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
JUSTICE
– Concept of rightness basedon law, fairness, and equity
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 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
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
• 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
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
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
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.
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