Abstract：
Rice is a major dietary staple in many communities owing to its high nutritional value and characteristic aroma. Oryzanol, a mixture of ferulic acid esters of triterpene alcohols and phytosterols, is a major group of phytochemicals found in rice. 24-Methylenecycloartanyl ferulate (24MCA-FA), cycloartenyl ferulate (CA-FA), and campestanyl ferulate (Camp-FA) have been identified as the primary components of oryzanol. At present, for the quantification of oryzanol in rice and rice products, UV spectroscopy or high performance liquid chromatography (HPLC) is widely employed. However, these methods cannot differentiate individual oryzanols, resulting in higher measured values. To extract oryzanol, methods including liquid-liquid extraction, acidulation extraction, and direct solvent extraction have been typically employed, as they do not require specific extraction instrumentation. However, there has been no systematic study on the direct solvent extraction and purification conditions of oryzanol in rice. In this study, a rapid and accurate analytical method based on HPLC-MS/MS and mixed-mode anion exchange (MAX) solid-phase extraction was established to determine the content of three oryzanols (24MCA-FA, CA-FA, and Camp-FA) in rice. The MS parameters, such as the collision energy of three ion pairs of each oryzanol, were optimized. Further, the chromatographic separation conditions and response intensities of the oryzanols in different mobile phases were compared. The effects of different pretreatment conditions on the extraction efficiency of the three oryzanols in rice samples and different purification conditions on their recovery were investigated. Combined with the external standard method, the three oryzanols in rice were successfully quantified. The results showed that the baseline separation and highest response for the three oryzanols were achieved using the Agilent Eclipse XDB-C8 chromatographic column (150 mm×2.1 mm, 3.5 μm) when methanol∶ acetonitrile in a 1∶1 ratio (v/v) and an aqueous solution of 5 mmol/L ammonium acetate were used as the mobile phases for gradient elution. The extraction rate of the three oryzanols was highest when using 2.5 g of the sample, adding 20 mL of methanol, soaking for 12 h, ultrasonicating at a temperature of 40 ℃ for 20 min, and centrifuging the extracted solutions at 4500 r/min for 10 min. The samples were purified by MAX, and the sample matrix effect was found to be lesser than 1.6%-10.8%. Under the optimum conditions, the calibration curves of the three oryzanols showed good linearity (correlation coefficients r2≥0.9983) within their respective linear ranges. The limits of detection were in the range of 0.5-1.0 μg/L, and limits of quantification were in the range of 2.0-3.5 μg/L. Accuracy and precision experiments were performed on rice samples spiked at three levels (2, 5, and 10 times the background concentration), with three replicates. The average recoveries of the three oryzanols ranged from 86.1% to 110.6%, and the relative standard deviations (RSDs) were between 0.9% and 3.2%. The method showed good performance when applied to the analysis of real samples. In conclusion, the developed method can determine the content of the three oryzanols in rice quickly and accurately, and can be used for the subsequent measurement of oryzanol compounds in rice.
摘要：
大米具有很高的营养价值和独特的香气，是许多社区的主要饮食主食。谷维素,三萜醇和植物甾醇的阿魏酸酯的混合物，是大米中发现的主要植物化学物质。24-亚甲基环烷醇阿魏酸酯（24MCA-FA），阿魏酸环烯酯(CA-FA)，阿魏酸菜烷酯（Camp-FA）已被确定为谷维素的主要成分。目前,用于定量大米和大米产品中的谷维素，UV光谱法或高效液相色谱法(HPLC)被广泛使用。然而,这些方法不能区分单个谷维醇，导致更高的测量值。为了提取谷维素，方法包括液-液萃取，酸化提取，通常采用直接溶剂萃取，因为他们不需要特定的提取仪器。然而,大米中谷维素的直接溶剂萃取和纯化条件尚未系统研究。在这项研究中,建立了基于HPLC-MS/MS和混合模式阴离子交换（MAX）固相萃取的快速准确的分析方法，用于测定三种谷维醇（24MCA-FA，CA-FA,和Camp-FA）在大米中。MS参数，例如每个谷维素的三个离子对的碰撞能量，进行了优化。Further,比较了谷维醇在不同流动相中的色谱分离条件和响应强度。研究了不同预处理条件对大米样品中3种谷维醇的提取效率和不同纯化条件对其回收率的影响。结合外标法，成功定量了水稻中的三种谷维醇。结果表明，使用AgilentEclipseXDB-C8色谱柱（150mm×2.1mm，3.5μm），当使用1∶1比例（v/v）的甲醇∶乙腈和5mmol/L乙酸铵水溶液作为梯度洗脱的流动相。当使用2.5g样品时，三种谷维醇的提取率最高，加入20毫升甲醇，浸泡12小时，40℃超声20min,并以4500r/min的速度将提取的溶液离心10min。样品经MAX纯化,样品基体效应小于1.6%-10.8%。在最佳条件下,三种谷氮醇的校准曲线在各自的线性范围内显示出良好的线性（相关系数r2≥0.9983）。检出限范围为0.5-1.0μg/L,定量限范围为2.0-3.5μg/L。对三个水平（背景浓度的2、5和10倍）加标的大米样品进行了准确性和精密度实验，有三个重复。三种谷氮醇的平均回收率为86.1%~110.6%,相对标准偏差（RSD）在0.9％至3.2％之间。该方法应用于实际样品分析时表现出良好的性能。总之,该方法可以快速准确地测定大米中三种谷维醇的含量，并可用于大米中谷维素类化合物的后续测定。
