Deciphering a novel RP-HPLC based bioanalytical method for Estimation of xanthohumol in rat plasma and postbiotic-based nanostructured lipid carriers

The study aimed to establish an accurate, specific, linear, precise, and robust method for quantifying xanthohumol loaded in nanostructured lipid carriers using the liquid chromatography. Sodium butyrate was added in nanostructured lipid carriers as postbiotic. Curcumin was taken as an internal standard. Pure xanthohumol and xanthohumol-loaded nanostructured lipid carriers were spiked separately in rat’s plasma and method was developed. Isocratic elution was done on Altin C-18 column having length 250 mm, pore size 5 μm and an internal diameter of 4.6 mm. The mobile phase used was a combination of acetonitrile and glacial acetic acid (0.1% in water) in the ratio of 65:35 w/v. The flow rate was kept at 1mL/minute and quantification was done at 370 nm. The retention times for xanthohumol and curcumin were found at 7.03 min and 4.5 min, respectively. The developed method demonstrated linearity between 2 and 10 ng/mL with an R² equal to 0.9992. The results of all validation parameters were within the accepted limits, with percent relative standard deviation below 2. The absence of any peak related to the plasma matrix, sodium butyrate, and placebo nanostructured lipid carriers over the retention time of xanthohumol indicated that the bioanalytical method was specific. Furthermore, short, long, and freeze-thaw stability were performed. The percentage recovery and % relative standard deviation of xanthohumol from plasma samples were within ± 5%. Additionally, the stability of xanthohumol-loaded nanostructured lipid carriers was assessed in plasma, where particle size, zeta potential, and entrapment efficiency were recorded as 120.27 nm, -11.7 mV, and 95%, respectively, indicating the integrity and stability of nanostructured lipid carriers in plasma.