LC/MS/MS and GC/MSApplications in Testing Illicit

SubstancesSubstances

Dr. Darcie Wallace DuckworthAssistant Director of TrainingAegis Sciences Corporation

March 17, 2009

What is forensic toxicology?

• ABFT defines as “the study and practice ofthe application of toxicology to thepurposes of the law”

• Purpose• Purpose

– Obtain analytical data on poisons

– Apply information to understanding of episodeof intoxication

• Toxicology

– Study of toxic or harmful effects of chemicals

– Concerned with how toxins act, when effectshappen, and what are the symptoms andhappen, and what are the symptoms andtreatments

Worked is performed in the interest of

TRUTH

– Accuracy

– Honesty

– Agreement with fact or reality

Worked is performed in the interest of

JUSTICE

– Concept of rightness basedon law, fairness, and equity

Worked is performed in the interest of

PUBLIC GOOD

Brief History of Toxicology• Greeks-hemlock for state sponsored execution• Middle Ages-opium, arsenic, and hydrocyanic

acid poisonings• 1836 James Marsh developed the Marsh Test to

detect arsenic trioxidedetect arsenic trioxide• 1840 Mathieu Orfila involvement in LaFarge

case• 1851Jean Servais Stas development of alkaloid

extraction method• Beginning of 20th century Dr. Alexander Gettler,

lab director for New York City medical examiner

What is the Marsh Test?• Arsenic trioxide, As2O3, was a favored

poison

• Treat sample with H2SO4 and As free Zn,zinczinc

• Zn reduces the trivalent As

As2O3 + 6 Zn + 6H+ → 2 As 3- + 6 Zn 2+ + 3 H2O

Mathieu Orfila

• Father of toxicology

• In 1840, Marie LaFarge case– Marie was tried for murdering her husband by

Arsenic poisoningArsenic poisoning

– Court asked Orfila to investigate

– Discovered the initial Marsh test improperlydone

– Found Arsenic in the body

– Convicted largely on the work of Orfila

Biological Aspects of Toxicology• Chemicals usually cause effects by

interacting with cells to change theirfunction

• Damage to cells• Damage to cells

– Explosive or corrosive

– Irritation

– Sensitization

– Toxicity

Factors that Influence Toxicity• How Much?

• How Long?

• How Often?

• Routes of exposure-oral, dermal,inhalation

• Other factors-species, sex, age, nutrition,state of health, sensitivity, presence ofother chemicals

How Much? The Dose

• All substances have the capability of beingtoxic, depends on the dose

• Dose-response relationship

– As dose increases, the effect increases– As dose increases, the effect increases

How Long?• Duration of the exposure

How Often?

• Frequency the exposure occurs

• Dose-time relationship

• Acute versus chronic toxicity

– Acute– Acute

• Local damage or systematic change as result ofONE exposure to a relatively large amount ofsubstance

– Chronic

• Damage as result of repeated exposure to relativelysmall amounts over a prolonged time period

• What is

– A matrix?

• Biological specimen such as body fluid or solidtissuetissue

– Agent of interest?

• Exists in matrix in a simple solution or may bebound to protein or other cellular constituents

Our challenge…..

Separate toxic agent in sufficient purity and quantityto permit it to be characterized and quantifiedto permit it to be characterized and quantified

Metabolism

• Terms– Metabolism

• Aggregate of all physical and chemical changes that occurwithin the living substance of an organism

– Xenobiotic metabolism

» metabolism of substances that are foreign to the body of» metabolism of substances that are foreign to the body ofan organism

» Results in detoxification and elimination from the body,toxification and distribution to receptors, chemicallyunmodified and distributed to receptors

» Many are lipophilic, increase water solubility, and facilitatesexcretion

– Metabolite• Product of metabolism

Parent Drug versus Metabolites

Common Characteristics ofAnalytical Assays

• S/N– compares the level of a desired

signal to the level of backgroundnoise

– High S/N lower LOD and LOQ

• LOD

• Accuracy– how close a measurement is to

the accepted value

• Precision– how close together or how

repeatable the results are• LOD

– Limit of Detection– lowest quantity of a substance

that can be distinguished from theabsence of that substance

• LOQ– Limit of Quantitation– limit at which we can reasonably

tell the difference between twodifferent values

• ULOQ– Upper limit of Quantitation

– how close together or howrepeatable the results are

• Interference

• Robustness

GC/MS in Testing Illicit Substance

• Analytes must be chemically extracted frommatrix

– Matrices• Urine

• Blood• Blood

• Oral Fluid

• Hair, tissue, etc.

• Most cases should be derivatized

– Why derivatize?• Affect volatility, improve chromatography, enhance MS

pattern

• Used to identify and quantify DOA and licitpharmaceuticals

• Typical LOQ 1-10 ng/mL

• ULOQ limited by IS concentration• ULOQ limited by IS concentration

– IS chemically similar to analytes of interest

– Addition of constant amount to specimens,standards, controls and calibrators

• Operated in SIM

– Determines presence of selected ions that arepresent in specific ratios and quantifies bydetermining ion abundances compared to ISdetermining ion abundances compared to ISabundances

– IR abundances are unique for givencompound

– Use 3 to 2 ion ratios

Cannabinoids

• Mostly widely used illicit drug in US• Carboxy THC major urine metabolite of THC• Highly lipophilic, readily soluble in nonpolar organic solvents, and

slightly soluble in water• Carboxy THC excreted as ester-linked glucuronide conjugate

– Hydrolyzed after addition of 6 M NaOH, 15 min at 25ºC or 5 minat 50ºCat 50ºC

– Neutral and basic compounds removed by adding hexane andagitating

– After centrifugation, hexane layer is removed, aqueous layer isacidified and extracted with hexane:ethyl acetate

– Followed by derivatization• Alkylation with tetrahexylammonium hydroxide and

iodomethane in toluene• Silylation with BSTFA

GC/MS of Cannabinoids

1. cannabidiol2. Δ8-tetrahydrocannabinol3. Δ9-tetrahydrocannabinol4. cannabinol5. 11-hydroxy-Δ9-

tetrahydrocannabinoltetrahydrocannabinol6. 11-nor-Δ9-tetrahydrocannabinol

carboxylic acid

http://www.restek.com/aoi_forensics_A006.asp

Amphetamines

• Methamphetamine-most commonlyabused class of drugs

• Amphetamine

• MDA-Adam• MDA-Adam

• MDMA-Ecstasy

• MDEA-Eve

Amphetamine

Methamphetamine

Interferences in Analysis• Labs reported methamphetamine in

samples which were actually negative

• Shown to contain high concentrations ofephedrine or pseudoephedrineephedrine or pseudoephedrine

– Urine can undergo periodate oxidation whichconverts ephedrine and pseudoephedrine tocompounds that do not interfere

– Test for presence of amphetamine

GC/MS of Amphetamines

Peak List: Tailing Factor:

1. amphetamine 1.109

2. methamphetamine 0.9922. methamphetamine 0.992

3. MDA 1.106

4. MDMA 1.068

5. MDEA 1.113

http://www.restek.com/aoi_forensics_A012.asp

Cocaine

Cocaine Ecgonine methyl esterNorcocaine

benzoylecgonine ecgonine

cocaethylene

Metabolite results in thepresence of Ethanol

LC/MS/MS in testing IllicitSubstances

Sample Preparation

Alprazolam-Xanax

Clonazepam

Diazepam-Valium

7-aminoclazepam 3.3 minOxazepam 4.2 minLorazepam 4.3 minClonazepam 4.7 minAlprazolam 4.8 minDiazepam 5.5 minDiazepam 5.5 minNordiazepam 5.0 min

Doping

• Doping (sports) is the use of drugs or othersubstances to improve athletic performance

• Androgenic- from the Greek word andros “man”and genein “to produce” androgens areand genein “to produce” androgens areresponsible for development and maintenanceof male sex characteristics

• Anabolic- from the Greek word anabole “to buildup” constructive metabolism aka anabolism

Doping Effects

In Men• Acne• Sleep apnea• Gynecomastia• Azoospermia

In Women• Hirsutism• Acne• Amenorrhea• Deepening of the voice

• Azoospermia• Decreased testicle

size• Stimulate renal EPO

secretion• Serum lipid changes

• Deepening of the voice• Clitoral enlargement• Serum lipid changes

LC/MS/MS of Steroids• Anabolic agents are banned by IOC and

WADA

• Difficult to detect, so have set lowdetection limitsdetection limits

Diuretics

Methylphenidate

SalmeterolAlbuterol, bronchodilatordrugs, anabolic sideeffects at highconcentrations

References

JAT, June 2007, v31, n5, 237-253.

JAT, May/June 2005, v29, n4, 217-222.

JAT, May/June 2005, v29, n4, 234-239.

JAT, Jan/Feb 2003, v21, n1, 15.

JAT March 2003, v27, n2, 106-109.

JAT April 2007, v31, n3, 125-131.JAT April 2007, v31, n3, 125-131.http://chromatographyonline.findanalytichem.com/lcgc/article/articleDetail.jsp?id=51260&

pageID=1&sk=&date=

Yinon, J. Forensic Applications of Mass Spectrometry, 1995, p. 1-59.

The Dope on Doping, Dr. Mindy Shelby, 2008.