(M1330-11-62) Simultaneous Detection of Camostat/Niclosamide Cocrystal and Its Related Impurities via Analytical Quality by Design

Purpose: In recent years, drug-drug cocrystals have garnered significant attention due to their potential to enhance physicochemical and biopharmaceutical properties. Moreover, the use of drug-drug cocrystals presents a low-risk, low-cost, and high-return strategy since the efficacy and safety of the individual drugs have already been established. As the combination of acidic and basic drugs is common in drug-drug cocrystals, it is essential to develop a robust method for the simultaneous detection of acidic and basic drugs. Camostat is a potent serine protease inhibitor used to treat certain viral infections, while niclosamide is commonly used to treat tapeworm. In recent years, both drugs have been the subject of active research due to their potential as treatments for COVID-19. However, niclosamide is a weak acid, while camostat is a strong base that can be unstable under stressed conditions, making it challenging to develop a simple assay that can detect both drugs simultaneously. Therefore, there is a need to develop a simple HPLC method that can identify camostat, niclosamide, and their related substances simultaneously for further development.

Methods: Forced degradation studies: Forced degradation studies including acid (0.1N HCl), alkali (0.1N NaOH), oxidation (3% H₂O₂), heat (60℃), and light (according to the ICH guideline) was conducted to identify potential impurities of Camostat/Niclosamide. Preparation of potential impurity mixture samples: All six camostat impurities can be prepared by combining the base-induced degradation sample and the thermal degradation study sample. To prevent further degradation, the base-induced degradation sample was neutralized by adding 0.1 N HCl. The final impurity mixtures were prepared by adding niclosamide dissolved in methanol to the camostat solution, followed by filtration for further analysis. AQbD method: The AQbD method was conducted in a series of steps, including the set-up of an analytical target profile (ATP), risk assessment, screening, optimization study, and verification study. To investigate the effect of various chromatographic parameters on the separation of camostat/niclosamide and potential impurities, Fusion X was used. During the screening process, the buffer pH values were adjusted to 3, 4.5, and 5.7, and the gradient time was screened between 2 and 9 min. The column was conditioned with mobile phases between runs for 9 to 15 min.

Results: Forced degradation studies: During the forced degradation studies, six camostat-related impurities and no trace of niclosamide impurity were found (Figure 1). The six impurities can be generated by base-induced degradation and thermal degradation study. During the AQbD process, an ATP was set as a resolution greater than 2, USP tailing between 0.8 and 1.2, and an S/N ratio of no less than 10 at 0.5 ppm of camostat/niclosamide. During risk assessment, the column stationary phase, pH, and gradient time were selected as critical method parameters (CMP) for the separation of camostat/niclosamide and six potential impurities. The effect of various chromatographic parameters on the separation was systematically investigated using Fusion X. After the optimization step, a design space was established to meet all ATP criteria. A three-dimensional graph was created to display the optimal gradient time and pH range to meet the ATP criteria (Figure 2). The left two-dimensional spectrum graphs show that the red area is the ideal region. On the right two-dimensional graphs, the blue area fails to meet the USP tailing criteria (0.8-1.2), the red area violates the USP resolution requirement ( < 2.0), and only the white region satisfies the ATP criteria. Based on design space analysis, ACN was selected as a mobile phase B, and C18, 2.1mm×100mm column with a particle size of 1.7 μm was chosen. The experimental conditions employed a gradient time of 2-4.5 min. and a pH range of 2.8-3.0. In the verification study, the camostat/niclosamide cocrystal sample was analyzed, and all ATP criteria were found to be within the required limits. Specifically, the sample met the resolution requirement of more than 2.0, the USP tailing criteria in the range of 0.8-1.2, and the signal-to-noise (S/N) ratio requirement of more than 10, with a camostat/niclosamide concentration of 0.5 ppm. The final selected UPLC method was converted to HPLC method which then, further optimized for simultaneous detection of camostat/niclosamide cocrystal and its related impurities (Figure 3).

Conclusion: Six camostat-related impurities and no impurity of nicalosamide were identified by two different HPLC methods. In order to develop the simultaneous determination method of camostat/niclosamide cocrystal and six camostat-related impurities, AQbd method using UPLC method was employed to meet ATP. AQbD analysis reveals that CMP are column stationary phase, pH, and gradient time on separations.ased on AQbD analysis, HPLC method which enables to simultaneously determine camostat/niclosamide cocrystal and six camostat-related impurities was developed. This study highlighted that AQbD method can be used as a useful tool to separate drug-drug cocrystals with different physicochemical properties, and provide deep understanding of the effects of pH of mobile phase, column, organic solvent, or gradient time on the separation of drug-drug cocrystals and their impurities.


Potential impurities of camostat and niclosamide identified by forced degradation studies.


Through the Fusion X software, a three-dimensional were obtained.


Method and chromatogram of Optimization study