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 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 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 detect arsenic trioxide • 1840 Mathieu Orfila involvement in LaFarge case • 1851Jean Servais Stas development of alkaloid extraction method • Beginning of 20 th century Dr. Alexander Gettler, lab director for New York City medical examiner
What is the Marsh Test? • Arsenic trioxide, As 2 O 3 , was a favored poison • Treat sample with H 2 SO 4 and As free Zn, zinc zinc • Zn reduces the trivalent As As 2 O 3 + 6 Zn + 6H + → 2 As 3- + 6 Zn 2+ + 3 H 2 O
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 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 – 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 • Accuracy – compares the level of a desired – how close a measurement is to signal to the level of background the accepted value noise • Precision – High S/N lower LOD and LOQ – – how close together or how how close together or how • • LOD LOD repeatable the results are repeatable the results are – Limit of Detection • Interference – lowest quantity of a substance that can be distinguished from the absence of that substance • Robustness • LOQ – Limit of Quantitation – limit at which we can reasonably tell the difference between two different values • ULOQ – Upper limit of Quantitation
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 2. methamphetamine 0.992 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 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 of male sex characteristics • Anabolic- from the Greek word anabole “to build up” constructive metabolism aka anabolism
Doping Effects In Men In Women • Hirsutism • Acne • Acne • Sleep apnea • Amenorrhea • Gynecomastia • Deepening of the voice • Deepening of the voice • Azoospermia • Azoospermia • Clitoral enlargement • Decreased testicle • Serum lipid changes size • Stimulate renal EPO secretion • 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
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