Development and validation of RP-HPLC method for 1΄-Acetoxychavicol acetate (ACA) and its application in optimizing the yield of ACA during its isolation from Alpinia galanga extract as well as its quantification in nanoemulsion

An isocratic RP-HPLC was developed and validated for isolation of 1΄-Acetoxychavicol acetate (ACA) from Alpinia galanga extract (AGE). Nucleodur C18 column was used as the stationary phase and mixture of acetonitrile/0.1% formic acid in water (60/40, v/v) were used as mobile phase. The elution was carried out with a flow rate of 1 mL/min. Further, the method was validated as per ICH Q2 [R1] guidelines in terms of linearity, LOD, LOQ, recovery, precision and specificity. The validated method was utilized for two purposes i.e. optimizing the isolation of ACA from AGE and quantifying ACA's concentration and release from its nanoemulsion. To achieve high yield of ACA, Central composite design (CCD) was used to optimize sieve number and solid to solvent ratio. The retention time of standard and isolated ACA were found at 5.63 min and 5.46 min respectively. The results indicated that the validated method was linear, accurate, precise and specific. During optimization, excellent correlation was observed between practical yield (3.89 ± 0.23% w/w of dried rhizomes) and predicted yield (3.73% w/w of dried rhizomes) of ACA with p value more than 0.05. The percentage of ACA loaded in the nanoemulsion was found to be 99.12 ± 0.39. The in vitro diffusion studies showed 90.12 ± 2.21% release of ACA at the end of 24 h. It was concluded that the validated method has been successfully utilized to isolate the ACA from AGE and quantify the amount of ACA present in nanoemulsion.