An Isocratic RP‐HPLC Method for Simultaneous Quantification of Nateglinide and Furosemide: Application to In Vitro Caco‐2 Intestinal Permeability Studies of Nanodrugs

In the booming era of nanomedicines, Caco-2 intestinal permeability experimental models allow characterizing intestinal permeabilities of nanopharmaceuticals and are conducted in the presence of cell culture media (CCM) and stabilizers. Methods for simultaneous quantification of furosemide (FRS) and nateglinide (NTG) in Caco-2 experiments are lacking. We developed and validated a novel, simple, and practical reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV detection to simultaneously quantify FRS and NTG in the presence of CCM and stabilizers (Povidone K-25, Pluronic F-38) for Caco-2 intestinal permeability experiments exploring nanoformulations. FRS and NTG nanosuspensions were prepared using high-speed ultrasonic homogenization top-down technique. Caco-2 cell culture studies were conducted. ICH Q2(R1) guideline requirements (i.e., selectivity, specificity, accuracy, precision, robustness, stability) were met. System suitability and linearity (FRS: 5.12-40.00 μg/mL; NTG: 7.31-50.00 μg/mL), with high determination coefficients (>0.999), and short retention times (FRS: 3.3 min; NTG: 5.3 min) were achieved. NTG and FRS in Caco-2 samples were successfully simultaneously quantified, proving usefulness for nanodrug research applications. Varying stabilizers impact intestinal permeability. This work simulates pragmatic conditions, as cardiovascular patients are co-administered these drugs, paving the pathway to using our novel method for a wide range of "bench-to-bedside" nanomedicine research designs to tackle the challenges of polypharmacy in this complex population.