混合型固相萃取-高效液相色谱-串联质谱法测定稻米中3种谷维素北大核心CSCD

建立了利用高效液相色谱-串联质谱结合混合型阴离子交换固相萃取柱测定稻米中3种谷维素(环木菠萝烯醇阿魏酸酯(CA-FA)、24-亚甲基环木菠萝烯醇阿魏酸酯(24MCA-FA)和菜油甾醇阿魏酸酯(Camp-FA))含量的分析方法。实验通过优化3种谷维素的多反应监测(MRM)质谱参数,比较了在不同流动相中的分离度以及响应强度,同时考察了不同提取条件、净化条件对3种谷维素提取率和净化效果的影响,再结合外标法定量,实现了对稻米中3种谷维素的定量分析。实验结果表明,采用5 mmol/L乙酸铵水溶液为流动相A,甲醇∶乙腈=1∶1(v/v)为流动相B,梯度洗脱,3种谷维素在Agilent Eclipse XDB-C8色谱柱(150 mm×2.1 mm,3.5μm)上基本分离且响应强度和峰形最佳;提取条件经正交实验优化后可得:料液比为1∶20(g/mL)、提取溶剂为甲醇、浸泡时间为12 h、超声温度为40℃以及超声时间为20 min时,3种谷维素提取率最高;对混合型阴离子交换固相萃取柱的上样溶剂以及洗脱溶剂优化后,样品基质效应为1.6%~10.8%。在各优化条件下,3种谷维素在各自的线性范围内线性良好,相关系数(r2)均≥0.9983,检出限(LOD)为0.5~1.0μg/L,定量限(LOQ)为2.0~3.5μg/L。在稻米样品本底浓度2、5和10倍的加标水平下3种谷维素的平均回收率为86.1%~110.6%,相对标准偏差(RSD)为0.9%~3.2%。该方法可快速准确测定稻米中3种谷维素的含量,为后续稻米中谷维素类化合物测定及鉴定奠定基础。

Determination of three oryzanols in rice by mixed-mode solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry

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.