Quality-by-design optimized HPLC approach for the therapeutic drug monitoring of isosorbide dinitrate and sildenafil in human plasma samples

Isosorbide dinitrate (ISDN) and sildenafil citrate (SIL) are reported to have a potential drug-drug interaction when co-administered clinically. ISDN is indicated as first-line therapy for the long-term management of chronic stable angina. Sildenafil is the first oral treatment for erectile dysfunction. It is contra-indicated in patients who may require organic nitrates, such as ISDN, because this combination may cause a sudden drop in blood pressure by inhibiting the phosphodiesterase type 5 enzyme. Additionally, sildenafil is effective in treating cardiovascular disorders secondary to endothelial dysfunctions. Therefore, a sensitive, simple, and selective HPLC method is urgently needed for their simultaneous determination in plasma samples for monitoring their therapeutic blood concentration levels in emergency cases. The current study introduces the first HPLC approach for their simultaneous estimation in less than 10 minutes. The optimal separation was achieved using Nova-Pack® C18, 4 µm column at room temperature, with a mobile phase consisting of acetonitrile and acetate buffer (5 mM; pH 5, 39:61 % v/v), delivered at a flow rate of 1.1 mL/min. The injection volume was set at 50 µL, and detection was accomplished at 214 nm. The method demonstrated excellent linearity for ISDN(0.01–10.0µg/mL) and SIL (0.025–10.0µg/mL), with LOQ of 0.01 µg/mL and 0.020 µg/mL, respectively. The proposed method was successfully applied to analyze spiked human samples with recovery rates (104.9 and 105.55%) for ISDN and SIL respectively, confirming its suitability for bio-analytical use. Utilizing the quality-by- design (QbD) approach adopting the two-level full factorial design allowed the optimization of the experimental conditions of the proposed method. The method’s greenness was evaluated using Analytical Eco-Scale, Green Analytical Procedure Index (GAPI), and Analytical Greenness (AGREE). Validation of the proposed method was performed in accordance with ICH and US FDA guidelines.