微藻功能脂质的超临界流体色谱-蒸发光散射检测方法的建立

Methodological Development of Supercritical Fluid Chromatography Coupled with Evaporative Light Scattering Detector for Determining Microalgal Functional Lipids

摘要:

微藻脂质成分复杂且极性差异大，目前缺乏一种既不依赖质谱又能同时测定其多种功能脂质的方法。为了解决该难题，该研究建立了一种绿色环保的超临界流体色谱-蒸发光散射检测法。采用Diol色谱柱，以超临界二氧化碳-甲醇为流动相，进行梯度洗脱，流量0.5 mL·min-1，ELSD检测器检测，外标法定量。结果表明，该方法可在35 min内实现8种功能脂质的分离测定。相比传统液相方法有机试剂约需40 mL，该方法仅需12.28 mL。在0.01~1.0 mg·mL-1线性关系良好（R2＞0.9902），检出限≤0.74 μg·mL-1，精密度≤1.19%，加标回收率92.12%~105.78%。六种微藻的功能脂质含量存在较为明显的差异。其中，小球藻和拟微球藻的甘油酯（分别约53.08、57.91 mg·g-1）、甘油糖脂（分别约39.90、32.76 mg·g-1）和磷脂含量（分别约19.40、29.54 mg·g-1）均较其它微藻高，有望作为甘油糖脂及磷脂的膳食补充来源。该方法灵敏、准确、绿色，可在一次进样中实现低极性至高极性脂质的分离测定。该方法能满足未来对微藻样本的快速精准脂质分析需求适用于各种含特定脂质成分的食品基质分析。

Abstract:

Due to the complexity of the lipid composition of microalgae and the large difference in polarity, there is a lack of a method to simultaneously determine its multiple functional lipids without relying on mass spectrometry. To tackle this challenge, a green and eco-friendly supercritical fluid chromatography-evaporative light scattering detection method had been developed. The separation was conducted using a Diol column through gradient elution with a mobile phase consisting of supercritical carbon dioxide, methanol. The flow rate was 0.5 mL·min-1. Detection was performed using an ELSD, and the compounds were quantified using an external standard method. The eight functional lipids could be separated and determined within 35 minutes. Compared with traditional liquid phase methods, which require approximately 40 mL of organic reagents, this method requires only 12.28 mL. The method exhibited a good linear range (0.01~1.0 mg·mL-1) with a correlation coefficient (R2) greater than 0.9902. The limits of detection were less than 0.74 μg·mL-1. The relative standard deviation of all the standards was less than 1.19%, and the spiked recovery rates ranged from 92.12% to 105.78%. There were more significant differences in the content of functional lipids in different microalgae. Among them, Chlorella vulgaris and Nannochloropsis sp. had the higher contents of glycerides (approximately 53.08 and 57.91 mg·g-1, respectively), glycerolipidsand (approximately 39.90 and 32.76 mg·g-1, respectively), phospholipids (approximately 19.40 and 29.54 mg·g-1, respectively) than the other microalgae. They were expected to be used as dietary supplement sources of glycerolipids and phosphatidylinositol. The established method is highly sensitive, accurate and environmentally friendly. It enables the separation and quantification of lipids spanning a wide polarity range, from low-polarity to high-polarity, within a single injection. The method fulfills the need for rapid and precise lipid analysis of microalgae samples, and is applicable for lipid profiling in diverse food matrices containing specific lipid components.