Academic Year 2018/2019 - 2° Year - Curriculum Tossicologia dell'Ambiente e degli Alimenti
Teaching Staff: Emanuele AMATA
Credit Value: 7
Scientific field: CHIM/08 - Pharmaceutical chemistry
Taught classes: 28 hours
Exercise: 36 hours
Term / Semester:

Learning Objectives

  • The course enables students to gain detailed knowledge and understanding of the theory and practice of analytical toxicology and its applications
  • To present the main methodologies for the evaluation, handling and preparation of the sample
  • To provide an insight on the main analytical techniques

Course Structure

  • Frontal lessons 28 hours
  • Laboratory practice 36 hours

Detailed Course Content

First module

Principles of Analysis of Toxicological Chemistry: general methodology. The analytical method. Definition of poison. General and specific analysis.

Extraction methods: Separative and not separative methods. Analytical classification of toxic substances:

Gaseous and volatile compounds

  • Headspace analysis. General principles. Equilibrium distribution, partition coefficient, phase ratio, salting out.
  • Microdiffusion extraction, General principles. Carbon monoxide microdiffusion extraction. Sodium cyanide microdiffusion extraction.

Strong acid and base: general principles. Metals: General principles, Reinsch test. Sample preparation. Anions: general principles. Miscellaneous and organic compounds not soluble in organic solvents: general aspects.

Solvent soluble drugs: general aspects

  • Solubility and partition coefficient. General principles and LogP
  • Electrolytic distribution equilibrium, ionization constant and ionization state. Distribution of acid, base and amphoteric compounds
  • Ion pair extraction. Cetrimonium bromide, Bromothymol blue, Bromocresol green and Bromophenol blue. Paraquat extraction
  • Internal standards
  • Analysis of glucoronated or conjugated metabolites: enzymatic and chemical hydrolysis
  • Sample treatment, homogenization and protein elimination: Chemical and enzymatic digestion.
  • Buffer. Buffer preparation at specific pH value and use of these buffers in the extraction process. One and two components buffers. Henderson-Hasselbalch’s equation application.
  • Solutions, use and preparation. Sodium hydroxide, Hydrochloric acid, sulfuric acid, sodium borate.
  • Type of solvents and solvent selection. Chlorobutane, ethyl acetate, hexane, cyclohexane, methanol, ethanol, acetonitrile, chloroform, dichloromethane, diethyl ether.

Liquid-liquid Extraction

  • Extraction yield. Equilibrium distribution, Electrolytic dissociation equilibrium, phase ratio, number of extraction. Salting out. Type of solvent and selection. Extraction scheme of a basic compound. Extraction scheme of an acid and neutral compound. Separation of Acidic, Basic and Neutral Compounds. Aspirin extraction procedure. Caffeine extraction procedure.

Solid-phase extraction (SPE)

  • General considerations. Application fields. Types of interactions with the solid phase. Types of solid phases. Type of solvents and selection. Chemical mechanism of separation of acid/basic/neutral substances. Sample treatment. Stationary phase treatment. Sample application. Washing phases. Elimination and collection of contaminants. Drug recovery. Diphenhydramine SPE procedure.

Color tests: introduction and interpretation of color tests, color test types and methods. Color tests: ferric chloride, Marquis reagent, Mandelin reagent, McNally’s test.

Second module

UV – Visible spectrophotometry: general principles, the electromagnetic radiation (amplitude, period, frequency, wavelength). Chromophore. Quantitative analysis, transmittance, absorbance, Beer-Lambert law, auxochrome. Solvent effect. Instrumentation: Generalities on spectrophotometers, the source, the monochromator, the cell, the cuvette, detectors. Spectrophotometers type: single and double beam spectrophotometers and diode array.

Chromatography methods: general principles. Chromatogram. Migration rate, retention and dead time, retention and dead volume. Column efficiency. The theoretical plates. The Van Deemter equation, turbulent diffusion, longitudinal molecular diffusion, mass transfer. Efficiency optimization. Selectivity. Resolution. Separation mechanism: adsorption, partition, ion exchange, exclusion

  • Thin-Layer Chromatography. Instrumentation and execution. Retention factor. TLC plate and stationary phases. Mobile phases and eluotropic series. TLC plate visualization. Derivatization, Dragendorff reagent. Selectivity, capacity, efficiency and resolution.
  • High performance liquid chromatography (HPLC). General characteristics and advantages. Equivalent height to a theoretical plate. Direct and reverse phase chromatography. The HPLC column. The mobile phase and solvents in HPLC. Mobile phase modifiers, buffers, acidifiers, ion pair reagents. Solvents filtration and degassing. Isocratic and gradient elution. HPLC instrumentation: pumps, manual injector. Detectors. UV-Vis detector, the flow cell, diode array detector.

Third module

Mass spectrometer (MS). General principles. The mass spectrum. Ion mass and Dalton. Isotopic peaks and intensity. Exact and nominal mass. Resolution and resolving power. Instrumentation:

  • Sample introduction: direct and indirect manner
  • Ionization. The ionization process and its classification. Hard and soft techniques, vaporization, desorption and spray.
    • Electron ionization. General principles, the ionization chamber, molecular ion formation and fragmentation. The electron ionization source, features, limits and advantages.
    • Chemical ionization. General principles. Differences between chemical and electron ionization. Reagent gases. Adducts formation. Methane, ammonia and proton affinity. How to choose the reagent gas.
    • FAB and MALDI. General principles and application fields. Matrix types.
    • Elettrospray ionization. General principles. Spray process. Solvent evaporation. Electrospray chamber, the capillary, the cone and the counter electrode. Ions formation. Molecular ion and multicharged ions.
  • Mass Analyzers: types of mass analyzer
    • Magnetic Sector Mass Analyzer: features and operative conditions. Ions kinetic energy. Ions trajectory. Fundamental equations
    • Quadrupole mass analyzer: features and operative conditions. Stable and unstable oscillations
    • Time-of-flights: features and operative conditions. Ion velocity and time. Fundamental equations
  • Detectors: features and operative conditions. Conversion dynode and continuous dynode particle multiplier detector
  • Computer: features and operative conditions

Types of ions: molecular ion, fragmentation ion, rearrangement ion, multicharged ion, metastable ion, negative ion, ion to molecule interactions. The molecular ion recognition. Differences in molecular ion based on ionization source used. The molecular ion intensity and nitrogen rule.

Liquid chromatography-Mass spectrometry (LCMS). Features and operative conditions.

Textbook Information

  • Lecture notes and slides
  • Renato Cozzi, Pierpaolo Protti, Tarcisio Ruaro, Elementi di analisi chimica strumentale, seconda edizione, Zanichelli 2013
  • Moffat A.C., Osselton M.D., Widdop B. - Clarke’s analysis of drugs and poisons in pharmaceuticals, body fluids and postmortem material - Pharmaceutical Press 2011
  • Flanagan R.J., Taylor A., Watson I.D., Whelpton R. - Fundamentals of analytical toxicology - John Wiley & Sons Ltd 2007