Development and validation of a stability‐indicating reversed‐phase–high‐performance liquid chromatography method for quantification of 2‐[1‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a from lipid‐polymeric hybrid nanoparticles

2‐[1‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a is a second‐generation, lipophilic, chlorin‐based photosensitizer currently used for various types of solid cancer including breast, esophageal, neck, and head cancer, etc. The aim of the present work was to develop a sensitive and robust reversed‐phase–high‐performance liquid chromatography–ultraviolet detection method for the determination of 2‐[1‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a to explore in vitro‐based applications including encapsulation, loading, drug release, and stability studies, and also could be utilized for the selective analysis of 2‐[1‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a from developed lipid polymer hybrid nanoparticle. The developed reversed‐phase–high‐performance liquid chromatography–ultraviolet detection method was validated in terms of linearity, accuracy, precision, stability, and robustness as per standard guidelines. The developed method exhibited linearity ranging from 50 to 2000 ng/ml with an R2 value of 0.999. The limit of detection and limit of quantitation value of the developed method was found to be 14.03 and 42.80 ng/ml, respectively. Also, degradation studies revealed that 2‐[1‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a degraded quickly under basic conditions compared to other stability conditions (i.e., acidic, oxidative, temperature, etc.). Furthermore, the developed drug‐loaded lipid polymer hybrid nanoparticle showed an average size of 123.03 nm (polydispersity index = 0.253) with an encapsulation efficiency of 70.23%. Cell cytotoxicity assay of lipid polymer hybrid nanoparticle showed significant cancer cell death equivalent to 2‐fold higher than the free drug. In conclusion, the developed reversed‐phase–high‐performance liquid chromatography–ultraviolet detection analytical method was found to be sensitive and reliable for the determination of free 2‐[1‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a and 2‐[1‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a content in loaded lipid polymer hybrid nanoparticle. Thereby, opening the avenues for future research to explore the detailed physiochemical and formulation designing for the 2‐[1‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a.