A Northwest company in the pursuit of excellence
The Seventh Annual Providers Conference
Lynnwood Convention Center
Lynnwood, WA
April 18, 2013
Unraveling the Mysteries of
Urine Drug Testing
Jim Heit, BS, MT(ASCP)
Technical Support Manager
STERLING Reference Laboratories
Unraveling the Mysteries of Urine
Drug Testing
COMMON TOXICOLOGY
QUESTIONS
DRUG TESTING
How are Drug testing results Obtained?
• Screening Assays – indicate the
presumptive presence of drugs.
• Confirmation Assays – identify the drug
detected in the screening assay
DRUG TESTING
Immunoassay Screening tests
• What are they?
• How do they work?
• How accurate are they?
DRUG TESTING
U + R
=
UR
(Urine) + (Reagent) = (Reaction Product)
–Appropriate reagents
–Method for recognizing or
measuring the reaction product
DRUG TESTING
• Screening Tests for Drug Class
– Enzyme Immunoassay
– Presumptive Presence of Drugs
– Indicates the presence of a drug by recognizing
that substance’s unique structure.
– Relatively Inexpensive, easily automated
• False Positives are Possible
– Essential to Confirm all POSITIVE Screens
• False Negatives are Rare
THE QUESTION
“Paul’s explanation for his positive THC
result of 45 ng/mL was because he was in
his friend’s car. He wasn’t smoking but two
of his buddies were.”
PASSIVE INHALATION
WEIGHT LOSS??
• My client, who is very much over weight, has a
history of heavy use of marijuana for many
years. He recently started exercising and lost a
lot of weight. He claims he tested POSITIVE for
THC because THC was released from his fat
cells.
• There is no evidence that rapid weight loss
results in release of THC from adipose tissue.
CONFIRMATION ASSAYS
What are the criteria?
• Better specificity and sensitivity than the
screening test
• The “Gold Standard” - Gas
Chromatography/Mass Spectrometry
(GC/MS)
CONFIRMATION TESTING
• Gas Chromatography/Mass Spectrometry
–
–
–
–
Gold Standard for Confirmation
Chemical “Fingerprint” of Drugs
Sensitive and Specific
Legally Defensible
• Liquid Chromatography/Tandem Mass
Spectrometry (LC/MS/MS)
– Emerging Standard for Confirmation
Confirmation Testing
• Quantitative Results ???
– The higher the result, the more recent the
use or a much larger dose of drug was used.
– Debate on use of quantitative results.
THE EXCUSE
My client tested POSITIVE for morphine at
739 ng/mL. He has no history of opiate
abuse. He claims that he tested positive
because he ate a large poppy seed muffin
for breakfast on the morning of the day of
the specimen collection.
• Poppy seeds contain morphine. Morphine
levels up to 5,000 ng/mL are possible from
ingestion of poppy seeds in baked goods.
WINDOW of DETECTION
• Depends on Drug Class
• Amphetamines
– 2 - 3 Days
• Cocaine
– 2 - 4 Days, Longer for Chronic Use
• Opiates
– 3 - 4 Days
• PCP
– 5 – 8 Days
• THC
– Less than 2 Weeks most people
– Heavy, Chronic use, up to 6 – 8 Weeks
Specimen Validity Testing
• Is the specimen sufficiently concentrated
to interpret negative screening results?
• Has the specimen been tampered with or
adulterated in some manner to make
negative screening results invalid?
SPECIMEN VALIDITY TESTING
Components
 Visual examination
 Olfactory examination
 Chemical Evaluation




Creatinine
Specific Gravity (S.G.) if creatinine is < 20 mg/dL
General oxidant
pH
SPECIMEN VALIDITY TESTING
• Dilution
– Creatinine <20 mg/dL
• Inert metabolite from skeletal muscle,
concentration dependent on hydration status
• Most sensitive indicator of dilution
– Specific Gravity <1.003
• Measurement of dissolved solids
• Determined only if Creatinine <20 mg/dL
SPECIMEN VALIDITY TESTING
There is ABSOLUTELY NOTHING
that can be taken by mouth,
except WATER or other fluids,
that will produce a Negative Urine Drug Test.
Excessive fluid intake results in Low Creatinine
levels.
Creatinine Distribution
Creatinine Distribution
45
40.12
40
N = 11,141
35
Median = 119.3 mg/dL
Mean = 130.3 mg/dL
25
22.34
20
15.14
13.93
15
10
3.87
5
0.04
0.03
3.13
0.83
0.45
0.10
0.03
Creatinine mg/dL
>6
00
-6
00
50
0.
1
-5
00
40
0.
1
-4
00
30
0.
1
-3
00
20
0.
1
-2
00
10
0.
1
50
.
1
-1
00
0
-5
20
.
1
0.
0
-2
10
.
1
-1
0.
0
5.
1
2.
1
-5
.0
0
<2
.0
Percent
30
THE QUESTION
So what is the big deal about a dilute
specimen? Why should I care that it is
dilute?
Adequate Fluid Intake
Excessive Fluid Intake
Kidney
Kidney
BLADDER
THE QUESTION
I received the report that said that the
urine was “dilute”. How much water did
the person drink?
URINE SPECIMEN DILUTION
 Pre-Collection Dilution
• consumption of large quantities of fluids prior
to collection
 Post-Collection Dilution
• adding fluid to specimen at the time of
collection
PRE-COLLECTION DILUTION
• High-volume ingestion of fluids (water
loading, flushing, hydrating, etc.)
• Flushing or detoxifying products
– Gold Seal, Clean ‘n Clear, Test-Free, etc
• No evidence these products have any
additional effect on drug elimination
SPECIMEN VALIDITY TESTING
Medical Causes for Dilute Urines
• Diabetes Insipidus
• Anorexia Nervosa or other muscle wasting syndromes
• Kidney Disease
• Diuretics
• Pharmaceutical Toxicity
• Lithium, others
SPECIMEN VALIDITY TESTING
pH Testing – SAMHSA Guidelines
• Acceptable pH:
4.5 to 9.0
• SAMHSA Guidelines for Adulteration:
≤3.0 or ≥11.0
• SAMHSA Guidelines for Invalid Result:
>3.0 to <4.5 or >9.00 to <11.00
SPECIMEN VALIDITY TESTING
• Iodine Producing Adulterants (Urine Luck
6.5)
• Strong Acid and Fluorine (Urine Luck 6.3
and 6.4)
• Chromium VI (various formulations of PCC
and potassium dichromate)
• Peroxidase/Peroxide (Stealth)
• Bleach
• Nitrite (Klear, Whizzies)
• NaCl (table salt)
SPECIMEN VALIDITY TESTING
• Oxidants
– Hypochlorite (Bleach)
– Persulfate
– Fluorine
• Others
– Vinegar
– Sodium Hydroxide (Drano®)
– Soap
SPECIMEN VALIDITY TESTING
pH
• The uses of Iodine and Fluorine containing compounds
results in pH of 2.6 – 5.5
• Drano (NaOH) is the only common adulterant that can
raise the pH
EXAMPLES
• Creatinine <2 mg/dL
• Specific Gravity 1.0005
• pH 6.5
• Interpretation – Substituted – Creatinine
<2.0, S.G. <or= 1.001
– Most likely pure water
EXAMPLES
• Creatinine <2 mg/dL
• Specific Gravity 1.032
• pH 3.2
• Interpretation – Substituted/invalid;
Creatinine <2.0, S.G. =or> 1.020; pH
invalid 3.2
– Fruit juice?
EXAMPLES
• Creatinine <1 mg/dL
• Specific Gravity 1.011
• pH 7.8
• Interpretation – Creatinine <2.0, specific
gravity acceptable
– Actual results from an artificial urine
encountered frequently in Northern WA
SPECIMEN VALIDITY TESTING
• Substitution is now more prevalent
than adulteration
Quick Fix
Clear Test
Ultra Pure
WHIZZINATOR AD
 Available in a variety of natural lifelike skin tones
 Fully adjustable latex belt
 4 oz vinyl bag
 One dehydrated, toxin free urine specimen
 Four organic heat pads
 $150.00
SPECIMEN VALIDITY TESTING
• Synthetic Urine
– Mimics normal human urine
• Creatinine
• Electrolytes (Na ,K, Cl, Ca, Mg)
• Urea, Phosphate
– Difficult to detect by standard testing
• Depends on knowledge and skill of chemist
• Non-Human Urine
– Difficult to Detect
SYNTHETIC URINE
• Is it legal to make or sell synthetic urine?
• YES
• Is it Illegal to substitute synthetic urine?
– In most states NO
• WA has no statute
– Illegal in ten states
• PA, TX, NE, NC, SC, NJ, VA,OR, MD, AL
ARTIFICIAL URINE TEST
• STERLING has found a unique analyte
that is lacking in synthetic urine.
• Five Synthetic Urines Purchased
– 12 Components Screened
– 3 Compounds Studied
– 1 Analyte Chosen
• Missing in all synthetic urines studied.
• Unobserved Employment Urines Screened
– 5.7% of 567 specimens synthetic urine
ALCOHOL TESTING
• Blood or Breath Alcohol
– Gold Standard
– Legally Defined Limit of Impairment
– 12 Hour Window of Detection
• Urine Alcohol
– Does not correlate with Blood Alcohol
– 14 Hour Window of Detection
– Fermentation is Potential Problem
ALCOHOL TESTING
• Ethylglucuronide
–
–
–
–
Direct Bio-Marker of Ethanol Exposure
Stable, Water Soluble
Minor Metabolite of Ethanol
Synthesis in Liver
• EtOH +glucuronic acid = EtG
– Window of Detection 72 – 96 Hours
• Dependent on amount and frequency of consumption
– No False Positives
– Fermentation NOT a Factor
ETHYLGLUCURONIDE
• Not a marker of impairment
– Does not correlate with BAC
• Not a marker for amount of alcohol
consumption
ETHYLGLUCURONIDE:
ANALYSIS
• Screening Assays
– LC/MS/MS
– Immunoassay
• Confirmation Assays
– LC/MS/MS
• EtS Detected and Quantified
• No False Positives
• Legally Defensible
ETHYLGLUCURONIDE
• Voluntary Exposure to Ethanol
– Voluntary consumption of alcoholic beverage
• Incidental Exposure to Ethanol
– Alcohol exposure without intent
• Not a FALSE POSITIVE Result
– Alcohol exposure in both situations
ETHYLGLUCURONIDE
• SAMHSA Advisory
• Analytical Methods are Valid!
• What are Appropriate Cut-Offs?
• Interpretation of Low Positive Results?
• Incidental Exposure?
Ethylglucuronide-Incidental Exposure
10%
26.9%
0.2 - 0.8 % 14%
62%
0.5%
35%
3–6%
Ethylglucuronide
Effect of Nyquil on EtG
900
800
766
700
673
EtG ng/mL
600
500
400
300
200
176
178
182
189
BF
AM
RM
MD
131
100
1
1
1
1
WS
NS
BS
DB
60
62
CAH
BT
83
98
0
DE
TS
Subject
MM
JH
VR
Ethylglucuronide
Effect of Purell on EtG
600
513
EtG ng/mL
500
400
300
200
100
153
50
50
55
70
B
C
D
E
176
183
G
H
0
Subject
F
I
THE EXCUSE
“My job requires me to wash dirty car parts
in denatured alcohol. Every 20 – 30
minutes I have my hands in the alcohol.
That is why my EtG level was 2600 ng/mL”
ETHYLGLUCURONIDE
CUT-OFFS
• Common Positive Cut-Off Values
• 100 ng/mL
– Used in “zero tolerance” programs
– Susceptible to incidental exposure
• 250 ng/mL
– Used in most programs
– Less susceptible to incidental exposure
• 500 ng/mL
– Used in more “liberal/tolerant” programs
– Least susceptible to incidental exposure
Alternate Matrices
• Hair
• Oral Fluid
• On Site Devices
HAIR
ADVANTAGES
• Provides a longer estimate of time of drug use
• Observed collection
• Ease of obtaining, storing, and shipping
specimens
• Second specimen can be obtained from original
source
HAIR
DISADVANTAGES
•
•
•
•
Recent use not detected
Casual user may not be detected
Possible hair type biases
Possible false positives from external
contamination
• Expensive
ORAL FLUID
ADVANTAGES
• Gender neutral collections
• Non-invasive collection
• Detects recent use
• Possible correlation with state of
impairment
ORAL FLUID
DISADVANTAGES
• Short window of detection
• Quality of specimen is collection device
dependent
• Specimen recovery from stimulant abusers
difficult
• Drug recovery, especially cocaine, is pH
dependent
• Limited test menu
Onsite Devices
 Advantages
Instant Results
Ability to confront donor
May be cheaper
 Disadvantages
Qualitative results and subjective interpretation
No accurate cut-offs
High false positive rate
Specimen validity tests unreliable
Others
•
•
•
•
Sweat Patches
Finger/toe nails
Skin scrapings
Meconium
SPICE
SPICE and BATH SALTS
• Calls to Poison Control Centers
• SPICE
– 2010-- 2,870 calls about spice
– Jan. 2011-- 385 calls 2011 Projected = 4,620
– 2011 6,890 actual calls
• BATH SALTS
– 2010-- 236 calls about bath salts
– Jan. 2011--251 calls 2011 Projected = 3,012
– 2011
6072 actual calls
SPICE: Synthetic Cannabinoids
• Synthetic cannabinoids started out as
legitimate scientific endeavors.
• More than 250 synthetic cannabinoids
synthesized.
SPICE: HISTORY
•
•
•
•
2004
2008
2008
2008
First Appearance in Europe
Wide Spread use in Europe
First Appearance in United States
First Analysis of SPICE at University
of Freiburg, Germany
• 2009 Wide Use in United States
• 2010 Laboratory Testing of Spice
Available
SPICE: Preparation
Botanical Material
Herbal Incense
• Synthetic cannabinoids
sprayed on herbs,
allowed to dry
• Residual solvent
• No quality control
• Batches inconsistent
• Expensive compared to
marijuana
• Caveat emptor
SPICE: Pharmacology
•
•
•
•
•
•
•
•
Euphoria (“high”) similar to Marijuana
Relaxation
Altered state of consciousness
Distortion of sensory experiences
Impaired motor control
Increased reaction time
Decreased cognition
NO effect on appetite
SPICE: Adverse Side Effects
•
•
•
•
•
•
•
•
Elevated Blood Pressure
Increased Heart Rate
Hyperventilation
Anxiety and Agitation
Paranoia
Seizures
Vomiting
Death (unsubstantiated media reports)
SPICE: International Legal Status
• All or some Synthetic Cannabinoids Banned
Australia
Austria
France
Germany
Japan
New Zealand
Poland
Romania
Russia
Slovak Republic
South Korea
Switzerland
United Kingdom
SPICE: Legal Status in U.S. States
• Some or all Synthetic Cannabinoids Banned
Alabama
Georgia
Iowa
Kentucky
Michigan
Nevada (?)
New Mexico
Oregon
Utah (?)
West Virginia
Arkansas
Illinois
Kansas
Louisiana
Missouri
New Hampshire
Oklahoma
Tennessee
Washington
SPICE: Legal Status U.S. Military
• Banned in All Branches of Military
– U.S. Army
– U.S. Air Force
– U.S. Coast Guard
– U.S. Marine Corps
– U.S. Navy
SPICE: Legal Status U.S. Federal
• Schedule Status
November 24, 2010 DEA published in
Federal Register intent to place five
synthetic cannabinoids on Schedule 1 of
CSA.
March 1, 2011 final rule published.
STATUS: Schedule 1, therefore ILLEGAL
SPICE: Laboratory Testing
• Synthetic Cannabinoids not detected in THC
screening immunoassays or THC confirmatory
GC/MS testing.
• Immunoassays now available.
• On-Site screening tests now available, but high
cutoff severely limits usefulness.
• LC/MS/MS technology available.
• Few labs perform testing.
• Expensive $30 - $100 per sample
SPICE: Lab testing
• Current Metabolites STERLING screens
for:
– JWH018 (2 metabolites)
– JWH019 (1 metabolite)
– JWH073 (2 metabolites)
– JWH250 (2 metabolites)
– AM2201 (1 metabolite)
SPICE: ANALYSIS
• Future of Spice Testing
– More compounds to be tested as SPICE formula
evolves.
– New metabolites.
• Alternative Matrix
– Blood
• Measure Parent Drug
– Oral Fluid
• Measure Parent Drug
– Hair
• Enhanced Window of Detection
DESIGNER DRUGS: BATH SALTS
CATHINONES
•
•
•
•
Fresh leaves chewed or consumed as tea
Originated in Ethiopia
Causes release of dopamine from brain areas
Sale of Khat legal in 



Australia by permit
Oman
Yemen
United Kingdom
DESIGNER DRUGS: Bath Salts
•
•
•
•
•
•
•
Central Nervous System Stimulants
Similar in action to methamphetamine
Thought to be highly addictive
MDPV is 5–8x potency of methylphenidate
Available as research chemical in 2007
Popular in Europe and Australia
Legal
– Banned in Louisiana and Florida Jan. 2011
BATH SALTS: Adverse Side Effects
•
•
•
•
•
•
•
•
Elevated Heart Rate and Blood Pressure
Anxiety and agitation
Hallucinations
Extreme Paranoia
Delusions
Seizures
Nausea and Vomiting
Death
PREVALENCE: Urine
• MDPV
88.0%
• Methylone
20.7%
• Mephedrone
3.1%
• Butylone
1.2%
BATH SALTS: ANALYSIS
• Bath Salts are not detected in
Amphetamine Screening Immunoassays
or Confirmatory GC/MS assays
• Immunoassays available, but expensive.
• Rapid On-Site Tests not available
• LC- or GC/MS assays available for MDPV
and Others
• Available at STERLING March 1, 2011
SPICE and BATH SALTS
• FUTURE OF DESIGNER DRUGS
• They are here to stay.
• The problem will get worse long before it
gets better. DEMAND = SUPPLY
• Analytical Labs Will Always Lag
Behind the Synthetic Chemists
Contact Information
QUESTIONS???
jheit@regtox.com
dbaker@regtox.com
dricketts@regtox.com
certifying@regtox.com