Characterization of Palm Fatty Acid Distillate, Diacylglycerol Regioisomers, and Esterification Products Using High-Performance Size Exclusion Chromatography.

High-performance size exclusion chromatography (HPSEC) equipped with an evaporative light scattering detector (ELSD) was utilized for characterization of palm fatty acid distillate (PFAD) and its esterified products, with a particular focus on lipid profiles and diacylglycerol (DAG) regioisomers. The separation of triacylglycerol (TAG), DAG, monoacylglycerol (MAG), and free fatty acid (FFA) was achieved through a single 100-Å Phenogel column, coupled with a 2-cm C18 guard, utilizing toluene/acetic acid (100:0.25, v/v) as the mobile phase. This separation was based on size sieving principles and the interactions between the hydroxyl group(s) and the Phenogel matrix. The limit of detection (LOD) and limit of quantification (LOQ) for the esterified PFAD products analyzed by this method fell within the range of 4.8-5.5 µg/mL and 14.7-16.7 µg/mL, respectively. Additionally, the same column, paired with a 2-cm silica guard and a mobile phase comprised of toluene/isooctane/acetic acid (35:65:0.15, v/v/v), was used for the characterization of DAG regioisomers within the esterified PFAD. LODs and LOQs for sn-1,3-DAG and sn- 1,2-DAG were determined to be 39.2 and 118.7 µg/mL, and 32.8 and 99.5 µg/mL, respectively. Investigation of esterified PFAD products prepared using 4% H2SO4 at 120°C. After 2 h, the analysis revealed the highest MAG content at 31.85%, accompanied by 51.54% DAG, 2.35% TAG, and a residual 14.27% FFA. Notably, as the reaction time extended, the MAG content decreased, while both DAG and TAG levels exhibited an increasing trend. Further examination of DAG regioisomers during PFAD esterification, under varying catalyst concentrations (2-10%) and reaction temperatures (80-140°C), demonstrated a significant increase in the percentage of sn-1,3-DAG, inversely correlated with the reduction in FFA from 2% H 2 SO 4 and 80°C onwards. Remarkably, the percentage of sn-1,2-DAG remained relatively stable regardless of changes in catalyst concentrations or temperatures, confirming its susceptibility to isomerization into the thermodynamically more stable sn-1,3-DAG form. This study provides valuable insights into the composition and behavior of esterified PFAD products.

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.

A Simple and Efficient Method for Synthesis and Extraction of Ethyl Ferulate from γ-Oryzanol.

Ethyl ferulate (EF) is a ferulic acid (FA) derivative with high commercial value. It is not found naturally and is mostly synthesized from FA via esterification with ethanol. The present work aimed to synthesize the EF from γ-oryzanol, a natural antioxidant from rice bran oil via acid-catalyzed transethylation at refluxing temperature of ethanol. The reaction was optimized by central composite design (CCD) under response surface methodology. Based on the CCD, the optimum condition for the synthesis of EF from 0.50 g of γ-oryzanol was as follows: γ-oryzanol to ethanol ratio of 0.50:2 (g/mL), 12.30% (v/v) H2SO4, and a reaction time of 9.37 h; these conditions correspond to a maximum EF yield of 87.11%. Moreover, the optimized transethylation condition was further validated using 12.50 g of γ-oryzanol. At the end of the reaction time, distilled water was added as antisolvent to selectively crystallize the co-products, phytosterol and unreacted γ-oryzanol, by adjusting the ethanol concentration to 49.95% (v/v). The recovery yield of 83.60% with a purity of 98% of EF was achieved. In addition, the DPPH and ABTS assays showed similar antioxidant activities between the prepared and commercial EF.

Recovery of γ-Oryzanol from Rice Bran Acid Oil by an Acid-base Extraction Method with the Assistance of Response Surface Methodology.

A rapid and low energy consumption method for the recovery of γ-oryzanol from rice bran acid oil (RBAO), a byproduct of rice bran oil (RBO) refining, is presented. The RBAO was converted to the fatty acid ethyl ester (FAEE) and was used as the starting material. The dissolved γ-oryzanol was separated from the FAEE using an acid-base extraction method with alkaline aqueous ethanol and hexane as extraction media. A systematic investigation of the extraction yield was carried out by applying response surface methodology (RSM) based on central composite design (CCD) and Derringer's desirability function. The concentration of NaOH, the percentage of ethanol in water, the hexane content and their interactions showed significant effects on the yield of γ-oryzanol and FAEE. The optimal extraction conditions were as follows: extraction time of 1 min at room temperature (28-32°C); extraction medium: 1.855 M NaOH; 75.91% ethanol in water and 20.59% hexane in the total volume of the extractant; and FAEE to extractant ratio of 1:10 corresponding to a maximum γ-oryzanol yield of 75.82±3.44% and the desired FAEE yield of 54.42±7.80%. The γ-oryzanol-rich fraction was further purified by washing with a 2% Na2CO3 solution, obtaining 69.94% recovery yield with 89.90% purity of γ-oryzanol. The purified γ-oryzanol showed good scavenging activity on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and the ABTS radical and was comparable to the commercial product, clearly suggesting that the presented process was efficient and feasible.