Week 3: Analytical techniques and instrumentation/toxicology

Clinical chemistry - 4 basic disciplines

• spectrometry

• luminescence

• electroanalytic methods

• chromatography

Spectrometry measurement

• measure either absorption or emission of radiant energy to determines the concentration of atoms or molecules

• characteristics of absorption/emission spectra-line (atoms), band (molecules), and continuous (solids)

Spectrophotometer

used to measure light transmitted by a solution to determine concentration of the light-absorbing substance in solution

Luminescence

• process of emitting light from a substance

• bioluminescence is the emission of light by living organisms

• chemiluminescence is a result of a chemical rxn

  • ex. luminol test gives blue glow in detecting blood

Fluorescence

• fluorescence in several wavelengths can be detected by an array detector to detect compounds using HPLC

• TLC (thin layer chromatography) plates can be visualized under UV if the compounds or a coloring reagent is fluorescent

Electroanalytic methods

• pH electrode is universally used in clinical labs

• ion-selective electrodes measure the hydrogen-ion concentration to indicate its acidity or alkalinity

• acidosis and alkalosis describe the abnormal conditions that result from an imbalance in the pH of blood

• excess acid or alkali in blood is typically caused by an underlying condition or disease

Electroanalytic methods - pH

• any disease or condition that affects the lungs, kidneys, metabolism or breathing has the potential to cause acidosis or alkalosis

• acidosis: blood pH <7.35 (diabetes)

• alkalosis: blood pH >7.45 (severe vomitting)

• body balances between incoming acids and bases (faucet on) and elimination of acids and bases (drain on)

Electroanalytic methods - Urinalysis pH

• benefits:

  • indicates metabolic acidosis or alkalosis

  • precipitation of crystals

  • treatment of UTIs

• ex. formation of various types of kidney stones strongly influenced by urinary pH

• by modifying urine pH through diet or meds, formation of crystals can be reduced or eliminated

Chromatography

• involves a group of techniques used to separate complex mixtures on the basis of different physical interactions between the individual compounds and the stationary phase of system

  • Mobile phase: gas or liquid

  • Stationary phase: solid or liquid; column holds stationary phase

  • Complex mixture: sample.

  • Separated components: eluate

Thin layer chromatography (TLC)

• variant of column chromatography

• thin layer of sorbent like silica gel is coated on glass

• sample applied on the spot and the mobile phase (solvent) separates the sample into molecules

• retention factor (Rf) is compared to standard known Rf

High performance liquid chromatography

• separation and analysis of mixed compounds

• mobile phase is liquid

• thermally unstable compounds can be analyzed

• qualitative and quantitative analysis can be performed

TLC vs. HPLC

Gas chromatography mass spectrometry (GCMS)

• gas mobile phase

• complex samples separated in column

• each component enters mass analyzer where it’s fragmented into unique and repeatable pattern

Mass spectrometry

• analytical technique that sorts ions according to their mass to charge ratio

• ionization source - small sample is ionized, usually to cations by loss of an electron

• mass analyzer - ions are sorted and separated according to their mass and charge

• detector - separated ions measured and results displayed on a chart as a spectrum (Fragmentation pattern)

Mass spectrometry - cocaine

• fragment pattern is considered to be the fingerprint of the compound

• the more abundant ions (82, 182, 303) can be rationalized by certain cleavages and eliminations occurring to the structure of cocaine

Presumptive (screening)

test indicates the sample is probably the substance

• Kastle-Meyer test will show that a sample is not blood or that the sample is probably blood

  • chemical indicator phenolphthalein is used to detect the possible presence of hgb (+ = pink)

• Marquis reagent used as a simple spot-test to presumptively ID alkaloids (drugs/poisons) as well as other compounds

confirmatory testing

verify the substance’s identity, or to measure the % purity or other quantitative analysis

1. high specificity

2. high sensitivity

3. quantitative and qualitative

4. legally defensible results

5. avoids false pos/negs

Marijuana analysis

• hemp plant - Cannabis sativa

• plant contains mind-altering chemical delta-9-tetrahydrocannabinol (THC)

• analysis of cannabis via color test, urinalysis, hair analysis, saliva test, comprises of presumptive testing and necessary confirmatory tests are required for ID

• THC’s major metabolite is 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH). not psychoactive

Marijuana analysis - screening

• urinalysis: provide info to past exposure, but tests don’t necessarily differentiate between exposure to second hand smoke and actually smoking marijuana

• false pos are common

Duquenois-Levine color test

• Marijuana becomes purple with addition of Duquenois reagent and hydrochloric acid

• upon addition of organic solvent, purple color transfers to organic layer, indicating that cannabinoids may be present

• color gives 2 layers: purple (top) and purple/violet (bottom)

Marijuana analysis - confirm

• TLC

  • Marijuana is a mixture of compounds

  • components spread out in TLC plate as solvent travels

  • chromatography occurs and a visualizing agent along with THC standard is used for confirmation

  • Rf calculated

• GC

  • peaks are ID’d by their retention times (time of elution)

Chemical components of marijuana

Metabolite of Marijuana

• metabolism of THC results in glucuronide

• common soluble conjugates formed as a step towards metabolism and excretion

Drugs of abuse in urine

Clinical toxicology labs

Forensic toxicology labs

Xenobiotics

• xenos - greek for ‘stranger’

• xenobiotic compound foreign to the body

  • artificial components such as chemicals, pesticides and drugs

  • they are not normally found or produced in body

  • exogenous agents that may have adverse effects on a living organism

Toxicology

• study of the adverse effects of xenobiotics in humans

• scope is very broad

• 3 major disciplines

  • Mechanistic

  • descriptive

  • regulatory

Mechanistic toxicology

elucidates the cellular, molecular, and biochemical effects of xenobiotics

Descriptive toxicology

uses results from animal experiments to predict what level of exposure will cause harm in humans

regulatory toxicology

interpretation of the combined data from mechanistic and descriptive studies is used to establish standards that define the level of exposure that will not pose a risk to public health

ex. FDA, US environmental Agency, OSHA

Risk Assessment

RISK - the harm a hazard can do

HAZARD - object that can potentially hurt someone

EXPOSURE - takes into account the amount, frequency, route, and duration

RISK = HAZARD x EXPOSURE

Forensic toxicology

• primarily concerned with medicolegal consequences of exposure to chemicals or drugs

• focuses on establishing and validating the analytic performance of methods used to generate evidence in legal situations

Clinical toxicology

• study of interrelationships between xenobiotics and disease states

• emphasizes diagnostic testing and therapeutic interventinos

Environmental toxicology

• evaluation of environmental chemical pollutants and their impact on human health

• sources include organic and inorganic pollutants, pesticides and biological agents, all of which can have harmful effects on living organisms

Clinical poisoning facts

1. intentional suicide - 50% of poisoning cases

• suicide - highest mortality rate

• therapeutic or illicit drug accidental overdose common in adolescents and adults

2. accidental exposure - 30% of cases (most frequent in children)

3. remaining cases - homicide and occupational exposure

Routes of exposure

• ingestion, inhalation, transdermal absorption most common

• most often observed - ingestion

• systemic effect occurs when the toxin is absorbed into circulation

Ingestion

• toxins enter through pharmacokinetic processes

• in most cases, passive diffusion occurs where toxic substances that are hydrophobic cross cell barriers

• toxins not absorbed from GI tract don’t produce systemic effects but local effects

  • ex. diarrhea, bleeding and malabsorption of nutrients

• local effects can cause systemic effect secondary to toxin exposure

Factors affecting absorption

• pH

• rate of dissolution

• gastric motility (movement)

• resistance to degradation in GI tract

Dose-response relationship

• dose-response relationship correlates the dose of a xenobiotic that will result in a harmful response

• ED₅₀ is the effective dose that produces a therapeutic effect in 50% of population

• TD₅₀ is the toxic in 50% of population. Associated with an early pathologic effect at lower than lethal doses

• LD₅₀ is the lethal dose in 50% of population

Individual dose-response

changing health effects based on change in xenobiotic exposure levels

Quantal dose-response

change in health effects of a defined population based on changes in exposure to xenobiotics

Acute toxicity

single, short term exposure to a substance, the dose of which is sufficient to cause immediate toxic effects

Chronic toxicity

repeated frequent exposure for extended periods for greater than 3 months and possibly years, at doses that are insufficient to cause an immediate acute response