食品科学 ›› 2021, Vol. 42 ›› Issue (20): 216-221.doi: 10.7506/spkx1002-6630-20200616-209

• 成分分析 • 上一篇    下一篇

高效液相色谱法测定母乳及牛乳中总核苷酸含量

杨璐桐,于苗,郭志恒,田芳,毛颖异,蔡小堃,Philip HASELBERGER,赵艳荣,谢林   

  1. (1.吉林大学公共卫生学院营养与食品卫生学教研室,吉林 长春 130021;2.吉林大学第一医院产科,吉林 长春 130021;3.雅培营养中国研发中心,上海 200233;4.雅培营养品,美国 俄亥俄 哥伦布 OH43219)
  • 出版日期:2021-10-25 发布日期:2021-11-12
  • 基金资助:
    雅培贸易(上海)有限公司资助横向课题(2019YX078)

Determination of Total Nucleotide Contents in Breast Milk and Cow Milk by High Performance Liquid Chromatography

YANG Lutong, YU Miao, GUO Zhiheng, TIAN Fang, MAO Yingyi, CAI Xiaokun, Philip HASELBERGER, ZHAO Yanrong, XIE Lin   

  1. (1. Department of Nutrition and Food Hygiene, School of Public Health, Jilin University, Changchun 130021, China; 2. Department of Obstetrics, First Hospital, Jilin University, Changchun 130021, China; 3. Abbott Nutrition R&D, Shanghai 200233, China; 4. Abbott Nutrition, Columbus OH43219, USA)
  • Online:2021-10-25 Published:2021-11-12

摘要: 建立高效液相色谱测定母乳和牛乳中总核苷酸含量和组成的分析方法。总核苷酸包括游离核苷酸、游离核苷、核苷聚合物及加合物等不同来源的可利用核苷总量。利用酶解法释放出母乳中的核苷,以硼化聚丙烯酰胺凝胶(Affi-Gel 601)作为固相萃取介质进行样品净化,用反相高效液相色谱仪检测,内标法定量,检测结果以核苷酸计。且针对母乳样品的特性,增加高温灭活步骤进行样品前处理,以检测母乳中核苷酸的天然含量及组成。对所建方法进行全面验证,结果表明其具有良好的线性,相关系数(r2)均在0.999以上;母乳中各核苷酸的检出限和定量限分别在0.18~0.45 mg/L和0.60~1.51 mg/L之间;母乳基质中3 种不同加标水平下总核苷酸的回收率均在99%~108%之间,连续3 d 6 次检测结果的相对标准偏差在0.8%~7.8%之间,显示了良好的准确性及精密度。然后进一步利用所建检测方法,评估母乳及5 种市面常见的牛乳中的核苷酸总量及组成用以验证该方法的适用性。该方法的灵敏度、准确性及精密度均可满足对母乳和牛乳样品中天然的核苷酸含量及组成的测定要求。本研究为今后大样本量调查母乳及牛乳样品中核苷酸的含量和组成提供了更加科学准确的方法。

关键词: 总核苷酸;含量和组成;高效液相色谱法;母乳;牛乳

Abstract: A method for the determination of the contents and composition of total nucleotides in breast milk and cow milk by high performance liquid chromatography (HPLC) was established. The total nucleotides detected included free nucleotides, free nucleosides, nucleoside polymers and nucleoside adducts. The nucleosides in breast milk were released by enzymatic hydrolysis, purified by polyacrylamide gel (Affi-Gel 601) as a solid phase extraction medium, detected by reverse-phase-high performance liquid chromatography (RP-HPLC) and quantified by an internal standard method. The results were calculated as nucleotides. According to the characteristics of breast milk, a high-temperature inactivation step was introduced into sample pretreatment before the detection of nucleotides in breast milk. The proposed method was systematically validated. The results showed that this method had good linearity with a correlation coefficient (r2) higher than 0.999. The limit of detection (LOD) and the limit of quantification (LOQ) for all nucleotides in breast milk were 0.18–0.45 and 0.60–1.51 mg/L, respectively. The recoveries of total nucleotides in breast milk samples spiked at three concentration levels ranged from 99% to 108%, with relative standard deviations (RSDs) 0.8%–7.8% for six replicate determinations in three consecutive days, suggesting good accuracy and precision. The HPLC method was used to evaluate the composition and contents of total nucleotides in breast milk and five types of commercial cow milk in order to verify its applicability. The sensitivity, accuracy, and precision of this method can meet the requirements for the determination of natural nucleotides in breast milk and cow milk. This study should provide a more scientific and accurate method for the determination of nucleotides in massive samples of breast milk and cow milk in the future.

Key words: total nucleotides; content and composition; high performance liquid chromatography; breast milk; cow milk

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