Perspectives on urine adulteration before testing for drugs of abuse

P A T H O L O G I S T S
LANCET
L A B O R A T O R I E S
N E W S L E T T E R
<strong>Perspectives</strong> on urine adulteration
before testing for drugs-of-abuse
Reviewed by Dr. T Laurens, MFSS,FRSC
Lancet Toxicology Centre - August 2007
E-mail laurensj@lancet .co.za
PAGE 1
Introduction
Sample adulteration is a serious potential problem in forensic urine drug
testing. A variety of substances have been employed to interfere with testing
procedures in hopes of causing the sample to yield a negative result. Many
of these substances have been shown to produce false negative results
however, some substances, depending on the methodology, cause false
positive results. Some adulterants cause samples to be negative since the
drug or metabolite of interest is changed or destroyed while others affect the
assay itself. Although the adulteration of urine samples to avoid detection of
drug use is not a recent phenomenon, in the last several years a number of
cottage industries have sprung up to assist in this endeavor, providing
everything from negative urine for substitution, to additives that cause
negative results in the immunoassay tests. This work describes many of the
methods and substances used in adulteration of urine samples. It also
describes the effect of the adulterants on the immunoassay screening test
along with the influence on the confirmatory testing.
Definition of Sample Adulteration
An adulteration can be defined as any process by which an individual
knowingly interferes with (or attempts to interfere with) the processes of
specimen collection, transport or analysis with the intention of avoiding a
legitimate test result. The actions undertaken can include (but are not limited
to) addition of water or foreign substances to the specimen, specimen
substitution, damaging bottle seals or packaging and the deliberate
consumption of interfering substances or copious volumes of water prior to
specimen collection.
Adulteration practices
The adulteration practices are summarized in Table 1.
Table1: Forms of adulteration for urine testing
Form of adulteration
Substitution
In vivo
In vitro
Inadvertent
Description
Replacement of a freshly voided sample
with a drug free sample
Ingestion of water, substance, or drug to
hasten excretion or inhibit excretion
Addition of a foreign substance directly to a
sample and to invalidate test results
Ingestion or exposure to food or preparation
that results in a positive urine test result in
an individual who is not abusing a drug.
Substitution
Substitution includes the practice by which a urine specimen from a drug
abusing donor is switched by urine from a drug free individual. Two
procedures are currently in use for detection of substituted urine
Monitoring urine temperature immediately after the
collection - Unfortunately, acceptable temperatures can be
achieved if the substituted urine is stored in the axils, vaginal
cavity, or next to the scrotum just prior to donation. It is of
prime importance for the collection officer to adopt procedures
to minimize the risk of adulteration of the specimen during the
collection procedure.
o
o
An essentially impossible to detect substitution, which
admittedly represents an extreme action on the part of
drug abusers, is the placement of drug free urine, saline,
or other fluids, into the bladder using a catheter.
The fluid is excreted in a normal fashion, and evades
even the strictest direct observation or temperature
measurement. This process holds a number of
potentially various serious health hazards to the
individual.
A less extreme method of substitution, although not in vivo,
is accomplished using a tube running to the genital area
from a concealed container. The liquid is being delivered
into the sample collection container. This method can be
detected with a stringent observation process or by placing
strict temperature requirements on the sample
1 News Letter No. 2 of Drugs-of-Abuse
o
A similar method is to place clean urine into a condom and to
tape it to the groin area, or to insert it into the vaginal cavity. The tip
of the condom is then pierced with a pre sharpened
fingernail, allowing the urine to flow into the container.
Careful witnessing of the collection of urine itself. Effective samegender
witnessed collection, requiring close
observation of urination, is an unpleasant duty for most
individuals. Even with close observation where the individual
is closely monitored during a void, substitution can still occur.
The donor can conceal a pouch of drug free urine and release
its contents directly to the urine cup.
In vivo adulteration
Dilutional: The intentional ingestion of fluids, substances, and/or
drugs designed to dilute urine or to hasten or increase the
metabolism and/or excretion of drugs in the body. Water is an
effective in vivo dilution adulterant and in the case of psychogenic
polydipsia a patient routinely consumes large volumes of water,
which can dilute urine or electrolytes by up to ten fold. Drugs will
present at or near the cut-off and negative results can be produced.
Phencyclidine (PCP) and tetrahydrocanabinol (THC) are not
excreted in very high oncentrations into urine and can be diluted by
excessive fluid intake. Other drugs such as opiates and cocaine
produce drug concentrations that can exceed ten times the cut off
concentrations, particularly when urine is donated soon after
the drug is used. For these individuals, dilutional adulterants
may not be effective in producing the desired negative result,
because it is not possible to consume enough water to reduce
urine drug concentrations to that extent. Diuretics are drugs
used for the treatment of heart failure, hypertension, hepatic
ascites, pulmonary edema, and renal edema. There are many
types of natural and synthetic diuretics. It should be noted that
some diuretics give the urine an unusual color, indicating the
presence of an unnatural condition. Xanthine compounds are
diuretics found in popular beverages like caffeine (coffee and tea),
theophilline (tea) and theobromine (cocoa). Xanthines increase
blood flow to the kidneys and may produce a diuretic action through
an increase in the glomerular filtration pressure.
Acidic or basic food drugs or liquids that are highly acidic (such as
vinegar) or basic (such as sodium bicarbonate) can produce
sufficient changes in urine pH to interfere with immunoassay
screening procedures. Acidification or alkalisation can affect the
metabolism and rate of clearance for drugs. For the basic drug
amphetamine, as much as 74% of the parent compound is excreted
in urine that is slightly acidic. In alkaline urine (intake of sodium
bicarbonate/baking soda), only 1% is excreted as the parent
compound. In a similar way, acidification has been reported to
enhance the excretion of weakly basic drugs.
Miscellaneous drugs and substances
There are many other drugs and substances that can cause negative
interferences with drugs that could be considered candidates for an in vitro
adulterant by a drug abuser. Compounds like salicylates (aspirin), Ibuprofen,
and fluorescein (used in retinal angiography) can invalidate the test such that a
re-collection may be required. Thus the drug-positive individual may escape
detection on that particular instance and might abstain from use before recollection.
Inadvertent adulterants
The consumption of certain foods and drinks can lead to the
inadvertent true-positive on a drug screen but a false positive
indication of drug abuse. The finding of positive urine results
for opiates by individuals eating bakery products containing
poppy seeds before a drug test is well known. Poppy seeds
contain varying concentrations of codeine and morphine. Poppy
seed consumption will produce a true-positive opiate result in
urine, and a false positive indication of drug abuse by the donor.
Y:\LANCET NEWS\2007\Perspective on urine adulteration before testing for drugs of abuse.CDR

P A T H O L O G I S T S
LANCET
L A B O R A T O R I E S
N E W S L E T T E R
Food produced from farm animals is naturally rich in
creatinine. This byproduct of muscle metabolism in the animal
is identical to that produced in humans. Therefore, the
consumption of meat temporarily increases serum and urine
creatinine concentrations. This may obscure the laboratory's
attempts to identify diluted human specimens if creatinine is
the only dilutional marker used.
There are several examples whereby prescription drugs can
produce falsely positive immunoassay results for targeted
drugs-of-abuse. Well known examples include:
o Sympathomimetic amines in the amphetamine assay.
o Hydrocodone and hydromorphone in the opiate
o Cyclobenzaprine in the tricyclic antidepressant.
These drugs have chemical structures similar to the target
compounds and cross-reactivity is due to the inability of the
antibody to ecognize the subtle differences.
In vitro adulterants
Household items and over the counter pharmaceutical products are
popular in vitro adulterants since they are readily available in bathroom
closets, pockets, and purses. Table 2 summarizes some of the effects that
household products can have on immunoassay screening.
Table 2: Effect of adulterants on immunoassays for drugs of abuse
Adulterant Amphetamines Barbiturates Benzodiazepines Cocaine Opiates PCP THC
Ammonia +R
-R
-R
-All
-C
-E;-C
-C
-R
-C
+F;-C
-E;-C
-E;-C
-C
-All
-F
-C
+F;-C
-E;-C
-E
-All
-C
-C
-F
-All
-C
-C
-E;-F;-C
-R;-E;-C
-R;-E
+F
+F
+F
+F
-E
+R;-E;+F
-E
-R;-E;-C
+R;-E;+F
-R
-R -R
Ascorbate
Bicarbonate
Bleach
Detergent
Drano
Golden seal
Hand soap
Joy
Lime
Peroxide
Phosphate
Salt
Vanish
Vinegar
Visine
R=RIA
E=EMIT
F=FPIA
C=CEDIA
-E
-R
-E
-E;+F
Commercial adulterants: The paranoia surrounding drug testing has
spurned a 'cottage industry' of companies that are formulating and
marketing in vivo and in vitro adulterants. These products are widely
available through the World Wide Web. Typical trade names are: 'UrinAid',
'Mary Jane's super Clean 13', 'Urine Luck' etc. All the commercial products
contain products acting as:
Surfactants (alkylephoxysulphonate)
Fixatives (Gluteraldehyde),
Acids (HCl 1.7 2.1 M),
Oxidants (Nitrite, Pyridinium chlorochromate, Chromate,
Peroxsidase, Chloride)
Mechanism of action for adulterants
Photometric interferences Causing an optical interference
during analysis.
Alterations of pH - Most enzymes have a narrow pH range by
which activity is optimized (vinegar, lemon juice,
bicarbonate, and alkaline detergents)
Antibody-antigen interactions - All immuno assays rely on specific
interaction between antibodies from the assay and the antigens
-E
-E
-R;-F
-E
<strong>Perspectives</strong> on urine adulteration
before testing for drugs-of-abuse
Reviewed by Dr. T Laurens, MFSS, FRSC
Lancet Toxicology Centre - August 2007
E-mail laurensj@lancet .co.za
PAGE 2
-F
-E
-R
-R
-E
-E
-R
-E;-F
-R;-E;-C
(drugs) from the specimens. This delicate balance can be disrupted
by changes in pH, ionic strength, viscosity, and surface tension.
Miscellaneous interactions - Benzalkonium chloride promotes
the sequestration of THC into micelle bodies, making it unavailable
for binding to THC specific antibodies
Interferences in Point-of-Care drug testing systems
Most of these assays make use of immuno chromatography,
whereby antibodies are immobilized onto a solid support in a strip,
to capture and detect drugs as they pass through in the aqueous
mobile phase.
To date, there have been no studies conducted on the performance
of these devices in the presence of in vitro adulterants. The course
of the designing of these assays is significantly different from the
solution based laboratory assays. It is possible that the adulterants
discussed in the previous section will not behave in the same
manner. For example, adulterants that retard or impede flow to the
targeted areas will produce interferences in point-of-care devices
that will not be observed on instrument based immunoassays.
Point-of-care devices have built in procedural positive controls
that enable the analyst to see whether the sample has migrated to
an area where the antibodies are immobilized.
Poit-of-care devices are designed to be used by individuals
with considerable less experience and training than qualified
technologists employed in accredited toxicology laboratories.
Adulterants that act by interfering with antibody-antigen reactions,
is likely to affect point-of-care testing in the same manner as
laboratory-based immunoassays.
Interference of adulterants in confirmation procedures
Confirmation of the presence of a drug or its metabolite in urine samples is
most often carried out using a sophisticated analytical procedure and
instrumentation such as GC-MS. With the absolute specificity of a properly
conducted assay, it is rare for an adulterant to interfere with the testing
process (unless the target compound has been altered by an adulterant e.g.
by oxidation). The entire analytical procedure must be sufficiently robust to
prevent extremes of pH to affect extraction, or loss of derivatizing reagent
due to reaction with a high concentration of adulterant (in which case a
competition between the derivatizing agent and analyte will result).
Laboratory detection adulterants
The aim of validity testing is to demonstrate that the sample submitted for
analysis is in fact urine. The validity of the sample must be checked before
the screening process commences. The minimum validity tests that must be
performed are pH and creatinine. The laboratory may also test for nitrite or
other adulterants. The following guidelines may serve as an indication of the
criteria for urine validity testing:
1. pH measurement: Results within the range pH 4 - 9 are deemed to
be within a normal range. Results less than 3 or greater than 11
should be considered to be adultered. Samples falling outside this
range should be reported as: ''Sample adultered, pH out of range''
2. If the creatinine concentration is less than or equal to 20 mg/dL
(1.77 mmol/L), the specific gravity must be determined. Acceptable
values for specific gravity are 1.001-1.020
3. Samples with creatinine results within the range 5-20 mg/dL (0.44 -
1.77 mmol/L) AND specific gravity results within range (1.001-
1.020), sample should be reported as ''dilute''.
4. Samples with creatinine results less than or equal to 5 mg/dL (0.44
mmol/L) OR specific gravity results out of the range are unsuitable
for testing and should be reported as e.g.: ''Sample not consistent
with normal human urine''.
5. A nitrite level equal to or above 500 μg/ml is conclusive proof of an
adultered sample. The result should be reported as e.g.: ''Sample
adultered Nitrite too high''.
See Lancet News Letter referring to the “Urine Collection Procedures An
example of a typical protocol”O' Connor E, Ostheimer D et al. Limitations of
forensic urine drug testing by methodology and adulteration. Therapeutic
Drug Monitoring and Toxicology In-Service Training and continuing education
Y:\LANCET NEWS\2007\Perspective on urine adulteration before testing for drugs of abuse.CDR