2.3. HPLC Analysis of γ-Oryzanol in Rice Bran Extract

The concentration of γ-oryzanol in rice bran extracts was determined using an HPLC 1200 series instrument (Agilent Technologies, Santa Clara, CA, USA). To extract γ-oryzanol, 1 g of rice bran extract was pretreated in a Soxhlet apparatus with 250 mL of petroleum ether at 80 °C for 8 h. Following the Soxhlet extraction, the solvent was removed from the extract using rotary evaporation. Hexane was added to the Soxhlet extracts and subjected to sonication. The resulting extract was then filtered using a syringe filter for HPLC analysis. Prior to analysis, the sample was diluted with hexane at a ratio of 1:50. Detection was performed at a wavelength of 325 nm. HPLC columns (5 μm) from GL Science Inertsil SIL 100 A (Gl Sciences Inc., Torrance, CA, USA) were employed, maintained at a temperature of 25 °C. The mobile phase consisted of a mixture of hexane, iso-propyl alcohol, ethyl acetate, and acetic acid (97.6:0.8:0.8:0.8 v/v/v/v), flowing at a rate of 1.0 mL/min. A 10 μL injection volume was used. To establish a standard curve, γ-oryzanol standards ranging from 100 ppm to 1 ppm were injected. The γ-oryzanol content in the rice bran extracts was calculated using the following formula:

A = concentration of test solution;

V = volume of filtered Soxhlet extract;

DF = dilution factor;

C = amount of rice bran prepared for Soxhlet extraction.