高效液相色谱-柱后衍生法测定特殊医学用途配方食品中生物素含量

Determination of biotin in formula food for special medical purposes by high performance liquid chromatography-post column derivatization

中文关键词:  柱后衍生  高效液相色谱法  特殊医学用途配方食品  生物素  异硫氰酸荧光素标记亲和素。

英文关键词:postcolumn derivatization  high performance liquid chromatography  formula food for special medical purposes  biotin  fluorescein isothiocyanate labeled avidin

中文摘要:

      目的 建立高效液相色谱-柱后衍生法测定特殊医学用途配方食品中生物素含量的检测方法。方法 样品加水溶解并经淀粉酶和木瓜蛋白酶在60 ℃水浴酶解1 h进行提取, 采用Zorbax SB-AQ色谱柱分离并进入柱后反应装置, 与异硫氰酸荧光素标记亲和素衍生; 衍生物采用荧光检测器检测, 激发波长为495 nm, 发射波长为525 nm, 外标法定量。结果 在优化条件下, 生物素在5.00~75.00 ng/mL线性范围内线性关系良好(r2>0.999), 平均回收率为97.5%~100.1%, 相对标准偏差为0.86%~2.96%, 检出限为12 μg/kg, 定量限为41 μg/kg。生物素标准溶液和样品待测液在24 h内的相对标准偏差分别为0.68%和1.08%, 实际样品测定中, 本方法与GB 5009.256—2016《食品安全国家标准 食品中生物素的测定》测定结果无显著性差异。结论 本方法前处理简单, 回收率高, 灵敏度和精密度好, 可用于特殊医学用途配方食品中生物素含量的测定。

英文摘要:

      Objective To establish a method for the determination of biotin in formula food for special medical purposes by high performance liquid chromatography-post column derivatization. Methods The biotin in the sample was dissolved in water and extracted by enzymatic hydrolysis with amylase and papain at 60 ℃ for 1 hour in a water bath. Using Zorbax SB-AQ chromatographic column separation and entering the post column reaction device, the biotin was derived from fluorescein isothiocyanate labeled avidin. Derivatives were detected using a fluorescence detector with an excitation wavelength of 495 nm and an emission wavelength of 525 nm. The results were quantified by the external standard method. Results Under the optimized conditions, the biotin showed good (r2>0.999) linear relationships within the concentration range of 5.00?75.00 ng/mL. The average recoveries were 97.5%?100.1%, and the relative standard deviations were 0.86%?2.9%. The limit of detection was 12 μg/kg, and the limit of quantification was 41 μg/kg. The relative standard deviations of the biotin standard solution and the sample solution to be tested within 24 h were 0.68% and 1.08%, respectively. There was no significant difference between the results of this method and GB 5009.256—2016 National food safety standard-Determination of biotin in food. Conclusion This method has simple pretreatment, high recovery, good sensitivity and precision, and can be used for the determination of biotin content in formula food for special medical purposes.