5 7 Vol. 5 No. 7 2014 7 Journal of Food Safety and Quality Jul., 2014 李 爽 1, 陈启 1, 蔡明明 1, 任一平 2* (1., 310057; 2., 310051) 摘要 : 目的 方法, C 18+ AQC, 结果, 4.65% 结论, 关键词 : ; ; ; Comparative study on quantification of hydrolysis amino acids in human milk using liquid chromatography and amino acid analyzer LI Shuang 1, CHEN Qi 1, CAI Ming-Ming 1, REN Yi-Ping 2* (1. Beingmate Baby & Child Food Co., Ltd, Hangzhou 310057, China; 2. Zhejiang Provincial Center for Disease Prevention and Control, Hangzhou 310051, China) ABSTRACT: Objective Comparison the methods of amino acid analyzer (AAA) and ultra-performance liquid chromatography (UPLC) for determination of hydrolysis amino acids in human milk. Methods Hydrolysis amino acids were analysed with C 18+ column and AQC precolumn derivation by UPLC method; and ion exchange chromatography column and ninhydrinpostcolumn derivation by AAA method. Results Two methods provided similar validation results. The difference of analysis result using two methods was below 4.65%. Conclusion Both methods can be used for the quantitation of hydrolysis amino acids. KEY WORDS: hydrolysis amino acid; derivative; ultra-performance liquid chromatography; amino acid analyzer 1 引言,,, [1], [2-5],, * 通讯作者 :,, E-mail: renyiping@263.net *Corresponding author: REN Yi-Ping, Senior Engineer, Zhejiang Provincial Center for Disease Control and Prevention, NO.3399, Binsheng Road, Binjiang District, Hangzhou 310051, China. E-mail: renyiping@263.net
2074 5,,, [6-8],,,, - - [9-12],, (FA) (PITC) 6- -N- - (AQC) (FMOC-Cl) (Dansyl-Cl) (OPA) 2, 4- (DNFB) [13,14] AQC,,,,, [15,16], AQC, AQC (UPLC) (AAA), 100, 15 2 材料与方法 2.1 仪器与试剂 L-8900 ( ), ACQUITY( Waters ),, 2.5 μmol/ml( Sigma-Aldrich ); AccQ Fluor ( Waters ); (, ); (, Merck ); (, ); (, Sigma-Aldrich ); (99.99%, ) 2.2 溶液配制 2.2.1 UPLC 衍生液配制 AccQ Fluor2B 1 ml, AccQ Fluor2A,, 10 s, 55 10 min,, 10 mmol/l AccQ Fluor, 2.2.2 AAA 缓冲液和茚三酮反应液的配制 (PH-1 PH-2 PH-3 PH-4) (PH-RG) L-8900 [17,18] 2.3 人乳取样方法 来源 保存条件,,,, -80 2.4 样品预处理方法 20 mg( 0.1 mg), 6 mol/l 1 ml, 30 s, (110±1) 22 h, 50, 2.5 衍生及分析 2.5.1 UPLC 柱前衍生及色谱条件 1 ml, 0.22 μm, 10μL, 70 μl, 20 μl AQC, 1 min 55 10 min, : CORTECS UPLC C 18 (1.6 μm, 2.1 150 mm); : 40 ; : 0.3 ml/min; : 260 nm; : 1 µl; A: 0.1%, B: ; : 0~6.0 min, B 3%~5%, 9.0~10.0 min, B 7%~12%, 13.8~14.5 min, B 12%~15%, 16.5~17.0 min, B 22%~60%, 17.2~20.0 min, B 3%~3% 2.5.2 AAA 柱后衍生及分析 1 ml 0.02 mol/l, 0.22 μm, 20 μl,, 135, 570 nm 440
7, : 2075 nm, 3 结果与讨论 3.1 UPLC 法色谱柱的选择 Waters UPLC,, 55, 0.7 ml/min, : 0.7 ml/min,,,,,,, (A : ACQUITY UPLC BEH C 18 1.7 μm, 2.1 mm 100 mm; B : ACCQ-TAG ULTRA C 18 1.7 μm, 2.1 mm 100 mm; C : CORTECS UPLC C 18+ 1.6 μm, 2.1 mm 150 mm), ( 1), C,,,, C,, 3.2 UPLC 法流动相的优化 ph,, - 0.05%TFA,, 0.07%TFA,,,,, 0.1%TFA, 15,, 0.1%TFA( 2) 3.3 方法学验证 ( 1), R 2 0.99; AAA UPLC, AAA UPLC 20,, UPLC, 3.4 两种方法的对比, ( 1),, (P<0.05), 4.65% 3-a, AAA, ;,,,, 3-b UPLC,, AAA,, 53 min UPLC 20 min, AAA, AAA UPLC, 2 1 Fig. 1 Optimization and selection of column (H S G R D E T A P C K Y M V I L F )
2076 5 2 Fig. 2 Optimization of mobile phase (H S G R D E T A P C K Y M V I L F ) Table 1 表 1 方法学及样品实测值比较 (n=6) Comparing of methodology and sample measured value (n=6) (mg/kg) (%) (%) (mg/100 g, n=6) AAA UPLC AAA UPLC AAA UPLC AAA UPLC P RSD(%) 6.69 14.91 98.29 93.02 1.13 1.14 25.82±0.33 23.09±0.37 0.00 4.25 4.69 33.25 89.81 98.82 2.35 4.83 49.96±0.58 49.97±1.63 0.99 2.33 3.24 2.51 98.30 98.50 1.41 3.04 25.49±0.23 24.77±1.21 0.21 3.61 13.20 6.81 96.72 91.75 0.92 1.61 38.67±0.54 37.63±0.31 0.00 1.80 6.28 16.66 100.37 98.43 3.74 3.59 96.17±0.97 96.40±1.40 0.74 1.20 8.36 21.04 99.20 95.71 3.23 4.55 192.25±2.06 192.87±2.46 0.65 1.14 5.41 9.88 96.38 96.05 2.52 2.47 49.57±0.48 48.45±0.87 0.03 1.81 4.84 4.65 98.59 95.75 1.57 2.23 40.38±0.42 38.24±0.57 0.00 3.09 47.96 6.95 96.69 98.52 1.03 3.13 96.24±0.70 95.79±1.15 0.43 0.98 4.30 7.37 99.23 103.68 1.35 1.82 73.00±0.84 74.90±1.00 0.01 1.79 7.31 17.76 94.18 90.32 4.11 1.18 46.00±0.94 42.84±1.58 0.00 4.65 4.07 11.39 98.85 90.22 1.72 2.62 59.77±0.81 58.75±1.52 0.19 2.16 8.63 9.50 100.49 90.48 2.19 2.33 57.75±0.87 54.08±1.18 0.00 3.85 7.81 9.27 100.48 94.43 2.86 2.90 107.60±1.40 104.69±1.39 0.00 1.91 7.00 11.95 101.85 93.85 4.60 5.55 41.03±0.60 39.49±0.58 0.00 2.44 3.5 人乳结果分析 100 15, 4,,,,,,
7, : 2077 Fig. 3-a 3-a AAA Chromatogram of human milk sample using amino acid analyzer Fig. 3-b 3-b UPLC Chromatogram of human milk sample using UPLC Fig. 4 4 15 15 kinds of Amino acids content in human milk sample from Hangzhou, Lanzhou and Beijing
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