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1 7 1 Vol. 7 No 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., ; 2., ; 3., ) 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, , China; 2. Technology Center of Ningbo Entry-Exit Inspection and Quarantine Bureau, Ningbo , China; 3. Ningbo Academy of Science and Technology for Inspection and Quarantine, Ningbo , 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 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 :,, mam@nbciq.gov.cn *Corresponding author: MA Ming, Senior Engineer, Technical Center of Ningbo Entry-Exit Inspection and Quarantine Bureau, Ningbo , China. mam@nbciq.gov.cn

2 引言 (methylamine, MA)(ethylamine, EA) (propylamine, PrA)(butylamine, BA) (pentylamine, PA)(hexylamine, HA), [1-3],,,, [4] [3] [5] [6],, (Dns-Cl) [7] , 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 方法荧光标记试剂的合成 6- -(N- )-9-(2 - )(SSMF) : Fig (N-)-9-(2 - ) The synthesis route of 6-oxy-(N-succinimidyl succinate acid monoester)-9-(2 -methoxycarbonyl) fluorescein

3 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,, h, 100 ml 2%NaHCO 3,, 0.1 mol/l, 1% 3, h, (2 - ) (2 - ) N-, 3 ml DMF, 0.5 g, (20 ),,,,,, h, 6- -(N- )-9-(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 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

4 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 衍生温度的选择 10 ~50,, 20,, 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= X EA 0.02~2 Y= X PrA 0.01~2 Y= X BA 0.02~2 Y= X PA 0.02~2 Y= X HA 0.05~2 Y= X

5 1, : Fig. 4 Chromatograms of 6 aliphatic amine derivatives 0: ; 1: ; 2: ; 3: ; 4: ; 5: ; 6: 0: hydrolyzed SIIA; 1: MA derivatives; 2: EA derivatives; 3: PrA derivatives; 4: BA derivatives; 5: PA derivatives; 6: HA derivatives. 参考文献 [1] Busto O, Guasch J, Borrull F, Improvement of a solid-phase extraction method for determining biogenic amines in wines [J]. J Chromatogr A, 1995, 718(2): [2] Cao LW, Wang H, Zhang HS, et al. 6-Oxy-(N-succinimidyl acetate)-9-(2 -methoxycarbonyl) fluorescein as a new fluorescent labeling reagent for aliphatic amines in environmental and food samples using high-performance liquid chromatography [J]. J Chromatogr A, 2005, 1063(1 2): [3] Cao LW, Wang H, Zhang HS, et al. Spectrofluorimetric determination of aliphatic amines using a new fluorigenic reagent: 2,6-dimethylquinoline-4-(N-succinimidyl)-formate [J]. Talanta, 2003, 59(5): [4] Zhao YY, Cai LS, Jing ZZ, et al. Determination of aliphatic amines using N-succinimidyl benzoate as a new derivatization reagent in gas chromatography combined with solid-phase microextraction [J]. J Chromatogr A, 2003, 1021(1 2): [5],,.. - [J]., 2007, 35(6): Zhao XE, Li YL, You JM, et al. Pre-column derivatization-high performance liquid chromatography for the determination of aliphatic amines with fluorescence detection and mass spectrometric identification [J]. Chin J Anal Chem, 2007, 35(6): [6] Cao LW, Wang H, Zhang HS, Analytical potential of 6-oxy-(N-succinimidyl acetate)-9-(2 - methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser -induced fluorescence detection [J]. Electrophoresis, 2005, 26: [7] Yamada K, Aizawa Y. Determination of catecholamines in rat tissue by precolumn dansylation using micro high-performance liquid chromatography with fluorescenece detection [J]. J Pharmacol Meth, 1983, 9(1): 1 6. [8],,,. [J]., 2006, 34(12): Yuan GX, He QH, Chen HW, et al. Rapid separation and determination of ephedrine dopes by chip-based capillary electrophoresis and laser induced fluoresence detection [J]. Chin J Anal Chem, 2006, 34(12): [9] Lange J, Thomas K, Wittmann C. Comparison of a capillary electrophoresis method with high-performance liquid chromatography for the determination of biogenic amines in various food samples [J]. J Chromatogr B, 2002, 779(2): [10],,,. AQC [J]., 2006, 34(10): Hou SM, Sun J, He HB, et al. Simultaneous determination of amino acids in soil by reversed phase high performance liquid chromatography by using 6-aminoquinoly-N-hydroxysuccinimidyl carbamate as a precolumn derivatization reagent [J]. Chin J Anal Chem, 2006, 34(10): [11] Mattush JJ, Dittrich KJ. Improvement of laser-induced fluorescence detection of amino acids in capillary zone electrophoresis [J]. J Chromatgr A, 1994, 680: [12] Lau Shung Kei, Zaccardo Fabian, Little Michael, et al. Nanomolar derivatizations with 5-carboxyfluorescein succinimidyl ester for fluorescence detection in capillary

150 7 electrophoresis [J]. J Chromatogr A, 1998, 809(1 2): 203 210. 作者简介 [13],,. - [J]., 2008, 36(1): 24 28. Cao LW, Meng JX, Liu YJ.

xiaolingyan2008@163.com detection [J]. Chin J Anal Chem, 2008, 36(1): 24 28.

6 150 7 electrophoresis [J]. J Chromatogr A, 1998, 809(1 2): 作者简介 [13],,. - [J]., 2008, 36(1): Cao LW, Meng JX, Liu YJ. Determination of biogenic amines using capillary electrophoresis with laser-induced fluorescence 肖玲艳, 硕士, 工程师, 主要研究方向为化学品和消费品安全分析检测 xiaolingyan2008@163.com detection [J]. Chin J Anal Chem, 2008, 36(1): [14] Adamczyk M, Grote J, Moore JA, Chemoenzymatic synthesis of 3'-O-(carboxyalkyl) fluorescein labels [J]. Bioconjugate Chem, 1999, 10(10): 马明, 博士, 高级工程师, 主要研究方向为色谱分离技术 mam@nbciq.gov.cn ( 责任编辑 : 白洪健 )

DOI /j.issn Food Research And Development UPLC-MS/MS 5 0.

DOI /j.issn Food Research And Development UPLC-MS/MS 5 0. 118 217 5 38 1 DOI 1.3969/j.issn.5-6521.217.1.28 Food Research And Development 1 2 1 2 1 2 1 2 1 2 1 2 1. 731 2. 731 - UPLC-MS/MS 5. 1 mol/l ProElut PXC 5 % - Thermo Hypersil Gold mm 2.1 mm 1.9 μm ESI