7 1 Vol. 7 No. 1 2016 1 Journal of Food Safety and Quality Jan., 2016 肖玲艳 1, 李赛花 1 2,, 马明 3* 2,, 肖道清 3, 陈丹超 2, 俞雄飞 (1., 315040; 2., 315012; 3., 315012) 2 摘要 : 目的, N-, - 方法 ph 8.5 Na 2 B 4 O 7 -H 3 BO 3, 25, 50 μmol/l, 10 min, 结果, 0.01~2 μmol/l, 0.999, 0.1~1 μg/l -, 6,, 关键词 : ; ; Synthesization of novel aliphatic amine fluorescence labeling reagents and derivative determination of micromolecule aliphatic amine XIAO Ling-Yan 1, LI Sai-Hua 1, MA Ming 2, 3*, XIAO Dao-Qing 2, 3, CHEN Dan-Chao 1, YU Xiong-Fei 1 (1. Societe Generale de Surveillance S.A.-China Standard Technology Development Corp. Standards Technical Services Co., Ltd. Ningbo Branch, Ningbo, 315040, China; 2. Technology Center of Ningbo Entry-Exit Inspection and Quarantine Bureau, Ningbo 315012, China; 3. Ningbo Academy of Science and Technology for Inspection and Quarantine, Ningbo 315012, China) ABSTRACT: Objective To synthesize a new aliphatic amine labeling reagent, using fluorescein as fluorophore and N-hydroxysuccinimidyl acetive ester as reaction group, and establish a separation method by labeling short chain aliphatic amine with new reagent and using high performance liquid chromatographyfluorescence detection. Methods The derivatization reaction was achieved with the buffer of Na 2 B 4 O 7 -H 3 BO 3 at ph 8.5, the reaction temperature at 25, the reagent solution at 50 μmol/l and the reaction time of 10 min. Results The linear range from 0.01 μmol/l to 2 μmol/l was observed for the proposed method. The limit of detection (LOD) of developed method was between 0.1 μg/l and 1 μg/l with the linear regression coefficient above 0.999. Conclusion The baseline separation of 6 small molecule aliphatic amine derivatives is achieved using optimized HPLC fluorescence detection. The method has the advantages of high derivative speed and sensitivity. KEY WORDS: aliphatic amine; fluorescence labeling; high performance liquid chromatography 基金项目 : (2011IK217)(2010A610032)(2014C50050) Fund: Supported by Scientific Research Project of National Quality Supervision, Inspection and Quarantine Bureau (2011IK217), Ningbo Natural Science Foundation (2010A610032), and Ningbo Social Development Research Project (2014C50050) * 通讯作者 :,, E-mail: mam@nbciq.gov.cn *Corresponding author: MA Ming, Senior Engineer, Technical Center of Ningbo Entry-Exit Inspection and Quarantine Bureau, Ningbo 315012, China. Email: mam@nbciq.gov.cn
146 7 1 引言 (methylamine, MA)(ethylamine, EA) (propylamine, PrA)(butylamine, BA) (pentylamine, PA)(hexylamine, HA), [1-3],,,, [4] [3] [5] [6],, (Dns-Cl) [7] 4- -7- -2, 1, 3- 噁 (NBD-Cl) [8] (OPA) [9] 6- -N- (AQC) [10] (FITC) [11] 5(6)- (CFSE) [12] 6- -[(1- ) ]- (SOFE) [13], OPA, FITC, CFSE, N- : 6- -(N- )-9-(2 - )(SSMF),,, 2 材料与方法 2.1 仪器与试剂 Alliance e2695, 2475 ( Waters ) (), (Sigma ), N- (DCC) N,N-, milli-q 2.2 方法 2.2.1 荧光标记试剂的合成 6- -(N- )-9-(2 - )(SSMF) : Fig. 1 1 6- -(N-)-9-(2 - ) The synthesis route of 6-oxy-(N-succinimidyl succinate acid monoester)-9-(2 -methoxycarbonyl) fluorescein
1, : 147 [14] 10 mmol 10 ml, 2.5 ml 12 h,, 10 g NaHCO 3,, 2% 50 ml NaHCO 3,, 3 1%,,,, 10 ml DMF,, 75 10 h, 100 ml 2%NaHCO 3,, 0.1 mol/l, 1% 3, 40 24 h, 6- --9-(2 - ) 6- --9-(2 - ) N-, 3 ml DMF, 0.5 g, (20 ),,,,,, 40 24 h, 6- -(N- )-9-(2 - ) 2.2.2 衍生反应 10 μl 1.5 ml, 200 μl Na 2 B 4 O 7 -H 3 BO 3 (ph 8.5), 10 μl SSMF, 1 ml, 10 min 0.45 μm 2.2.3 色谱条件 Xbridge C 18 (250 mm 4.6 mm, 5 μm), -, 1.0 ml/min, : 0~5 min: - (30:70, V:V); 5~15 min: - (80:20, V:V); 15~20 min: - (80:20, V:V) 25 3 结果与分析 3.1 色谱条件的优化 -, -, :, 80%(V:V), 6 ;, 50%(V:V), 6 ;, 30%(V:V), 6, 70min,,,, : 0~5 min: - (30:70, V:V)5~15 min: - (80:20, V:V)15~20 min: - (80:20, V:V) 22 min 3.2 衍生条件的优化 3.2.1 衍生试剂用量的选择 N-,, N-,,, 0.50 µmol/l,, 2 2 SSMF Fig. 2 Effect of SSMF concentration on the peak area of amine derivatives : ; : ; : ; : ; +: ; :. : MA; : EA; : PrA; : BA; +: PA; : HA. 2, 10~100 µmol/l, 7.5 µmol/l, 10 µmol/l,,,, 50 µmol/l
148 7 3.2.2 缓冲溶液 ph 的选择 N-, H 3 BO 3 -Na 2 B 4 O 7 H 3 PO 4 -NaOH NH 4 Cl-NH 3, H 3 PO 4 -NaOH, NH 4 Cl-NH 3 N-, H 3 BO 3 -Na 2 B 4 O 7 ph 7.5~9.0, 3, ph 8.0~9.0,, ph 8.5 ph 3.2.3 衍生温度的选择 10 ~50,, 20,, 25 3.2.4 衍生时间的选择 25,,, 5 min,,, 10 min 3.3 方法的线性范围 标准曲线 相关系数 相对标准偏差 检出限,,, 0.01~2 µmol/l, 1, 4 4 结论 3 ph Fig. 3 Effect of ph on the peak area of amine derivatives : ; : ; : ; : ; +: ; :. : MA; : EA; : PrA; : BA; +: PA; : HA., N- : 6- -(N- )-9-(2 - ),, HPLC,,,, Table 1 表 1 方法的线性范围 标准曲线 相关系数 相对标准偏差以及检出限 Linear calibration ranges, regression equations, correlation coefficients, relative standard deviations and detection limits of amino derivatives (µmol/l) r 2 RSD(%, n=6) LOD(µg/L) MA 0.01~2 Y=40991231X+40349 0.9991 1.4 0.1 EA 0.02~2 Y=15901932X-19464 0.9997 2.3 0.2 PrA 0.01~2 Y=165964578X-80448 0.9995 2.7 0.1 BA 0.02~2 Y=353399742X+883636 0.9982 3.0 0.4 PA 0.02~2 Y=39356643X-53210 0.9976 1.9 0.4 HA 0.05~2 Y=16783784X-42577 0.9979 2.6 1
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