Analytical Strategy for Low-Level Estimation of Unstable Genotoxic Boronate Ester Impurities

Abstract

Boronate esters are commonly used starting materials in the Suzuki–Miyaura coupling reaction for the C–C bond formation due to their cost-effectiveness and ease of manufacturing process. However, most of them are highly sensitive to moisture and pose challenges during their in-process analysis, with conversion to acids under reversed-phase analytical conditions. They are prone to hydrolysis under moisture, pH, and even on-column stationary phases under neutral conditions. At the same time, boronate esters are considered as potential genotoxic substances; hence, their estimation is very important from the patient safety perspective. There are inherent challenges in the existing methods of analysis of these compounds. In this paper, a convenient, simple, highly sensitive, and greener SFC-MS method was developed for the screening of such unstable boronate esters. The optimized method consisted of Celeris Arginine column (250 mm × 4.6 mm; 5 μm) with CO₂ (A) as a solvent with a cosolvent of ACN: MeOH (80:20) containing 0.2% 7N methanolic ammonia (B) in gradient mode [T_min/B %: 0.01/05, 0.50/05, 5.00/50, 7.00/50, 7.10/05, and 10.00/05]. Critical method parameters such as ABPR pressure, makeup solvent, additives, and pump flow rate were optimized to enhance the sensitivity with a model compound, i.e., 1-(benzenesulfonyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrrolo[2,3-b]pyridine (CAS no. 886547-94-0). The robustness of the method was demonstrated by validating the method as per the ICH guidelines on the same model compound in the concentration range of 0.03–0.3 ppm. The LOD and LOQ for this compound were determined as 0.01 and 0.03 ppm (1 and 3 ppm with respect to API concentrations of 10 mg/mL), respectively. The method was successfully applied for the estimation of 16 structurally different boronate esters with no chemical derivatization or hydrolysis.