7 7 Vol. 7 No. 7 2016 7 Journal of Food Safety and Quality Jul., 2016-19 胡 青 1, 林媛 2, 张甦 1, 郑希望 2, 崔亚君 2, 季申 1* (1., 201203; 2., 201203) 摘要 : 目的 - 方法 -0.1% (V:V=3:2) 3,, WAX WCX, - 结果 19 62.3% 128.2%, 0.5 345.5 µg/kg, 结论, 关键词 : ; ; ; - Detection of 19 dark pigments illegally added in Mumu Fructus by ultra high performance liquid chromatography-tandem mass spectrometry HU Qing 1, LIN Yuan 2, ZHANG Su 1, ZHENG Xi-Wang 2, CUI Ya-Jun 2, JI Shen 1* (1. Shanghai Institute for Food and Drug Control, Shanghai 201203, China; 2. Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China) ABSTRACT: Objective To establish a method for detection of 19 dark pigments illegally added in Mumu Fructus by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Methods Samples were extracted with methanol-0.1% formic acid solution (V:V=3:2) by ultrasonic 3 times, cleaned up with WAX and WCX columns to obtain acidic and alkaline pigments tested solution, respectively. Finally, the analytes were detected by UHPLC-MS/MS. Results Recoveries of 19 kinds of dark pigments in Mumu Fructus were 62.3% 128.2%, and the limits of detection were 0.5 345.5 µg/kg. This method could be applied to sample analysis, and the results found some partial illegally added pigments in Mumu Fructus. Conclusion The established method is fast and sensitive, which can meet the determination requirements of dark pigments illegally added in Mumu Fructus. KEY WORDS: Mumu Fructus; dark pigment; dye; ultra high performance liquid chromatography-tandem mass spectrometry 基金项目 : (14DZ2294000) Fund: Supported by Science and Technology Commission of Shanghai Municipality (14DZ2294000) * 通讯作者 :,, E-mail: jishen2013@163.com *Corresponding author: JI Shen, Chief Pharmacist, Shanghai Institute for Food and Drug Control, No.1500 Zhangheng Road, Pudong New District, Shanghai 201203, China. E-mail: jishen2013@163.com
2664 7 1 引言,,,,,,, [1-3],,,, 1~5,,,, 3 [4] [5-8],,,,,,, (MRM), 2 材料与方法 2.1 试剂与耗材 2.1.1 前处理试剂 ( Merck ); ( ) 2.1.2 质谱试剂 ( Merck ); ( Merck ); ( Fluka ) 2.1.3 色谱柱及固相萃取柱 ACQUZTY UPLC HSS T3 (3.0 mm 100 mm, 1.8 μm); Waters ACQUITY Guard Filter (0.2 μm); Waters ACQUITY UPLC HSS T3 (1.8 μm); ((PA) ; oasis WAX 6cc WAX ; oasis WCX 6cc WCX 2.1.4 色素品种及分组, 3 21 X J, : X : (B4) (G2) 25(G3) 1(H1) G(B1) V(B2) (B6) 7(G5) (B8) (B7) (G7) B(G8) FCF(G9) SF(G10) 1(H2) J : 9(B9) A(B10) (G1) (G6) 1 Table 1 表 1 19 种色素对照品信息 Reference substance information of 19 pigments 1 G1 malachite Green 20501 C 23 H 25 ClN 2 Dr.E 96.9% 2 B4 methyl Blue SLBB4827V C 37 H 27 N 3 Na 2 O 9 S 3 Sigma 60.0% 3 G2 green S 00624 C 27 H 25 N 2 NaO 7 S 2 Dr.E 96.0% 4 G3 25 acid Green 25 13227JOV C 28 H 20 N 2 Na 2 O 8 S 2 Sigma 75.0% 5 H1 1 naphthol Blue Black MKBK0226V C 22 H 14 N 6 Na 2 O 9 S 2 Sigma 6 B1 G brilliant Blue G SLBB221V C 47 H 48 N 3 NaO 7 S 2 Sigma 7 B2 V patent Blue V 00728 C 27 H 31 N 2 O 6 S 2 Dr.E 88.5% 8 B6 brillinant Blue FCF 40710 C 37 H 36 N 2 O 9 S 3 Na 2 Dr.E 98.0% 9 G5 7 pyranine GJ01-HBKA C 16 H 7 Na 3 O 10 S 3 85.0% 10 B8 bromothymol Blue MKBR4619V C 27 H 28 B r2 O 5 S Sigma-Aldri ch 95% 11 B7 indigotine BCBD1142V C 16 H 8 N 2 O 8 S 2 2Na Fluka 98% 12 B9 9 methylene Blue 426046 C 16 H 18 N 3 SCl Polyscience 13 G7 acid green 9 KNCQG-DC C 37 H 34 ClN 2 O 6 S 2 Na
7, : - 19 2665 续表 1 14 G8 B guinea Green B 04423HNV C 37 H 35 N 2 O 6 S 2 Na Aldrich 50% 15 G9 FCF food green 3 BCBD0137V C 37 H 34 N 2 O 10 S 3 2Na Fluka 98% 16 G10 SF light Green SF 451782 C 37 H 34 N 2 O 9 S 3 2Na Polyscience 17 H2 1 brilliant black PN BCBN2507V C 28 H 17 N 5 O 14 S 4 4Na Fluka 96% 18 G6 leucomalachite green 1405114V C 23 H 26 N 2 Fluka 98% 19 B10 A azure A 609044 C 14 H 14 ClN 3 S Polyscience 2.2 样品, 2 表 2 样品基本信息 Table 2 Information of samples / 131119 13072202 130716 / 2.3 仪器 Agilent 1290 Infinity ( Agilent ); Agilent 6495, ( Agilent ); Millipore Milli-Q Gradient A10 ( Millipore ); Starorius CP225D ( Sartorius ); BRANSON B9500S-DTH ( ); eppendorf centrifuge 5810R ( eppendorf ); HWS-28 ( ); IKA HS 260 ( ) 2.4 对照品溶液的制备, X J 10 mg, ( 0.1 mg),, 20 ml, 4, 2.5 供试品溶液的制备 1 g, -0.1% (V:V=3:2) 20 10 10 ml 3, 30 min, 50 ml 10 ml, WAX ( ), 8 ml 4 ml,, - - (V:V:V=10:80:10) 8 ml,, ph, 10 ml,, 14000 r/min 10 min, 10 ml, WCX ( ), 5%,, - - (V:V:V=2:90:8) 8 ml,, ph, 10 ml,, 14000 r/min 10 min, 2.6 色谱条件 3( : A: 10 mmol/l, B: ; : A: 10 mmol/l +0.1%, B: ) : 0.550 ml/min; : 30 ; : 1 μl (EIC) 1 2 表 3 流动相梯度洗脱条件 Table 3 Gradient elution of liquid chromatography (min) (min) A% B% A% B% 0 95 5 0 50 50 7 60 40 5 30 70 10 20 80 8 5 95 12 5 95 10 5 95 20 5 95 11 95 5 21 95 5 12 95 5 23 95 5 13 50 50
2666 7 2.7 串联质谱条件 : m/z 100~1000; : (N 2 ): 20 psi; (N 2 ): 14 L/min, 200 ; (N 2 ): 11 L/min, 350 ; 3.5 KV; : 1.5 KV, RF: 150 V, RF: 60 V : ; : ( 4 5) 1 ( ) Fig. 1 Chromatogram of acidic pigments (ESI - ) 2 ( ) Fig. 2 Chromatogram of alkaline pigments (ESI + ) Table 4 表 4 酸性色素主要参考质谱参数 Parameters for the analysis of acidic pigments (RT, min) (m/z) (m/z) (ev) (m/z) (ev) G5 0.9 228.0[M+H] 2+ 188.1 14 295.9 22 H1 7.1 285.0[M+H] 2+ 163.9 14 238.9 14 B6 5.6 747.0 683.1 34 / / B4 4.9 376.6[M+H] 2+ 336.4 20 502.9 40 G9 5.7 381.0[M+H] 2+ 170.0 34 497.3 22 B1 9.3 414.6[M+H] 2+ 169.9 40 644.2 20 B7 2.5 421.0 340.9 34 260.9 46 H2 3.3 518.4[M+H] 2+ 388.2 20 777.5 20 G2 5.9 553.0 511.1 34 523.1 46 B2 7.9 559.0 479.2 38 435.3 54 G3 7.5 577.1 479.1 38 392.1 42 B8 9.7 621.0 542.1 30 463.3 42 G8 8.2 667.0 587.2 38 497.3 42 G7 8.3 701.0 621.1 34 531.1 46 G10 6.0 747.0 341.1 14 683.2 42
7, : - 19 2667 3 结果 3.1 线性关系的考察,,, 6 3.2 回收率和重复性试验 1 g,, 12, 3 1 ( 6 ), 3 ( ),,,, 7 Table 5 表 5 碱性色素主要参考质谱参数 Parameters for the analysis of alkaline pigments (RT, min) (m/z) (m/z) (ev) (m/z) (ev) B10 1.1 256.1 214.1 38 241.1 30 G1 3.2 329.2 313.2 46 165.0 74 G6 8.4 331.0 239.0 42 223.0 58 B9 1.5 284.1 268.0 46 252.0 66 Table 6 表 6 线性方程及相关系数 Regress equations and correlation coefficients (µg/ml) (r) G5 50~1000 Y=214425.484089X-3753.759085 0.9985 H1 50~1000 Y=212685.543964X-302.857449 0.9999 B6 50~5000 Y=76610.627488X-2161.025386 0.9999 B4 50~1000 Y=2.749775X-85.483769 0.9922 G9 50~5000 Y=41335.850693X-862.118642 0.9999 B1 50~1000 Y=87.985046X-760.403969 0.9999 B7 50~1000 Y=12730.036993X+289.815307 0.9985 H2 500~5000 Y=4.661650X-2724.106755 0.9964 G2 50~5000 Y=23795.479836X-547.217145 0.9999 B2 50~5000 Y=90422.704800X-3262.536087 0.9998 G3 50~5000 Y=66453.076887X-665.601914 0.9999 B8 50~5000 Y=375629.356684X-69844.836841 0.9957 G8 50~5000 Y=24031.146107X+569.041018 0.9997 G7 50~5000 Y=21053.202348X-816.903949 0.9998 G10 50~5000 Y=63603.508123X+604.697071 0.9997 B10 8~1000 Y=46049.751064X-1321904.180081 0.9981 G10 8~5000 Y=7134.152920X-44648.565407 0.9999 G1 1.6~1000 Y=91759.172253X-1022981.896076 0.9994 B9 1.6~1000 Y=148549.522504X-3066961.176182 0.9981
2668 7 表 7 加样回收率及重复性 Table 7 Recoveries and repeatability mg/kg /%(n=3) mg/kg /%(n=3) mg/kg /%(n=3) RSD/%(n=6) G5 5 97.1 10 93.2 15 104.2 6.0 B7 15 89.2 30 82.3 45 92.0 3.2 H2 25 72.5 50 68.5 75 81.0 3.5 B4 25 100.1 50 94.1 75 128.2 8.2 B6 10 65.3 20 87.1 30 85.0 5.7 G10 10 66.0 20 94.5 30 80.0 6.6 G9 5 84.2 10 83.2 15 91.0 1.7 G2 10 66.0 20 68.6 30 77.5 5.2 H1 5 80.3 10 81.2 15 89.3 4.0 G3 10 74.4 20 70.7 30 83.2 3.7 B2 5 65.2 10 71.5 15 79.1 5.4 G8 5 62.3 10 71.5 15 74.2 4.2 G7 5 63.3 10 62.3 15 72.3 8.6 B1 5 71.2 10 75.5 15 84.2 2.0 B8 5 65.6 10 72.5 15 86.1 2.7 B10 5 98.2 10 94.4 15 93.5 1.1 B9 5 92.2 10 88.6 15 89.2 1.3 G1 5 104.5 10 98.2 15 99.1 0.8 G10 5 98.5 10 94.3 15 89.1 4.3 3.3 方法检测限,, S/N 3, 8 3.4 样品测定结果, 2.3 2.4,, G( 9) 表 8 检测限 Table 8 The limits of detection /pg (µg/kg) G5 0.4 19.3 H1 0.3 13.7 B6 1.3 66.7 B4 0.5 23.0 G9 2.4 119.0 续表 8 /pg (µg/kg) B1 0.5 22.8 B7 0.9 46.7 H2 4.7 236.8 G2 0.01 0.5 B2 1.5 76.5 G3 2.6 132.2 B8 0.2 8.3 G8 2.2 109.1 G7 0.1 5.5 G10 6.9 345.5 B10 0.2 7.5 G1 0.05 2.7 G10 0.1 4.0 B9 0.5 23.5
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