叶黄素、β-隐黄质及其异构体检测方法的研究

Separation and Identification of Lutein, β-Cryptoxanthin and Their Isomers

摘要:

应用C30-HPLC-DAD技术，建立一种同时分离全反式叶黄素、全反式β-隐黄质及其异构体的检测方法。采用APCI-MS和光谱技术对分离出化合物进行定性分析。最佳色谱条件：1.5%乙酸铵水-甲基叔丁基醚（MTBE）-甲醇（5:25:70，V/V/V）为流动相A，1.5 %乙酸铵水-甲基叔丁基醚（MTBE）-甲醇（5:85:10，V/V/V）为流动相B，线性梯度洗脱；流速：0.6 mL/min；洗脱时间：24 min；进样量：20 μL；柱温：25 ℃。混合物中的全反式叶黄素及其15-顺式、13/13’-顺式、9-顺式、9’-顺式异构体，全反式β-隐黄质及其15-顺式、13-和13’-顺式、9-顺式、9’-顺式异构体均得到较好分离。全反式叶黄素、全反式β-隐黄质分别在2~150 ng和5~250 ng范围内峰面积与进样量呈良好线性关系。该方法能够快速、准确地同时分离全反式叶黄素、全反式β-隐黄质及它们的异构体，可用于果蔬中全反式叶黄素和全反式β-隐黄质及其异构体混合物的定性定量分析。

Abstract:

A method to simultaneously detect all-trans-Lutein, all-trans-β-cryptoxanthin and their stereoisomers was developed using C30-column, high-performance liquid chromatography, diode array (C30-HPLC-DAD) technique. The structures of isolated compounds were elucidated by atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) and ultraviolet/visible (UV/Vis) spectroscopy. The optimal chromatographic condition was as follows: mobile phase A: 1.5% ammonium acetate:water:methyl tert-butyl ether (MTBE)-MeOH (5:25:70, V/V/V), mobile phase B: 1.5% ammonium acetate:water:MTBE-MeOH (5:85:10, V/V/V); flow rate: 0.6 mL/min; elution time: 24 min; injection volume: 20 μL; column temperature: 25 ℃. Using this novel method, 15-cis-lutein, 13/13′-cis-lutein, 9-cis-lutein, 9′-cis-lutein, 15-cis-β-cryptoxanthin, 13- and 13′-cis-β-cryptoxanthin, 9-cis-β-cryptoxanthin and 9′-cis-β-cryptoxanthin were successfully separated from the mixture. The peak areas of all-trans lutein and all-trans β-cryptoxanthin showed a good linear relationship with their injection volumes in the range of 2 to 150 ng and 5 to 250 ng, respectively. The proposed method accurately and rapidly separated all-trans lutein, all-trans β-cryptoxanthin, and their cis-isomers simultaneously. This method has potential applications in qualitative and quantitative analysis of all-trans lutein, all-trans β-cryptoxanthin, and their stereoisomers in fruits and vegetables.