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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2013, Vol. 39 Issue (2): 185-190    DOI: 10.3785/j.issn.1008-9209.2012.11.611
论文     
固相萃取二维液相色谱法测定婴幼儿配方食品中的维生素B12
储小军1， 陶保华1， 赖世云1， 张京顺2， 宋亿文2， 任一平2*
1.浙江贝因美科工贸股份有限公司，杭州 310057；2.浙江省疾病预防控制中心，杭州 310054
Quantification of vitamin B12 in infant formula by dimensional liquid chromatography with solid phase extraction. Journal of Zhejiang University
CHU Xiaojun1, TAO Baohua1, LAI Shiyun1, ZHANG jingshun2, SONG Yiwen2, REN Yiping2*
1. Zhejiang Beingmate Co. Ltd., Hangzhou 310057, China; 2. Zhejiang Provincial Center for Disease Prevention and Control, Hangzhou 310054, China
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摘要： 采用固相萃取(solid phase extraction, SPE)与二维液相色谱联用技术测定婴幼儿配方食品中的维生素B12；试样经淀粉酶酶解、HLB SPE净化，以双三元液相色谱仪用双柱切换法经ZORBAX GF250柱进一步净化和浓缩样品，用ZORBAX BonusRP 柱分析，在550 nm波长处进行检测，外标法定量.结果表明，维生素B12在2~40 ng/mL范围内线性良好，其线性决定系数大于0.999，方法检出限为1.0 μg/kg，定量下限为2.5 μg/kg，在加标量为5.0、10.0和50.0 μg/kg时的回收率为88.0%~94.8%.本方法定量准确、重现性好、灵敏度高、实验成本小，为婴幼儿配方食品中维生素B12的定量测定提供了较为灵敏、实用的方法.
Abstract: Only a small amount of vitamin B12 is absorbed and utilized by human body. The suitable vitamin B12 levels can improve the utilization efficiency of folic acid. Excessive intake of vitamin B12 may cause allergic reactions, while vitamin B12 deficiency in infancy can lead to anemia. At present, most of the infants are not breastfeeding. Formula food supplemented with a suitable level of vitamin B12 is an important nutrient guarantee for healthy growth and development of infants. Thus, it is very necessary and important to develop a sensitive, efficient and accurate method for determination of vitamin B12. Currently, the commonly used methods for the determination of vitamin B12 in infant formula foods are microbial analysis methods. But they usually lead to get higher results than the real amounts because they do not distinguish vitamin B12 from its analogues. In addition, liquid chromatography technologies are also applied to analyze vitamin B12 in infant formulas. However, they have some disadvantages including long sample preparation time and high analysis costs. In the present study, a new method using solid phase extraction (SPE) coupled to twodimensional liquid chromatography with ultraviolet detector was developed for the quantification of vitamin B12 in infant formulas. The different forms of vitamin B12 were transformed to cyanocobalamin by reacting with potassium cyanide solutions after the dissolved samples were digested with amylase. The extracts of samples were purified through the HLB SPE cartridges before they were injected into a dual gradient pump series liquid chromatography system in large volume injection. The samples were separated and enriched in ZORBAX GF250 column (9.4 mm × 250 mm, 4 μm) with the mobile phase of 7.5% acetonitrile in water. Afterwards they were switched into Agilent ZORBAX BonusRP column to analyze in gradient elution using 0.4% triethanolamine in water (pH=6) and 75% acetonitrile in water (including 0.4% triethanolamine, pH=6) as the mobile phases. The contents of vitamin B12 in samples were determined at the wavelength of 550 nm using external standard method. The developed method showed a good linearity (R2>0.999) when the calibration curve ranged from 2 to 40 ng/mL. The limit of detection and the limit of quantitation were evaluated as the signaltonoise 3∶1 and 10∶1, and they were 1.0 and 2.5 μg/kg, respectively. The accuracy of the method was evaluated by employing the standard addition method. The spiked samples with 5.0, 10.0 and 50.0 μg/kg vitamin B12 standard (six portions for each spike level) were prepared and analyzed. The results showed that the spike recoveries were 88.0%94.8% with the relative standard deviation (RSD) of 2.54%4.87% at the three spiked levels. The sample pretreatment of the established method involved enzymic hydrolysis using amylase, in order to maximize the extraction of vitamin B12. The transformation of vitamin B12 from different forms to cyanocobalamin with the best stability ensured the accuracy of quantitation. Most of the impurities were removed by employing HLB SPE cartridges during the cleanup of sample extracts. Compared with the purification technology using immunoaffinity cartridges, the experimental costs were significantly reduced and the time of sample preparation was shortened. After the sample extracts were further purified and enriched through the first dimensional liquid chromatography by large volume injection, they were analyzed at 550 nm by the dual column switching method. The wavelength of 550 nm minimized the signal interferences from impurities and further improved the accuracy of results. In conclusion, the twodimensional liquid chromatography method with online column switching shows better sensitivity and reproducibility than the traditional liquid chromatography methods, and it is verified to be suitable for the determination of vitamin B12 in infant formulas.
出版日期: 2013-03-20
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储小军1
陶保华1
赖世云1
张京顺2
宋亿文2
任一平2*

引用本文:

储小军1， 陶保华1， 赖世云1， 张京顺2， 宋亿文2， 任一平2*. 固相萃取二维液相色谱法测定婴幼儿配方食品中的维生素B12[J]. 浙江大学学报(农业与生命科学版), 2013, 39(2): 185-190.

CHU Xiaojun1, TAO Baohua1, LAI Shiyun1, ZHANG jingshun2, SONG Yiwen2, REN Yiping2*. Quantification of vitamin B12 in infant formula by dimensional liquid chromatography with solid phase extraction. Journal of Zhejiang University. Journal of Zhejiang University (Agriculture and Life Sciences), 2013, 39(2): 185-190.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2012.11.611    或    http://www.zjujournals.com/agr/CN/Y2013/V39/I2/185

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