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The Seventh Annual Providers Conference Lynnwood Convention Center
Lynnwood, WAApril 18, 2013
A Northwest company in the pursuit of excellence
Unraveling the Mysteries of Urine Drug Testing
Jim Heit, BS, MT(ASCP)
Technical Support Manager
STERLING Reference Laboratories
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
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.”
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 DistributionCreatinine Distribution
0.04 0.03 0.83
3.87
13.93
22.34
40.12
15.14
3.13
0.45 0.10 0.030
5
10
15
20
25
30
35
40
45
<2.0
2.1 -
5.0
5.1 -
10.0
10.1
- 20.
0
20.1
- 50
50.1
- 100
100.
1 - 20
0
200.
1 - 30
0
300.
1 - 40
0
400.
1 - 50
0
500.
1 - 60
0>60
0
Creatinine mg/dL
Per
cen
t
N = 11,141
Median = 119.3 mg/dL
Mean = 130.3 mg/dL
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?
EthylglucuronideEffect of Nyquil on EtG
1 1 1 1
60 6283
98131
176 178 182 189
673
766
0
100
200
300
400
500
600
700
800
900
WS NS BS DB CAH BT DE TS MM BF AM RM MD JH VR
Subject
EtG
ng
/mL
Ethylglucuronide
Effect of Purell on EtG
50 50 55 70
153176 183
513
0
100
200
300
400
500
600
B C D E F G H I
Subject
EtG
ng
/mL
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”
ETHYLGLUCURONIDECUT-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
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
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 First Appearance in Europe
• 2008 Wide Spread use in Europe
• 2008 First Appearance in United States
• 2008 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 BannedAlabama Arkansas
Georgia Illinois
Iowa Kansas
Kentucky Louisiana
Michigan Missouri
Nevada (?) New Hampshire
New Mexico Oklahoma
Oregon Tennessee
Utah (?) Washington
West Virginia
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
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
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???