Isolation And Purification Of ?-oryzanol From Rice Bran Oil By High-speed Countercurrent Chromatography And Preparation Of Two Reference Materials

As the largest food crop in China,rice has a large planting area and high yield.At present,the research on rice is mainly focused on increasing its yield.For rice bran and other by-products are not in-depth study.Rice bran is mainly eaten as an animal by the animal and is not fully utilized.Scientific research shows that the nutritional value of rice bran is extremely high.So strengthening the comprehensive application of rice bran,studying its nutrient composition and separating can turn rice bran into treasure.As a new type of liquid-liquid partition chromatography,high-speed countercurrent chromatography has many advantages such as wide separation range and high separation purity.This paper combines high-speed countercurrent chromatography to separate and purify y-oryzanol in rice bran oil.At the same time,the reference materials play a very important role in scientific research,production and life.The development of relevant reference materials can meet the needs of the industry.Therefore,this paper has carried out research on these two aspects,the main contents are as follows:(1)The crude extraction process of rice bran oil and liquid chromatography analysis method were studied.The high-speed countercurrent chromatography combined with ionic liquid was used to separate and purify the four main components of y-oryzanol in rice bran oil(cycloartenyl ferulate,24-methylene cycloartanyl ferulate,campesteryl ferulate,sitosteryl ferulate).The effects of three different ratios of n-hexane-dichloromethane-acetonitrile solvent system on the partition coefficients of four target compounds were investigated.Based on this,the effects of different types of ionic liquids on the separation efficiency of four target materials were studied.The separation was performed with a solvent system of n-hexane-dichloromethane-acetonitrile-[CMIM][PF6]at a volume ratio of 10:1:10:0.15.As a result,the four triterpene alcohol ferulates including 10.7 mg of cycloartenyl ferulate,11.9 mg of 24-methylene cycloartanyl ferulate,1.6 mg of campesteryl ferulate and 3.1 mg of sitosteryl ferulate were successfully purified in one run from 400 mg rice bran oil with the purities of 96.98%?93.39%?94.11%and 95.47%,respectively.(2)The national secondary reference material for auramine O was developed.The auramine O was separated and purified by preparative high performance liquid chromatography using commercially available standard materials with purity of 80%as raw materials.After qualitative analysis of four major spectra(ultraviolet,infrared,mass spectrometry and nuclear magnetic spectroscopy),high performance liquid chromatography for fixed value analysis,uniformity test and stability investigation,8 laboratory settings,the final determination of the purity of auramine O is 97.02 ± 0.36%.An impurity was identified in the auramine O sample using the optimized high performance liquid chromatography-ion trap-time of flight mass spectrometry method.According to the exact mass of each fragment ion measured by multistage MS,the structure of the impurity was determined to be that of 4-(imino(4-(methylamino)phenyl)methyl)-N,N-dimethylaniline hydrochloride.(3)The reference material for p-coumaric acid was developed.The crude p-coumaric acid obtained by separating and purifying the sugarcane skin by pH-zone countercurrent chromatography was further purified by recrystallization.After qualitative analysis of four major spectra(ultraviolet,infrared,mass spectrometry and nuclear magnetic spectroscopy),high performance liquid chromatography for fixed value analysis,uniformity test and stability investigation,8 laboratory settings,the final determination of the purity of p-coumaric acid is 99.87±0.22%.At the same time,the national standard for HPLC detection of p-coumaric acid in sugarcane skin residue was established.The linear relationship of the concentration of p-coumaric acid in the concentration range of 4.0-2000 ?g/mL was well.The sampling amount of the sugarcane skin residue sample was 1 g,the detection limit was determined to be 0.33 mg/kg,and the limit of quantification was 1.09 mg/kg.The content is 13292.88 mg/kg.