Development and validation of an HPLC-fluorescence method for the quantification of IR780-oleyl dye in lipid nanoparticles
Résumé
A reversed-phase (RP) high-performance liquid chromatography (HPLC) method for the content determination of IR780-oleyl (IRO) dye in lipid nanoparticles was developed and validated. Chromatographic separation was performed on a RP C18 column with a gradient program of water and acetonitrile both with 0.1% (v/v) TFA, at a flow rate of 1.0 mL/min and a total run of 21 min. IRO dye detection was made by fluorescence at emission wavelength of 773 nm (excitation wavelength 744 nm). According to ICH guidelines, the developed method was shown to be specific, linear in the range 3-8 mg/ mL (R-2 = 0.9998), precise at the intra-day and inter-day levels as reflected by the coefficient of variation (CV <= 1.98%) at three different concentrations (4, 6 and 8 mg/mL) and accurate, with recovery rates between 98.2-101.6% and 99.2-100.5%. The detection and quantitation limits were 0.41 and 1.24 mg/mL, respectively. Stability studies of sample processing showed that IRO dye was stable after 24 h in the autosampler or after three freeze/thaw cycles. Combined with fluorescence measurements, the developed method was successfully applied to optimize the loading capacity of IRO dye in the core of lipid nanoparticles.