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Challenging Toxicology:

Beyond the Lab

Presented by

James L. Ferguson, DO, DFASAM

jferguson@fssolutions.com

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What do test results really tell us?

• Specimen Selection

• What happens when different specimens sing different songs?

• Validity Testing/Dilution• When is a result not to be trusted?

• Alcohol Biomarkers

• What is your definition of “Beverage Alcohol”?

• Interpreting unexpected metabolites that show up

• Follow up recommendations

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8 Cases

• Cases 1&2: Equivocal hair test results

• Case 3: Invalid specimen (creatinine)

• Cases 4,5,6: Unexpected metabolite positives

• Cases 7&8: Alcohol biomarker results

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Screening and Confirmation• Screening: The first step (Either in the lab or at Point of Collection-POCT)

- Sensitive but not specific

- Less expensive

- Most specimens are negative

- Sensitivity: The proportion of truly positive results, as measured by the gold standard, that are identified as positive by the test under study

• Confirmation: The second step (In the lab)

- Specific

- More expensive

- Most specimens that go from screening to confirmation are positive, and results must be quantitative

- Specificity: The proportion of truly negative results, as measured by the gold standard, that are identified as negative by the test under study

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Forensic Drug Testing Has Limits

• Both screening and confirmation must be positive for the test to be

reported positive

• A negative result does not guarantee the absence of alcohol or drug

• Positive alcohol or drug levels do not differentiate acceptable use

from abuse even if elevated

• Positive alcohol or drug levels do not indicate impairment

• There is no dose/result relationship because there is no way to tell if

level is rising or falling

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CutoffsCutoffs are quantitative levels. If alcohol or drug is present above cutoff, the

specimen is positive, if not, the specimen is negative.

• Three Types:

- Limit of Detection (LOD): The lowest concentration at which a measurand can

be identified, but (for quantitative assays) the concentration cannot be

accurately calculated.

- Limit of Quantitation (LOQ): For quantitative assays, the lowest concentration

at which the identity and concentration of the measurand can be accurately

established.

- Administrative: Above both LOQ, and LOD. Balances detection and forensic

defensibility requirements

• Cutoffs may vary

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Drug Effects And Detection Periods

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Effects:� Intoxication Minutes to Hours� Impairment Minutes to Hours� Under Influence Minutes to Hours

Detection Periods:� Blood Minutes to Days

• (Exception: Peth is 2-4 Weeks)� Oral Fluid (Saliva) Minutes to Days� Urine Hours to Days � Hair Days to Years

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Interpretation Guidelines

• Generally speaking, the longer the detection period of the

specimen, the more drug usage required to produce a positive

result

• All specimen results are two dimensional:

- Is the analyte concentration going up or down?

- Results from different collections using the same specimen matrix

within the detection window can help answer that question

- Multiple results using alternative specimens may be less clear,

but may hint at total amount of usage

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Matrix Differences

• Urine (aqueous)

• Oral Fluid (aqueous, mucus, adsorption on collection device)

• Hair (dry, protein-complex)

• Drug uptake, retention, possible metabolism is different in each matrix

A positive in one matrix is not invalidated by a negative in another

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Hair Testing for Drugs of Abuse

• Long time window for drug

detection

• Easy to collect, handle,

store

• Noninvasive

• “Beating” a hair test may be

more difficult than with

urine

• Does not detect recent use

• Environmental contamination is a

concern

• Controversial hair color bias potential

unresolved

• Mechanism(s) of drug deposition not

understood

• Dose/time relationships not established

• Larger drug panels use more hair: qns

concerns

Advantages Disadvantages

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Hair Stages of Growth

• Anagen Phase: Growing

• Catagen Phase: Transition

• Telogen Phase: Resting

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Anagen Phase: Hair Growth Rates (mm/day)

Scalp Crown 0.35

Scalp Vertex 0.44

Beard 0.27

Chin /Lip 0.38

Axilla 0.30

Chest 0.40

Arm 0.20

Thigh 0.20

Pubic 0.40

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Telogen (Resting) vs Anagen(Growth)

Body

Area

% Telogen

Hair

% Anagen

Hair

Telogen

Duration

Follicles

Density (1/Cm² )

Depth of

Follicle

Scalp 13 85 3-4 Months 350 3 - 5 mm

Beard 30 70 10 Weeks 500 2 - 4 mm

Upper Lip 35 65 6 Weeks 500 1 - 2.5 mm

Axillae 70 30 3 Months 65 3.5 - 4.5 mm

Trunk 70 2 - 4.5 mm

Pubic Area 70 30 12 Weeks 70 3.5 - 4.5 mm

Arms 80 20 18 Weeks 80

Legs and Thighs 80 20 24 Weeks 60 2.5 - 4 mm

Breasts 70 30 65 3 - 4.5 mm

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Case 1

• 24 year old male medical student with history of alcohol abuse and experimentation with other substances (MDMA, Adderall®, marijuana) referred by medical school after 2 alcohol-related arrests

• All UDS and PEth tests negative. On random hair test, positive result for amphetamine. He claimed last use of Adderall® was 8 months prior to test

• Hair specimen had been taken from pubic area because he has insufficient head hair

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Case 1 (Q&A)

• What do you think?

• Pubic area hair grows more quickly but has a longer resting phase so it may be

positive for 12-18 months.

• Repeat hair sample taken from leg was negative

• Still monitoring closely because of past stimulant misuse

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Case 2

• 42 year old female physician with history of prescription opioid dependence, depressive disorder and anxiety disorder

• Completed a 3-month residential treatment and signed 5-year monitoring contract

• Provided several abnormal UDS specimens in monitoring (2 dilute, 1 over-concentrated) which led to hair test

• Hair test was positive for oxycodone, hydrocodone and hydromorphone

• Participant claimed that the hair was taken from the ends of her long, uncolored/ untreated but plaited hair, about 5 inches out from scalp

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Case 2

• Participant was referred for a recovery status evaluation which

did not uncover any other concerns from treatment providers,

family, sponsor

• Repeat hair test 2 weeks later was negative

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Case 2 (What Happened?)

• We may never know for sure:

- Collectors should always be interviewed in cases like this

- If she told the truth about the collection issue:

• It may boil down to donor’s word vs collector’s word

• Even though the laboratory attempts to identify hair orientation and length, there is no guarantee

- If the collection was done properly

• A second collection is always a brand new drug test

• Results of the second collection do not invalidate the first collection

• Hair in the second test may be from a different part of the body and/or in different stges of growth

than the hair in the first specimen

• Participant was found safe to practice with continued monitoring including

observed urine collections

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Urine and Alternative Specimens in Monitoring

• Urine Testing is the Forensic Backbone of the Program

• Follow up invalid or indeterminate results with an alternative

specimen (hair, nails, PEth, with appropriate detection period

• Randomness is Key

- Random testing frequency

- Random choice of specimen

- Follow-up to all unexpected results

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Action on Alternative Specimen Results?

• Action based solely on a hair or nail test result should be

taken with caution.

• So why do we bother with alternative specimens?• They provide a beneficial back-up approach to strong forensic urine based

monitoring programs

• One drug test result by itself does not make a diagnosis, but it can provide a

helpful perspective of the total picture

• Alternative specimen testing may be able to do the same thing for “gray area” urine results

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Validity Testing (SVT)

• HHS mandated since 11/1/2004 on all federal urine specimens

• SVT should be done on all urine specimens, and

• Appropriate SVT should be developed for all forensic specimens,

BUT IT CURRENTLY DOES NOT EXIST FOR HAIR, NAILS, OR ORAL FLUID

Validity testing is the evaluation of a specimen to determine if it is

consistent with normally expected values for humans and if the

observed properties allow detection of target analytes

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Urine Validity Testing Includes

• Measures of urine concentration/dilution

- Creatinine on all specimens

- Specific gravity only if creatinine <20 mg/dL

• pH

• General screen for oxidants

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The Solution to Pollution is Dilution

• Average creatinine concentration range is 100 mg/dL -150 mg/dL

• Creatinine not considered low if above 20 mg/dL

• 4460 workplace specimens studied, creatinine corrected to 100

mg/dL:

– Opiates positives increased 18%

– Amphetamines positives increased 58%

– THCA positives increased 105%

J Price, J, J. Addict. Med. Volume 7, Number 2, March/April 2013

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“False” Positives and Negatives

• “False” Positives:

- Poppy seeds

- Incidental alcohol exposure

• “False” Negatives:

- Dilute negatives

- Non specific test panels

◦Tests looking for parent drugs not metabolites◦Tests looking for different drug isomers

- Drug present but below cutoff

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Urine Testing Issues

• The best way to pass a drug test is to not take one

• Short detection period• If dilution or tampering was intended to hide a drug, the drug may be gone by the time re-

collection is performed

• Recommendation: Test an alternative specimen with a longer detection period• Hair/nails for drug/s

• PEth for alcohol

• Sensitivity• Urine is a more sensitive matrix

• Alternative specimens less sensitive: More ingestion needed before hair, nails, PEth become positive.

• Using all available matrices gives better perspective on what is actually happening, but

• A negative hair test does not invalidate a positive urine test and vice-versa

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Case 3

A certified laboratory reports the results of a urine collection as having a creatinine of 1.5 mg/dL, specific gravity of 1.0035.

How should these results be interpreted?A. Negative specimen

B. Substituted specimen

C. Adulterated specimen

D. Invalid Specimen

E. Dilute Specimen

What should you do?

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Case 3 (answers)

D. Invalid Specimen

You should consider the “Myth of the Observed Collection”

• Creatinine and specific gravity values should approximate each other

• If they don’t the specimen is inconsistent with normal urine

• Absent a very unusual medical condition, there was a collection issue

• The donor tampered with the specimen and avoided a fair drug test

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Weird Metabolites and Interpretations (Role of the MRO)

• The role of the metabolite

• Metabolite presence confirms drug ingestion

• For hair and nail, confirmed presence of only parent compound may indicate

contamination not ingestion

• Not true for urine

• Opioids

• Benzodiazepines

• Methamphetamine

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CODEINE*

MORPHINE*

HEROIN(DIACETYLMORPHINE)

6-ACETYLMORPHINE(6-AM OR 6-MAM)

OXYCODONE*

OXYMORPHONE* Noroxycodone

Noroxymorphone

NormorphineMorphine Gluduronide

Norcodeine

HYDROCODONE*

HYDROMORPHONE

Dihydrocodeine

Norhydromorphone Nordihydroisomorphine

*Forms glucuronide metabolites

Opiate/Opioid Metabolism

Poppy Seeds HYDROMORPHONE/codeine (minor)

Norhydrocodone

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Interpretive Implications - 1

• Parent compound eliminated more quickly than metabolites

• We now test for the ‘nor’ metabolites of both

• A person taking oxycodone could have drug test positive only

for oxymorphone or noroxycodone

• Someone taking hydrocodone could have drug test positive

only for hydromorphone or norhydrocodone

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Interpretive Implications - 2

• Important not to rely on parent/metabolite ratios, which change

throughout metabolic cycle, except

• Both codeine and more commonly morphine may produce

minor metabolites

• When a large amount of morphine is present, hydromorphone may be seen

in a quantity up to 12% of the total morphine

• Smaller amounts of codeine have also been reported

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Case 4

Urine tests positive for hydromorphone at 225 ng/mL.

Hydrocodone is negative. Donor denies use of morphine or

hydromorphone. Donor presents valid hydrocodone RX; says

only takes in the evenings at bedtime

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Case 4

• What are possible reasons hydrocodone was negative?

A. Present but below cutoff?

B. Metabolized more quickly than metabolite?

C. Donor took Dilaudid but submitted hydrocodone RX?

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Case 4 (answers)

• All are possible reasons hydrocodone was negative

A. Present but below cutoff

B. Metabolized more quickly than metabolite

C. Donor took Dilaudid but submitted hydrocodone RX

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Case 5

• Laboratory confirmed positive for oxazepam.

• Participant presents a prescription for Klonopin ® (clonazepam) that is current, and also states he had sedation for a dental procedure one month previously.

• Did clonazepam cause this result?• Clonazepam did not cause this result

• What is the best action? • Ask the participant to verify the medication used for the dental sedation

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Case 6

• Confirmed positive methamphetamine 2256 ng/mL; amphetamine 1012 ng/mL. Donor denies use or RX:

• Your first thought?• Do you have the results of a d/l separation?

• ‘d’ meth is illicit meth “d=drug”

• ‘l’ meth is not psychoactive “l=legal”

• If ‘l’ methamphetamine is present greater than 80% source may be generic dry nasal inhaler or prescription selegilene.

• Donor just remembered he used a dry nasal inhaler.

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Case 6

• What do you make of this?• If ‘d’ methamphetamine is present at greater than 20% of the total the result is consistent with illicit methamphetamine use

• If ‘l’ methamphetamine is greater than 80% of the total the result is not consistent with illicit methamphetamine use.

• It may be consistent with either dry nasal inhaler use or ingestion of selegilene.

• Selegilene is pharmaceutically pure ‘l’ methamphetamine, not commonly abused but still requires an RX and the RX should be verified.

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Urine Alcohol Interpretation Issues

• Alcohol can form in vitro after the urine has been collected:

- Glucose present

- Microorganisms present in the urine

- Urine stored at room temperature without preservative for 1 or more days

• Urine alcohol testing is very problematic in workplace/deterrent testing

• Urine alcohol should not be used to verify alcohol abstinence by itself

• Confirm EtG/EtS even if no glucose is present.

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Alcohol Monitoring: Direct Biomarkers

• So named because these are direct products of ethanol (ethyl

alcohol) metabolism

• Not produced by any other form of alcohol (isopropyl, cetyl, sugar,

etc.)

• Produced by very small percentage of the total ethyl alcohol

consumed

• Includes:

- Urine: EtG, EtS,

- Blood/blood spot: PEth

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Ethyl Glucuronide, EtG

• Direct metabolite of ethanol

• Non-volatile, water-soluble

• Conjugation of ethanol with activated glucuronic acid in the presence of membrane bound mitochondrial UDP glucuronyl transferase (Stephan Seidlet al)

• Can be detected for up to 80 hours after alcohol elimination (depending on cutoff, use patterns, metabolism)

• It has been reported that as little as 0.02 to 0.06% of the ethanol ingested is recovered as EtG. (Dahl et al. 2002, Goll et al. 2002)

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EtG is not stable

• Postcollection synthesis and metabolism of EtG by bacteria in urine may

cause wildly fluctuating EtG levels

• High concentrations of EtG (24-h range 0.5–17.6 mg/L) were produced

during storage in 35% of E. coli-infected urines containing ethanol. In some

specimens that were initially EtG positive because of recent alcohol

consumption, EtG was also sensitive to degradation by bacterial hydrolysis

• EtG used alone can result in both false positive and false negative

indications of ethanol presence

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Ethyl Sulfate, EtS

• In contrast to the previous slide, EtS was completely stable under those conditions.

• Formed by a different pathway

• Ethanol undergoes sulfate conjugation through the action of

sulfotransferase to produce ethyl sulfate (EtS)

• EtS is excreted in urine mainly during the first 24 hours

• Because EtG and EtS are formed via different pathways they can and should be used conjointly, thereby increasing sensitivity.

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EtG/EtS Interpretation Issues

• EtG can be both formed and degraded in a urine sample – retests often differ greatly in concentration due to bacterial contamination

• EtS is not produced in vitro and is stable

• EtG is one factor used to suggest alcohol ingestion but, • A thorough Medical Review is recommended to identify other potential

sources for the alcohol exposure

• Results are affected by hydration: lower creatinine leads to lower EtG results

• All results require EtS to be present with EtG to confirm the presence of EtG from alcohol ingestion.

• This information has been successful in defending positive EtG results.

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Phosphatidyl Ethanol (PEth)

• A direct biomarker that incorporates into cellular membranes

• Long lifespan - t1/2 4.4 days

• Stable molecule, minimally metabolized

• Stays in red cell membrane until it decomposes or cell dies.

• 2-4 week window of detection

• 5 molecular fractions comprise 80% of total PEth in blood• 16:0/18:1, 16:0/18:2, 16:0/20:4, 18:1/18:1, 18:1/18:2

• Only 16:0/18.1, 16:0/18.2, 18:1/18:1 are routinely tested

• Only 16:0/18.1 routinely reported

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PEth Indications

• Testing blood for phosphatidyl ethanol (PEth) is a reliable way to

determine alcohol ingestion of at least 2-3 standard drinks or more

occurring in the 2-4 weeks prior to the test

• PEth testing is ordered when there is significant concern about

possible alcohol use

- Following a low positive EtG/EtS test when participant denies alcohol

consumption

- After a third dilute UDS or any UDS with extremely low creatinine

- As part of an initial, recovery status or appropriateness for completion evaluation

- Following any reported suspicion of drinking

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Case 7

• 57 year old female nurse treated for diabetes and hypertension had the

following results on 12/29/18:

• Urine alcohol 22 mg/dL, glucose negative, EtG at 1310 ng/mL, EtS at 123 ng/mL, creatinine 243 mg/dL.

• During MRO review she told me she may have ingested a small amount of an otc alcohol containing medication.

• 1/11/19 negative PEth.

• She had multiple positive urine alcohol results during the succeeding

months, all with negative EtG’s until 5/23/19:

• Urine alcohol 39 mg/dL, glucose negative, EtG 4734 ng/mL, EtS 386 ng/mL, creatinine 222.7 mg/dL.

• During MRO review she told me she ate alcohol containing barbeque sauce

• 6/3/19 PEth negative.

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Case 7 Questions & Answers

• Are the 12/29 lab results definitive for alcohol ingestion?• No

• Creatinine corrected EtG is 539 ng/mL but EtS is 50 ng/mL

• Studies show EtS <100 ng/mL may be from skin absorption

• Diabetics frequently produce positive urine alcohols from fermentation

• Are the 5/23 lab results definitive for alcohol ingestion?• Maybe

• Creatinine corrected EtG is 2118 ng/mL, EtS is 173 ng/mL

• What do the negative Peths tell us?• The amount of alcohol that might have been ingested was not enough to make

PEth positive.

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PEth Only Formed in the Presence of Ethanol

• Research ongoing since 1983

• No false positives have been reported (Kechagias, et al, 2015)

• Single ethanol dose of 30-47gm (2-3 drinks):

• did not produce measurable amounts of total PEth at high cutoff(Varga, et al,

1998)

• but would be expected to produce positive 16:0/18:1 at 20 ng cutoff (Javors, et

al, 2016)

• Statistically significant differences in the mean values and confidence

intervals of total PEth concentrations in heavy drinkers(>60g/day) and

social drinkers(<60g/day) (Wurst, et al, 2010)

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More Research

• 32 males, 12 females studied for 3 months:1-2 glasses of wine/day

produced PEth up to 60 ng/mL (Kechagias, et al, 2015)

• 80 females aged 18-35: 2+ glasses of wine a day produced PEth 127

ng/mL (Stewart, et al 2010)

• 252 participants: PEth results in combination with previous EtG

results allow differentiation between exposure and drinking (Skipper,

et al, 2013)

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Case 8

• 5/5/19

• EtG 481 ng/mL

• EtS 72 mg/mL

• Creatinine 19.3 mg/dL

• Are these lab results definitive for alcohol ingestion?

• Yes

• Creatinine corrected EtG is 2492 ng/mL, EtS is 373 ng/mL

• Denies drinking beverage alcohol but admits to daily

consumption of fermented pro-biotics

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Case 8 (cont)

• 5/17/19

• PEth 32.9 ng/mL

• So, what is the definition of beverage alcohol?

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Conclusion

Each drug test result stands on its own:

- A second result does not invalidate the first result

- An alternative specimen result does not invalidate a urine result

- In either case the second result may support the first

- BUT: Failure of a split specimen to reconfirm the original result does invalidate the original result

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No matter how hard we

work,

No matter how right we

are…..

Just Remember -

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Questions?

Thank You!

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