Microsoft Word 胡 青-二校

Transcription

1 7 7 Vol. 7 No Journal of Food Safety and Quality Jul., 胡 青 1, 林媛 2, 张甦 1, 郑希望 2, 崔亚君 2, 季申 1* (1., ; 2., ) 摘要 : 目的 - 方法 -0.1% (V:V=3:2) 3,, WAX WCX, - 结果 % 128.2%, 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 , China; 2. Shanghai University of Traditional Chinese Medicine, Shanghai , 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 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 : (14DZ ) Fund: Supported by Science and Technology Commission of Shanghai Municipality (14DZ ) * 通讯作者 :,, [email protected] *Corresponding author: JI Shen, Chief Pharmacist, Shanghai Institute for Food and Drug Control, No.1500 Zhangheng Road, Pudong New District, Shanghai , China. [email protected]

2 引言,,,,,,, [1-3],,,, 1~5,,,, 3 [4] [5-8],,,,,,, (MRM), 2 材料与方法 2.1 试剂与耗材 前处理试剂 ( Merck ); ( ) 质谱试剂 ( Merck ); ( Merck ); ( Fluka ) 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 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 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 C 27 H 25 N 2 NaO 7 S 2 Dr.E 96.0% 4 G3 25 acid Green JOV 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 C 27 H 31 N 2 O 6 S 2 Dr.E 88.5% 8 B6 brillinant Blue FCF 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 % 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 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

3 7, : 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 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 V C 23 H 26 N 2 Fluka 98% 19 B10 A azure A 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 / / 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) ml 3, 30 min, 50 ml 10 ml, WAX ( ), 8 ml 4 ml,, - - (V:V:V=10:80:10) 8 ml,, ph, 10 ml,, r/min 10 min, 10 ml, WCX ( ), 5%,, - - (V:V:V=2:90:8) 8 ml,, ph, 10 ml,, r/min 10 min, 2.6 色谱条件 3( : A: 10 mmol/l, B: ; : A: 10 mmol/l +0.1%, B: ) : 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%

4 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) G [M+H] H [M+H] B / / B [M+H] G [M+H] B [M+H] B H [M+H] G B G B G G G

5 7, : 结果 3.1 线性关系的考察,,, 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) B G G B 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= X H1 50~1000 Y= X B6 50~5000 Y= X B4 50~1000 Y= X G9 50~5000 Y= X B1 50~1000 Y= X B7 50~1000 Y= X H2 500~5000 Y= X G2 50~5000 Y= X B2 50~5000 Y= X G3 50~5000 Y= X B8 50~5000 Y= X G8 50~5000 Y= X G7 50~5000 Y= X G10 50~5000 Y= X B10 8~1000 Y= X G10 8~5000 Y= X G1 1.6~1000 Y= X B9 1.6~1000 Y= X

6 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) G B H B B G G G H G B G G B B B B G G 方法检测限,, S/N 3, 样品测定结果, ,, G( 9) 表 8 检测限 Table 8 The limits of detection /pg (µg/kg) G H B B G 续表 8 /pg (µg/kg) B B H G B G B G G G B G G B

7 7, : 表 9 样品测定结果 Table 9 Detection results of samples (mg/kg) / G(6.66) / (5.21) 4 讨论 4.1 供试品溶液提取方法研究,,,,, 4,, 4,,,,, -0.1%, -0.1% (V:V=3:2) min, 30 min 2 3 4, 3 4, 供试品溶液净化方法研究,, [9],,,,,,,, (WAX), - (5:80:15 10:30:60 10:40:50 10:50:40 10:60:30 10:70:20 10:80:10 10:90 15:80:5 1:80:19), - (V:V:V=10:80:10), WAX - (V:V:V=10:80:10) (WCX), - - (30:2:68 40:2:58 50:2:48 60:2:38 70:2:28 80:2:18 90:2:8 90:1: 9 90:0.1:9.9 90:4:6), - (V:V:V=90:2:8), WCX - (V:V:V=90:2:8) 4.3 色谱条件优化, B9 B10, 2,,,,,, 质谱条件优化,, G5 H1 B3 B4,, 种未成功纳入方法的色素 19, ,, WAX, 19 5 结论,,, [16-18], 2015 [19], 参考文献 [1],,,. [J]., 2011, 18(20): 46. Jiang L, Rao WW, Peng FY, et al. Studies on illegally addition in decoction pieces [J]. Cap Food Med, 2011, 18(20): 46. [2],,,. [J]., 2015, 50(4): Wei F, Liu W, Yan H, et al. National wide quality surveillance and analysis

8 of Chinese material medical and decoction pieces [J]. Chin Pharm J, 2015, 50(4): [3]. [J]., 2012, 20(3): Hu HB. Three concocting behavior of Chinese material medical and decoction pieces [J]. Pharm Clin Res, 2012, 20 (3): [4] [S] State supplemental testing method for drug [S]. [5],,,. HPLC-DAD 20 [J]., 2015, 34(5): Li QY, Zhu RR, Sun P, et al. Simultaneous determination of 20 pigments in illegally dyed Chinese herbs by HPLC-DAD [J]. Herald Med, 2015, 34(5): [6],,,. HPLC-PDA [J]., 2010, 20(11): Zou YH, Yin HM, Guo YB, et al. Determination of eleven colouring agent in Saffron and Safflower with HPLC-PDA [J]. Chin J Health Lab Technol, 2010, 20(11): [7],. HPLC-PDA [J]., 2011, 21(5): Zheng J, Zou YH. Determination of ten colouring agent in pollen typhae and pollen typhae with HPLC-PDA [J]. Chin J Health Lab Technol, 2011, 21(5): [8],. HPLC-PDA 6 [J]., 2013, 36(1): Wang JJ, Chen HL. Determination of six pigments illegally added in zhengtian pill and zhengtian capsule by HPLC-PDA [J]. Drug Eval Res, 2013, 36(1): [9] GB/T [S]. GB/T Determination of synthetic color in foods [S]. [10] He GG, Wei JG, Yong GZ. Rapid method for quantification of seven synthetic pigments in colored Chinese steamed buns using UPLC-MS/MS without SPE [J]. Anal Sci, 2015, 31(3): [11] Ping Q, Zi HL, Gui YC. Fast and simultaneous determination of eleven synthetic color additives in flour and meat products by liquid chromatography coupled with diode-array detector and tandem mass spectrometry [J]. Food Chem, 2015, 181(15): [12] Feng F, Zhao YS, Wei Y, et al. Highly sensitive and accurate screening of 40 dyes in soft drinks by liquid chromatography electrospray tandem mass spectrometry [J]. J Chromatogr B, 2011, 879(20): [13],,,. / 32 [J]., 2012, 31(6): Guo XD, Xian YP, Luo HY, et al. Determination of 32 industrial dyes in seasoning paste by gel permeation chromatography and ultra performance liquid chromatography tandem mass spectrometry [J]. J Instrum Anal, 2012, 31(6): [14],,, [J]., 2011, 29(7): Zhao YS, Yang ML, Zhang F, et al. Screening method for 29 forbidden or limited synthetic pigments in cheese by liquid chromatography/quadropole time-of-flight mass spectrometry [J].Chin J Chromatogr, 2011, 29(7): [15],,, [J]., 2015, 33(08): Wang PY, Zhou Y, Dai YF, et al. Rapid screening of 28 acidic artificial dyes in fishery products by ultra performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry [J]. Chin J Chromatogr, 2015, 33(08): [16],, 甦,. 48 TLC [J]., 2015, 46(7): Hu Q, Sun J, Zhang S, et al. Simultaneous detection of 48 banned pigments in traditional Chinese medicine by TLC [J]. Chin J Pharm, 2015, 46(7): [17],,,. -HPLC 16 [J]., 2015, 37(5): Sun J, Feng R, Hu Q, et al. Simultaneous detection of sixteen synthetic acid pigments in medicinal Chinese herbs by polyamide SPE-HPLC [J]. Chin Tradit Pat Med, 2015, 37(5): [18]. [D]. :, Fang L. Simultaneous detection of banned dyes in traditional Chinese medicine [D]. Shanghai: Shanghai University of Traditional Chinese Medicine, [19] 2015 [S]. Chinese Pharmacopoeia 2015 IV [S]. 作者简介 ( 责任编辑 : 白洪健 ) 胡青, 副主任药师, 主要研究方向为中药质量控制和安全性检测技术研究 [email protected] 季申, 主任药师, 主要研究方向为中药 天然药物及保健食品和有害残留物的质量标准研究 [email protected]