chemosensors Article Expediting Disulfiram Assays through a Systematic Analytical Quality by Design Approach João Basso 1,2 , Maria Luísa Ramos 2 , Alberto Pais 2 , Rui Vitorino 3,4,5 , Ana Fortuna 1,6 and Carla Vitorino 1,2, *   Citation: Basso, J.; Ramos, M.L.; Pais, A.; Vitorino, R.; Fortuna, A.; Vitorino, C. Expediting Disulfiram Assays through a Systematic Analytical Quality by Design Approach. Chemosensors 2021, 9, 172. https://doi.org/10.3390/ chemosensors9070172 Academic Editor: Cláudia Maria Rosa Ribeiro Received: 30 April 2021 Accepted: 3 July 2021 Published: 6 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal; joaobasso@ff.uc.pt (J.B.); afortuna@ff.uc.pt (A.F.) 2 Coimbra Chemistry Centre, Department of Chemistry, University of Coimbra, 3004-504 Coimbra, Portugal; mlramos@ci.uc.pt (M.L.R.); pais@qui.uc.pt (A.P.) 3 iBiMED—Department of Medical Sciences, University of Aveiro, 3810-193 Aveiro, Portugal; rvitorino@ua.pt 4 UnIC—Cardiovascular Research & Development Centre, Department of Surgery and Physiology, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal 5 QOPNA & LAQV-REQUIMTE, Chemistry Department, University of Aveiro, 3810-193 Aveiro, Portugal 6 Coimbra Institute for Biomedical Imaging and Translational Research, University of Coimbra, 3000-548 Coimbra, Portugal * Correspondence: csvitorino@ff.uc.pt Abstract: An Analytical Quality by Design (AQbD) approach is presented, aiming at the development and validation of an HPLC method for the quantification of disulfiram and copper diethyldithio- carbamate in lipid nanoparticles. Following the definition of the analytical target profile (ATP), encompassing the critical analytical attributes (CAA), a two-level risk assessment strategy (Ishikawa diagram—failure mode and effect analysis (FMEA)) was employed to identify the critical method parameters (CMPs) with an extensive impact on method performance. The behavior of the CMPs (flow rate and mobile phase composition) was further characterized by experimental design, resorting to a face-centered central composite design (FcCCD). Statistical modeling, response surface analysis, and Monte Carlo simulations led to the definition of the Method Operable Design Region (MODR), associated with a negligible risk of failing the predefined CAA specifications. The optimal method was validated according to international regulatory recommendations. Apart from guaranteeing linearity, accuracy, precision, specificity, robustness, and stability, these conditions were found to be suitable for analysis using a different HPLC column and equipment. In a nutshell, the development and optimization strategies, under the comprehensive framework of AQbD, provided an effective, simple, rapid, reliable, and flexible method for routine analysis of the compounds in research or industrial environments. Keywords: AQbD; cancer; central composite design; copper diethyldithiocarbamate; disulfiram; lipid nanoparticles; liquid chromatography; method optimization; method robustness; MODR 1. Introduction Disulfiram (DSF) is a dithiocarbamate derivative with clinical application to treat alcohol addiction. It blocks ethanol metabolism by inhibiting hepatic aldehyde dehydroge- nases 1 and 2, thus increasing acetaldehyde blood levels. Consequently, alcohol consumers experience nausea, sweating, hypotension, respiratory difficulties, and other alcoholic intoxication symptoms, thereby rejecting additional beverages [1]. In parallel, DSF shows promising in vitro/in vivo results against several types of neoplastic diseases, as it may act over 19 different targets/pathways to reduce cancer cell viability [2]. DSF is also a chelating compound, producing copper (II) diethyldithiocarbamate (Cu(DDC) 2 ) in the presence of endogenous or exogenous copper (II) ions. Alternatively, Cu(DDC) 2 can be synthesized by chelating copper with sodium diethyldithiocarbamate (Figure 1)[3,4]. Interestingly, this Chemosensors 2021, 9, 172. https://doi.org/10.3390/chemosensors9070172 https://www.mdpi.com/journal/chemosensors