快速溶剂萃取-超高效液相色谱法测定螺旋藻中β-胡萝卜素的含量

Determination of β-Carotene in Spirulina by ASE-UPLC

摘要:

本文采用快速溶剂萃取技术结合超高效液相色谱仪，建立了一种快速提取和测定螺旋藻中β-胡萝卜素含量的分析方法。利用单因素试验和正交试验对快速溶剂萃取法从螺旋藻中提取β-胡萝卜素的工艺条件（萃取溶剂、温度、静态萃取时间和循环次数）进行优化，结果表明最佳工艺为：石油醚（30~60 ℃）为萃取溶剂，温度120 ℃，萃取时间5 min、静态循环3次。萃取液无需净化，经稀释过滤后用Agilent Eclipse Pluse C18色谱柱（1.8 μm，2.1 mm×50 mm）分离，以甲醇:乙腈（85:15）为流动相，柱温为25 ℃，采用超高效液相色谱法，利用二极管阵列检测器于450 nm波长处检测。结果表明：β-胡萝卜素在1~50 μg/mL范围内呈良好线性关系，相关系数0.999，加标回收率为98.65~103.18%，相对标准偏差为0.68~1.74%，检测限为0.10 mg/kg。该方法简便、快速、准确、灵敏度高、重现性好，适用于螺旋藻中β-胡萝卜素的含量测定及质量评价。

Abstract:

A novel method has been developed for the rapid extraction and determination of β-Carotene in Spirulina by accelerated solvent extraction (ASE) coupled with ultra performance liquid chromatography (UPLC). The ASE parameters (extraction solvent, temperature, static time and cycles) were optimized by Single factor experiment and Orthogonal experiment design in order to maximize the extraction efficiency. The result showed that the optimum condition for extraction was as followed: petroleum ether (30~60 ℃) as solvent, extraction temperature of 120 ℃, extraction time of 5 min and static loop of three times. The ASE extract was then diluted and determined by UPLC on the Agilent Eclipse Pluse C18 column (1.8 μm, 2.1 mm×50 mm) at 25 ℃, using 85% CH3OH-15% CH3CN as mobile phase and a Diode array detector at 450 nm. The results showed that the linear ranges of β-Carotene was 1~50 μg/mL with correlation coefficient higher than 0.999. The recoveries ranged from 98.65% to 103.18% with RSDs of 0.68~1.74% and the limit of detection was 0.10 μg/kg. The method was simple, rapid and accurate, with high sensitivity and reproducibility, which was suitable for the determination of β-Carotene in Spirulina.