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

LC/MS/MS and GC/MS
Applications in Testing Illicit
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
– Obtain analytical data on poisons
– Apply information to understanding of episode
of intoxication
• Toxicology
– Study of toxic or harmful effects of chemicals
– Concerned with how toxins act, when effects
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
on 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
• 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
• 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
– 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
– 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
other 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
How Long?
• Duration of the exposure
How Often?
• Frequency the exposure occurs
• Dose-time relationship
• Acute versus chronic toxicity
– 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
– 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
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
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
•
LOQ
– Limit of Quantitation
– limit at which we can reasonably
tell the difference between two
different values
•
ULOQ
– Upper limit of Quantitation
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
•
•
•
Interference
•
Robustness
GC/MS in Testing Illicit Substance
• Analytes must be chemically extracted from
matrix
– Matrices
•
•
•
•
Urine
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
– 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
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
– 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.
2.
3.
4.
5.
cannabidiol
Δ8-tetrahydrocannabinol
Δ9-tetrahydrocannabinol
cannabinol
11-hydroxy-Δ9tetrahydrocannabinol
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
• 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
– 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
3. MDA
1.106
4. MDMA
1.068
5. MDEA
1.113
http://www.restek.com/aoi_forensics_A012.asp
Cocaine
Norcocaine
Cocaine
Ecgonine methyl ester
benzoylecgonine
ecgonine
Metabolite results in the
presence of Ethanol
cocaethylene
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
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
responsible for development and maintenance
of 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
• Decreased testicle
size
• Stimulate renal EPO
secretion
• Serum lipid changes
In Women
•
•
•
•
•
•
Hirsutism
Acne
Amenorrhea
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
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.
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.