Development and validation of a stability indicating UPLC-DAD method coupled with MS-TQD for ramipril and thymoquinone in bioactive SNEDDS with in silico toxicity analysis of ramipril degradation products

Abstract

The identification of degradation products of therapeutic molecules in pharmaceutical formulations has gained significant attention due to their potential impact on patient safety. Ramipril (RP), an antihypertensive agent, was incorporated into a self-nanoemulsifying drug delivery system (SNEDDS), which greatly enhanced its bioavailability. However, none of the previous studies have investigated the toxicological effects of these degradation products that may form during storage. Moreover, a bioactive SNEDDS containing black cumin oil (BCO) and its bioactive ingredient, thymoquinone (TQ), was used to further enhance the therapeutic activity of RP. To assess the stability of the proposed formulation, a validated ultrahigh-performance liquid chromatography (UPLC) method was developed to simultaneously measure the concentrations of RP and TQ. The formulation was subjected to accelerated stress conditions to facilitate drug degradation. The resulting degradation products were analyzed using mass spectroscopy (MS) to determine their molecular mass, and their chemical structures were in silico predicted using Zeneth Nexus software, while their toxicity was assessed using in silico Derek Nexus software. RP and TQ, along with their degradation products, were separated using an HSS T3 column at a flow rate of 0.25 mL/min. The detection wavelengths for RP and TQ were 210 and 254 nm, respectively. The developed UPLC method exhibited acceptable linearity for both RP and TQ, with correlation coefficient (r 2) values exceeding 0.9995 and 0.9998, respectively. The method provided accurate, precise, and high-resolution analysis of both drugs and their degradation products within a short run time of less than 3.2 min. The toxicity and mutagenicity of two alkaline degradation products of RP were predicted using in silico software Derek Nexus (version 6.3). Several toxicity endpoints, including chromosomal damage, skin sensitization, and hepatotoxicity, were predicted. Overall, the developed method can be used to evaluate the stability and integrity of RP and TQ during the development of the proposed antihypertensive formulation.