgonine (BE), a cocaine
metabolite; 60 µg/L 9-carboxy-THC (THC-COOH); and 30 µg/L
phencyclidine (PCP). Except for the opiates, for which the cutoff
concentration has been raised to 2000 µg/L, these concentrations
exceeded the current Substance Abuse and Mental Health Services
Administration cutoff concentrations by ~20–25%. Because the
control material did not contain codeine, we supplemented a drug-free
urine sample (pH 6.0) with a codeine calibrator to a final
concentration of 500 µg/L and examined the effect of PCC.
Immunoassays for barbiturates, benzodiazepines, methadone,
propoxyphene, and other drugs of interest were not tested because these
drugs are not part of the Federal workplace drug testing panel. A 200
mmol/L solution of PCC (Sigma Chemical Co.) was prepared in saline. The
pH of this solution was <1.0. Control samples were supplemented with
10, 50, and 100 g/L PCC. These concentrations were selected because the
instructions accompanying Urine Luck call for dilution of 90–150 mL
(3–5 oz) of urine with the vial containing 7 mL of the adulterant
(final urine PCC concentration, 47–78 g/L). To identify the active
component of Urine Luck, 200 mmol/L potassium chlorochromate (Sigma)
and pyridine (Aldrich) were also obtained and prepared separately.
Pyridine (10 g/L) was added to one vial of the Bio-Rad Level II
control. Chlorochromate (10 g/L) was also added to a separate vial of
the Level II control.
immunoassays
Unadulterated and adulterated quality controls were tested for
five drug classes with the Emit II (Syva) and Abuscreen Online (Roche
Diagnostics) assays on Hitachi 717 and Cobas Integra chemistry
analyzers, respectively. Both reagents were used according to the
manufacturers' recommendations (no extra dilutions made). For Emit II,
the absorbance rates were recorded relative to the cutoff calibrator,
which was set to zero. Under these conditions, any positive absorbance
rate was indicative of a positive drug result. For Abuscreen, the
approximate concentrations of drugs were recorded in µg/L. To
simulate routine workplace drug testing procedures, adulterated samples
were stored at room temperature for a minimum of 2 days before
screening or confirmation testing.
spectrometric analysis and spot test for the adulterant
Absorbance curves for blank urine and that adulterated with 50 g/L
PCC were measured on a scanning ultraviolet-visible spectrophotometer
(Cary 219; Varian). The wavelength was scanned from 380 to 620 nm,
using a 1-cm pathlength quartz cuvette. A spot test was developed using
an indicator solution of 10 g/L 1,5-diphenylcarbazide in methanol. This
indicator detected the presence of chromate anions and was colorless
when first prepared (6). Two drops of the indicator were
added to 1.0 mL of urine. A positive result was the production of a
reddish-purple color.
gc/ms
Samples were assayed quantitatively by GC/MS analysis in the
selected-ion monitoring mode, using procedures in routine use at the
Hartford Hospital Forensic Toxicology Laboratory. A summary of the
specific conditions used is shown in Table 1
. Drug assays were conducted on a Hewlett-Packard 5890A gas
chromatograph 5970B mass selective detector.
A GC/MS assay was developed for the detection of pyridine. To a 5-mL
urine sample, 4.0 g of potassium carbonate was added with
intermittent mixing. Acetone (0.5 mL) was added and mixed for 5 min.
Under these high salt conditions, acetone forms a separate (top) layer
with aqueous urine. The samples were centrifuged at 1000g. A
100-µL aliquot of the acetone layer was placed into a vial for GC/MS
analysis. A Finnigan Mat ITS40 GC/ion trap mass spectrometer was used
for pyridine analysis. A 1-µL sample was analyzed (splitless
injection) on a 0.25 mm diameter DB-5 capillary GC column (J&W
Scientific). The initial column temperature was 35 °C, which was
ramped to 120 °C at 15 °C/min, and then to 280 °C at
50 °C/min, and held for 1 min. The temperatures of the injector and
transfer lines were 275 and 280 °C, respectively. Full-scan electron
ionization spectra were collected from m/z 30 to 200 atomic
mass units at a scan rate of 600 ms/scan. All processed spectra were
background subtracted. A drug-free urine sample supplemented with 2.5
and 5g/L PCC was used to make the reference calibrators.
urine samples
Fifty urine samples submitted for routine workplace drugs-of-abuse
analysis were assayed using the spot test. Some of these samples were
suspected of having been adulterated with PCC on the basis of an
unusual color or low pH. Samples positive for the spot test (putatively
because of chromate ions) were assayed by GC/MS for pyridinium. As
controls, 50 urine samples submitted for routine urinalysis testing at
Hartford Hospital and 50 urine samples submitted for compliance
toxicology testing were also assayed using the spot tests. There was no
reason for donors of these samples to adulterate their urine with PCC.
nitrite and chromium analysis
Four urine samples positive for the spot test and negative for
pyridinium by GC/MS were further tested for nitrites, using a
qualitative urinalysis dipstick (Chemstrip; Roche Diagnostics) and
quantitative nitrite assay (nT; Chimera Chemical) on an automated
chemistry analyzer (Hitachi 717; Roche). Nitrite is absent in
unadulterated urine. These samples were also sent to National Medical
Services (Willow Grove, PA) for chromium analysis using graphite
furnace atomic absorption spectroscopy.
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Results
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The spectrophotometric absorbance curve of a 50 g/L solution of
PCC in saline is shown in Fig. 1
(spectrum A). A clearly defined inflection occurs in this
spectrum at 440 nm. Spectrum B in Fig. 1
is the spectrum of an
unadulterated urine specimen. Spectrum C in Fig. 1
is the spectrum of
the same specimen adulterated with 50 g/L PCC. Other than a large
increase in the absorbance at 440 nm, there are no distinguishing
features in the absorbance spectrum of the adulterated sample. A
similar spectrum would be expected with a urine sample that contains
hemoglobin because of the presence of the Soret band at 400–440 nm
(7). The adulterated urine itself appears a little more
orange than normal, but was within the realm of possible urine colors.
Spectrum D in Fig. 1
is the spectrophotometric absorbance curve for a
urine sample adulterated with 50 g/L PCC and reacted with the
indicator. A major absorbance peak is present at 550 nm, which does not
interfere with the native absorbance of the urine or urine to which the
adulterant was added (Fig. 1
, spectra A and C). The indicator is
colorless and contributes nothing to the visible spectrum. In the
absence of the adulterant, the spectrum of a urine specimen containing
the indicator is identical to that of urine without the indicator.
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Figure 1. Spectrophotometric absorbance of a 50 g/L solution of PCC
in saline (spectrum A), drug-free urine (spectrum
B), drug-free urine adulterated with 50 g/L PCC
(spectrum C), and urine after addition of the spot test
indicator (spectrum D).
The absorbance maxima are at 550 nm. The absorbance spectrum of the
drug-free urine (spectrum B) did not change with the
addition of the colorless spot-test indicator.
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The effects of PCC adulteration on immunoassay screening using the Emit
II and Abuscreen systems are shown in Tables
2 and
3. There was no effect in either system for the BE assay except
for a minor (10%) drop in the absorbance rate and concentration at the
highest adulteration concentration (100 g/L). There was no significant
change in the results of the BE GC/MS analysis (Fig. 2
). Only at high PCC adulterant concentration (100 g/L) did the
amphetamine assay give a response rate less than the threshold value
for Emit II (Table 2
). For Abuscreen, the apparent concentration was
actually higher with increasing PCC concentrations. Although we were
not able to determine why this occurred, the increase had been reported
previously with the in vitro adulterant nitrite (8). A
reduction in the absorbance rate was observed for the Emit II PCP
assay. GC/MS analysis showed <10% interference for methamphetamine
and no interference for PCP (Fig. 2
). These data suggest that the low
pH of the highly adulterated samples was likely the mechanism for the
interference in the Emit II assay, rather than a chemical alteration to
the target drug. The Emit II package insert list an operational
immunoassay pH range of 4.5–8.0. Urine specimens with pH values below
(or above) this range will interfere with the activity of the enzyme
label, glucose-6-phosphate dehydrogenase. It is unclear why low pH did
not interfere with the BE assay to the same extent. The Abuscreen
apparently was less subject to interferences by extremes in pH. (No
restrictions for pH were listed on the manufacturer's package insert.)
Urine Luck had a greater effect on the results of the opiate and THC
immunoassays. For morphine, there was a significant drop in the
morphine signal at 50 g/L PCC, whereas at 100 g/L PCC, the result was
below the 300 µg/L cutoff concentration. A similar effect in the Emit
absorbance rate was also observed for codeine. GC/MS analysis (Fig. 2
)
confirmed these findings: morphine decreased in concentration from 391
µg/L (blank) to 11 µg/L (100 g/L PCC, with ion ratios outside
acceptable limits), whereas a more modest drop was observed for codeine
(589 to 368 µg/L, respectively). The recoveries of the deuterated IS
for morphine and codeine were unaffected by the presence of PCC. We
were not able to determine what morphine or codeine were converted to
by the PCC adulterant.
For THC, the destruction of the drug begins at the lowest adulterant
concentrations and was complete at 50 g/L for both the Emit II and the
Abuscreen assays (Tables 2
and 3
). GC/MS results also confirmed that
the drug was degraded by PCC. Moreover, the deuterated IS added to the
GC/MS assay was also progressively destroyed in the presence of
increasing concentration of the adulterant, as shown in Table 4
. Unlike the other assays, the mechanism of interference for
this adulterant appeared to be a combination of a suboptimum pH and
oxidation effects. The GC/MS assay for the THC-COOH metabolite was
particularly vulnerable to degradation because the solid-phase
extraction for THC metabolites was conducted at low pH (2.0 ±
0.5), further facilitating the oxidation of the drug and its IS.
The effect of 10 g/L PCC adulterant on the Emit II results as a
function of time after adulteration are shown in Table 5
. There was no additional degradation in the resulting
absorbance rates beyond the first few hours.
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Table 5. Absorbance rates ( A/min) for control
samples supplemented with 10 g/L PCC vs time using the Emit II
assay.
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The colorimetric spot test (screening assay) for PCC detects the
presence of chromate. In unadulterated urine, the total chromium
concentration (Cr+3 and
Cr+6) is 1.9–38.4 nmol/L (9). Even in
toxic overdoses, urine concentrations are far below the values found in
PCC-adulterated samples (10 mmol/L for a 50 g/L solution). The spot
test was not sensitive enough to detect chromate ions in unadulterated
urine samples. Under physiologic urine pH conditions, the color
development took 5–7 min for completion and remained stable for hours
(data not shown). Under increasing acid conditions, the reaction was
faster, although the resulting color faded after 1 h. When this
spot test was used on 50 samples submitted for toxicologic analysis, a
positive result was obtained for 9 urine samples (Table 6
). Sufficient amounts of urine for GC/MS analysis were available
on six of these samples. Pyridine was identified in two of these six
samples. Fig. 3
is a representative GC/MS analysis of an adulterated urine
sample submitted for toxicology testing. Under alkaline extraction
conditions, pyridinium was dissociated from the metal complex to form
free pyridine. The major features were the M+1 ion at m/z
80, the M ion at m/z 79, and a fragment ion at
m/z 52. Ions at m/z 43 and 59 were produced from
residual bleed from the acetone solvent, which could not be background
corrected. The spot test was negative for all 100 samples submitted for
routine urinalysis and compliance drug testing.
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Table 6. Results of the spot test and GC/MS confirmation for PCC in
urine samples submitted for workplace drug
testing.
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Figure 3. Gas chromatogram and mass spectrum (inset)
of a urine sample submitted for toxicologic analysis and containing
pyridine putatively adulterated with Urine Luck.
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We determined that the active ingredient of Urine Luck was the
chlorochromate anion, and not pyridine or its combination with
chlorochromate. A 10 g/L solution of either PCC or potassium
chlorochromate reduced the THC Emit II response for the control from
-31 A/min for the unadulterated urine, to -60 and -54
A/min, respectively, for the
chlorochromate-adulterated samples. A 10 g/L solution of pyridine alone
produced no change in response relative to the unadulterated urine
(-25 A/min).
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Discussion
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Detecting the presence of adulterants is essential in reducing the
rate of false-negative results for urine drugs-of-abuse testing.
Nitrites can be readily detected by a urinalysis dipstick test or
through direct chemical analysis. Because nitrites do not interfere
with immunoassays, samples containing concentrations of THC above the
threshold will be routinely assayed for the THC-COOH metabolite by
GC/MS. A urine sample that exhibits little or no recovery of a
deuterated IS or target drug is highly suggestive of an adulterant such
as nitrite (4).
Using elemental analysis, mass spectrometry (for pyridine), ion
chromatography (for chloride), and inductively coupled plasma atomic
emission spectroscopy, the active ingredient in Urine Luck was
determined to be PCC. Urine samples adulterated with PCC tested by
GC/MS will produce results that are similar to those for nitrite, i.e.,
low or no recovery of the IS or target drug. This adulterant differs
from nitrite, however, because at high concentrations, the chemical
also produces negative immunoassay screening results. Thus, drug
testing laboratories unaware of the presence of PCC will not perform
GC/MS analysis, and the use of PCC may escape detection. Its presence
can be suspected by the presence of an abnormally low pH or the
appearance of an orange tint to the urine.
The spot test described in this report may be a simple screening test
that can be used for further testing of suspected urine samples. This
test is not specific for chromate ions because molybdenum, mercury, and
vanadium salts can also produce a positive reaction (6).
However, it is unlikely that a spot test will be positive under normal
physiologic or pathologic conditions or after environmental exposure
because the expected metal concentrations in these urines would be
substantially lower than those in situations of in vitro adulteration
(9). For laboratories performing a large volume of forensic
urine drug testing, an instrument-based colorimetric assay may be more
convenient, although none are commercially available at this time. The
specificity of the spot test for PCC is of concern because we were able
to confirm only one-third of the positively screened samples for
pyridine. It may be possible that other oxidizing adulterants yet
unknown to toxicology laboratories are being used that contain metal
ions or substances capable of oxidizing drugs of abuse. Both PCC and
potassium chlorochromate react with the nitrite urinalysis dipstick to
produce a purple color that is darker than the color that occurs for
nitrites; therefore, the dipstick method could be used as an
alternative or in addition to the spot test.
Confirmation of the presence of pyridine may be performed by direct
GC/MS analysis for pyridine. When the DB-5 column was used, pyridine
eluted very early in the gas chromatogram. Use of a more polar column,
such as carbowax, will likely enable solvents such as pyridine to be
retained longer on the column. However, these polar columns are not
routinely used for drug-of-abuse testing and, therefore, are less
readily available. Although the molecular ion of pyridine is
m/z 79, we also consistently observed the M+1 ion. This
suggested that some chemical ionization putatively occurred, with
saturation of ions within the source. Because the adulterant was not
tested on the mass selective detector, it is unknown whether this
phenomenon was specifically related to testing using the ion trap.
The combination of a colorimetric assay for chromate and GC/MS assay
for pyridine is forensically acceptable for the detection of PCC. Each
uses a different analytical technique, and each targets a different
chemical aspect of the adulterant. With routine screening,
manufacturers might opt to remove Urine Luck from the market, as was
the case for UrinAid. Undoubtedly it will be replaced with another
adulterant.
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Footnotes
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1 Nonstandard abbreviations: GC/MS, gas chromatography/mass spectrometry; THC, tetrahydrocannabinol; IS, internal standard; PCC, pyridinium chlorochromate; BE, benzoylecgonine; THC-COOH, 9-carboxy-THC metabolite; and PCP, phencyclidine. 
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References
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Top
Abstract
Introduction
, methamphetamine; 
, benzoylecgonine; 
, 9-carboxy-THC, pH 8.0;
, 9-carboxy-THC, pH 6.0; 
, PCP; •, morphine.