Lecture 3 - Analytical Techniques and Intrumentation

General Disciplines and Clinical Applications

Four basic disciplines of clinical chemistry:

• Spectrometry

• Luminescence

• Electroanalytic methods

• Chromatography

Spectrometry - Measurement:

• Instruments measure either absorption or emission of radiant energy to determine concentration of atoms or molecules

Characteristics of absorption/emission on spectra:

• Line (atoms)

• Band (molecules)

• Continuous (solids)

Spectrophotometer:

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

Luminescence:

• Process of emitting light from a substance

• Bioluminescence is the emission of light by living organisms (example: fireflies)

• Chemiluminescence is a result of a chemical reaction (example: luminol test gives a blue glow in detecting blood)

Fluorescence:

• Can be detected by an array detector, to detect compounds using HPLC (High performacne Liquid Chromatography)

• TLC (Thin Layer Chromatography) plates can be visualized under UV if the compounds or a coloring reagent is fluorescent

Electroanalytic Methods: pH Meter:

• Ion selective electrodes measures 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 the blood

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

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

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

• Lungs and kidneys are major organs that regulate the blood pH

Acidosis:

• Occurs when blood pH falls below 7.35 (untreated diabetes)

Alkalosis:

• Occurs when blood pH rises above 7.45 (severe case of vomiting)

Urinalysis pH:

• Indicates metabolic acidosis (acid production)

• Indicates metabolic alkalosis (acid loss)

• Precipitation of crystals

• Treatment of urinary tract infections

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

• By modifying urine pH through diet or medications, the formation of these 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 the system

Chromatography: Mobile phase:

• Gas or liquid

Chromatography: Stationary phase:

• Solid or liquid; column holds the stationary phase

Chromatography: Complex mixture:

• Sample

Chromatography: Separated components:

• Eluate

Thin Layer Chromatography:

• Variant of column chromatography

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

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

• Retention factor (Rf) is compared to a standard (known) Rf

Basic Components of High Performance Liquid Chromatography (HPLC):

• Separation and analysis of mixed compounds

• Mobile phase is liquid

• Thermally unstable compounds can be analyzed

• Qualitative and quantitative analysis can be performed

Chromatography: TLC vs. HPLC:

Basic Components of Gas Chromatography:

Gas Chromatography Mass Spectrometry (GCMS):

• Mobile phase is gas

• Complex samples are separated in the column

  • Example: A mixture of Caffeine, Cocaine, and Lidocaine will separate and three peaks will be observed

  • Each component enters the mass analyzer where it is fragmented into a unique and repeatable pattern

Mass Spectrometry:

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

Mass Spectrometry: Ionization Source:

• A small sample is ionized, usually to cations by loss of an electron

Mass Spectrometry: Mass Analyzer:

• The ions are sorted and separated according to their mass and charge

Mass Spectrometry: Detector:

• The separated ions are measured and the results displayed on a chart as a spectrum (the fragmentation pattern)

Mass Spectrometry: Cocaine

• The 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 probably is the substance

Confirmatory testing:

• Verify the substance’s identity, or to measure the percentage purity or other quantitative analysis

• Confirms or indetifies a substance has a confidence level attached to it

  • High specificity

  • High sensitivity

  • Qualitative and Quantitative

  • Legally defensible results

  • Avoids false positives and false negative

Presumptove Kastle-Meyer test:

• Shows that a sample is not blood, or that the sample is probably blood

• Chemical indicator is phenolphtalein is used to detect the possible presence of hemoglobin

• Positive: Swab turns pink

Conrimatory Kasle-Meyer test:

• Further chemical testing required to prove that the substance is blood

Presumptive: Marquis reagent:

• Used as a simple spot test to presumptively identify alkaloids (drugs and posions fall into this category) as well as other compounds

• Different compounds produce different color reactions

Confirmatory: Marquis reagent:

• Further testing using GCMS or HPLC is necessary to confirm the substance is an illicit drug

Marijuana:

• Hemp plant: Cannabis sativa

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

Marijuana analysis:

• Via color test, urinalysis, hair analysis, saliva test, comprises of presumptive testing and necessary confirmatory tests are required for identification

• Major metabolite is 11-nor-9-carboy-delta-9-tetrrahydrocannabino (THC-COOH). Not psychoactive

Marijuana Urinalysis:

• Screening Test

• Can provide info to past exposure, but does not necessarily differentiate between exposure to second hand smoke and actually smoking marijuana

• False positives are common

Duqenois-Levine Color Test:

• Screening Test

• Marijuana as well as a variety of other plant substances becomes purple with addition of this reagent and hydrochloric acid

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

• Gives two layers: Purple (top layer) and Purple/Violet (bottom layer)

Marijuana Analysis: TLC:

• Confirmatory Test

• Components spread out in plate as the solvent travels

• Chromatography occurs and visualizing agent along the THC standard is used for confirmation

• Rf calculated

Marijuana Analysis: GC:

• Components separate

• Peaks are identified by their retention times (time of elution)

Chemical Components of Marijuana:

Cannabinoids are a class of diverse chemical compounds that act on cannabinoid receptors in cells that repress neurotransmitter release in the brain

Marijuna Analysis: GCMS:

Metabolite of Marijuana:

Drugs of abuse in urine

Clinical Toxicology: Screening and confirmation of the most commonly abused OTC Meds and DOAs:

Forensic Toxicology Labs: Screening and conformation of the most commonly abused OTC meds and DOAs: