The Seventh Annual Providers Conference Lynnwood Convention Center Lynnwood, WA April 18, 2013

1. A Northwest company in the pursuit of excellence The Seventh Annual Providers Conference Lynnwood Convention CenterLynnwood, WAApril 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 (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.

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.

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.

15. 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

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.

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. 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 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

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 • 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 • LC/MS/MS • 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% 26.9% 62% 35% 0.2 - 0.8 % 14% 0.5% 3 – 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. 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