高效液相色谱检测红球藻中虾青素的技术研究

Technical Study on the Detection of Astaxanthin in Haematococcus by High Performance Liquid Chromatography

摘要: 优化高效液相色谱法检测红球藻中虾青素的条件和方法。使用乙醇–甲醇(1:3)提取红球藻样品，经0.1 mol/L氢氧化钠–甲醇皂化后，用0.2 mL 2%磷酸–甲醇中和剩余碱后，采用高效液相色谱法测定。总虾青素在0.1~10 μg/mL范围内线性良好(R² ≥ 0.999)，方法定量限10 mg/kg。在添加水平为10 mg/kg和50 mg/kg时，回收率为91%~100%，相对标准偏差低于1.0%。该方法较GB/T 31520-2015中使用甲醇–叔丁基甲醚–磷酸为流动相，二氯甲烷–甲醇(1:3)作为提取液，经0.1 mol/L氢氧化钠–甲醇皂化后，用0.4 mL 2%磷酸–甲醇中和剩余碱后，结果更准确、稳定、灵敏，且更为环保，能够满足红球藻中总虾青素检测与确证需要。

Abstract: To optimize the conditions and methods for the determination of astaxanthin in Haematococcus by high-performance liquid chromatography (HPLC). The Haematococcus samples were extracted using ethanol-methanol (1:3), saponified by 0.1 mol/L sodium hydroxide-methanol, and then determined by high-performance liquid chromatography (HPLC) after neutralization of the remaining alkali with 0.2 mL 2% phosphoric acid-methanol. The linearity of total astaxanthin was good in the range of 0.1~10 μg/mL (R2 ≥ 0.999), and the limit of quantification of the method was 10 mg/kg. The recoveries ranged from 91% to 100% at the spiked levels of 10 mg/kg and 50 mg/kg with the relative standard deviations (RSDs) lower than 1.0%. Compared with GB/T 31520-2015, the method used methanol-tert-butyl-methyl ether-phosphoric acid as mobile phase, dichloromethane-methanol (1:3) as extraction liquid, was saponified by 0.1mol/L sodium hydroxide-methanol, and neutralized by 0.4mL 2% phosphoric acid-methanol, the results were more accurate, stable, sensitive, and environmentally friendly. It can meet the need for the detection and confirmation of total astaxanthin in Haematococcus.