drugsofabuseII2012

CLSC 4440 Clinical Chemistry II – Drugs of Abuse

Kathleen Schulman Ph.D., MT(ASCP)

Susan T. Smith Ph.D., MT(ASCP)Rev 2012

Drug Analysis

This topic is covered in K & P in chapter 55, starting on page 1085

Drug Analysis

Purpose of Drugs of Abuse (DOA) testing – A. Emergency Room- ID toxic substance in ER- eg. Accident related

injury. Need rapid ID method B. Workplace drug screening- 1. for employer- pre-employment 2. federal regulations – DOT and NRC; cover

employees in commercial transportation, intrastate transportation

emphasis here is on accuracy and sensitivity 3. athletes- required for championship events

Analytes most commonly encountered in ER -

Good Resource

Table 55-9 K & P, p. 1102 – This table lists: A. commonly used drugs of abuse B. how long they can be detected in the

body C. what metabolite is actually detected in lab

tests D. legal cutoffs for calling results positive

using screen and confirmation tests established by SAMSHA.

Analytes most commonly encountered in ER -

Clinical features of overdose: CNS depression (COMA) common,

patient usually unable to tell doctor what they took

Specimen Types Used

Drug Analysis

Handling “Legal” specimens – Must be able to establish “chain of custody” Usually requires witness for specimen collection,

signature for all possession transfers Use “proper, recognized” procedure Analytical method used and name of analyst Date and time of analysis Document testing used to detect adulteration of

sample In short- Document EVERYTHING

Drug Screening - Overall plan

If urine sample used for testing, evaluate sample first and then use a broad screen followed by specific confirmatory tests

A. Adulteration – if sample is urine B. Screen – constructed so there are no

false negatives C. Confirm – constructed so there are no

false positives

Drug Screening

Note: when the sample to be screened for drugs of abuse is sent as a clinical drug screen from the ER, the results are often not confirmed.

This is an example of the analytical processin DAU testing.

Image source: http://www.csc-scc.gc.ca/text/rsrch/reports/r149/r149_ex51x1.jpg

Urine Sample Evaluation Tests

Sample evaluation

Urine sample is evaluated prior to testing to insure it is appropriate for analysis and to detect possible presence of adulterants.

Adulterant- substance added to urine sample to mask presence of drugs of abuse.

Sample evaluation

Do adulteration tests- 1. pH – should be 5-9 2. specific gravity – 1.003 – 1.030 3. creatinine – should be 25 – 400

mg/dL 4. color – usual, not odd 5. odor – usual; may smell adulterants

Sample evaluation

Products used to mask positive urine tests-

Add bleach, ammonia, oxidizing agents such as potassium dichromate, commercial products such as UrinAid, Klear, Whizzies, Sweet Pee’s Spoiler, Urine Luck

Often detect by visual checks

Drug Screen Tests

Drug Screening

Procedure used may vary depending on the type of testing

A. pre-employment screen B. ER patient with possible overdose C. Athlete in major competition Drugs usually screened for include – cocaine,

opiates, amphetamines, cannbinoids, PCP, bartituates, tranquilizers

This group required by Fed gov for workplace testing

Cutoff limits for detecting positives set by state and Federal laws

Drug Screening

Three main types of screens done: A. Spot tests B. comprehensive drug screen C. immunoassay

Drug Screening- Spot Tests

Test for acetaminophen, salicylate, alcohols Confirmation – immunoassay, spectroscopy,

GLC, HPLC Available for salicylates, acetominophen,

phenothiazines, tricyclic anti-depressants Advantages – these tests are quick and

easy to do; get quick negative result Disadvantages – non-specific, must

confirm positive results

Drug Screening- Comprehensive

drug screen

Single pH solvent extraction of urine Do TLC for preliminary ID Confirm with GC-MS, immunoassay,

UV spectroscopy Quantitate when indicated Advantage – comprehensive Disadvantage – TLC not quantitative,

time consuming, difficult to interpret

Drug Screening- Immunoassay

available for drugs like benzodiazepines and opiates

Confirmation can be done with TLC, GC-MS

Advantage – quick and easy Disadvantage – may not be specific,

or must run separate tests for individual drugs

Screen Tests

Common causes of false positives or false negatives

False positives- Poppy seeds – opium, morphine Herb tea- mate de coca- cocaine False negatives – Urine too dilute Adulteration of sample

Confirmation Testing

Classification of Drugs

Urine Extraction for Drug Assays

Purpose: to improve the specificity of assay by removing metabolites and interfering substances

Types – multiple pH extraction preferred; single pH – use 9 or 3

Typical Extraction Diagram

Sample acidified to pH 3.0

Extraction with methylene chlorideCH3Cl

Organic Layer(A)Neutral & Acid Drugs

Aqueous Layer(A) Alkaline drugs

Organic Layer (A) Neutral & Acid Drugs

NaOH, pH 11

Organic Layer(B) Tranquilizers Neutral Drugs

Aqueous layer (B) Barbiturates

Salicylates

Organic Layer A Extraction

Organic Layer A Extraction Aqueous layer (B) Barbiturates

Salicylates

pH 7.4 Buffer +CH3Cl

AqueousSalicylates

Organic Layer Barbiturates

Organic Layer A Extraction

End result of Organic Layer A extraction: Have separated out – A. tranquilizers and neutral drugs B. Salicylates C. Barbiturates A specific assay is used on each fraction to

ID and quantitate the drugs there

Aqueous Layer A Extraction

Aqueous Layer (A)Alkaline drugs

KOH, CHCl3

OrganicAmphetamines, narcotics,

alkaloids

Aqueous Layer Waste

Use specific assays on the organic fraction to ID and quantitate

Assays for Specific Drugs

This will include: A. barbiturates B. salicylates C. phenothiazines D. acetaminophen The K & P CD ROM has information

on analytical techniques used for these and other drugs.

Assays for Specific Drugs

A. Barbiturates

Barbiturate solubility is pH dependent. Increased lipid solubility results in inc onset of action, dec duration,inc potency. Lipid solubility also leads to inc hepatic clearance and metabolism of barbiturate.

Barbiturates

Short to Moderate acting barbiturates are used as sedative-hypnotics

Longer acting barbs used as anticonvulsants Barbiturate types – A. Short acting – Nembutal, Seconal B. Moderate – Amytal, pentobarbital C. Long Acting- Phenobarbital, Luminol Street names of barbs – barbs, sleepers,

downers, yellow jackets, rainbow

Barbituates

Because of their low therapeutic levels and high potential for abuse, barbs have been replaced largely by benzodiazepines for sedative and hypnotic purposes

Analysis of Barbiturates

A. semiquantitative immunoassays – Use secobarbital as calibrator at cutoff conc

of either 200 or 300 ng/mL Assays can be done on either urine or

serum B. TLC – used on urine samples C. GC-MS- used as confirmation test for

positive immunoassay result in urine

Barbiturate Analysis

D. GLC for Barbs – can both ID and quantitate mixture

Is considered reference method for barb analysis

Barbiturate Analysis GLC chromatogram of barbiturates 1- Butalbital2. Amobarbital 3. Pentobarbital 4. Secobarbital 5. Phenobarbital 6. Phenytoin

Web Sites for Clinical Toxicology

Good web site with info on common toxins http://www.atsdr.cdc.gov/toxfaq.html#-A-

http://emedicine.medscape.com/emergency_medicine -Good web site on clinical toxins commonly seen in the ER.

Salicylates

Are a common toxic agent in children Causes respiratory alkalosis which converts

to metabolic acidosis, because salicylate produces acidic metabolites

Is ingested as acetyl salicylate – rapidly hydrolyzed in stomach and absorbed as salicylic acid

Therapeutic range – 15 – 30 mg/dL Toxic levels - >30mg/dL Fatal levels - >60mg/dL

Salicylates

Can asses severity of poisoning by using nomogram

To use nomogram, must know time of ingestion or obtain at least two samples to plot a disappearance curve

Salicylates

Assay methods – A.GLC or HPLC reference method – not

usually available for ER use B. Spectrophotometric – has specific UV

spectrum C. Immunoassay – FPIA (TDX), EMIT;

most commonly used today D. Colorimetric – still in common use,

inexpensive, automated on ACA

Salicylates

Colorimetric method (Trinder) Serum or urine + “Trinder Rgt” Trinder reagent – iron (III) nitrate, mercurous

chloride Fe(NO3)3, HgCl2 Salicylate + Fe+3 -------------------violet color

(prop to salicylate conc) Test measures total salicylate Need to precipitate protein and use serum blank

to reduce interferences

Salicylates

False positive results seen with phenylpropanolamine (PPA), other phenolic compounds

Can eliminate by boiling urine Keller modification – is a direct

method, no HgCl2, so protein does not precipitate

Phenothiazines

are tri-cyclic antidepressants , used as tranquilizers and antihistamines

Phenothiazines

Screening test- uses Forrest reagent

FeCl3 oxid Urine + HClO4------ salmon Violet HNO3 pink Reagent is very toxic False positives – PPA, estrogens, bile

metabolites Helpful when used with TLC

Acetaminophen

Common analgesic agent – Phenacetin, Tylenol,Paracetamol

Common cause of overdose ; very toxic to liver, especially in combination with alcohol

Acetaminophen nomogram – see K & P page 1101, fig 55-7 for nomogram

Nomogram is only for acute ingestion Treatment with N-acetylcysteine may be

required; should be given within 8 hours of ingestion

Acetaminophen

Assay methods – only methods specific for parent acetaminophen should be used

A. UV spectrophotometric – insensitive; measure metabolites and parent

B. HPLC – reference method C. Spot test – NH4OH + o-cresol + HCl -- blue

color Is most commonly performed test, has good

sensitivity E. immunoassay- FPIA, EMIT; rapid, easy,

accurate

Acetaminophen

Preferred sample for assay – no earlier than four hours post-ingestion to insure complete absorption of the dose

It may take 48 – 72 hours for toxic symptoms to appear

Acetaminophen

Interpretation of results – Therapeutic levels – 10 – 20 ug/mL Toxic values vary with time since dose To interpret results, must know time of

ingestion or draw 2 samples and plot an elimination curve

Confirmation Methods

Confirmation Tests

These include: A. Thin Layer Chromatography – TLC B. Gas Chromatography/Mass

Spectrometry- GC-MS

TLC – Thin Layer Chromatography

Is the best system for ID large variety of drugs and their metabolites

Requires great technical skill Toxilab system is an example of a

highly standardized TLC system with 24 hour consultation service


After identifying drug with TLC, must confirm by :

A. eluting compound and testing with specific assay

B. testing another aliquot of sample with specific test


Application of sample to plate Migration of sample contents In solvent medium


Example ofToxilab TLCresult. All fourcards are thesame TLC cardshown at dif-ferent stages inthe staining process.

Toxilab Cards

Toxilab

Basic procedure – A. extract sample with 2 different solvents and

concentrate standardized amount onto filter paper disc

B. place disc in chromatogram with standards C. develop in appropriate solvent, stain and

record results D. interpret by comparison to Photograms E. identification is based on Rf values obtained

in different stains

Gas Chromatography/Mass

Spectrometry GC/MS

GC/MS – see this resource http://ull.chemistry.uakron.edu/gcms/ This web site has a great tutorial on the

principles of GC/MS analysis. There is also a test to review your knowledge.GC-MS analysis is the most commonly used confirmation method, because multiple criteria have to be met in order to call a sample result positive.

Sample GC/MS Scan

Image source: http://pubs.rsc.org/ej/AN/2000/a906316a/a906316a-f1.gif

Sample of GC/MS results for Sample that contains morphine.

Image source: http://www.aafs.org/images/resources/toxfig3b.jpg

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