Simultaneous Determination of Vitamin E and γ-Oryzanol in Rice Bran Oil via HPSEC-PDA without Sample Pretreatment

Vitamin E (tocopherols and tocotrienols) and γ-oryzanol are two minor constituents of rice bran oil (RBO) and are known to be potential bioactive compounds. The content of γ-oryzanol, a unique antioxidant found only in RBO, is a key factor in determining the retail price of the oil. Limitations of conventional HPLC columns for vitamin E and γ-oryzanol analysis are the alteration of these components and the time-consuming need for pretreatment of samples by saponification. High-performance size exclusion chromatography (HPSEC) equipped with a universal evaporative light scattering detector (ELSD) is a versatile tool for screening optimum mobile phase conditions because components of the sample can be separated and detected in the same run. In this work, the RBO components (triacylglycerol, tocopherols, tocotrienols, and γ-oryzanol) assessed on a single 100-Å Phenogel column using ethyl acetate/isooctane/acetic acid (30:70:0.1, v/v/v) as the mobile phase provided baseline separations (R _s >1.5) with a total run time of 20 min. The HPSEC condition was then transferred to determine the content of tocopherols, tocotrienols, and γ-oryzanol in RBO products using a selective PDA detector. The limit of detection (LOD) and limit of quantification (LOQ) of α-tocopherol, α-tocotrienol, and γ-oryzanol were 0.34 and 1.03 μg/mL, 0.26 and 0.79 μg/mL and 2.04 and 6.17 μg/mL, respectively. This method was precise and accurate, with a percentage of relative standard deviation (%RSD) of the retention time of less than 0.21%. The intra-day and inter-day variations were 0.15-5.05% and 0.98-4.29% for vitamin E and γ-oryzanol, respectively. The recoveries of tocopherols, tocotrienols, and γ-oryzanol ranged between 90.75% and 107.98%. Thus, the developed HPSEC-ELSD-PDA method is a powerful analytical tool for determining the vitamin E and γ-oryzanol present in oil samples without requiring any sample pretreatment.