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| Author(s): |
Juliana Caldeira Abreu
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2020-12-14 |
| Examining board members: |
Claudimir Lucio do Lago;
Carla Beatriz Grespan Bottoli;
Felipe Rebello Lourenço;
Liliana Marzorati
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| Advisor: | Claudimir Lucio do Lago |
| Abstract | |
The drug stability profile is related to its potency, purity and safety. Samples from the stability study must be analyzed using a stability-indicating method. The degradation products (DP) identified in the stress testing study are used to develop this method, which can be developed varying one factor at a time, or applying a chemometric approach to understand how the input parameters variation affect the results of the analytical method. Our aim was to use the concepts of Analytical quality by design (AQbD) to explore the separation and selectivity principles of the active pharmaceutical ingredient bromopride and its impurities, and thus develop a new stability-indicating method via UHPLC-DAD. The bromopride stress testing study and DP identification were systematically presented. In addition, a rationale was presented for each stage of the AQbD approach and how this concept contributed to a better method understanding, performance and robustness. Initially, the active pharmaceutical ingredient (API) was exposed toward different stress conditions. The degradation pathways in which the molecule was more labile (degradation ≥ 10%) were hydrogen peroxide, acid hydrolysis and UV-A light. The target degradation products, with relative area ≥ 0,2%, were identified via UHPLC-UV-ESIQToF, with impurities BIB (4-amino-5-bromo-2-methoxybenzoic acid) and BID (2-bromo- 5-methoxyaniline) originated via acidic degradation, BIE (2-(4-amino-5-bromo-2- methoxybenzamido)-N,N-diethylethanamine oxide) via oxidation and BIC (4-amino-5- bromo-N-(2-(ethylamino)ethyl)-2-methoxybenzamide) via oxidation and photodegradation conditions. BIB and BID were commercially adquired, BIC and BIE were isolated using semi-preparative HPLC. The impurity BIA (4-acetamido-5-bromo-N-(2- (diethylamino)ethyl)-2-methoxybenzamide), a synthetic intermediate of bromopride, was synthesized.The chromatographic separation was evaluated through two screening tests and an optimization design, including in the last step Monte Carlo simulation to access method robustness. Prior knowledge on analytes was used in the initial risk assessment contributing to assign Critical Method Parameters (CMP). In the first screening, a wide range of mobile phase pH, organic solvent and six LC columns with different selectivities were defined as CMPs and 87 experiments were performed. The second screening test, with 76 experiments, considered the CMPs with best performance and included column temperature and two other columns. The final optimization study, with 42 experiments, enabled the acquisition of a knowledge space, delimited by evaluating regions with acceptable mean performance and robustness to access the method operable design region (MODR). The Stability-indicating method for bromopride assay and related impurities was efficiently developed, evidencing to be selective, linear (r2 = 0.9999) and reproducible, proving that AQbD approach stands out as an efficient concept to detect the method limitations and to define the region with best performance, contributing to the assignment of appropriate control strategies (CS) and facilitating the method lifecycle management. The final method presented the experimental conditions: column YMC Triart Phenyl at 30 °C, flow rate of 0.3 mL.min-1, Ammonium bicarbonate 5 mmol.L-1 pH 8.9 (± 0.1) and Acetonitrile / Tetrahydrofuran (7: 3) ± 3% in gradient mode (AU) | |
| FAPESP's process: | 16/05633-0 - Vanadium haloperoxidase: searching for new sources and their application in continuous flow system aiming selective reactions |
| Grantee: | Juliana Caldeira Abreu |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |