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

2. Unraveling the Mysteries of Urine Drug Testing
Jim Heit, BS, MT(ASCP)
Technical Support Manager
STERLING Reference Laboratories

3. Unraveling the Mysteries of Urine Drug Testing
COMMON TOXICOLOGY QUESTIONS

4. 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

5. DRUG TESTING Immunoassay Screening tests What are they?
How do they work?
How accurate are they?

6. DRUG TESTING U + R = UR Appropriate reagents
(Urine) + (Reagent) = (Reaction Product)
Appropriate reagents
Method for recognizing or measuring the reaction product

7. 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

8. 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.”

9. PASSIVE INHALATION

10. 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. 10

11. CONFIRMATION ASSAYS What are the criteria?
Better specificity and sensitivity than the screening test
The “Gold Standard” - Gas Chromatography/Mass Spectrometry (GC/MS)

12. 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

13. 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. 13

14. 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. 14

15. WINDOW of DETECTION Depends on Drug Class Amphetamines Cocaine Opiates
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

16. 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?

17. SPECIMEN VALIDITY TESTING
Components
Visual examination
Olfactory examination
Chemical Evaluation
Creatinine
Specific Gravity (S.G.) if creatinine is < 20 mg/dL
General oxidant pH

18. 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

19. 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. 19

20. Creatinine Distribution

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

22. Excessive Fluid Intake
Adequate Fluid Intake
Excessive Fluid Intake
Kidney
Kidney
BLADDER

23. THE QUESTION
I received the report that said that the urine was “dilute”. How much water did the person drink?

24. 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

25. 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

26. SPECIMEN VALIDITY TESTING
Medical Causes for Dilute Urines
Diabetes Insipidus
Anorexia Nervosa or other muscle wasting syndromes
Kidney Disease
Diuretics
Pharmaceutical Toxicity
Lithium, others

27. 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

28. 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)

29. SPECIMEN VALIDITY TESTING
Oxidants
Hypochlorite (Bleach)
Persulfate
Fluorine
Others
Vinegar
Sodium Hydroxide (Drano®)
Soap

30. SPECIMEN VALIDITY TESTINGpH
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

31. 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

32. 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?

33. 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

34. SPECIMEN VALIDITY TESTING
Substitution is now more prevalent than adulteration
Quick Fix Clear Test Ultra Pure

36. 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

37. 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

38. 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

39. 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

40. ALCOHOL TESTING Blood or Breath Alcohol Urine 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

41. 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

42. ETHYLGLUCURONIDE Not a marker of impairment
Does not correlate with BAC
Not a marker for amount of alcohol consumption

43. ETHYLGLUCURONIDE: ANALYSIS
Screening Assays
LC/MS/MS
Immunoassay
Confirmation Assays
EtS Detected and Quantified
No False Positives
Legally Defensible

44. 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

45. ETHYLGLUCURONIDE SAMHSA Advisory Analytical Methods are Valid!
What are Appropriate Cut-Offs?
Interpretation of Low Positive Results?
Incidental Exposure?

46. Ethylglucuronide-Incidental Exposure
10% % % %
% 14% % – 6 %

47. Ethylglucuronide

48. Ethylglucuronide

49. 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”

50. 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

51. Alternate Matrices
Hair
Oral Fluid
On Site Devices 51

52. 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 52

53. HAIR Recent use not detected Casual user may not be detected
DISADVANTAGES
Recent use not detected
Casual user may not be detected
Possible hair type biases
Possible false positives from external contamination
Expensive 53

54. ORAL FLUID ADVANTAGES Gender neutral collections
Non-invasive collection
Detects recent use
Possible correlation with state of impairment 54

55. 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 55

56. 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 56

57. Others
Sweat Patches
Finger/toe nails
Skin scrapings
Meconium

58. SPICE

59. SPICE and BATH SALTS Calls to Poison Control Centers SPICE BATH SALTS
,870 calls about spice
Jan calls Projected = 4,620
,890 actual calls
BATH SALTS
calls about bath salts
Jan calls Projected = 3,012
actual calls

60. SPICE: Synthetic Cannabinoids
Synthetic cannabinoids started out as legitimate scientific endeavors.
More than 250 synthetic cannabinoids synthesized.

61. 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

62. 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

63. 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

64. SPICE: Adverse Side Effects
Elevated Blood Pressure
Increased Heart Rate
Hyperventilation
Anxiety and Agitation
Paranoia
Seizures
Vomiting
Death (unsubstantiated media reports)

65. 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

66. SPICE: Legal Status in U.S. States
Some or all Synthetic Cannabinoids Banned
Alabama Arkansas
Georgia Illinois
Iowa Kansas
Kentucky Louisiana
Michigan Missouri
Nevada (?) New Hampshire
New Mexico Oklahoma
Oregon Tennessee
Utah (?) Washington
West Virginia

67. 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

68. 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

69. 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

70. SPICE: Lab testing Current Metabolites STERLING screens for:
JWH018 (2 metabolites)
JWH019 (1 metabolite)
JWH073 (2 metabolites)
JWH250 (2 metabolites)
AM2201 (1 metabolite)

71. SPICE: ANALYSIS Future of Spice Testing Alternative Matrix
More compounds to be tested as SPICE formula evolves.
New metabolites.
Alternative Matrix
Blood
Measure Parent Drug
Oral Fluid
Hair
Enhanced Window of Detection

72. DESIGNER DRUGS: BATH SALTS

73. 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

74. 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

75. BATH SALTS: Adverse Side Effects
Elevated Heart Rate and Blood Pressure
Anxiety and agitation
Hallucinations
Extreme Paranoia
Delusions
Seizures
Nausea and Vomiting
Death

76. PREVALENCE: Urine MDPV 88.0% Methylone 20.7% Mephedrone 3.1%
Butylone 1.2%

77. 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

78. 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

79. Contact Information
QUESTIONS???