Chemicals and reagents
Apigenin was a gift sample from Aktin Chemicals Ltd., China. Methanol, acetonitrile, and formic acid (HPLC grade) were purchased from Merck Limited (Mumbai, India). Other common laboratory reagents were of analytical grade.
Shimadzu HPLC (LC- 2010) (Kyoto, Japan), pump system (LC-20 AD), degasser (DGU-20A5), a column oven (CTO-10ASVP), an auto-injector (SIL-20ACHT), PDA Detector (SPD-M20A), and a computer software (lab solutions, version 1.25) were used.
The chromatographic separation was carried out by injecting (10 μL) a sample to the HPLC system connected to a C-18 analytical column (Phenomenex Luna 5 μm, 250 mm × 4.6 mm) (set at 35 °C) operating at 1 ml/min flow rate, and detection was done at 269 nm. Acetonitrile and 0.1% formic acid, 55:45 (v/v) at pH = 7.4, was used as a mobile phase.
Standard solution preparation
Stock solution was prepared (100 μg/mL) in the mobile phase. Further dilutions were carried out to get the calibration curve ranging from 0.5 to 16 μg/mL.
Preparation of stealth apigenin-nanoliposomes
Formulating the stealth apigenin-nanoliposomes, an ethanol injection technique was used in the preparation of nanoliposomes as described by Sudhakar et al. . Briefly, apigenin (10 mg) and stearic acid (10 mg) were dissolved in 2 ml of absolute ethanol with gentle heating (< 50 °C) on a hot plate. Then, lipid di-stearoyl-sn-glycero-phosphoethanolamine–N (polyethyleneglycol) (DSPE-PEG) (0.003%) and cholesterol (0.0015%) were added into the same mixture with stirring. Phosphate-buffered saline (PBS) (pH 7.4) (10 ml) was added to mixture dropwise with constant stirring for 90–120 min to remove ethanol. The nanoliposomes were optimized for various concentrations of lipid. The optimized batch was homogenized at 10,000×g for 10 min followed by sonication for 15 min, which were further filtered through 0.45-μm and 0.2-μm sterile syringe filters and were allowed to equilibrate for 1 day and then stored at 4 °C. In the time, apigenin-loaded nanoliposomes were also assessed for their average particle size, poly dispersity index (PDI) according to Sharma et al. . Moreover, encapsulation efficiency of nanoliposome was also assessed by centrifugation at 10,000×g for 30 min, and supernatant solution obtained after centrifugation was mixed with mobile phase and analyzed by using HPLC [9, 10].
The chromatographic conditions were optimized and steady baseline was obtained. Chromatogram was assessed by injecting standard apigenin solution, and analyses were repeated in a similar way for six times.
Linearity studies reveal the standard calibration curve was plotted from 0.5 to 16 μg/mL concentrations. Standard calibration curves were determined for concentrations versus peak area. Sample run in triplicate of all the sample solutions was assessed, and a chromatogram was recorded.
Precision and accuracy
The apigenin samples were subjected to three levels of quality control; namely low, medium, and high respective concentrations of 0.5, 4, and 16 μg/mL were prepared and used in determining the precision and accuracy. For intra-day precision and accuracy, triplicates of standard solutions were injected on the same day and also for interday; triplicates of standard solutions (50%, 100%, and 150%) were injected over three consecutive days. %relative standard deviation (RSD) and % RE were calculated respectively.
Limit of detection (LOD) and limit of quantification (LOQ)
The LOD was determined by a series of dilutions of apigenin stock solutions to find a signal to noise (S/N) ratio of at least 3.3:1 for LOD and 10:1 for LOQ. Both parameters obliged the quantity of analyte that can be quantified.
To validate the chromatographic system, suitability test is carried out, and various factors such as tailing factor, theoretical plates, and peak area and resolution data were also considered.
The robustness of the method was evaluated by deliberately making a slight change in the optimized value. The evaluated parameters were as follows: detector wavelength (± 1 nm), flow rate (± 0.2 ml/min), and oven temperature (± 2 °C). Apigenin peak areas and the %RSD of robustness testing were statistically measured.
The specificity study chance of interferences from PBS and apigenin-loaded nanoliposomes at the retention time of apigenin was analyzed by comparing the chromatograms obtained from the standard apigenin solution and PBS.
Force degradation studies
The stability-indicating property of the HPLC method which was developed was carried out as per the International Conference on Harmonization (ICH) guidelines . Forced degradation studies of apigenin were conducted by exposing the working sample to oxidation, photolytic, acidic, and alkaline and heat conditions.