LC/MS/MS and GC/MS Applications in Testing Illicit Substances - - PowerPoint PPT Presentation

LC/MS/MS and GC/MS Applications in Testing Illicit Substances Substances

• Dr. Darcie Wallace Duckworth

Assistant Director of Training Aegis Sciences Corporation March 17, 2009

What is forensic toxicology?

• ABFT defines as “the study and practice of

the application of toxicology to the purposes of the law”

• Purpose
• Purpose

– Obtain analytical data on poisons – Apply information to understanding of episode

• f intoxication

• Toxicology

– Study of toxic or harmful effects of chemicals – Concerned with how toxins act, when effects happen, and what are the symptoms and happen, and what are the symptoms and treatments

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 based

• n 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 trioxide detect 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,

zinc zinc

• 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 poisoning Arsenic poisoning – Court asked Orfila to investigate – Discovered the initial Marsh test improperly done – 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 their function

• 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 of

• ther chemicals

How Much? The Dose

• All substances have the capability of being

toxic, 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 of

ONE exposure to a relatively large amount of substance

– Chronic

• Damage as result of repeated exposure to relatively

small amounts over a prolonged time period

• What is

– A matrix?

• Biological specimen such as body fluid or solid

tissue tissue

– Agent of interest?

• Exists in matrix in a simple solution or may be

bound to protein or other cellular constituents

Our challenge…..

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

Metabolism

• Terms

– Metabolism

• Aggregate of all physical and chemical changes that occur

within 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 of an organism » Results in detoxification and elimination from the body, toxification and distribution to receptors, chemically unmodified and distributed to receptors » Many are lipophilic, increase water solubility, and facilitates excretion

– Metabolite

• Product of metabolism

Parent Drug versus Metabolites

Common Characteristics of Analytical Assays

• S/N

– compares the level of a desired signal to the level of background noise – 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 the absence of that substance

• LOQ

– Limit of Quantitation – limit at which we can reasonably tell the difference between two different values

• ULOQ

– Upper limit of Quantitation – how close together or how repeatable the results are

• Interference
• Robustness

GC/MS in Testing Illicit Substance

• Analytes must be chemically extracted from

matrix

– 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 licit

pharmaceuticals

• 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 are present in specific ratios and quantifies by determining ion abundances compared to IS determining ion abundances compared to IS abundances – IR abundances are unique for given compound – 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 min at 50ºC at 50ºC – Neutral and basic compounds removed by adding hexane and agitating – After centrifugation, hexane layer is removed, aqueous layer is acidified 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. cannabidiol
• 2. Δ8-tetrahydrocannabinol
• 3. Δ9-tetrahydrocannabinol
• 4. cannabinol
• 5. 11-hydroxy-Δ9-

tetrahydrocannabinol tetrahydrocannabinol

• 6. 11-nor-Δ9-tetrahydrocannabinol

carboxylic acid

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

Amphetamines

• Methamphetamine-most commonly

abused 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 of

ephedrine or pseudoephedrine ephedrine or pseudoephedrine

– Urine can undergo periodate oxidation which converts ephedrine and pseudoephedrine to compounds that do not interfere – Test for presence of amphetamine

GC/MS of Amphetamines

Peak List: Tailing Factor:

• 1. amphetamine

1.109

• 2. methamphetamine

0.992

• 2. 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 ester Norcocaine benzoylecgonine ecgonine cocaethylene

Metabolite results in the presence of Ethanol

LC/MS/MS in testing Illicit Substances

Sample Preparation

Alprazolam-Xanax

Clonazepam

Diazepam-Valium

7-aminoclazepam 3.3 min Oxazepam 4.2 min Lorazepam 4.3 min Clonazepam 4.7 min Alprazolam 4.8 min Diazepam 5.5 min Diazepam 5.5 min Nordiazepam 5.0 min

Doping

• Doping (sports) is the use of drugs or other

substances to improve athletic performance

• Androgenic- from the Greek word andros “man”

and genein “to produce” androgens are and genein “to produce” androgens are responsible for development and maintenance

• f male sex characteristics
• Anabolic- from the Greek word anabole “to build

up” 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 low

detection limits detection limits

Diuretics

Methylphenidate

Salmeterol Albuterol, bronchodilator

drugs, anabolic side effects at high concentrations

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.