作者：范志颖1,2, 王奇2, 吴益淳2, 罗敏2, 肖伟敏2, 包跃超1, 蓝雄2, 陈佳平2, 杨国武2, 杨燕3
作者单位：1. 内蒙古科技大学包头医学院, 内蒙古 包头 014040;
2. 深圳市计量质量检测研究院, 广东 深圳 518109;
3. 中山大学深圳校区公共卫生学院(深圳), 广东 深圳 518107
关键词：天冬酰胺;谷氨酰胺;特殊医学用途配方食品;丹磺酰氯;灰色链霉菌蛋白酶
摘要：
建立酶水解-丹磺酰氯柱前衍生高效液相色谱法检测特殊医学用途配方食品（FSMP）中天冬酰胺（Asn）和谷氨酰胺（Gln）的方法。样品经灰色链霉菌蛋白酶（Pronase E）酶解后，取上清液与Dns-Cl进行柱前衍生，衍生化产物经Agilent Zorbax SB-C18色谱柱（250 mm×4. 6 mm，5 μm）分离，以10 mmol/L磷酸氢二钠溶液(pH 6.5)和甲醇作为流动相进行梯度洗脱，外标法定量。结果表明，两种氨基酸在0.500~50.0 μg/mL浓度范围内线性良好（R≥0.999），回收率为95.3%~105%，RSD为0.95%~3.22%。方法用2种蛋白标准品进行酶切效率与实测含量验证，准确度良好（δ＜10%），可用于FSMP中Asn和Gln含量检测，并正在用于制定食品安全国家标准。
Enzymatic hydrolysis pre-column derivatisation UPLC method for the detection of asparagine and glutamine in FSMP
FAN Zhiying, WANG Qi, WU Yichun, LUO Min, XIAO Weimin, BAO Yuechao, LAN Xiong, CHEN Jiaping, YANG Guowu, YANG Yan
1. Baotou Medical College, Inner Mongolia University of Science and Technology, Baotou 014040, China;
2. Shenzhen Academy of Metrology & Quality Inspection, Shenzhen 518109, China;
3. School of Public Health (Shenzhen), Sun Yat-sen University, Shenzhen 518107, China
Abstract: A high performance liquid chromatography method was established for the detection of asparagine (Asn) and glutamine (Gln) in special medical purpose food (FSMP) using enzyme hydrolysis-dansyl chloride pre-column derivatization. After the sample was enzymatically hydrolyzed by streptomyces griseus protease (Pronase E), the supernatant was collected and subjected to pre-column derivatization with Dns-Cl. The derivatized product was applied to an Agilent Zorbax SB-C18 chromatography column (250 mm × 4.6 mm，5 μm).The separation was performed using a 10 mM solution of disodium hydrogen phosphate (pH 6.5) and methanol as the mobile phase for gradient elution and quantification using an external standard method. The results indicate that the two amino acids are present at a concentration of 0.500-50.0μg/mL. The linearity is good within the concentration range of g/mL (R≥0.999), the recovery rate is 95.3%-105% and the RSD is 0.95%-3.22%. The method was validated for enzymatic digestion efficiency and measured content using two protein standards, with good accuracy (δ<10%).The method fills the gap in detection methods for Asn and Gln in FSMP, can be used to detect Asn and Gln content in FSMP, and is currently being used to develop national food safety standards.
Keywords: asparagine;glutamine;food for special medical purposes;dansulfonyl chloride;streptomyces griseus protease
2023, 49(11):127-131 收稿日期: 2023-04-29;收到修改稿日期: 2023-06-25
基金项目: 国家重点研发计划科研项目(2021YFF0701900，2021YFF0701903）
作者简介: 范志颖(1996-),女,内蒙古自治区通辽市人,研究生,专业方向为公共卫生。
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