First-in-class HPLC-fluorescence method for quantification of roscovitine in plasma: Greenness assessment and application to pharmacokinetic studies in rats

This research introduces a first-in-class high-performance liquid chromatography with fluorescence detection (HPLC-FD) method for the reliable quantification of ROS in plasma. The method relies on the native fluorescence of ROS, with excitation and emission set at 280 nm and 340 nm, resp. Sample preparation involved a simple one-step protein precipitation with methanol. Chromatographic resolution of ROS and pemigatinib (PEM), which served as the internal standard (IS), was achieved using Zorbax Eclipse Plus C18 HPLC column. The mobile phase consisted of methanol and formate buffer solution. The method was validated in compliance with ICH M10 guidelines. It showed linearity across concentration range of 50–1,500 ng mL⁻¹ (R² = 0.9991), with a lower limit of quantitation of 50 ng mL⁻¹. Accuracy ranged from 96.8 to 103.4 %, and intra- and inter-day precision (RSD) were between 3.2 and 6.1 %. The method was successfully applied to quantify ROS in plasma samples from rats following administration of a single oral dose (25 mg kg⁻¹), enabling the determination of key pharmacokinetic parameters, including C_max (361.31 ng mL⁻¹), t_max (1.75 h, t_½ (2.867 h), V_d (0.077 L kg⁻¹), CL (0.018 L h⁻¹ kg⁻¹), AUC (ng h mL⁻¹) and and MRT (h). The greenness and practical applicability of the method were confirmed using four comprehensive independent metric tools. In comparison to previously reported bioanalytical approaches, the proposed method offers several practical advantages, such as reduced complexity and cost, a shorter run time, full validation, established greenness, and suitability for the clinical laboratory setting.