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  • Title: [Determination of 18 amino acids in three different kinds of milk powder by ultra performance liquid chromatography coupled with pre-column derivatization].
    Author: Qu L, Gu S, Zhang J, Zhao C, Deng X.
    Journal: Se Pu; 2021 May; 39(5):472-477. PubMed ID: 34227331.
    Abstract:
    In recent years, goat milk powder and camel milk powder have gained popularity among consumers. Due to their potential low allergenicity, these milk powders have become a substitute for breast milk, especially for infants, and for people with lactose intolerance. In this paper, a method was developed for the simultaneous determination of 18 amino acids (AAs), histidine (His), serine (Ser), arginine (Arg), glycine (Gly), aspartic acid (Asp) combined with asparagine (Asn), glutamic (Glu), glutamine (Gln), threonine (Thr), alanine (Ala), proline (Pro), lysine (Lys), tyrosine (Tyr), methionine (Met), valine (Val), isoleucine (Iso), leucine (Leu), and dimer of cysteine (Cys) combined with cysteine (L-Cys-Cys), phenylalanine (Phe), taurine (Tau) in milk, goat milk, and camel milk power. The aim of the research was to compare the three kinds of milk powder from the perspective of the constituent amino acids. Therefore, the amino acid compositions and contents were compared. Thus, 2.0 g of the sample was accurately weighed, added to 16 mL H2O, and mixed thoroughly. Then, 200 mg of the sample was weighed in a glass tube with a stream of nitrogen to displace oxygen. The samples were hydrolyzed in HCl for 24 h at 110 ℃. Then, the amino acids were pre-column derivatized by 6-aminoquinoline-n-hydroxysuccinimide carbamate (AQC). In precolumn derivatization combined with reverse-phase chromatography, both 2,4-dinitrofluorobenzene (DNFB) and phenylisothiocyanate (PITC) can react with primary amines and secondary amines. However, the derivatization time is approximately 1 h. In contrast, the derivatization time of AQC was greatly shortened. Derivatization led to the conversion of free amino acids into highly stable derivatives, which were separated by ultra performance liquid chromatography (UPLC) with UV detection at 260 nm and quantified by the external standard method. The samples were separated on a BEH C18 column (150 mm×2.1 mm, 1.7 μm) at a flow rate of 0.4 mL/min. The calibration curves showed good linearity, with correlation coefficients greater than 0.999. The limits of detection (LODs) and limits of quantification (LOQs) of the 18 amino acids were 1.3-2.5 (mg/100 g) and 3.9-7.5 (mg/100 g), respectively. Quality control samples of SRM 1849a were used as the reference material. The results were in accordance with the content range. The RSDs ranged from 2.04% to 3.65%. Furthermore, the developed method was successfully applied to determine the types and concentrations of amino acids in 11 samples purchased from local markets in Shanghai and online shops. Abundant amino acids were detected in the three types of milk powder. While all the milk powder samples contained 18 types of amino acids, Tau was not detected in some of the goat and camel milk powder samples. Total essential amino acids (TEAA) in total amino acids (TAA) of milk powder was the highest of all. The TEAA values of TAA in the goat and camel milk powders were similar. The developed method requires only 22 min for the separation of 18 amino acids. This method is suitable for the large-scale analysis of milk powder samples, and it demonstrates high sensitivity and accuracy for the determination and confirmation of the 18 amino acids in different types of milk powders. 近年来羊奶粉和骆驼奶粉备受消费者青睐,它们具有潜在的低致敏性,因此成为牛乳不耐受人群尤其是婴幼儿的母乳替代品,其营养价值备受关注。牛奶粉、羊奶粉和骆驼奶粉中氨基酸含量的比较研究鲜有报道。利用酸水解得到游离氨基酸,选择6-氨基喹啉-N-羟基琥珀酰亚胺氨基甲酸酯(AQC)进行柱前衍生,超高效液相色谱分离并检测,外标法定量。18种氨基酸在各自线性范围内线性关系良好,相关系数(r 2)大于0.999;以3倍和10倍信噪比(S/N)确定方法的检出限(LOD)和定量限(LOQ),分别为1.3~2.5 (mg/100 g)和3.9~7.5 (mg/100 g)。方法验证采用奶粉标准参考物质SRM 1849a,测定值符合其含量范围,6次测定值的相对标准偏差(RSD)为2.04%~3.65%。采用建立的方法分别对市售和网购的牛奶粉、羊奶粉和骆驼奶粉进行18种氨基酸成分和含量分析,旨在从氨基酸角度对这3种不同来源乳品进行对比。该方法快速,灵敏度高,准确可靠,适用于不同基质乳粉中18种氨基酸成分和含量的确定。
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