Ateneo - UPO

During the course, the following topics related to the methodologies used in quantitative pharmaceutical analysis will be covered. • Classification of various types of samples and techniques used for their preparation for analysis. • Principles of volumetric analysis (titrations): reagents; chemical reactions; titration curves; indicators. Acid-base, precipitation, redox, and complexometric titrations; overview of non-aqueous titrations. • Chemical stability studies of drugs: forced degradation studies and stability studies on drug substances and their formulations. • Instrumental analytical methods for qualitative and quantitative drug analysis: UV-Vis spectrophotometry and spectrofluorimetry; fundamentals and practical aspects; method development. • Chromatographic methods: HPLC and GC – fundamentals and practical aspects; method development. • Use of mass spectrometry in qualitative and quantitative pharmaceutical analysis. • Calculations in pharmaceutical analysis. • Case studies from relevant scientific literature. • Laboratory practices: volumetric analysis by titration; analysis of active ingredients and pharmaceutical formulations. Determination of the active ingredient content in bulk substances and finished drug products.

• European Pharmacopoeia (Ph. Eur.) current edition • Analisi Chimico Farmaceutica F. Savelli, O. Bruno – Piccin • Analisi Farmaceutica D.G. Watson – EDRA LSWR • Analisi Farmaceutica Qualitativa A. Carta, M.G. Mamolo, F. Novelli, S. Piras – Edises • Manuale di Analisi Qualitativa G. Caliendo – Edises • Principi di Analisi Farmaceutica V. Cavrini, V. Andrisano – Società Editrice Esculapio. • Principi di Analisi Quantitativa dei Medicinali E. Abignente, D. Melisi, M.G. Rimoli – Loghìa • Chimica Analitica Quantitativa D.C. Harris – Zanichelli • Chimica analitica e analisi quantitativa D.C. Harris – Piccin. • Fondamenti di Chimica Analitica (Skoog & West) – EdiSES.

Attending the course will enable students to acquire the basic skills needed to use the qualitative/quantitative analytical techniques commonly employed in pharmaceutical analysis laboratories to assess drug identity, purity and stability in compliance with the regulatory guidelines. Laboratory practices and the critical reading of pharmacopoeia monographs and selected articles from the scientific literature will allow students to apply, or observe the application of, the analytical methods described. Finally, the laboratory activities and the calculation exercises will enable students to present and correctly express the results obtained.

To attend the course successfully, the prerequisites established by the degree program regulations in CTF must be met (Article 18): 1) in order to enrol in the third year, all first-year exams must have been passed. 2) to access the course in Methodologies in Pharmaceutical Analysis 2 (both theoretical and practical parts), students must have attended the Methodologies in Pharmaceutical Analysis 1 course (theoretical and practical parts), and to take the exam, they must have passed the Methodologies in Pharmaceutical Analysis 1 exam. To follow the course effectively, students are expected to have basic knowledge provided by the courses in General and Inorganic Chemistry, Organic Chemistry, and Analytical Chemistry. In addition, a basic working knowledge of Microsoft Excel is recommended.

Frontal lessons and laboratory activities. The theoretical lessons will be held mainly with the support of slides, videos and scientific articles projected in the classroom. The exercises related to calculations in pharmaceutical analysis will be treated in the classroom by resolution of real problems taken from the pharmaceutical field. The laboratory activities will be carried out in laboratories equipped with the analytical instrumentation suitable for the pharmaceutical analysis in the field of quality control: Burettes and volumetric flasks for volumetric analysis, UV-Vis spectrophotometers, HPLC systems, Karl Fischer titrator, etc.

Students with physical disabilities, Learning Disabilities or Special Education Needs can request specific services and tools via the “Staff Sviluppo e Coordinamento Carriere e Servizi alle Studentesse e agli Studenti”, consulting the University webpage: https://www.uniupo.it/en/services/services-students-physical-or-learning-disabilities Students with disabilities, learning disabilities or special education needs, once they have contacted the University Staff, can refer to the tutor in charge of the course to define the examination modalities, concerning academic aspects.

Assessment of learning will take place through two separate exams plus evaluation of the laboratory activities. A maximum total score of 31 points (30 cum laude) will be assigned. *PART A* (6 CFU, 21 points, Prof. Silvio Aprile): 1. Written exam (2 hours, maximum 16 points, minimum passing score 12 points) consists of true/false or multiple-choice questions, open-ended questions, and calculation exercises. At the end of the course, a simulation of the written exam will be provided. 2. Evaluation of laboratory experiment results (maximum 5 points). *PART B* (3 CFU, 10 points, Prof. Erika Del Grosso): Learning assessment will be conducted through an oral exam. During the oral exam, the student’s ability to read and interpret a scientific article related to the analytical techniques presented during the course and their applications will be evaluated. Knowledge of the basic principles of these techniques will also be assessed. The student will receive the scientific article to comment on at the conclusion of the chosen exam session and will have to present it in a maximum of 10 minutes using any tools deemed useful. To achieve the final course grade, the student must demonstrate understanding and the ability to use the fundamental concepts of each topic. The evaluation, expressed on a scale of 1 to 10, will be based on the following criteria: 1) Knowledge and theoretical and practical understanding of the studied topics; 2) Ability to connect the studied topics; 3) Communication skills and appropriateness in the use of technical language.

• General aspects of the pharmaceutical analysis: concept of sample, analyte, matrix, interfering substances, types of samples and objectives in drug analysis. Criteria for the selection of the analytical technique based on the sample to analyse. • Volumetric analysis: balances, glassware for volume determination (burette, volumetric flask, graduated pipettes). • Titration techniques (direct and indirect titration), titration curves, equivalence point, end point, titration error. • Primary standards and preparation of standard solutions. • Acid-base titrations, titrants used in acidimetry and alkalimetry, acid-base indicators. • Precipitation titrations (argentometric methods: Mohr, Volhard, Fajans). • Redox titrations (permanganometry, iodimetry, iodometry, cerimetry, bromometry), factors influencing redox processes. • Complexometric titrations, stability of metal-titrant complexes. • Drugs assayed by titration.• Introduction to non-aqueous titrations. • Exercises on volumetric analysis. • Analytical methods based on light absorption, UV-Vis spectrophotometry: properties of the electromagnetic waves and the chromophores. Fundamentals: sources, wavelength selectors, cuvettes, and detectors. Beer's law and its deviations, isosbestic point, calibration of a spectrophotometer according to the European Pharmacopoeia. Operating procedures for qualitative-quantitative analysis (calibration curves), Methods based on light emission; photoluminescence: fluorescence and phosphorescence, Jablonsky diagram. Fluorescence spectroscopy: fundamentals, Beer's law applied to the fluorimetry, fluorescence measurements (emission, excitation, fixed wavelength, synchronous spectra). • Generalities on the assays and analytical controls of drug substances and drug products: average mass and uniformity of mass, uniformity of content, loss on drying, determination of moisture by Karl-Fischer method. Definition of impurities and degradation products, main reactions involved in the degradation of drugs. • HPLC chromatographic methods: fundamentals, constituent parts of an HPLC system, types and features of the stationary phases (silica and chemically modified silicas to obtain the different selectivity), mobile phase (eluent system), elution in isocratic and gradient modes, control of the pH of the mobile phase using buffers (ion suppression and ion pairing), principal detectors (UV-Vis, PDA, FLD, MS, RID, electrochemical, ELSD, CAD). Quantitative analysis: internal standard, external standard, standard additions. Parameters for the evaluation of the chromatographic system performance: capacity factor, selectivity, efficiency, theoretical plates, resolution, asymmetry and tailing factor, band broadening theory for liquid chromatography. • Generalities on sample preparation techniques for pharmaceutical analysis: liquid-liquid extraction (LLE), liquid-liquid extraction on solid support (SLLE), solid phase extraction (SPE), other techniques. • Stability studies of drug substances and drug products: purpose of long-term and accelerated stability studies and their implementation according to ICH guidelines, on-going stability studies, forced degradation studies. • Validation of analytical procedures according to the ICH guidelines. • Pharmaceutical analysis calculations: titrations, development of formulas for determining the content of the active ingredient in pharmaceutical formulations, determination of the drug substances purity, buffer solutions preparation. • Principles of Gas Chromatography. Gas chromatograph: injectors, columns, detectors. Selectivity, efficiency, resolution. Van Deemter equation applied to gas chromatography. • Retention indices: Kovats indices. Headspace techniques: static, dynamic, SPME (Solid Phase Micro Extraction). • Principles of Mass Spectrometry. Ion sources, resolving power and resolution. Tandem mass spectrometry. High-resolution mass spectrometry. • Mass spectrum. Molecular ion, isotopic abundance, fragmentation pathways. • Acquisition methods in Full Scan, SIM (Selected Ion Monitoring), MS/MS. Use of databases. Qualitative and quantitative analysis. Gas Chromatography coupled with Mass Spectrometry (GC-MS). Liquid Chromatography coupled with Mass Spectrometry (HPLC-MS). Interface challenges. Qualitative and quantitative analysis. LABORATORY ACTIVITIES. Volumetric analysis using titration techniques (acid-base, redox, precipitation, complexometric). Determination of average weight and weight uniformity. Quantitative determination UV-Vis of the active ingredient (Fe2+) and the related impurity (Fe3+) in a formulation of ferrous gluconate. Determination of the purity (Fe2+ assay) of a batch of ferrous gluconate. Determination of loss on drying using thermogravimetric balance. Determination of pH of a pharmaceutical powder when reconstituted in aqueous solution. Quantitative HPLC-UV determination of levodropropizine and preservatives in a syrup formulation. Determination of moisture by Karl-Fischer method.

At the end of the course, which also includes laboratory activities, the student will have the knowledge of the main analytical techniques used to determine the purity of active pharmaceutical ingredients and their potency in the corresponding formulations also in the context of stability studies. Furthermore, the student will be able to calculate, interpret and evaluate the analytical data obtained.