Development of a High-Performance Liquid Chromatography - Mass Spectrometry Method for Qualitative and Quantitative Analysis of Arsenoplatin Compounds

Abstract

Arsenoplatin compounds, dual-pharmacophore anticancer agents containing platinum and arsenic pharmacophores, were developed due to the synergy between FDA-approved drugs cisplatin and arsenic trioxide in various cancer cell lines. These compounds have demonstrated potent in vitro activity, particularly in leukemia, breast, and ovarian cancer cell lines, as confirmed by the National Cancer Institute\u27s Developmental Therapeutic Program. Due to upcoming in vivo studies, it is essential to ensure the purity and stability of arsenoplatins in physiological solutions. The High-Performance Liquid Chromatography - Mass Spectrometry (HPLC-MS) method was developed to separate, identify, and quantify arsenoplatin compounds and their degradation products. An initial HPLC method was tested using ZORBAX Eclipse XBD-C18 4.6x100mm,3.5µm column as stationary phase and PBS (Phosphate Buffered Saline) solution (99.8%) and 1,2-hexandiole (0.2%) as the mobile phase, as PBS is commonly used in drug formulations for efficacy studies in vivo. However, since PBS was incompatible with the mass spectrometer due to phosphate salts being non-volatile, a new method was developed using ammonium acetate (96%) and methanol (4%) as the mobile phase and ZORBAX Eclipse XBD-C18 4.6x100mm,3.5µm column as stationary phase to detect arsenoplatins by ESI-MS (electrospray ionization mass spectrometry). This modified HPLC-MS method effectively separates and detects arsenoplatins and their degradation products. All arsenoplatins analyzed by ESI-MS exhibited distinctive cluster ions in their mass spectra due to five naturally occurring platinum isotopes. For all arsenoplatins, molecular ions are observed in negative mode. Based on the m/z values, the fragmentation pattern for arsenoplatins is proposed. The newly developed HPLC-MS method will be validated according to ICH (The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) and FDA guidelines. It will ensure accurate qualitative and quantitative analysis of arsenoplatin compounds and their degradation products, which is critical for maintaining arsenoplatins efficacy and safety in pharmaceutical formulations