Forensic Drug Testing Part 1

7/25/2019 Forensic Drug Testing Part 1

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Forensic Drug Testing

Part 1: ScreeningRoger L. Bertholf, Ph.D.

Associate Professor of Pathology

Chief of Clinical Chemistry & Toxicology



What is forensic drug testing?• MDs order drug tests to evaluate the

medical condition of a patient

– Medical drug testing or – !linical To"icolog#

• $mplo#ers order drug tests to determine

%hether someone uses illegal drugs – Drug testing for legal purposes or

– Forensic Drug Testing



Medical vs& forensic drug testing• Patient consent not

re'uired

• (dentit# of specimen is presumed

• Screening result issufficient for medical

decision• )esults are used for

medical evaluation

• Su*+ect must consentto *e tested

• (dentit# of specimenmust *e proved

• ,nl# confirmed resultscan *e considered

positive• )esults are used for

legal action



(llegal Drug -se in the -&S&

.1//0 ousehold Surve#2

• 13&4 million 5mericans use illicit drugs

– 67 million in 1/8/

• 0&39 of #ouths age 1618 use mari+uana

– 1;&69 in 1/8/

• 1&0 million 5mericans use cocaine

– 7&8 million in 1/07



T#pes of drugs used

Marijuana

an some

other rug

!"#

Drug other

than

marijuana"$#

Marijuana

only

%&#

Marijuana

an some

other rug

!"#

Drug other

than

marijuana"$#

Marijuana

only

%&#



T#pes of drugs used

&

"

!

'

(

)

%

*

P e r c e n t u s i n g

i n

+ r e , i o u s ' &

a y s

All rugs T-C PsyRx Cocaine LD, etc. /nhalants

&

"

!

'

(

)

%

*

P e r c e n t u

s i n g

i n

+ r e , i o u s ' &

a y s

All rugs T-C PsyRx Cocaine LD, etc. /nhalants



istor# of %or<place drug testing• 1/4=s – 1/8=s: The Department of Defense

*egins testing militar# personnel for illegal

drug use&• 1/04: President )eagan esta*lishes the

>Federal DrugFree Wor<place&

• 1/00: Mandator# @uidelines for FederalWor<place Drug Testing Programs is pu*lished in the Federal Register.



The >A(D5 program• A(D5 .no% S5MS52 re'uirements for

drug testing %ere drafted *# )esearch

Triangle (nstitute• The )T( esta*lished the Aational Ba*orator#

!ertification Program .AB!P2

• Drug testing for federal agencies .D,T A)! etc&2 must *e performed in a AB!Pcertified la*orator#



Florida DrugFree Wor<place• The Florida )S .no% 5!52 esta*lished a

drugfree %or<place program in 1//=

• Specifications for the State of Florida program are similar to federal re'uirements *ut there are nota*le differences

• $mplo#ees of Florida DrugFree Wor<placecompliant *usinesses must *e tested in5!5licensed la*oratories



!omparison of AB!P !ertified

and 5!5 BicensedBa*oratories

• Florida Drug FreeWor<place Program

• 1= drugs C ethanol

• (nspected ever# 4 months

• uarterl# proficiencies

• Director must *e *oard

certified

• Federal emplo#eesfederall#regulated +o*s

• 7 drugs

• (nspected ever# 4 months

• uarterl# proficiencies

• Director must *e *oard

certified

5!5 AB!P



Screening• Sensitivit# vs& specificit# of anal#tical

methods



Performance characteristics of

screening tests

1 :

S e n s i t i v i t #

Specificit#

)eceiver ,perator !haracteristic

.12

.62

.72

.1=2.162

.172 .6=2 .7=2 .0=2 .1==2



Screening• Procedure is designed to eliminate all

negatives

• Positive screens are presumptive

• Aegative screens can *e revie%ed and

released *# a Scientific )evie% ,fficer

• Positive screens are su*mitted for

confirmator# testing



!hallenge 'uestion & & &• We regularl# use immunochemical methods

for 'uantif#ing therapeutic drugs *ut

consider them >screening methods fordrugs of a*use&

Why?



(ntroduction to omogeneous

(mmunoassa#• What is the distinguishing feature of homogeneous

immunoassa#s? – They do not require separation of bound and free

ligands

• Do homogeneous methods have an# advantage.s2over heterogeneous methods? – Yes

• What are the#? – Speed

– Adaptability



$nE#melin<ed immunosor*ent

assa#

Microtiter %ell

E E E E E

Specimen 6nd anti*od#

E

Su*strate

S P



omogeneous immunoassa#s• irtuall# all homogeneous immunoassa#s

are one-site

• irtuall# all homogeneous immunoassa#sare ompetitive

• irtuall# all homogeneous immunoassa#s

are designed for small antigens – Therapeuti!abused drugs

– Steroid!peptide hormones



T#pical design of a homogeneous

immunoassa#

Ao signal

Signal



$nE#memultiplied immunoassa#

techni'ue .$M(TG2

• Developed *# S#va !orporation .Palo 5lto !52

in 1/8=sno% o%ned *# Hehring Diagnostics• ,ffered an alternative to )(5 or PB! for

measuring therapeutic drugs

• Spar<ed the %idespread use of TDM

• 5dapta*le to virtuall# an# chemistr# anal#Eer

• as *oth 'uantitative .TDM2 and 'ualitative.D5-2 applicationsI forensic drug testing is themost common use of the $M(T methods



$M(TG method

$nE#me

S

S P

Ao signal

Signal$nE#me

S



$M(TG signalJconcentration

curve

S i g n a l

. e n E # m e a c t i

v i t # 2

5ntigen concentration

0unctional concentration

range



Fluorescence polariEation

immunoassa# .FP(52• Developed *# 5**ott Diagnostics a*out the same

time as the $M(T %as developed *# S#va

– )oche mar<eted FP(5 methods for the !o*as F5)5anal#Eer *ut not have a significant impact on the

mar<et

• Bi<e the $M(T the first applications %ere for

therapeutic drugs• !urrentl# the most %idel# used method for TDM

• )e'uires an 5**ott instrument



Molecular electronic energ#

transitions

$=

$;

$3

$6

$1

Singlet

Triplet

5

1R

F

/C

P

1=41=/ sec

1=;1= sec



PolariEed radiation "

y

#

PolariEing

filter




OHO OH

C

O

O

Fluoresceinλin

$rientation of polari"ed radiation is maintained%

λout .1=41=/ sec2




O

H O

O H

C

O

O

)otational fre'uenc# ≅ 1=1= sec1

λin

$rientation of polari"ed radiation is &$T maintained%

λout .1=41=/ sec2

'ut. . .




immunoassa#

OHO OH

C

O

O

PolariEation maintained

lo2 rotation

OHO OH

C

O

O

Ra+i rotation

PolariEation lost



FP(5 signalJconcentration curve

S

i g n a l . (

J ( ⊥ 2



range



!loned enE#me donor

immunoassa# .!$D(5G2

• Developed *# Microgenics in 1/0=s

.purchased *# HM! then divested *#)oche2

• Hoth TDM and D5- applications areavaila*le

• 5dapta*le to an# chemistr# anal#Eer

• !urrentl# trails $M(T and FP(5applications in mar<et penetration



!loned enE#me donor

Donor

Acce+tor

Monomer

3inactie4

Actie tetramer

+ontaneous

β@alactosidase



!loned enE#me donor

immunoassa#

Donor

Acce+tor

Donor

Acce+tor

Ao activit#

5ctive enE#me



!$D(5G signalJconcentration

curve

S i g n a l

. e n E # m e a c t i v i t # 2



range



Screening thresholds• Why do (e need sreening thresholds?

– To ensure that results in all participating

la*oratories agree

• Who determines the thresholds?

– The agenc# sponsoring the drug testing

program .e.g. S5MS5 State of Florida orindividual emplo#er2



Screening thresholds for

S5MS5 drugsDrug ng5mL urine

5mphetamines 1===

!ocaine .as *enEo#lecgonine2 3==

,piates .morphine codeine2 6===

Phenc#clidine 67

T! 7=



)o sreening thresholds have any

quantitative relevane?

• !rossreactivit# of anti*odies

– 5mphetamines – !anna*inoids

– ,piates

– HenEodiaEepines *ar*iturates

• Ph#siological factors

– Diuresis



H

N

CH3

Methamphetamine

CH3

CH3

5mphetamine

NH2

5mphetamines

• !lassified as s#mpathomimetic amines .or

phen#leth#lamines2• !AS stimulants Schedule (( drugs .high

a*use potential2



S#mpathomimetic amines

CH3

NH2

H

N

CH3

CH3

CH3

NH2

H

N

CH3

CH3

OH

OH

CH3

NH2

H

N

CH3

CH3

H3C

H3C

C ,

C ,C !3

C !3

5mphetamine Phen#lpropanolaminePhentermine

Mephentermine Methamphetamine $phedrine



5mphetamine stereochemistr#

• Pharmacological preparations of amphetamine

can *e racemic d*l mi"tures .HenEedrine2 or pure d amphetamine .De"edrine2

• Most immunoassa#s are cali*rated %ith d*l

amphetamine

NH2

H3C H

NH2

H CH3

d 5mphetamine l 5mphetamine



Methamphetamine stereochemistr#

• d Methamphetamine is 1= times more

potent than the l isomer

• lDeso"#ephedrine is used in some non

prescription nasal decongestants

H

N

H3C H

H

N

H CH3

d Methamphetamine l Deso"#ephedrine



5mphetamine derivatives:

>Designer Drugs

NH2

CH3

O

O

H

N

CH3

O

O

Meth#lenedio"#amphetamine Meth#lenedio"#methamphetamine



!ocaine

N

H3C

O

O

CH3

O

O



!ocaine meta*olism

N

H3C

O

O

CH3

O

O

N

H3C

O

O

CH3

OH

N

H3C

OH

O

O

O

H

N

O

O

CH3

O

O

$cgonine meth#l ester HenEo#lecgonine Aorcocaine

!47!,,

!3

!3



Phenc#clidine

N

Phenc#clidine



∆/Tetrah#drocanna*inol .T!2

O

CH3

OH

H3C

H3C

O

COOH

OH

H3C

H3C

,"idation

∆/T! ∆/T!!,,



,piates

OHO OH

N

H3C

H

OO OH

N

H3C

H

H3C

Morphine !odeine

!3



eroin meta*olism

OO O

N

H3C

H

eroin

OHO OH

N

H3C

H

Morphine

H3C

O

CH3

O

O

HO O

N

H3C

H

4Monoacet#lmorphine CH3

O

!3!,

!3!,



Summar#

• Screening is the first step of a t%ostep

process in forensic drug testing

• Screening methods are designed toeliminate negative specimens

• Positive screens are presumptive

• Several homogeneous immunoassa#s have

*een developed for drug screening



Thank You!

Documents

Forensic Drug Testing Part 1