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    微波辅助酶萃取-高效液相色谱-电感耦合等离子体质谱法测定灵芝中6种硒形态

    Determination of Six Selenium Species in Ganoderma Lucidum by HPLC-ICP-MS After Microwave-Assisted Extraction with Enzyme

    • 摘要: 在0.5 g灵芝样品中加入10 mL水和20 mg蛋白酶XIV,在300 W微波功率下37℃反应30 min,以萃取出样品中的6种硒形态,包括硒酸Se (Ⅵ)、亚硒酸Se (Ⅳ)、硒代蛋氨酸(SeMet)、硒代胱氨酸(SeCys2)、硒代乙硫氨酸(SeEt)、甲基硒代半胱氨酸(SeMeCys),萃取液经离心10 min后,过0.22 μm滤膜,滤液供高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)分析硒形态。以安捷伦ZORBAX SB-Aq C18色谱柱为固定相,以含5 mmol·L-1庚烷磺酸钠的20 mmol·L-1柠檬酸溶液(pH 4.5)为流动相进行等度洗脱,在优化的仪器条件下,6种硒形态的保留时间分别为3.21,4.06,6.41,4.38,7.38,6.05 min。在0.2 g灵芝样品中加入3 mL市售过氧化氢溶液和5 mL硝酸,置于1 200 W微波功率下160℃消解30 min,用5%(体积分数)盐酸溶液将消解液定容至25.0 mL,供电感耦合等离子体质谱(ICP-MS)分析硒总量,检测同位素为82Se。结果表明:6种硒形态的质量浓度在0.5~100.0 μg·L-1内与其对应的峰面积呈线性关系,检出限(3S/N)为0.03~0.15 μg·kg-1;以灵芝样品为基质进行3个浓度水平的加标回收试验,回收率为91.7%~98.7%,测定值的相对标准偏差(n=6)为0.031%~3.2%。采用本方法对市售赤芝、紫芝、无柄紫芝、富硒赤芝等进行了硒形态分析,发现富硒赤芝中硒含量最高且种类最齐全。

       

      Abstract: 0.5 mL of Ganoderma lucidum sample was mixed with 10 mL of water and 20 mg of protease XIV, and 6 selenium species in the sample, including selenic acidSe(Ⅵ), seleniteSe(Ⅳ), selenomethionine (SeMet), selenocystine (SeCys2), selenoethionine (SeEt) and methylselenose semi Cystine (SeMeCys), were extracted at 37℃ and the microwave power of 300 W for 30 min. After the extract was centrifuged for 10 min, the supernatant was passed through a 0.22 μm filtering membrane and the filtrate was analyzed by HPLC-ICP-MS. The Agilent ZORBAX SB-Aq C18 chromatographic column was selected as the stationary phase, and the 20 mmol·L-1 citric acid solution (pH 4.5) containing 5 mmol·L-1 sodium heptane sulfonate was used as the mobile phase for isocratic elution. Under optimized instrument conditions, retention times of the six selenium species were found to be 3.21, 4.06, 6.41, 4.38, 7.38, 6.05 min, respectively. 3 mL of commercially available hydrogen peroxide solution and 5 mL of nitric acid were mixed with 0.2 g of Ganoderma lucidum sample,and the mixture solution was digested under 1 200 W microwave power at 160℃ for 30 min. The volume of digestion solution was made up to 25.0 mL with 5% (volume fraction) hydrochloric acid solution, and then the total selenium in the above solution was determined by ICP-MS with the detected isotope of 82Se. The results showed that the mass concentrations of the 6 selenium species were linearly related to their corresponding peak areas in the range of 0.5-100.0 μg·L-1, with detection limits (3S/N) in the range of 0.03-0.15 μg·kg-1. The recovery test was made by addition of standard solutions to the Ganoderma samples at three concentration levels, giving recoveries ranged from 91.7% to 98.7%, with RSDs (n=6) of the measured values in the range of 0.031% to 3.2%. The prescribed method was used to analyze the selenium species of commercially available Ganoderma lucidum, Ganoderma sinense, stalkless Ganoderma sinense and selenium-enriched Ganoderma lucidum, and the highest selenium content and the most complete species were found in the selenium-enriched Ganoderma lucidum of the 4 samples.

       

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