Purity determination of a new antifungal drug candidate using quantitative ¹H NMR spectroscopy: Method validation and comparison of calibration approaches

Abstract

Quantitative nuclear magnetic resonance (qNMR) is an analytical technique that offers numerous advantages in pharmaceutical applications including minimum sample preparation and rapid data collection times with no need for response factor corrections, being a powerful tool for assaying drug content in both drug discovery and early drug development. In the present work, we have applied qNMR, using both the internal standard and the electronic reference to access in vivo concentrations 2 calibration methods, to assess the purity of RI76, a novel antifungal drug candidate. NMR acquisition and processing parameters were optimized in order to obtain spectra with intense, well‐resolved signals of completely relaxed nuclei. The analytical method was validated following current guidelines, demonstrating selectivity, linearity, accuracy, precision, and robustness. The calibration approaches were statistically compared, and no significant difference was observed when comparing the obtained results and their dispersion in terms of relative standard deviation. The proposed qNMR method may, therefore, be used for both qualitative and quantitative assessments of RI76 in early drug development and for characterization of this compound.

Recent Development in Nuclear Magnetic Resonance as a Technique for Quantitative Method Validation

“…The remaining 30 % of the mass comprises impurities. The study revealed that equivalent results were observed with both IS and ERETIC-2 calibration approaches yield the same result [67] .…”

“…In the experiment, 5 mg of acetanilide and 12 mg of RI76 (fig. 1) were accurately weighed and dissolved in 600 µl of Deuterated dimethyl sulfoxide (DMSO-d 6 ), which was accurately transferred to NMR tubes for evaluating the purity of RI76 [67] .…”

“…Fig. 1: Chemical structure of the compound RI76 and acetanilide [67] A stock solution of α-bisabolol (105.0 mg/ml) and octamethylcyclotetrasiloxane (OMCTS; 1.9 mg/ml) were prepared to measure linearity, accuracy, precision and stabilities of α-bisabolol and OMCTS in solution (fig. 2).…”

One Century of Study: What We Learned about Paracoccidioides and How This Pathogen Contributed to Advances in Antifungal Therapy

“…Simulations based on the target structure bring a greater chance of success for drug development in less time [136] (Figure 3). The target prediction for drug discovery can be based on structural models available in databases, resulting from X-ray crystallography or NMR spectroscopy [137]. The best binding sites are identified by computational tools, according to physical-chemical properties, and thus the docking for selection of inhibitors can start by virtual screening [138].…”

Stability‐indicating method for the novel antifungal compound RI76: Characterization and in vitro antifungal activity of its active degradation product

“…RI76 increased the survival of Galleria mellonella larvae infected with Candida albicans to 81.3%, and it also proved to be effective in the murine model of oral and systemic candidiasis when tested at 10 mg/kg (Cruz et al, 2018). The purity of RI76 synthesized in‐house was assessed by quantitative nuclear magnetic resonance using two different calibration methods, which provided similar results for RI76 content (Franco, Braga, Oliveira, & César, 2020). Based on these results, RI76 was selected as a possible drug candidate.…”