Beating Drug Tests: Methods and Myths

By
Sarah Hamed Nasreldin
Assistant Lecturer of Forensic Medicine and
Clinical Toxicology, Cairo University
 Introduction

 A national survey conducted by the Anti-Addiction Fund in 2014

indicated that nearly 24 percent of Egyptian drivers operate their
vehicles under the influence of drugs.  

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 Introduction

 Drug testing programs in many countries are established to help

achieve a drug-free work environment.
 Professions such as police/defense force, mining industry,
transportation industry, and industries engaged in heavy machine
operations, drug testing is mandatory.

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 Samples for Drug Testing

 Drug testing can be completed on various biological specimens:

A. Urine
B. Blood
C. Saliva
D. Hair
E. Sweat

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 A. Urine

 Advantages:
– Non invasive
– Cost effective and reliable.
– Ease of sample collection.
– High drug concentrations
– Drug detection times are longer in urine than in blood or serum
 Disadvantages:
– Easy to adulterated
– Can not indicate blood level or toxicity
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 B. Blood

 Advantages:
– Correlates with level of impairment and toxicity.
 Disadvantages:
– Highly invasive
– potential danger of infection
– Detection time is short compared to urine

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 C. Saliva

 Advantages:
– Correlates with level of impairment and toxicity.
– Non invasive.
– Not easily to adulterate or dilute.
 Disadvantages:
– Subject to contamination from smoking
– Changes in Ph may alter the sample
– Concentrations of drugs are low
– Narrow window of detection
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 D. Hair

 Advantages:
– Non invasive.
– Long term detection of drug exposure
– Difficult to adulterate.
 Disadvantages:
– Can not be used for recent exposure
– Potential racial bias
– High cost to test

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 E. Sweat
 Advantages:
– Non invasive.
– Long term detection of drug exposure (days to weeks)
– Difficult to adulterate.
 Disadvantages:
– Can not be used for recent exposure
– High variability in the rate of sweat production
– Possible environmental contamination
– Risk of accidental removal of test patch
– High cost to test
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 Samples for Drug Testing Approximate Detection Period
Urine hrs 24-72
Blood hrs 8-36
Saliva hrs but only for 6-8hrs for THC 12-36
Hair Head hair: 14-90 days prior
Body hair: 30-365 days prior
Sweat weeks 1-4

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 Methods of drug testing

 There are 2 main types of urine drug tests (UDT)

A. Screening tests
B. Confirmatory tests.

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 A . Screening tests

 Initial drug screen is performed using immunoassay technology

 Immunoassays use antibodies to detect the presence of a drug or drug
metabolites in the urine

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 A . Screening tests (continued)

 Advantages of immunoassays include large-scale screening and rapid

detection with relatively cheap cost.
 The main disadvantage false-positive results due to cross reactivity.

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 A . Screening tests

1) On-Site Drug Testing

2) Laboratory Based Drug Testing

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 1) On-Site Drug Testing

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 1) On-Site Drug Testing

 Advantages:
– Rapid and easy
– Low Cost
– Elimination of specimen transport and storage issues.
 Disadvantages:
– Increased cross-reactivity and interference
– Does not include testing for diluted samples and adulteration testing
– Does not include quality control
– Potential privacy or conflict-of interest concerns.
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 1) On-Site Drug Testing

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 2) Laboratory Based Drug Testing
 Advantages:
– Use of approved scientific methods
– Quality assurance
– Confirmation testing more readily available.
– adulteration testing more readily available.
 Disadvantages:
– Potential increased cost per test
– The need for high cost instruments

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 2) Laboratory Based Drug Testing

 A range of immunoassay-based screening methods are currently used

by drug testing laboratories, and include:
– Enzyme-multiplied Immunoassay Technique (EMIT)
– Cloned Enzyme Donor Immunoassay (CEDIA)
– Fluorescence Polarization Immunoassay (FPIA)
– Kinetic Interaction of Microparticles in Solution (KIMS)

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 Enzyme-multiplied Immunoassay Technique (EMIT)

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 Cloned Enzyme Donor Immunoassay (CEDIA)

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 Fluorescence Polarization Immunoassay (FPIA)

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 Kinetic Interaction of Microparticles in Solution (KIMS)

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 B .Confirmatory tests

 Immunoassays are considered presumptive and confirmatory testing is

necessary either by (GC-MS) or (LC-MS).
 Accurate, sensitive, and reliable
 Time-consuming, requires a high level of expertise to perform, and is
costly.

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 Liquid Chromatography

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 Gas Chromatography

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 Mass Spectrometry

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 Beating drug tests

 Fearing a positive result with significant consequences, people abusing

drugs attempt to tamper urine specimens to escape positive drug
testing.
 There are a variety of in vivo and in vitro techniques designed to beat
urine drug screens by
A. Dilution
B. Adulteration
C. Substitution

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 A . Dilution

 Diluting a urine sample is probably the most common form of urine

adulteration.
 There are two methods of urinary dilution:
– Internal dilution
– External dilution

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 Internal dilution

1) Use of copious amounts of fluids

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 Internal dilution

2) Flushing and detoxification agents are frequently advertised as

effective means of passing drug tests eg:
– Herbal Teas
– Water pills
– Herbal Nutrients and Supplements

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 Internal dilution

 Many of these products contain caffeine or other diuretics to increase

urine output and lower the concentration of drugs below the detectable
limit.
 a lot of companies have now started to add creatine and B vitamins to
their formula which retains the natural color, odor and texture of urine.
 detox drinks for drug test works by stopping the metabolism of fat
cells that are passed in the urine for up to 5 hours.

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 External dilution

 add water directly to the urine sample after it is collected.

 So, some testing laboratories shut down access to their faucets and add
dye to their toilets to catch people who add water to their drug samples

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 B . Adulteration

 Adulterants are foreign substances that are added to a urine specimen

after micturition.
 These products work by:
– Either interfering with the immunoassay detection.
– Converting a target drug to compounds that are not detected in screening or
confirmation procedures.

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 B . Adulteration

Common Household Chemicals used as Urinary Adulterants:

1) Table salt (NaCl)
2) Vinegar
3) Bleach
4) Soup/detergent
5) Visine Eye drops

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 1. Table salt (NaCl)

 It interferes more with the EMIT than with other immunoassays

 It affect immunoassay results by changing protein structures, which
then alter enzyme activity and drug binding

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 1. Table salt (NaCl)

Drug Method Effect

Amphetamine EMIT Decreased immunoassay sensitivity

Cannabis EMIT Decreased immunoassay sensitivity

Cocaine EMIT Decreased immunoassay sensitivity

Opiates EMIT Decreased immunoassay sensitivity

Benzodiazepines EMIT,FPIA Decreased immunoassay sensitivity

Barbiturates EMIT Decreased immunoassay sensitivity

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 2. Vinegar

 Interferes with the detection of cannabinoids

 Lower pH levels, which can affect binding, reaction times, and drug
solubility

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 2. Vinegar

Drug Method Effect

Cannabis EMIT Decreased immunoassay sensitivity

CEDIA
FPIA
Cocaine CEDIA Decreased immunoassay sensitivity

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 3. Bleach (sodium Hypochlorite)

 One of the most effective and most popular oxidizing agents used as
urine adulterants.
 Bleach has demonstrated false negative results across several different
immunoassay and in some cases during GC/MS testing

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 3. Bleach (sodium Hypochlorite)

Drug Method Effect

Amphetamine EMIT,CEDIA,FPIA Decreased immunoassay sensitivity
GC-MS,LC-MS Major decrease in target analyte recovery

Cannabis EMIT,CEDIA,FPIA Decreased immunoassay sensitivity

GC-MS Complete degradation of target analyte

Cocaine EMIT,CEDIA Decreased immunoassay sensitivity

Opiates EMIT,CEDIA,FPIA,KIMS Decreased immunoassay sensitivity

Benzodiazepines CEDIA Decreased immunoassay sensitivity

Barbiturates CEDIA Decreased immunoassay sensitivity

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 4. Soup/detergent

 soap may alter pH levels in urine samples and may also interfere with
drug binding on immunoassays.
 Soaps and detergents have been reported to create both false negative
and false-positive results on several different immunoassays.

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 4. Soup/detergent

Drug Method Effect

Amphetamine CEDIA Decreased immunoassay sensitivity

Cannabis EMIT,CEDIA,FPIA Decreased immunoassay sensitivity

Cocaine CEDIA,FPIA Decreased immunoassay sensitivity

Opiates CEDIA Decreased immunoassay sensitivity

Barbiturates CEDIA Decreased immunoassay sensitivity

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 5. Visine Eye drops

 the mechanism of adulteration is most likely due to the inactive

ingredients benzalkonium chloride.
 benzalkonium chloride reduces the binding in immunoassay drug
screens, causing false-negative results on immunoassays

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 5. Visine Eye drops

Drug Method Effect

Cannabis EMIT,CEDIA Decreased immunoassay sensitivity

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 B . Adulteration

 Common urinary adulterants available through the Internet:

1) Stealth (peroxidase and peroxide)
2) Klear (nitrite)
3) Instant Clean ADDITive (glutaraldehyde)
4) Urine Luck (pyridinium chlorochromate [PCC])
5) Papain

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 1. Stealth (peroxidase and peroxide)

 Combination of two different compounds: peroxidase and peroxide

 Two separate vials that are combined with urine at the time of
sampling
– one containing a powder (peroxidase)
– a liquid (hydrogen peroxide).
 Works by oxidizing different drugs and their metabolites, which
renders them undetectable

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 1. Peroxidase and peroxide (Stealth)

Drug Method Effect

Cannabis CEDIA,KIMS Decreased immunoassay sensitivity

Opiates CEDIA,KIMS Decreased immunoassay sensitivity

GC-MS Complete degradation of target analyte

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 2. Nitrite (Klear)

 The product Klear comes in 2 microtubes containing 500mg of white

crystalline material.
 This product is a powerful oxidant which dissolves in urine without
affecting color or temperature.
 effective in masking the detection of cannabinoids in urine by both
screening and confirmatory assays.
 Nitrite successfully masked the detection of BZE by immunoassay
screening methods, but did not interfere with GC–MS confirmation

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 2. Nitrite (Klear)

Drug Method Effect

Amphetamine KIMS Decreased immunoassay sensitivity

Cannabis EMIT,KIMS Decreased immunoassay sensitivity

GC-MS,LC-MS Strong decrease in target analyte recovery

Cocaine GC-MS Slight decrease in target analyte recovery

Opiates LC-MS Strong decrease in target analyte recovery

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 3. Glutaraldehyde (UrinAid)

 Each kit contains 4 to 5 mL of glutaraldehyde solution, which is added

to 50 to 60 mL of urine.
 Glutaraldehyde solutions are available in hospitals and clinics as a
cleaning or sterilizing agent.
 Amphetamine, cannabis, benzodiazepine, opiate, and cocaine
metabolite with EMIT immunoassays.

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 3. Glutaraldehyde (UrinAid)

Drug Method Effect

Amphetamine EMIT,FPIA Decreased immunoassay sensitivity

Cannabis EMIT,CEDIA,FPIA Decreased immunoassay sensitivity

Cocaine EMIT,CEDIA,FPIA Decreased immunoassay sensitivity

Opiates EMIT,FPIA Decreased immunoassay sensitivity

Benzodiazepin EMIT Decreased immunoassay sensitivity

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 4. Pyridinium chlorochromate PCC (Urine Luck)

 A strong oxidizing agent, which at a concentration of 100 mg/ml,

decreased response rate for all EMIT drug screens, indicating the
possibility of false-negative results.

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 4. Pyridinium chlorochromate PCC (Urine Luck)

 concentrations of opiates and THC as determined by GC-MS were

reduced
 Amphetamine, barbiturates, benzodiazepines, cannabinoids, cocaine,
opiates

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 4. Pyridinium chlorochromate PCC (Urine Luck)

Drug Method Effect

Cannabis EMIT Decreased immunoassay sensitivity

GC-MS Strong decrease in target analyte recovery

Cocaine EMIT Decreased immunoassay sensitivity

Opiates EMIT,KIMS Decreased immunoassay sensitivity

GC-MS,LC-MS Strong decrease in target analyte recovery

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 5. Papain

 a cysteine protease found in papaya that is the major ingredient of

some meat tenderizers
 decrease both EMIT and FPIA immunoassay
 sensitivities and to lower the THC-COOH concentrations in GC–MS
confirmatory testing.

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 5. Papain

Drug Method Effect

Cannabis EMIT,FPIA Decreased immunoassay sensitivity

GC-MS Decreased analyte concentration

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 B. Substitution

 The donor provides urine that did not originally come from his or her
body which may be :
– Natural urine
– Synthetic urine

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 Natural urine

 A clean specimen from a friend will often pass integrity checks, as

well as checks for any of the adulterants
 However, it is usually hard to assure your friend is drug free or not
pregnant

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 Synthetic urine

 Fake urine for used by drug users as a substitute for their urine
samples during a drug test.
 Difficult to detect because it has similar pH, creatinine, and specific
gravity to normal urine.

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 B. Substitution

 The First challenge for the cheater when using substitute urine is
keeping the urine at the correct temperature. 
 However, those determined to do so may use several methods to keep
the sample warm
– Holding it close to their bodies in an armpit or the groin area.
– Digital Heating pad
– Heat activation powder

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 B. Substitution

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 B. Substitution

 The second challenge facing substitution is avoiding detection in case

of observed urine specimen
 Cheaters usually try to sneak in someone else’s urine or synthetic urine
by using several methods:
– Urine is stored in a condom strapped to the genitalia
– Device such as the Whizzinator, the Urinator, and the Butt Wedge

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 B. Substitution

 The Whizzinator: a synthetic urine belt with fake penis

 The Urinator: a flexible plastic container that is strapped to the body,
with a small plastic tube placed near the urethra.
 The Butt Wedge: wedge-shaped container to store and warm urine
between the user’s buttocks

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 B. Substitution

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 B. Substitution

 Patients may try to avoid detection by voiding before testing, then

refilling their bladder with clean urine using a catheter

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 Detecting dilute, substituted, and adulterated
urine samples

A. Observation of the patient

B. Visual inspection of the urine
C. Laboratory analyses

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 A. Observation of the patient

 Observation is most useful in preventing in vitro adulteration and

dilution during the collection process.
 Sources of running water
 Outer garments must be removed, and personal belongings
 wash their hands prior to providing a specimen

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 B. Visual inspection of the urine

 Soaps and detergents will generally produce an unusually and

persistently frothy or bubbly specimen.
 The use of additives such as table salt can be detected by the presence
of undissolved crystals in a specimen.

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 C. Laboratory analyses

1) Urine Integrity Tests

2) Color Tests
3) Dipstick devices
4) Immunoassay
5) Capillary Electrophoresis and Electrospray Ionization–MS
6) Polyethylene Glycol Urine Marker System

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 1. Urine Integrity Tests

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 1. Urine Integrity Tests

Specimen integrity tests

Tampering method pH Creatinine Temperature Specific gravity

Diluted urine

Sodium chloride

Vinegar
Sodium hypochlorite

Soup/detergent Cloudy

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 1. Urine Integrity Tests

Specimen integrity tests

Tampering method pH Creatinine Temperature Specific gravity

Visine Eye drops Cannot be detected by routine urine specimen integrity testing

Peroxidase and peroxide Cannot be detected by routine urine specimen integrity testing

Nitrite Cannot be detected by routine urine specimen integrity testing

Glutaraldehyde Cannot be detected by routine urine specimen integrity testing

Pyridinium chlorochromate Cannot be detected by routine urine specimen integrity testing

Papain Cannot be detected by routine urine specimen integrity testing

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 2. Color test

 Spot tests give rapid results and are simple to perform.

 PCC, nitrite, and Stealth can be detected using various spot tests,
however, false positive results are common.

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 Color test for PCC

 1 mL urine + 2 drops of a 1% 1,5-diphenylcarbazide solution in

methanol reddish purple color indicates a positive result.

 200 μL of urine + 4-5 drops of 3% hydrogen peroxide dark

brown color and precipitate indicates positive results.

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 Color test for nitrite

 few drops of urine specimen + 0.5 mL of 1% potassium permanganate

solution + few drops of 2N HCL pink permanganate solution
becomes colorless with effervescence.
 The presence of very high glucose in urine (>1,000 mg/dL) and ketone
bodies may cause a false-positive result. However, it takes
approximately 2 to 3 minutes for the solution to turn colorless.

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 Color test for Peroxidase and peroxide

 10 μL of urine + 50 μL of tetramethylbenzidine + 500 μL of 0.1 M

phosphate buffer dark brown color in case of positive result.

 Another reagent, acidified potassium dichromate will give a deep blue

color change that fades over time

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 3. Dipstick devices

 Portable and allow for on-site testing to detect adulterated specimens

 Nitrite, pH, and specific gravity can be tested using the widely
available urinalysis test strips

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 3. Dipstick devices

 Multistix (Bayer), and Combur-Test (Roche Diagnostics).

® ®

 Can indirectly detect Stealth®, as strong positive readings for glucose

and blood, due to the peroxidase activity of the oxidant that the test is
actually detecting.

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 3. Dipstick devices

 Designed for forensic toxicological purposes:

– Adultacheck® 4 (creatinine, pH, nitrite, glutaraldehyde)
– Adultacheck® 6 (like 4 + PCC + oxidants)
– the Intect®7 (creatinine, pH, nitrite, glutaraldehyde, PCC,bleach,specific
gravity)
– adultaceck® 10
 Intect 7 test strip has an advantage of detecting specific gravity and the
presence of bleach in urine at only 10 μL/mL urine

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 4. Immunoassay

 An immunoassay-based test specifically used for the detection of urine

adulteration by oxidizing compounds
 Based on the reaction between a tetramethylbenzidine reagent and any
oxidant present in the specimen
 Not be implemented in routine drug testing due to cost

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 5. Capillary Electrophoresis and Electrospray Ionization–MS

 Capillary electrophoresis has been used to detect the chromate ions

found in PCC and nitrite ions found in nitrite-based oxidants
 The chromium species present in PCC can also be detected using mass
spectrometric techniques such as LC–MS, GC–MS

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 6. Polyethylene Glycol Urine Marker System

 Ingestion by patients of polyethylene glycols that are excreted in urine

after oral ingestion.
 This allows reliable specimen identification by the successful detection
of the specific marker substance in urine samples.
 the method is effective in detecting urine substitution based on the
absence of the polyethylene glycol marker in the urine specimens.
However,
 the method is not effective in detecting in vitro adulteration by
chemicals
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 References
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samples after manipulation by oxidizing chemicals: Technical considerations. Bioanalysis, 6(11),
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 References
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