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1 3 3 Vol. 3 No Journal of Food Safety and Quality Jun., 2012 李玉玲 1,2, 李卫华 (1., ; 2., ) 1* 摘要 : 目的 (DTT), 方法,,,, 结果 (Met) [(Cys) 2 ] (Cys) 0.7 μmol/l 0.3 μmol/l 0.5μmol/L, 10~750 μmol/l, DTT 1 ml 100 μl 0.1 mol/l DTT, 1 h Met (Cys) g/100 g 0.14 g/100 g, 82%~87%, (RSD) 1.5% 结论 DTT, 关键词 : (DTT); ; ; (HPLC) Application of dithiothreitol in the detection of sulfur amino acids in milk powder LI Yu-Ling 1,2, LI Wei-Hua 1* (1. Shanxi Entry-Exit Inspection and Quarantine Bureau, Taiyuan , China; 2. Bio-technology Institute, Shanxi University, Taiyuan , China) ABSTRACT: Objective To develop a new method to detect sulfur amino acids by reverse-phase high performance liquid chromatography (HPLC) in milk powder reacted with dithiothreitol in pretreatment. Methods Milk powder was hydrolyzed by acid, further reduced by dithiothreitol, and then detected by HPLC. The optimum reduction conditions and chromatographic separation conditions were investigated. Results Detection limits of methionine, cystine and cysteine were 0.7 μmol/l, 0.3 μmol/l and 0.5 μmol/l, respectively, and all of the quantitation range were 10~750 μmol/l with the correlation coefficients of , and , respectively. The optimum operating conditions were as follows: 100 μl dithiothreitol at the concentration of 0.1 mol/l was added in 1 ml hydrolyzate and reacted for 1h at room temperature. The contents of methionine and cystine in milk powder were 0.37 g/100 g and 0.14 g/100 g, respectively. The recoveries ranged from 82% to 87% with the relative standard deviation (RSD) of less than 1.5%. Conclusion Applying optimum dithiothreitol in pretreatment and using the reverse phase HPLC method could detect the content of sulfur amino acids in milk powder accurately. KEY WORDS: dithiothreitol(dtt); milk powder; sulfur amino acids; high performance liquid chromatography 基金项目 : (2010IK157). * 通讯作者 :,,,, aplab@163.com

2 引言,,, [1], [2,3],,,,, [4,5],,, [6] ( ), [7],,, [8-14] [15-20], 4- -3, 5- (4-chloro-3,5-Dinitro trifluoromethylbenzene, CNBF),, 2 材料与方法 2.1 材料与试剂 ; ( Fapas ); L- (>98%): (Met) [(Cys) 2 ] (Cys) ( ); (DTT) ( ); 4- -3,5- (CNBF, ); (, ); (, ); ( Milli-Q ); 25 mmol/l 100 mmol/l 4- -3, 5- (CNBF) ; 40 mmol/l ; 2.2 仪器与设备 Waters Alliance2690 ( Waters ); CAPCELL PAK C 18 ( ); DF-15 L ( ); 101A-3 ( ); Minispin ( Eppendorf ); 1008 ( GFL ); Sartorious PB-10 ; Vortex-Genie2- G-560E( Scientific Industries ); 0.22 μm ( ) 2.3 方法样品前处理, 3.0 ml 6 mol/l HCl, 20, N 2, N 2, h,,, ph ml,,,, 1.0 ml DTT,, 40 N 2, ph 9.0 -,, CNBF 样品衍生 1.0 ml, 5.0 ml ph ml CNBF,,, min,, 17 ml ph 7.0, 0.5 min, 10 min, 0.22 μm, HPLC 反相 HPLC 法检测条件 A: ; B: ph ; : 240 nm; : 0.32 ml/min; : C 18 MGⅡ(5 μm, 4.6 mmi.d 250 mm); : 20 μl; [21] 3 结果与分析 3.1 色谱峰的定性及色谱图 1 mmol/l Met (Cys) 2 Cys 0.1 mol/l DTT 2.3.2, 20 μl,, 1, 0.1 mol/l DTT 2 Met (Cys) 2 Cys DTT min min min min,

3 3, : 173 1, Met (CNBF)OH (Cys) 2,, Met (Cys) 2, 19~21, 2 1,, DTT 1 Met (Cys) 2 (Cys) Fig. 1 Chromatogram of standard amino acids of methionine, cystine and cysteine Fig. 2 2 DTT Chromatogram of dithiothreitol solution 3.2 检测限及线性范围 Met (Cys) 2 Cys, μmol/l, 20 μl 3,,, 1 10~750 μmol/l Met (Cys) 2 Cys (S/N) 3, (LOD) 0.5 μmol/l,, ; (S/N) 10,, 10 μmol/l 3.3 水解反应前后加入 DTT 对含硫氨基酸回收率的影响 40 mmol/l Met (Cys) μl,, 6 mol/l HCl 3 ml, 0.1 mol/l DTT 100 μl,, 2.3 DTT, 1 ml, 0.1 mol/l DTT 50 μl, 2 h, N 2,,, 2, 0.1 mol/l DTT, Met,, (Cys) 2 Cys 35% ; DTT, Met Cys DTT ph,, DTT, 表 1 标准氨基酸的方法检测限及线性范围 (n=3) Table 1 Method detection limits and linear ranges of standard amino acids(n=3) (μmol/l) R 2 LOD(μmol/L, S/N 3) LOQ(μmol/L, S/N 10) Met 10~750 Y= X (Cys) 2 10~750 Y= X Cys 10~750 Y= X Table 2 表 2 水解前后加入 DTT 溶液对含硫氨基酸检测的影响 (n=3) Effect of dithiothreitol s addtion time on the detection of sulfur amino acids(n=3) Met (Cys) 2 Cys Met (Cys) 2 Cys (mmol/l) (%) RSD(%)

4 不同量 DTT 对 Met (Cys) 2 回收率的影响 40 mmol/l Met (Cys) μl, 2.3, 1.0 ml, 0.1 mol/l DTT, 2 h, Met 3, (Cys) 2 4 表 3 不同量 DTT 对 Met 的影响 (n=3) Table 3 Effect of different contents of dithiothreitol on the detection of methionine(n=3) DTT(µL) (mmol/l) (%) RSD(%) , 0~100 μl 0.1 mol/l DTT, Met (85%~99%), DTT, Met,, 0.1 mol/l DTT Met, 4, (Cys) 2, 40%, 20~200 μl 0.1 mol/l DTT, 20 μl,, 90%~113%, DTT (Cys) 2 Cys, DTT 100 μl, 100 μl 0.1 mol/l DTT 表 4 不同量 DTT 对 (Cys) 2 的影响 (n=3) Table 4 Effect of different contents of dithiothreitol on the detection of cystine(n=3) DTT(µL) (mmol/l) (%) RSD(%) DTT 不同作用时间对 (Cys)2 的影响 40mmol/L (Cys) μl, 2.3, 1.0 ml, 0.1 mol/l DTT 100 μl, 1~5 h, 5 1~5 h, (Cys) 2 95%~115%,, 1 h,, 1 h 表 5 Table 5 不同反应时间对 (Cys) 2 的影响 (n=3) Effect of different reaction time on the detection of cystine(n=3) (h) (mmol/l) (%) RSD(%) 精密度 40 mmol/l (Cys) μl, 2.3, 1 ml, 0.1 mol/l DTT 100 μl, 1 h (Cys) 2 Cys, mmol/l, 94.4%, RSD 2.6%, DTT 1 ml 0.1 mol/l DTT 100 μl, 1 h 3.7 重复性 g( g), 2.3, DTT, Met 85%, RSD 2.7%, (Cys) 2 106%, RSD 2.3% Met 奶粉含硫氨基酸的检测 g( g)15,, 40 mmol/l Met (Cys) 2 25 μl 50 μl 75 μl,, 3.7, 6, 3 6, Met mmol/l( 0.37 g/100 g), (Cys) /2 mmol/l mmol/l( 0.14 g/100 g);,

5 3, : 175 Met Cys 80%~85%, RSD 1.0%, 表 6 Table 6 奶粉含硫氨基酸检测结果 (n=6) Results of sulfur amino acids in milk powder(n=6) (mmol/l) (mmol/l) (%) RSD(%) Met Cys a DTT b 3 DTT Fig. 3 Detection chromatogram of milk powder with optimum dithiothreitol followed the acid hydrolysis 3, DTT Met Cys, DTT min ( ),, Cys,,, 4 结论 DTT, 1.0 ml 0.1 mol/l DTT 100 μl, 1 h, HPLC,,,,, 参考文献 [1]. [M]. :, 2004: [2],,,. [J]., 2009, 35(4): [3] GB/T , [S]. :, [4],. [J]., 2010, 31(9): [5]. HPLC [J]., 2006, 6: [6],,,. [J]., 1994, 9(1): 49. [7]. [M]. :, 2002: [8],. [J]., 1994, 13(1): [9],,,. [J]., 1994, 27(5): [10],,. GB/T , - [S]. :, [11],. [J]., 1999, 1: [12],. [J]., 1996, 5(4): [13],. [J]., 2009, 2: [14],,,. - [J]., 2011, 29(3): [15] Lopez del Castillo Lozano M, Tâche R, Bonnarme P, et al. Evaluation of a quantitative screening method for hydrogen sulfide production by cheese-ripening microorganisms: The first step towards L-Cysteine catabolism [J]. J Microbiol Methods, 2007, 69: [16] Pinto JT, Khomenko T, Szabo S, et al. Measurement of sulfur-containing compounds involved in the metabolism and transport of cysteamine and cystamine. Regional differences in cerebral metabolism [J]. J Chromatogr B, 2009, 877: [17] Suh JH, Kim R, Yavuz B, et al. Clinical assay of four thiol amino acid redox couples by LC-MS/MS: Utility in thalassemia[j]. J Chromatogr B, 2009, 877:

6 176 3 [18],,,. HPLC-ECD [J]., 2010, 3: [19],,,. [J]., 2008(24)3: [20],. [J]., 1993, 3: [21],,,. 18., : CN A, ( 责任编辑 : 张宏梁 ) 作者简介李玉玲,,, sxlclyl1986@163.com 李卫华,,,, aplab@163.com ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆ 快速检测与无损检测技术专题约稿函,,,,,,,, ,, jfoodsq@126.com

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min 25 4 2013 8 监测技术 5 1 2 1 ( 1. 江苏省疾病预防控制中心, 江苏南京 210009; 2. 南京医科大学第二附属医院, 江苏南京 210029) 5 Phenomenex kinetex C 18 2. 6 μm 4. 6 mm 150 mm 10 mmol /L ph 2. 5 + 60 40 DAD 280 nm 5 20. 0 mg /L ~ 100 mg /L 0.