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The Seventh Annual Providers Conference Lynnwood Convention Center Lynnwood, WA April 18, 2013 A Northwest company in the pursuit of excellence

The Seventh Annual Providers Conference Lynnwood Convention Center Lynnwood, WA April 18, 2013 A Northwest company in the pursuit of excellence

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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

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 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

So what is the big deal about a dilute specimen? Why should I care that it is dilute?

Adequate Fluid Intake Excessive Fluid Intake

BLADDER

Kidney Kidney

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% 62% 35%

0.2 - 0.8 % 14% 0.5% 3 – 6 %

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

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 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

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