Forensic & Analytical Toxicology PDF

Graham R. Jones, Ph.D. Former Chief Toxicologist Office of the Chief Medical Examiner & Clinical Professor, Faculty of Medicine and Dentistry [email protected] Forensic - for legal purpose; pertaining to the law. Deals with the living and the dead:  Postmortem cases - cause & manner of death ...

Graham R. Jones, Ph.D. Former Chief Toxicologist Office of the Chief Medical Examiner & Clinical Professor, Faculty of Medicine and Dentistry [email protected] Forensic - for legal purpose; pertaining to the law. Deals with the living and the dead:  Postmortem cases - cause & manner of death  Impaired driving; sexual assault  Workplace drug testing  Sports (e.g. Olympics; horse-racing)  Criminal cases: ◦ Impaired driving  Not just motor vehicles ◦ Homicides  Victim, perpetrator ◦ Drug facilitated assault  Assailant, victim  Civil issues/cases: ◦ Non-criminal accidents (e.g. falls, drowning) ◦ Workplace drug testing (Usually urine)  Condition of Employment / Site access  Witness in court ◦ Usually only testifies to facts, observations ◦ Medical technologists usually in this category ◦ What they did (or would normally have done)  Expert witness in court ◦ Must be “qualified” by the judge to give “opinion” evidence (based on education, training, experience) ◦ Doesn’t have to have done any lab work on the case  Clinical: ◦ emergency screening (e.g. overdose) ◦ therapeutic drug monitoring (TDM) (limited menu of drugs, defined concentration range) Testing only done if it is likely to influence treatment of the patient.       (Immunoassay) (HPLC/UV) GC (mainly alcohols) GC/MS(/MS) LC/MS(/MS), LC/(Q)TOF (AA; ICP-MS) (rare!) Similar instrumentation to clinical labs, but: 1. Forensic labs do NOT rely only on immunoassay. 2. Must use better extraction and chromatography methods than clinical labs because using whole blood – sometimes decomp!  Testing not usually ‘limited’ ◦ Drugs (prescription, non-prescription and ilicit) and other poisons (e.g. pesticides, other chemicals) ◦ May be “high” concentration – e.g. overdose ◦ May be very low concentration - e.g. sexual assault ◦ May use many methods for a single “drug screen”  e.g. immunoassay, GC-MS, LC-(Q)TOF, LC-MS/MS  Most forensic testing performed on whole blood or urine ◦ Most clinical testing performed on serum, plasma or urine  Whole blood specimens more difficult and give more interference than fresh serum or plasma  Matrix: is the fluid or tissue in which the analyte is contained: ◦ ◦ ◦ ◦    Proteins (blood or may be solid tissue) Lipids, phospholipids Electrolytes Water Analysis can be difficult or impossible without extraction of the analyte out of the matrix But it depends on the instrumentation Most forensic toxicology is performed on whole blood and/or tissue (e.g. criminal, postmortem)   Most samples have to be processed or extracted in some way prior to analysis There are two main purposes: ◦ Minimize or eliminate matrix interference (often involves extracting out the analyte) ◦ Concentrate the analyte to improve sensitivity of the assay  For immunoassay, the degree of sample processing depends on the assay design (e.g. homogenous enzyme immunoassay versus ELISA). Most clinical immunoassays are designed for urine or serum/plasma.  ELISA plates (96 wells per plate) are best suited for forensic work as they are optimised to work with diluted whole blood. Tissues must be homogenized and diluted.  Tissue can rarely be analyzed directly – need to make fluid sample  Use homogenizer ◦ e.g. Polytron homogenizer ◦ 1+3 tissue:water The type of column inlet is critical!!  GC ◦ Column inlet is heated to vaporize the solvent and the analyte; many drugs will not ‘chromatograph’ ◦ Will cause pyrolysis of some analytes and the matrix, causing interference ◦ More sample prep. needed than for LC-based methods  LC ◦ Inlet is cool (on-column), causing little or no destruction of the analyte or matrix ◦ Sample is introduced to MS spray chamber as liquid where it is then vaporized and ionized ◦ Less sample preparation may be needed  1. Extract the analyte from the sample and leave the matrix behind ◦ For example, liquid-liquid or SPE extractions ◦ Most common approach for GC-based assays  2. Remove the interferences from the sample and leave the analytes behind ◦ For example, protein ‘crash’ extracts ◦ Increasingly common approach for LC-MS (and LC-TOF) ◦ MUST CONSIDER THE ANALYTICAL INSTRUMENTATION  Dilution ◦ To “dilute out” proteins in plasma or blood sample to allow direct analysis  e.g. Immunoassay   Headspace analysis  e.g. Alcohols by GC Protein ‘crash’ (e.g. acetonitrile, methanol)  Some highly selective LC-MS(MS) and LC-(Q)TOF assays    Used for “volatiles” (alcohols and solvents) Only volatiles from air above the sample get injected on to the GC Very clean with little interference Headspace GC Diagram Step 1 Sample reaches equilibrium Step 2 Sample is extracted from headspace Step 3 Sample is injected into GC Can use simpler methods such as a “protein crash” if the analytical methods are specific enough!  Add cold acetonitrile and/or methanol to the blood ◦ Can also use for serum or plasma    Proteins and peptides in whole blood are denatured; centrifuge to form a pellet Clear supernatant injected (sometimes after a concentration step) (aka “dilute and shoot”) Increasingly used for LC-MS/MS and LC-(Q)TOF analysis  Caution: analytes can be trapped in the precipitation matrix  Need good, validated methods with deuterated internal standards  Does NOT remove salts, lipids or phospholipids 4) Remove supernatant 5) Analyze supernatant, often after dry down and reconstitution     Principle: analyte more soluble in solvent than aqueous specimen Add solvent to liquid specimen Mix to temporarily “blend” phases Centrifuge to separate     Un-ionized analyte is more lipid-soluble Ionized analyte (e.g. salt form such as hydrochloride or sulfate) is more water soluble Need to get analyte into UN-ionized form in order to extract into organic solvent Use buffers to do this (i.e. adjust pH) Cantharadin Ibuprofen Methamphetamine Specimen + pH 12 Buffer + Solvent Mix & Centrifuge Organic to GC/LC Aqueous (waste) To extract acidic drugs add an acidic buffer instead of pH 12 buffer Specimen (+ pH 12 buffer) (+ solvent) Mix & Centrifuge Organic Aqueous (waste) + Acid pH 2 Mix & Centrifuge Organic (waste) This extraction scheme extracts basic drugs, but leaves neutrals (e.g. fats) and acidic drugs behind Aqueous + Base pH 12 + Solvent Mix & Centrifuge Organic (to GC/LC) Aqueous (waste)  Amphoteric Functions ◦ Phenolic ◦ Amino  e.g. morphine  Zwitterion Functions ◦ Carboxylic ◦ Amino  e.g. GABA OH OH OH H CH3 N CH3 pH 2 N CH3 N O O O OH OH O pH 8 (e.g. morphine) pH 12  Depends on: ◦ ◦ ◦ ◦ ◦ ◦ ◦ Specimen type (e.g. urine vs. whole blood) Instrumentation used/available (e.g. GC/MS vs. LC/MS) Robustness and precision of the assay Purpose of the test (quantitative vs. quick screen) Processing time (urgency and speed of test) Type of analyte/drug being tested Cost of reagents (SPE uses less solvent than liquid/liquid, but SPE columns can be expensive)             GC – gas chromatography LC – liquid chromatography (aka HPLC) MS – mass spectrometry MS/MS – tandem MS LC-MS(MS) refers to single MS or MS/MS TOF – time of flight (accurate mass MS) QTOF – combination “MS” and TOF (highly selective) FID – flame ionization detection NP(D) – nitrogen phosphorus (detection) UV/VIS – ultraviolet/visible detector ELISA – enzyme linked sorbent immunoassay (96-well plates) SPE – solid phase extraction          B.Sc. Pharmacy 1971 Registered as U.K. Pharmacist 1972 Ph.D. Drug Metabolism and Drug Analysis 1976 Post-doctoral Fellowship U. of Alberta Pharmacy 1976-78 Head, Commonwealth Games Drug Testing Lab 1978 Clinical Toxicologist, Univ. Hospital Edmonton 1978-81 Toxicology Lab Director /Chief Toxicologist, Alberta Medical Examiner’s Office 1981 – 2018 Chair, Universiade Games Drug Testing Program1983 Now semi-retired but very active professionally plus private consulting 33