Analysis of Isobaric Pesticides in Pepper with High-Resolution Liquid

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29 473-475 j461 食品安全与检测 3-12 液相色谱-电喷雾串联质谱法测定豇豆中的水胺硫磷

液相色谱-电喷雾串联质谱法测定豇豆中的水胺硫磷邹树丹，黄金凤，罗东辉，罗海英，陈立伟，吴玉銮(广州市质量监督检测研究院，国家加工食品质量监督检验中心(广州)广州市食品安全检测技术重点实验室，广州市食品安全风险动态监测与预警研究中心，广东广州 510110)摘要：建立了豇豆中水胺硫磷的液相色谱-电喷雾串联质谱分析方法。

样品经乙腈超声提取后进行液相色-电喷雾串联质谱分析。

方法检出限为0.1 μg/kg（S/N=3）。

空白样本添加水平在0.1、1.0、5.0 μg/k g时，平均回收率为80.1~87.9%，相对偏差（n=6）为6.7~8.1%。

使用MRM-IDA-EPI的模式，一次进样可得到MRM色谱图及MS2质谱图。

该方法具有前处理简单、回收率高、精密度好，选择性强，灵敏度高的优点，符合农药残留的分析要求。

关键词：水胺硫磷；分析；残留；质谱；豇豆文章篇号：1673-9078(2012)4-473-475Determination of Isocarbophos in Cowpea by LC/MS/MS withMRM-IDA-EPIZOU Shu-dan, HUANG Jin-feng, LUO Dong-hui, LUO Hai-ying, CHEN Li-wei, WU Yu-luan (Guangzhou Quality Supervision and Testing Institute, National Centre for Quality Supervision and Testing of Processed Food (Guangzhou), Guangzhou City Key Laboratory of Detection Technology for Food Safety, Guangzhou City Research Center of Risk Dynamic Detection and Early Warning for Food Safety,Guangzhou Guangdong 510110, China) Abstract: An analytical method for the determination of isocarbophos residues in cowpea with HPLC/MS/MS was established.Samples were extracted by acetonitrile with sonic extracting and detected LC/MS/MS. The LODs (S/N=3) were 0.1 μg/kg. For spiking level at 0.1, 1.0, and 5.0 μg/k g, the average recoveries were 80.1%~87.9% and RSD (n=6) values ranged from 6.7% to 8.1%. Using the MRM-IDA-EPI model, MRM chromatograms and the MS2 mass spectrum of the samples can be obtained by only one single injection. The method was simple, precise and had high recoveries, which can meet the requirement for residues analysis.Key words: isocarbophos; analyst; residue; MS; cowpea水胺硫磷（图1）为高毒杀虫剂，对皮肤有一定的刺激作用，但在试验剂量下无致突变和致癌作用、无蓄积中毒作用。

高效液相色谱内标法测定辣椒制品中苏丹红

关键词：内标法；橘红２号；ＨＰＬＣ；辣椒制品中图分类号：ＴＳ２０Ｉ．６／ＴＳ２０７．５文献标识码：Ａ文章编号：１００６—２５１３（２０１６）０３—０１７１—０４
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ＤＩＷａｎ—ｓｈａｎ
（ＬｉａｏｎｉｎｇＰｅｔｒｏ－ｃｈｅｍｉｃａｌＶｏｃａｔｉｏｎａｌＴｅｃｈｎｉｃａｌＣｏｌｌｅｇｅ，Ｊｉｎｚｈｏｕ１２１００１）
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AOAC 2007 美国农药残留检测标准方法

AOAC Official Method 2007.01Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium SulfateGas Chromatography/Mass Spectrometry and Liquid Chromatography/Tandem Mass SpectrometryFirst Action 2007[Applicable for the following pesticides in grapes, lettuces, and oranges: atrazine, azoxystrobin, bifenthrin, carbaryl, chlorothalonil, chlorpyrifos, chlorpyrifos-methyl, l -cyhalothrin (incurred in lettuces), cyprodinil, o,p ¢-DDD, dichlorv os, endosulfan sulfate,ethion (incurred in oranges), imazalil, imidacloprid,kresoxim-methyl (incurred in grapes), linuron, methamidophos,methomyl, permethrins (incurred in lettuces) procymidone,pymetrozine, tebuconazole, thiabendazole (incurred in oranges),tolylfluanid (degraded in lettuces), and trifluralin. These were representative pesticide analytes chosen in representative matrixes,and the method is expected to be applicable to many other similar pesticides and matrixes. Limits of quantitation were demonstrated to be <10 ng/g.]See Tables 2007.01A–E for the results of the interlaboratory study supporting acceptance of the method.A. PrincipleThe QuEChERS (quick, easy, cheap, effective, rugged, and safe)method uses a single-step buffered acetonitrile (MeCN) extraction and salting out liquid–liquid partitioning from the water in the sample with MgSO 4. Dispersiv e-solid-phase extraction (dispersive-SPE) cleanup is done to remove organic acids, excess water, and other components with a combination of primary secondary amine (PSA) sorbent and MgSO 4; then the extracts are analyzed by mass spectrometry (MS) techniques after a chromatographic analytical separation. Figure 2007.01 outlines the protocol in a box format. In brief, a well-chopped food sample alongwith 1 mL of 1% acetic acid (HOAc) in MeCN and 0.5 g anhydrous MgSO 4/NaOAc (4/1, w/w) per g sample are added to a centrifuge tube or bottle, which is shaken and centrifuged. A portion of the MeCN extract (upper layer) is added to anhydrous MgSO 4/PSA sorbent (3/1, w/w; 200 mg per 1 mL extract), mixed, and centrifuged. This final extract is transferred to autosampler vials for analysis by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) to identify and determine a wide range of pesticide residues. To achieve <10 ng/g detection limits in modern GC/MS, large volume injection (LVI) of 8 m L is typically needed, or the final extract can be concentrated and solvent exchanged to toluene (4 g/mL), in which case 2 m L splitless injection is used.Both GC/MS and LC/MS/MS techniques are prone to matrix effects in pesticide residue analysis, albeit for different reasons [Erney, D.R., Gillespie, A.M., Gilvydis, D.M., & Poole, C.F. (1993)J. Chromatogr. 638, 57–63; Hajslova, J., & Zrostlikova, J. (2003) J.Chromatogr. A 1000, 181–197; Alder, L., Luderitz, S., Lindtner, K.,& Stan, H.J. (2004) J. Chromatogr. A 1058, 67–79]. To account for these effects, matrix-matched calibration was conducted (calibration standards in solvent solution may also be used if matrix effects are shown not to occur). Due to the situation that some laboratories had LVI capability and others did not, the necessary amounts of matrix blank(s) and final extract volume was different for some laboratories than others. Depending on the water content of the matrix, a 15 g sample typically yields 11–14 mL of initial MeCN extract after centrifugation. In dispersive-SPE, roughly half of the extract is lost to the powders, thus about 6–7 mL of final extract can be expected for a 15 g sample. Two options were provided in the protocol to account for the different situations among the laboratories.ã 2007 AOAC IN T ER N A T IONALTable 2007.01A. Interlaboratory study results for incurred pesticides (and chlorpyrifos-methyl)AnalyteMatrix Avg. concn s raRSD r b, %S R c, ng/g Rec., %RSD R d, %HorRat No. of labsOutlierlabs e Chlorpyrifos-methylGrapes 165 14 8.535 8321 1.00116-C, 4-C Lettuces 178 20 11 30 89170.811011-SGOranges174 25 14 36 87200.9812Kresoxim-methyl Grapes 9.2 1.921f3.2NA 35f1.0912Cyprodinil Grapes 112 NAgNA 18 NA 160.7313l -Cyhalothrin Lettuces 58 6.111 11 NA 200.80 911-C Permethrins Lettuces 112 9.88.741 NA 36f1.63 96-C, 1-C Imidacloprid Lettuces 12NA NA 1.6NA 140.4411Ethion Oranges 198 23 12 36 NA 180.891111-C Thiabendazole Oranges 53 3.87.27.6NA 140.5812ImazalilOranges13NANA4.7NA35f1.1587-SGa s r = Standard deviation for repeatability (within laboratory).b RSD r = Relative standard deviation for repeatability.c s R = Standard deviation for reproducibility (among laboratories).d RSD R = Relative standard deviation for reproducibility.e C = Cochran outlier; SG = single Grubbs outlier.f RSD r >15%; 120% < Rec. < 70%; RSD R >25%; HorRat >1.2; and fewer than 8 laboratories in an assessment.gNA = Not applicable.Analyte Avg. C, ng/g s r, ng/g RSD r, %s R, ng/g Rec., %RSD R, %HorRat No. of labs Outlier labs Atrazine9.30.6 6.9 2.093210.651345 3.27.1 5.790130.4913365 23 6.271 9119 1.0413Azoxystrobin9.40.6 6.6 2.094210.641392 8.79.411 92120.51128-SG182 17 9.226 91140.70128-SG Bifenthrin7.80.811 2.37830b0.89112-C, 10-C86 5.9 6.914 86170.73126-C923 71 7.7136 92150.9113Carbaryl12 1.211 2.8104 27b0.85125-SG50 6.413 11 100 220.87131003 70 7.0189 100 19 1.18125-C Chlorothalonil 6.30.914 2.163b33b0.97 810-C59 8.314 13 79230.9310140 19 13 38 7027b 1.27b10Chloropyrifos8.1 1.519b 3.08137b 1.121268 8.312 14 84200.8413396 25 6.450 79120.681211-SG Cyprodinil c123 13 10 26 101 210.9513240 20 8.363 9226b 1.32b13581 42 7.3110 9519 1.0913o,p¢-DDD8.9 1.416b 3.28936b 1.091242 3.17.37.084170.6512445 32 7.147 89100.58116-C Dichlorvos7.2 1.014 1.372180.53118-SG85 7.48.715 85180.77114-C294 25 8.562 9821 1.1012Endosulfan sulfate8.60.910 1.586170.52 7b10-C 115 14 12 21 77180.8111415 56 14 111 8327b 1.47b11Imazalil7.60.89.8 3.17641b 1.22b1150 2.5 4.915 67b30b 1.19108-C432 53 12 161 7837b 2.06b11Imidacloprid8.80.88.9 3.08834b 1.041345 3.57.78.999200.7813218 18 8.224 97110.56128-SG Linuron9.9 1.717b 2.99929b0.901199 7.47.415 99150.6712971 65 6.7191 9720 1.23b12Methamidophos10 2.929b 3.0101 30b0.95 95-SG80 8.010 14 80180.7712852 72 8.4119 85140.85118-SG Methomyl9.3 1.212 2.99332b0.981250 3.3 6.79.3100 190.7413204 10 4.926 102 130.6313Procymidone8.20.78.2 2.082240.74115-SG64 6.09.416 8524 1.0113428 16 3.870 86160.90129-C Pymetrozine 6.2 1.220b 1.662b27b0.771147 3.1 6.79.662b200.8111341 20 5.859 68b170.9211Tebuconazole9.2 1.112 1.292130.41123&4-DG63 5.58.78.884140.5813439 29 6.784 8819 1.0613Tolylfluanid7.9 1.012 3.17939b 1.191334 4.313 13 67b37b 1.41b13144 13 8.842 7229b 1.37b13Trifluralin7.80.78.5 1.878230.681210-C58 3.7 6.414 7725 1.0213379 19 5.148 76130.69106-C, 4-C, 11-SG aC = Cochran outlier; SG = single Grubbs outlier; DG = double Grubbs outliers.bRSD r >15%; 120% < Rec. < 70%; RSD R >25%; HorRat >1.2; or fewer than 8 laboratories in an assessment.cCyprodinil was incurred in the grapes and affected quantitation.ã 2007 AOAC IN T ER N A T IONALAnalyte Avg. C, ng/g s r, ng/g RSD r, %s R, ng/g Rec., %RSD R, %HorRat No. of labs Outlier labs Atrazine9.9 1.515 1.899180.561170 7.9111593210.8812930 50 5.4166 9318 1.11115-C Azoxystrobin10 0.8 7.6 1.8102 180.561247 2.4 5.2 6.593140.5512531 32 6.188106 170.9412Bifenthrin9.10.8 9.1 1.491160.481166 8.012 9.488140.5911217 27 123387150.7711Carbaryl9.4 1.112 2.094220.671292 6.1 6.7 9.092 9.80.43118-SG589 38 6.4127 9822 1.24b12Chlorothalonil 6.20.814 2.062b32b0.93 6b28 10 37b147048b 1.77b 7b684 134 20b205 68b30b 1.77b 6bChloropyrifos9.0 2.124b 2.39026b0.79 912-SG, 10&11-DG86 9.411208623 1.011111-SG179 18 103090170.821111-SG Cyprodinil9.7 1.010 1.497140.441111-SG44 2.7 6.1 8.989200.791111-SG848 61 7.2117 85140.84108-SGo,p¢-DDD8.90.6 7.0 1.989210.66 881 4.8 5.91281150.63 911-C214 19 8.72786130.6210Dichlorvos 5.2 1.020b 2.452b45b 1.29b1258 6.6111277200.8112838 50 6.0224 8427 1.63b11Endosulfan sulfate 5.6 3.359b 2.556b45b 1.28b 2b38 9.625b157539b 1.48b 7b769 330 43b312 7740b 2.44b 7bImazalil7.60.3 3.5 3.57639b 1.18 82-C72 3.7 5.22457b33b 1.39b11589 47 7.9229 59b39b 2.25b11Imidacloprid c22 1.3 6.2 1.7100 7.90.28118-SG84 6.2 7.4 8.197 9.60.4112515 21 4.253101 100.58115-C Linuron8.6 1.112 1.586170.531146 2.2 4.9 7.491160.63102-C234 14 5.82594100.5311Methamidophos8.80.8 8.5 1.388150.46 86-C66 4.5 6.91282180.7211538 37 6.86384120.67 95-C, 8-SG Methomyl9.70.8 8.6 1.096100.321099 8.0 8.1 6.499 6.50.29102-SG997 24 2.4168 100 17 1.0511Procymidone10 0.6 6.2 2.2101 220.68 82-C92 8.5 9.21592170.7311967 118 12129 97130.8311Pymetrozine 6.90.4 6.1 1.469b200.591033 1.6 4.7 4.667b140.51 911-C127 8.5 6.71763b130.6110Tebuconazole9.70.7 6.9 1.297130.40114-C89 6.8 7.71189120.5212948 42 4.4226 9524 1.48b114-C Tolylfluanid 3.7 1.130b 2.237b59b 1.59b 4b9.3 3.740b 4.1 9.3b44b 1.37b 83-SG, 8-SG142 22 158614b61b 2.84b 812-C, 3&8-DG Trifluralin10 1.413 1.7103 170.541142 4.511 9.084220.8311169 25 153084180.8411aC = Cochran outlier; SG = single Grubbs outlier; DG = double Grubbs outliers.bRSD r >15%; 120% < Rec. < 70%; RSD R >25%; HorRat >1.2; or fewer than 8 laboratories in an assessment.cImidacloprid was incurred in the lettuces unbeknownst to the SD.ã 2007 AOAC IN T ER N A T IONALAnalyte Avg. C, ng/g s r, ng/g RSD r, %s R, ng/g Rec., %RSD R, %HorRat No. of labs Outlier labs Atrazine 8.9 1.011 1.989210.65112-C 908.29.11290130.5712187 19 10 2793140.6912Azoxystrobin 8.4 1.316b 1.884210.651165 5.28.0 8.186120.5212853 35 4.18285 9.60.591111-C Bifenthrin 9.7 2.324b 2.397240.75109-SG45 2.5 5.6 6.891150.59102-C488 51 10 7698160.8712Carbaryl 8.40.67.3 2.184250.771066 5.07.51488210.8811172 8.8 5.13486200.9512Chlorothalonil 4.80.816b 2.748b56b 1.57b 3b7014 20b297042b 1.74b 6b330 137 42b131 66b40b 2.09b 7bChloropyrifos11 1.614 5.0111 45b 1.58b 92-C82 4.5 5.61282150.64109-C953 97 10 284 9530b 1.85b1211-C Cyprodinil 8.70.910 2.087230.721256 4.58.0 9.075160.6512199 12 6.23580180.8612o,p¢-DDD 9.10.67.2 1.891200.60 99-C74 5.1 6.9 9.899130.561010-C967 81 8.41919720 1.22b11Dichlorvos 9.30.88.1 1.093110.35 7b12-C43 2.2 5.2 8.085190.73 812-SG446 22 5.05489120.6810Endosulfan sulfate12 5.444b 5.4124b43b 1.40b 4b3-SG 8319 23b198323 1.0110240 35 15 618025 1.28b10Imazalil c22 1.77.7 6.29628b0.98 87-C58 4.37.41392220.91 9186 9.7 5.2418722 1.0610Imidacloprid10 1.110 2.8104 27b0.861193 6.57.01293130.5711989 64 6.5124 99130.7811Linuron 7.8 1.317b 2.77835b 1.041160 3.0 5.01386210.8611387 26 6.64279110.59 9111-DG Methamidophos 9.2 1.112 1.592160.49 89-C42 3.58.2 5.685130.52 84-C211 12 5.53185150.73 94&9-DG Methomyl 8.50.88.9 2.88533b0.99 97-C68 4.87.0 8.791130.5412492 19 3.96098120.6912Procymidone110.98.1 3.9108 36b 1.15 812-C43 3.58.0 5.886140.531010-C170 16 9.72585150.7111Pymetrozine 7.5 1.318b 2.17528b0.821077 5.97.71077140.5710789 38 4.8117 79150.89 912-C Tebuconazole 8.70.78.0 1.287140.421141 2.2 5.4 6.282150.5812177 14 7.92888160.7612Tolylfluanid 5.8 1.220b 1.458b240.69 911-SG 467.516b1461b31b 1.21b119-C356 54 15 134 7138b 2.02b12Trifluralin 8.60.4 4.5 2.48628b0.87 99-C 928.69.41192120.5412915 60 6.5194 9221 1.31b116-CaC = Cochran outlier; SG = single Grubbs outlier; DG = double Grubbs outliers.bRSD r >15%; 120% < Rec. < 70%; RSD R >25%; HorRat >1.2; or fewer than 8 laboratories in an assessment.cImazalil was incurred in the oranges unbeknownst to the SD.ã 2007 AOAC IN T ER N A T IONALIn Option A, if the laboratory had LVI capability, then 1 or 2 mL extracts were taken for dispersive-SPE (the volume depended on the analyst preference and the type of centrifuge and tubes available in the laboratory). The final extract volume was 0.5 mL if 1 mL was taken for dispersive-SPE, and 1 mL if 2 mL underwent the cleanup step. In either case, two 15 g blank samples were used for the matrix blank (0-standard) and 6 matrix-matched calibration standards (5, 10, 50, 100, 250, and 1000 ng/g equiv alent concentrations). For dispersive-SPE of the matrix blanks, either 7 separate tubes using the same 1–2 mL extract volumes as the test samples could have been used, or 1–2 dispersive-SPE tube(s) with 7-fold greater extract volume(s).In Option B, if LVI is not available for GC/MS, then »30 mL of matrix blank extract was needed after dispersive-SPE cleanup to prepare the matrix-matched calibration standards (or $60 mL initial extract). In this case, 6 matrix blanks of 15 g each were extracted along with the test samples to provide enough blank extract volume, which were combined, and seven 8 mL aliquots were distributed to 7 dispersive-SPE tubes containing 0.4 g PSA + 1.2 g anhydrous MgSO4.B. Apparatus and ConditionsNote: Tables 4 and 5 of the collaborative study [J. AOAC Int. 90, 485(2007)] list the analytical instrumentation and sources of sample preparation materials used by each laboratory in the study. Further information appears in the full report. Since the time of the collaborativ e study, at least 3 v endors, United Chemical Technologies (Bristol, PA, USA), Restek (Bellefonte, PA, USA) and Supelco (Bellefonte, PA, USA) hav e introduced commercial dispersive-SPE products for QuEChERS and other applications. See Table 4 [J. AOAC Int. 90, 485(2007)] for sources of analytical instruments.(a) Gas chromatograph/mass spectrometer.—An ion trap, quadrupole, time-of-flight (TOF), or other GC/MS instrument may be used with electron impact (EI) ionization, an autosampler (AS), and computerized instrument control/data collection. Either LVI of 8 m L for a 1 g/mL MeCN extract (e.g., 75°C ramped to 275°C at 200°C/min) or 2 m L splitless injection of 4 g/mL extracts in toluene a t250°C m a y b e u s e d.A3–5m,0.25m m i d, phenylmethyl-deactivated guard column must be used as a retention gap in either case. The analytical column is a 30 m, 0.25 mm id, 0.25 m m film thickness (5%phenyl)-methylpolysiloxane (low bleed) analytical column (DB-5ms or equiv alent). Set He head pressure on the column to be 10 psi or constant flow to be 1.0 mL/min with systems capable of electronic pressure/flow control. After an appropriate time for solv ent delay, use an appropriate ov en temperature program, for example, starting at 75°C for MeCN extracts or 100°C for toluene ramped to 150°C at 25°C/min, then to 280°C at 10°C/min, and hold for 10 min. All collaborators had much experience in pesticide residue analysis and were free to use their own analytical conditions provided that peak shapes were Gaussian, peak widths at half heights were <5 s, and signal-to-noise ratio (S/N) of the quantitative ion for the pesticides at 10 ng/g equivalent concentrations in the sample were >10. For qualitative purposes (which were not the focus of this study), at least 3 ions yielding relativ e abundances that reasonably match a contemporaneously analyzed reference standard are typically needed to make an analyte identification.(b) Liquid chromatograph/tandem mass spectrometer.— A triple quadrupole, ion trap, or other LC/MS/MS instrument may be used provided it is capable of electrospray ionization (ESI) in the positive mode with computerized instrument control/data collection and has an AS. An injection volume (5–100 m L) will be determined for each instrument to achieve S/N > 10 for the quantitation ion for a 10 ng/g equivalent sample concentration. As in GC/MS, the collaborators had much experience in the analysis of pesticides and were free to use their own conditions. Suggested LC conditions, howev er, include a 15 cm long, 3.0 mm id, 3 m m particle size C18 column, flow rate of 0.3 mL/min, and gradient elution with an initial condition of 25% MeOH in 5 mM formic acid solution taken linearly in 15 min to 90% MeOH in 5 mM formic acid solution and held for 15 min. A short C18 guard column must be used to protect the analytical column, and a bypass valve must be used before the MS instrument to av oid introduction of the early and late eluting nonanalyte components into the detector. The MS/MS conditions were optimized in each laboratory using direct infusion into the ESI source to prov ide highest S/N for the quantitation ion of each LC-type analyte from a single MS/MS transition. A second transition with reasonably matching relative abundance ratios vs a contemporaneously analyzed reference standard is typically needed for qualitative purposes.(c) Centrifuge(s).—Capable of holding the 50 mL centrifuge tubes or bottles used for extraction and 10–15 mL graduated centrifuge tubes or 2 mL mini-tubes used in dispersiv e-SPE. Determine the rpm settings that yield a given relative centrifugal force (RCF), and ensure that maximum ratings of the centrifuge, tube/bottles, and rotors for the instrument are not exceeded.(d) Balance(s).—Capable of accurately measuring weights from0.05 to 100 g within ±0.01 g.(e) Freezer.—Capable of continuous operation <–20°C.(f) Furnace/oven.—Capable of 500°C operation.ã 2007 AOAC IN T ER N A T IONALTable 2007.01E. Averaged interlaboratory study results for the fortified and incurred pesticides aMatrix Recovery, %RSD r, %RSD R, %HorRat No. of labs (n) Grapes86 ± 1110 ± 422 ± 80.90 ± 0.2912 ± 1 Lettuces87 ± 1210 ± 720 ± 90.83 ± 0.4510 ± 1 Oranges87 ± 1510 ± 620 ± 80.84 ± 0.3710 ± 2 Overall87 ± 1110 ± 621 ± 80.86 ± 0.3711 ± 2Incurred NA b12 ± 422 ± 80.92 ± 0.3011 ± 2aData from fewer than 7 laboratories in an assessment were excluded.bNA = Not applicable.(g ) Food chopper and/or bl ender.—Preferably an s-blade vertical cutter (e.g. Stephan, Robotcoupe) and and probe blender (e.g. Ultra-Turrax, Propsep).(h ) Sol vent evaporator (optional ).—For the ev aporation of MeCN extracts, if LVI is not used in GC/MS.C. Reagents[See Table 5 [J. AOAC Int. 90, 485(2007)] for sources of chemicals.](a ) Anhydrous magnesium sul fate (MgSO 4).—Powder form;purity >98%; heated in bulk to 500°C for >5 h to remove phthalates and residual water.(b ) Acetonitril e (MeCN).—Quality of sufficient purity that is free of interfering compounds.(c ) Acetic acid (HOAc).—Glacial; quality of sufficient purity that is free of interfering compounds.(d ) 1% HOAc in MeCN.—Prepared on a v/v basis (e.g., 10 mL glacial HOAc in a 1 L MeCN solution).(e ) Anhydrous sodium acetate (NaOAc).—Powder form (NaOAc·3H 2O may be substituted, but 0.17 g per g sample must be used rather than 0.1 g anhydrous NaOAc per g sample).(f ) Primary secondary amine (PSA) sorbent.—40 m m particle size (Varian Part No. 12213024 or equiv alent). (Note : Premade dispersive-SPE tubes are now available from at least 3 vendors.)(g ) C 18 sorbent (optional ).—40 m m particle size, if samples contain >1% fat.(h ) G r a p h i t i z e d c a r b o n b l a c k (G C B ) s o r b e n t (optional ).—120/400 mesh size, if no structurally planar pesticides are included among the analytes.(i ) Helium.—Purity that has been demonstrated to be free of interfering compounds in GC/MS.(j ) Toluene (optional ).—Quality of sufficient purity that is free of interfering compounds; only needed if LVI is not used in GC/MS.(k ) Methanol (MeOH).—Quality of sufficient purity that is free of interfering compounds in LC/MS/MS prepared in mobile phase solution.(l ) Water.—Quality of sufficient purity that is free of interfering compounds in LC/MS/MS.(m ) Formic acid.—Quality of sufficient purity that is free of interfering compounds in LC/MS/MS prepared in mobile phase solution.(n ) Pesticide standards .—High purity reference standards of the pesticide analytes, and quality control (QC) and internal standards (ISs) prepared at highly concentrated stock solutions (e.g.,2000 ng/m L) in MeCN with 0.1% HOAc. Stored in dark vials in the freezer. Check annually for stability.(o ) Standard sol utions.—Prepared in MeCN for all collaborators: IS solution = 40 ng/m L of both d 10-parathion and d 6-a -HCH in MeCN; triphenylphosphate (TPP) solution = 2 ng/m L TPP in 1% HOAc in MeCN solution; QC-spike solution = 40 ng/m L of the 27 pesticide analytes in 0.1% HOAc in MeCN; and individual test solutions = 10 ng/m L of each of the 30 compounds to be detected (except 40 ng/m L TPP) in 0.1% HOAc in MeCN solution.Collaborators prepared a test mix and calibration standard spike solutions from those provided as described in E .(p ) Bl ank sampl e .—Verified to be free of analytes abov e the detection limit.(q ) Other reagents.—Certain instruments may require nitrogen or other materials/devices for their operation.D. Materials(a ) Fl uorinated ethyl ene propyl ene (FEP) centrifugetubes.—50 mL; e.g., Nalgene Part No. 3114-0050 or equivalent for <16 g sample (or 250 mL FEP centrifuge bottles for 16–75 g sample size).(b ) Spatul a/spoon and funnel .—For transferring sample into centrifuge tubes.(c ) Solvent dispenser and 1–4 L solvent bottle.—For transferring 15 mL 1% HOAc in MeCN per 15 g sample in FEP centrifuge tubes or bottles.(d ) Centrifuge tubes (optional ).—10–15 mL graduated. For evaporation and/or dispersive-SPE.(e ) Mini-centrifuge tubes (optional).—2 mL. For dispersive-SPE (use tubes with o-ring-sealed caps to avoid leaks).(f ) Syringes/pipets.—Capable of accurate sample introduction of 2 or 8 m L volume into GC/MS and appropriate volumes of matrix spike, IS, and calibration standard solutions (12.5–300 m L).(g ) Repeating or vol umetric pipets.—Capable of accurately transferring 0.5–8 mL solvent.(h ) Containers .—Graduated cylinders, volumetric flasks, weigh boats, v ials, and/or other general containers in which to contain samples, extracts, solutions, standards, and reagents.E. Preparation of Reagent Materials and Comminuted Sample(1) Prepare the necessary number of sealable vials/cups containing 6.0 ± 0.3 g anhydrous MgSO 4 + 1.5 ± 0.1 g anhydrous NaOAc (or 2.5ã 2007 AOAC IN T ER N A TIONALFigure 2007.01. Outline of the QuEChERS protocol used in the collaborative study.± 0.2 g NaOAc·3H2O) per 15 g sample. Scoops of appropriate volume can be used to speed the process, but weighing should still be done to check consistency. The containers should be sealed during storage and can be refilled and re-used without cleaning in between usages.(2) Prepare the necessary number of appropriate centrifuge tubes (2 mL mini-centrifuge tubes or 10–15 mL centrifuge tubes) containing 0.05 ± 0.01 g PSA sorbent + 0.15 ± 0.03 g anhydrous MgSO4 per 1 mL extract taken for dispersive-SPE cleanup. (Note: At least United Chemical Technologies, Restek, and Supelco now provide dispersive-SPE products commercially to replace this step.) If LVI is not available for GC/MS, then evaporation of the extracts will be needed, and 8 mL extract will be transferred to 10–15 mL sealable centrifuge tubes containing 0.40 ± 0.08 g PSA sorbent + 1.20 ± 0.24 g anhydrous MgSO4. For matrixes that contain >1% fat, add an additional 0.05 ± 0.01 g C18 sorbent per mL extract to the container. If no planar pesticides are among the analytes (e.g., thiabendazole, terbufos, quintozene, and hexachlorobenzene), then 0.05 ± 0.01 g GCB sorbent per mL extract can also be added to the tube. (Note: Final extract volume may have to be reduced to 0.4 mL per 1 mL aliquot in dispersive-SPE if all 4 powders are used.) (3) Prepare 1% HOAc in MeCN in dispenser bottle by adding 10 mL HOAc to 990 mL v olume of MeCN or different desired amount in the same ratio.(4) Label all vials and tubes appropriately that will be used in the method.(5) Note: Step 5 was conducted by the Study Director (SD) when preparing the test samples. An appropriate chopper must be used to comminute large, representative sample portions. An uncommon or deuterated pesticide standard may be spiked into the sample during homogenization to determine the effectiv eness of the procedure. Blend the sample until it gives a consistent texture. Transfer .200 g to a sealable container for freezer storage after further homogenization with a probe blender. Blend this subsample with the mixer until it is homogeneous. The test portion (e.g., 15 g) is taken for extraction immediately, and the container is then sealed and stored in the freezer in case re-analysis is necessary. The advantages of this approach are that the 15 g portion is highly representative of the original sample, the sample is well-comminuted to improv e extraction by shaking, less time is spent on the ov erall homogenization process than trying to prov ide equiv alent homogenization of the large initial sample with the chopper, and a frozen subsample is available for re-analysis if needed.To provide the most homogeneous comminuted samples, frozen conditions, sufficient chopping time, and appropriate sample size to chopper volume ratio should be used. Use of frozen samples also minimizes degradative and volatilization losses of certain pesticides. In this case, cut the sample into 2–5 cm3 portions with a knife and store the sample in the freezer prior to processing. Cryogenic blending devices, liquid nitrogen, or dry ice may also be used (but make sure all dry ice has sublimed before weighing samples and ensure that water condensation is minimal, especially in a humid environment). (6) For laboratories with LVI in GC/MS, prepare a test mix of the pesticides in MeCN + 0.1% HOAc to determine the retention times (t R) and MS quantitation/diagnostic ions at the particular GC/MS conditions to be used in the analysis [see Table 2 of the collaborative study (J. AOAC Int. 90, 485(2007)].The preparation of the test mix and calibration spiking standards are described as follows:(1) Test mix in MeCN + 0.1% HOAc.—4 ng/m L in 10 mL of all 30 compounds to be analyzed. Add 1 mL each of QC-spike solution + IS solution + TPP test solution + 1% HOAc in MeCN and fill to 10 mL with MeCN. Calibration spike standards in MeCN for 27 pesticide analytes (make 10 mL each in volumetric flasks, then transfer to 15 mL dark glass vials and store in freezer).(2) Cal-standard-1000.—20 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 5 mL QC-spike solution + 1 mL IS solution + 1 mL 1% HOAc in MeCN and fill to the mark with MeCN.(3) Cal-standard-250.—5 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 1.25 mL QC-spike solution + 1 mL IS solution + 1 mL 1% HOAc in MeCN and fill to the mark with MeCN.(4) Cal-standard-100.—2 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 500 m L QC-spike solution + 1 mL IS solution + 1 mL 1% HOAc in MeCN and fill to the mark with MeCN.(5) Cal-standard-50.—1 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 250 m L QC-spike solution + 1 mL IS solution + 1 mL 1% HOAc in MeCN and fill to the mark with MeCN.(6) Cal-standard-10.—0.2 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 50 m L QC-spike solution + 1 mL IS solution + 1 mL 1% HOAc in MeCN and fill to the mark with MeCN.(7) Cal-standard-5.—0.1 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 25 m L QC-spike solution + 1 mL IS solution + 1 mL 1% HOAc in MeCN and fill to the mark with MeCN. For laboratories without LVI in GC/MS, the preparation of the test mix and the calibration spiking standards are described below: (1a) Test mix for GC in tol uene.—4 ng/m L in 10 mL of all 30 compounds to be analyzed. Add 1 mL QC-spike solution + 1 mL IS solution + 1 mL TPP test solution and fill to 10 mL with toluene. Calibration spike standards in MeCN for LC/MS/MS (in dark glass AS vials stored in freezer).(2a) Cal-standard-1000.—20 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 500 m L QC-spike solution + 100 m L IS solution + 100 m L 1% HOAc in MeCN + 320 m L MeCN.(3a) Cal-standard-250.—5 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 125 m L QC-spike solution + 100 m L IS solution + 100 m L 1% HOAc in MeCN + 695 m L MeCN.(4a) Cal-standard-100.—2 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 50 m L QC-spike solution + 100 m L IS solution + 100 m L 1% HOAc in MeCN + 770 m L MeCN.Dilute QC-spike solution.—4 ng/m L. Transfer 100 m L QC-spike solution to AS vial and add 900 m L MeCN.(5a) Cal-standard-50.—1 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 250 m L dilute QC-spike solution + 100 m L IS solution + 100 m L 1% HOAc + 570 m L MeCN.(6a) Cal-standard-10.—0.2 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 50 m L dilute QC-spike solution + 100 m L IS solution + 100 m L 1% HOAc and 770 m L MeCN.(7a) Cal-standard-5.—0.1 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 25 m L dilute QC-spike solution + 100 m L IS solution + 100 m L 1% HOAc + 795 m L MeCN.Calibration spike standards in toluene.—Make 10 mL each in volumetric flasks, then transfer to 15 mL dark glass vials and store in freezer.(8a) Cal-standard-1000-tol.—20 ng/m L of each pesticide + 4 ng/m L IS in toluene. Add 5 mL QC-spike solution + 1 mL IS solution and fill to the mark with toluene.ã 2007 AOAC IN T ER N A T IONAL。

液相色谱-串联质谱法检测玩具中的3种异噻唑啉酮类防腐剂

液相色谱-串联质谱法检测玩具中的3种异噻唑啉酮类防腐剂杨荣静;卫碧文;于文佳;高欢;孙晓娟【摘要】建立了液相色谱-串联质谱快速测定玩具中异噻唑啉酮类防腐剂(2-甲基-4-异噻唑啉-3-酮、5-氯-2-甲基-4-异噻唑啉-3-酮和1,2-苯并异噻唑啉-3-酬)的分析方法.样品经去离子水超声提取后进行液相色谱-串联质谱分析.色谱流动相为甲醇-水(15：85,v/v),等度洗脱,在选择反应监测(SRM)模式下定性和定量分析.3种被分析物的工作曲线线性范围均为2.0～1 000μg/L,方法的定量限(信噪比大于10)为0.04 mg/kg,灵敏度优于欧盟玩具协调标准EN71-11-2005中推荐的方法.两种类型玩具样品中的加标回收率分别为95.9％～105.2％和94.7％～102.8％,精密度分别为3.04％～4.96％和2.36％～4.79％.应用本方法对10种玩具样品进行了测试,结果完全能满足欧盟玩具协调标准EN71-9-2005对玩具中异噻唑啉酮类防腐剂的检测要求.%A rapid analytical method for the determination of 3 isothiazolinone preservatives (2-methyl-4-isothiazolin-3-one, 5-chloro-2-methyl-4-isothiazolin-3-one and 1,2-benzylisothiazolin-3-one) in toys using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. After ultrasonic extraction with water, the analytes in the sample were separated and analyzed by LC-MS/MS under the isocratic elution of methanol and water (15: 85 , v/v) and in selected-reaction monitoring (SRM) mode. The linear ranges of calibration curves for the 3 analytes were 2. 0 - 1 000 jjig/L. The limit of quantification was 0. 04 mg/kg for all the 3 analytes, which was lower than that of the method recommended by the European Toy Safety Directive in EN71-11-2005. The recoveries of the spiked standards in the two.toy samples were 95. 9% -105. 2% and 94. 7% - 102. 8% with the relative standard deviation ranges from 3. 04% to 4. 96% and from 2. 36% to 4. 79%, respectively. The method was applied in the determination of 10 toy samples, and the results can meet the requirements of the European Toy Safety Directive in EN71-9-2005 for the determination of isothiazolinones in toys.【期刊名称】《色谱》【年(卷),期】2011(029)006【总页数】4页(P513-516)【关键词】液相色谱-串联质谱;异噻唑啉酮;防腐剂;玩具【作者】杨荣静;卫碧文;于文佳;高欢;孙晓娟【作者单位】上海出入境检验检疫局,上海,200135;上海出入境检验检疫局,上海,200135;上海出入境检验检疫局,上海,200135;上海出入境检验检疫局,上海,200135;赛默飞世尔科技(中国)有限公司,上海,201206【正文语种】中文【中图分类】O658异噻唑啉酮类化合物是一种广谱、高效、非氧化性杀菌防腐剂，通过杀灭细菌或者防止微生物繁殖而起到防腐的效果，在家用工业中常用作皮革清洗剂、黏合剂、纺织品柔软剂和上光剂，在水基工业中常用作杀黏菌剂［1－6］。

固相萃取-超高效液相色谱-串联质谱法快速测定环境水样中30种极性农药

赵彬1,张付海1,张敏1,姚帮本2
1. 安徽省生态环境监测中心( 安徽省重污染天气预报预警中心) ,安徽合肥 230071 2. 安徽省产品质量监督检验研究院食品化工质检所,安徽合肥 230051
摘要:建立了固相萃取-超高效液相色谱-串联质谱快速测定环境水样中 30 种极性农药的方法。 30 种极性农药经过固相萃取( SPE) 富集净化,以超高效液相色谱-串联质谱 ( UPLC-MS-MS) 多级监测模式 ( MRM) 外标法进行定性定量分析。结果表明:环境水样中 30 种极性农药的检出限为 0. 2 ~ 5 ng / L。对同一环境样品进行了低、中、高 3 个不同浓度水平的加标回收实验,平均回收率为 63. 7% ~ 105. 1% ,相对标准偏差为 4. 4% ~ 21. 2% 。该方法快速、灵敏、准确,可有效应用于环境水样中 30 种极性农药的快速监测。关键词:极性农药;固相萃取;超高效液相色谱-串联质谱中图分类号:X832. 02 文献标志码:A 文章编号:1002-6002( 2021) 03- 0169- 10 DOI:10. 19316 / j. issn. 1002-6002. 2021. 03. 20
采集 2 L 环境水样品于棕色广口玻璃瓶中, 用甲酸调至 pH 为 5 ~ 6,加入 0. 1 g 抗坏血酸除去残余氯,在 4 ℃ 冷藏避光保存,2 d 内分析。 1. 3 样品的提取与净化
用 10 mL 甲醇活化固相萃取柱,再用 10 mL 超纯水活化小柱, 保证小柱柱头浸润。量取 500 mL 混匀水样,以 8 mL / min 的流速通过小柱富集,再用 10 mL 纯水淋洗小柱,将小柱上保留较弱的杂质淋洗下来。用 10 mL 乙腈-二氯甲烷溶液(体积比为 4 ∶1)洗脱富集后的小柱,洗脱液收集于接收管中。加入 2 g 无水硫酸镁于上述洗脱液脱水,氮吹浓缩至近干,用乙腈-0. 1% 甲酸水溶

桃金娘花青素的抗氧化能力就和主要花色苷的鉴定

Antioxidant capacity of anthocyanins from Rhodomyrtus tomentosa (Ait.)and identiﬁcation of the major anthocyaninsChun Cui a ,Shaomin Zhang a ,Lijun You a ,Jiaoyan Ren a ,Wei Luo b ,Wenfen Chen a ,Mouming Zhao a ,⇑a College of Light Industry and Food,South China University of Technology,Guangzhou 510640,China bAnalysis and Testing Center,South China University of Technology,Guangzhou 510640,Chinaa r t i c l e i n f o Article history:Received 30October 2012Received in revised form 5January 2013Accepted 28January 2013Available online 10February 2013Keywords:Rhodomyrtus tomentosa AnthocyaninsAntioxidant activity Cyanidin-3-O-glucosidea b s t r a c tThe anthocyanins in the fruits of Rhodomyrtus tomentosa (ACN)were extracted by 1%TFA in methanol,and then puriﬁed by X-5resin column and C 18(SPE)cartridges.The puriﬁed anthocyanin extract (ART)from the fruits of R.tomentosa showed strong antioxidant activities,including DPPH radical-scavenging capacity,ABTS radical scavenging capacity,reducing power and oxygen radical absorbance capacity (ORAC).The puriﬁed anthocyanin extract was analyzed by high performance liquid chromatography (HPLC).The major anthocyanins were puriﬁed by semi-preparative HPLC and Sephadex LH-20column chromatography,and were identiﬁed as cyanidin-3-O-glucoside,peonidin-3-O-glucoside,malvidin-3-O-glucoside,petunidin-3-O-glucoside,delphinidin-3-O-glucoside and pelargonidin-3-glucoside by HPLC–ESI/MS and nuclear magnetic resonance spectroscopy (NMR).Cyanidin-3-O-glucoside was consid-ered as the most abundant anthocyanin,which was 29.4mg/100g dry weight of R.tomentosa fruits.Addi-tionally,all the major anthocyanins were identiﬁed from R.tomentosa fruit for the ﬁrst time.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionRhodomyrtus tomentosa (Ait.)Hassk,a member of the Myrtaceae family,commonly known as rose myrtle,is an abundant evergreen shrub native to southeast Asia,with rose-pink ﬂowers and dark-purple edible bell-shaped fruits (Amporn,Tony,&John,2005).The stem,leaf,fruits of the whole plant can be used as medical materials.The R.tomentosa fruit possesses excellent pharmacolog-ical properties,including antibacterial activity against Gram-posi-tive bacteria,such as Streptococcus pyogenes and Escherichia coli (Dachriyanus et al.,2002;Surasak &Supayang,2008).R.tomentosa fruit is widely distributed in south China;its bright purplish-red colour is due to anthocyanins.Anthocyanins,an important group of water-soluble pigments in natural products,are widely spread in ﬂowers,fruits and leaves.They usually link with sugar moieties and constitute ﬂavonoids,attracting more and more attention due to their usage as natural food additives and excellent functional properties for human health (Kaliora,Dedoussis,&Schmidt,2006;Li,Wang,Guo,&Wang,2011).On the basis of their structural characteristics,anthocyanins possess various biological activities,including antioxidant (Cerezo,Cuevas,Winterhalter,Garcia-Parrilla,&Troncoso,2010),anticancer (Wang &Stoner,2008),anti-inﬂammatory (Greenspan et al.,2005),anti-artery atherosclerosis,anti-hypertensive (Pinent et al.,2004)and antibacterial activities (Lacombe,Wu,Tyler,&Edwards,2010).In recent decades,the antioxidant activities of anthocyanin and its working mechanism have attracted growing global interest.As re-ported,anthocyanin might play its protective role through the working system of H atom transfer,single electron transfer and metal chelation (Monica,Nino,&Marirosa,2011).However,to our knowledge,the information regarding the antioxidant capacity and major anthocyanins of R.tomentosa is limited.The objectives of the present study were to extract and purify the anthocyanins from the fruit of R.tomentosa and to evaluate their antioxidant capacity.The major anthocyanins were further isolated by semi-preparative HPLC and column chromatography,and identiﬁed by HPLC–ESI–MS and NMR spectroscopy.2.Materials and methods 2.1.Plant materialThe wild-grown mature fruits of R.tomentosa (Ait.)Hassk were collected in Shanwei,Guangdong Province,China,in August (Fig.1),freeze-dried after being washed with clean sterile water,and then stored at À20°C prior to extraction.2.2.Chemicals2,20-Azobis (2-methylpropionamidine)dihydrochloride (AAPH),2,2-diphenyl-1-picrylhydrazyl (DPPH),2,20-azino-bis (3-ethylbenz-thiazoline-6-sulphonic acid)(ABTS),6-hydroxy-2,5,7,8-tetra-methyl-2-chromanecarboxylic acid (trolox),sodium ﬂuorescein,0308-8146/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.foodchem.2013.01.107Corresponding author.Tel./fax:+862087113914.E-mail address:femmzhao@ (M.Zhao).30,60-dihydroxyspiro[isobenzofuran-1[3H],90[9H]-xanthen]-3-one (FL),ascorbic acid,CF3COOD and CD3OD were purchased from Sig-ma Chemical Co.(St.Louis,MO,USA).X-5resins were obtained from Haiguang Chemical Co.Ltd.,(Tianjin,China).Sephadex LH-20was purchased from Pharmacia Fine Chemicals Co.(Uppsala,Sweden) and Sep-Pak cartridges were purchased from Waters Co.,(Milford, Bedford,MA,USA).All the solvents for HPLC analysis were of HPLC grade.All the other chemicals used were of analytical grade.2.3.Extraction of anthocyaninsFree-dried sample(300g)was macerated with1000ml of TFA (triﬂuoroacetic acid):methanol(1:99;v/v)for48h in the dark at room temperature,and the remaining residues were extracted by 400ml of the extract solvent to extract anthocyanin.The superna-tants were obtained by centrifugation(8000g,15min)in a GL-21M refrigerated centrifuge(Xiangyi Instrument Co.Ltd.,Changsha,Chi-na)andﬁltration.Finally,the acidic methanol extracts were com-bined and evaporated,using a rotary evaporator(RE-52AA, Yarong Instrument Factory,Shanghai,China)at40°C.2.4.Puriﬁcation of anthocyaninsThe concentrated crude extract was puriﬁed by partition(sev-eral times)against ethylacetate and chloroform to remove non-po-lar compounds.The partial puriﬁed aqueous(10ml each time) phase was then subjected to a X-5resin column(1.6Â40cm)to remove free sugars,aliphatic acids and other water-soluble com-pounds by washing(several times)with the column volume of dis-tilled water.The adsorbed anthocyanins were eluted using methanol containing0.1%triﬂuoroacetic acid(TFA,v/v).The concentrated anthocyanin extract was further reﬁned by solid phase extraction(SPE)in C18cartridges(Sep pak,Waters). The aqueous extract of anthocyanin was passed through a sorbent C-18Sep-Pak cartridge(Waters Associates,Milford,MA)previously activated with acidiﬁed methanol(0.01%HCl v/v)and equilibrated with water.The extract adsorbed onto the cartridge was rinsed with ultra-pure water to remove water-soluble impurities,and then eluted with acidiﬁed methanol(0.01%HCl,v/v).The acidiﬁed methanol solution was evaporated under vacuum,redissolved in water,and lyophilized(R2L-100KPS,Kyowa Vacuum Engineering, Tokyo,Japan).The dry fraction was dissolved with deionized water. Samples wereﬁltered through a0.45l mﬁlter before analysis. 2.5.Isolation and identiﬁcation of the main anthocyanins from the puriﬁed extract2.5.1.IsolationThe puriﬁed extract,containing the major anthocyanin-derived pigments,was isolated by semi-preparative HPLC,using a Waters X-bridge reversed-phase C18column(5l m,10Â150mm,i.d.)at 35°C with aﬂow rate of 4.5ml/min and was monitored at 520nm.The solvents were(A),water/formic acid(98:2),and(B), formic acid/methanol(2:98),with the following gradient:10–15%B over5min,15–18%B over5min,18–23%B over20min, from23%to25%B over10min.and then each fraction was further chromatographed on a Sephadex LH-20column(1.0Â60cm), eluting with a mixture of methanol/water/triﬂuoroacetic acid at a ratio of20:79.5:0.5to obtain compounds1–6,respectively.2.5.2.Molecular weight determinationA MS system(Esquire HCT PLUS,Phenomenex,Torrance,CA, USA),equipped with a Hewlett–Packard1100series liquid chroma-tography system,was used to determine the molecular weight of each compound.One hundred microlitres of sample solution (100l g/ml)was injected into the MS system.Mass spectra in the positive-ion mode were generated under the following conditions: HV capillary=2800V;HV end plate offset=À500V;nebulizer pressure=10psi;dry temperature=300°C;dry gas=5.00l/min; m/z range=100–1000.A reversed-phase column(250Â4.6mm, 5l m,C18),thermostatted at35°C,was used for separation;sol-vents were(A)aqueous0.1%triﬂuoroacetic acid,and(B)100%ace-tonitrile,establishing the gradient as described in2.10.2.5.3.NMR identiﬁcationThe NMR results were obtained at400MHz and100MHz for1H and13C,respectively,on a Bruker AVANCE Spectrometer(Bruker DRX400,Bruker Biospin Co.,Karlsruhe,Germany)in the solvent, CF3COOD–CD3OD(5:95;v/v);coupling constants were expressed in Hertz,and chemical shifts were given on a d(ppm)scale with TMS(tetramethylsilane)or solvent signals as an internal standard.2.6.Determination of total anthocyanin content(TAC)TAC was determined according to the pH differential method by Kim,Jeong,and Lee(2003).Absorbance was measured at520and 700nm in buffers at pH1.0and4.5,using A=[(A520ÀA700)pH1.0-À(A520ÀA700)pH 4.5]and expressed as mg of cyanidin-3-glycoside (molar extinction coefﬁcient of26,900and molecular weight of 449.2)equivalents per100g of dry fruit weight.TAC was calcu-lated using the following equation.Data were reported as means±standard deviation of triplicate determinations.TACðmg=100gÞ¼AÂMWÂDFÂ1eÂLÂVMÂ100ð1Þwhere A is absorbance,e is cyanidin-3-O-glucoside molar absor-bance(26,900),L is the cell pathlength(1cm),MW is the molecular weight of cyanidin-3-glucoside(449.2Da),DF is the dilution factor, V is theﬁnal volume(ml),and M is the dry weight(mg). Fig.1.The fruit andﬂower of Rhodomyrtus tomentosa(Ait.)Hassk collected in Guangdong province.2.7.DPPH radical-scavenging activity assayDPPH radical-scavenging activity was measured according to the method of Wu,Chen,and Shiau(2003)with a slight modiﬁca-tion.Aliquots(2.0ml)of0(control),2,4,6,8,10,12,20and30l g/ ml of anthocyanin extract(ART)dissolved in distilled water were added to2.0ml of0.2mM DPPHÅthat was dissolved in95%etha-nol.The mixture was then shaken vigorously using a mixer(QT-1 Mixer,Tianchen Technological Co.Ltd.,Shanghai,China).The reac-tion mixture was incubated for30min at30°C in the dark.The absorbance of the resulting solution was recorded at517nm by a spectrophotometer(UV2100,Unico Instrument Co.,Ltd.,Shanghai, China).The scavenging activity was calculated using the following equation:Scavenging activityð%Þ¼ðA DPPHÅsampleÀA sample controlÞÂ100=A DPPHÅblankð2Þwhere A DPPHÅsample=absorance of2ml of sample solution+DPPH solution;A sample control=absorance of2ml of sample solution+2ml of95%ethanol;and A DPPHÅblank=absorbance of2ml of95%etha-nol+DPPHÅsolution.Ascorbic acid was used as the reference.IC50 value(l g compound mlÀ1),the concentration of extract that was required to scavenge50%of radicals,was calculated.2.8.AbtsÅ+radical cation-scavenging activity assayABTS radical cation-scavenging activity of anthocyanins was determined as described by Wang and Xiong(2005)with a slight modiﬁcation.The ABTSÅ+solution was prepared withﬁnal concen-trations of7mM ABTSÅ+and2.45mM potassium persulfate.The solution was incubated for16h at room temperature in the dark until the reaction was completed.Prior to the assay,the absor-bance of the ABTSÅ+solution at734nm was adjusted to 0.70±0.02by dilution with0.2M sodium phosphate-buffered sal-ine(pH7.4).Then40l l of ART(30,50,80,100,120,160,200l g/ ml)were added to4ml of diluted ABTSÅ+solution.The mixture was shaken vigorously for30s and allowed to stand in the dark for6min.An equivalent volume of distilled water,instead of the sample,was used for the blank.The absorbance of the resultant solution was measured at734nm.A standard curve was obtained by using trolox standard solution at various concentrations(0.4, 0.8,1.2,1.6,2.0,2.4mM)with ethanol.The standard curve was prepared by reacting40l l of trolox(0.4,0.8, 1.2, 1.6, 2.0, 2.4mM)with4ml of diluted ABTSÅ+solution.The degree of ABTS radical-scavenging activity of anthocyanins was calculated,based on the trolox standard curve,and was expressed in terms of mg trolox equivalents(TE)/mg anthocyanin.2.9.Reducing power assayThe reducing power of anthocyanins was determined according to the method of Oyaizu(1988)with a slight modiﬁcation.ART were dissolved in distilled water to obtain various concentrations (10,20,40,60,80,100l g/ml)for analysis.Sample solution (1ml)was mixed with2.5ml of sodium phosphate buffer(0.2M, pH6.6)and2.5ml of1%(w/v)potassium ferricyanide.The mixture was incubated at50°C for20min.Then,2.5ml of10%trichloro-acetic acid were added.After centrifugating at1000g for10min, 2.5ml of the supernatant were collected and mixed with2.5ml of distilled water and0.4ml of0.1%(w/v)ferric chloride in a test tube.After incubation at room temperature for10min,the absor-bance was measured at700nm.Distilled water,instead of the sample,was used as the blank.The reducing power of ascorbic acid was also assayed for comparison.Decreased absorbance,at 700nm,of the reaction mixture indicated decreased reducing capacity.The concentration of the test sample needed to raise the absorbance at700nm to0.5was evaluated.2.10.ORAC assayThe peroxyl radical-scavenging activity of ART was measured according to the method of You,Zhao,and Liu(2011).2.11.HPLC analysis of puriﬁed anthocyanin extractHPLC analyses were performed using a Waters600pump (Waters,Milford,MA)equipped with a Waters2998photodiode array detector at520nm.Separation was performed with a Waters C18column(5l m,250mmÂ4.6mm,i.d.)at30°C.Elution was carried out by using a gradient procedure with a mobile phase con-taining solvent A(0.1%TFA in water)and solvent B(acetonitrile)as follows:0–5min,10–15%B;5–10min,15–18%B;10–20min,18–20%B;20–25min,20–23%B;25–30min,23–10%B;40–45min; 10%B(Paola-Naranjo,JoséSánchez-Sánchez,&González-Paramás, 2004).The solventﬂow rate was1.0ml/min,and the injection vol-ume was20l l.2.12.Statistical analysisThe total anthocyanin content was measured in triplicate and data represent mean values±standard deviation(n=3).Samples were analyzed in triplicate and one-way analysis of variance per-formed using SPSS11.5(SPSS Inc.,Chicago,IL,USA).Signiﬁcant dif-ferences were detected at P<0.05.3.Results and discussion3.1.Total anthocyanin contentThe total anthocyanin content of the puriﬁed R.tomentosa ex-tract,determined by the pH differential method,was 62.8±1.2mg/100g of freeze-dried weight of R.tomentosa fruits, expressed as cyanidin-3-O-glucoside and reported as the average of three determinations.Cyanidin-3-O-glucoside was the major anthocyanin detected in large amount(47%),followed by peoni-din-3-O-glucoside(34%)and malvidin-3-O-glucoside(8%).3.2.Antioxidant activity3.2.1.Radical-scavenging activityDPPH and ABTS radical-scavenging activities of the puriﬁed anthocyanin extracted from R.tomentosa and ascorbic acid control are shown in Table1and Fig.2,respectively.As shown in Fig.2A,the puriﬁed anthocyanin extract showed strong DPPH radical-scavenging activity in a dose-dependent man-ner,and exhibited good DPPH radical-scavenging activity at the concentration of2l g/ml and it almost completely inhibited DPPH radicals(>90%)at a concentration of20l g/ml.The IC50value of DPPH radical-scavenging activity was6.27±0.25l g/ml(Table1). Furthermore,DPPH radical-scavenging activity was linearly corre-lated(positive)with the concentrations of anthocyanin extracts from0to10l g/ml,while ascorbic acid exhibited the activity from 2to30l g/ml,respectively.ABTSÅ+is often used for in vitro determination of free radical activity and the relative ability to scavenge the ABTSÅ+radical has been compared with the standard trolox.Fig.2B shows a steady in-crease in ABTS radical-scavenging capacity,up to a concentration of0.20mg/ml,that is equivalent to1.32mg/ml of trolox.The IC50 value of ABTS radical-scavenging activity was90.3±1.52l g/ml.It is noteworthy that both DPPH and ABTS radical-scavenging activities of the tested extract were higher than those of V CC.Cui et al./Food Chemistry139(2013)1–83(IC50=6.27±0.25l g/ml,206±2.37l g/ml)which is always con-sidered as an excellent tool for determining the antioxidant activ-ity of hydrogen-donating antioxidants and of chain breaking antioxidants.3.2.2.Reducing power activityReducing power is often used as an indicator of electron-donat-ing activity,which is an important mechanism for testing antioxida-tive action of phenolics.The reducing powers of ART and ascorbic acid control are shown in Table1and Fig.2C,respectively.As shown in Fig.2C,the tested ART extract exhibited high reducing power in a dose-dependent manner,but showed a slightly weaker ability com-pared with those of ascorbic acid.The value,raising the absorbance at700nm to0.5,of reducing power was51.7±0.74l g/ml(Table1). Furthermore,the reducing power was linearly correlated(positive) with the concentrations of anthocyanin extracts and ascorbic acid from0to0.08mg/ml.The corresponding correlation coefﬁcients were0.996for anthocyanin extracts(Y=7.591X+0.107)and 0.995for ascorbic acid(Y=15.17X+0.026),respectively.3.2.3.ORAC capacity testThe ORAC assay is the only antioxidant test that combines both inhibition time and degree of inhibition into a single quantity.The assay uses a biologically relevant radical source,and is also an assay where an added antioxidant competes with a substrate(ﬂuores-cein)for the radicals generated by thermal decomposition of azo compounds,like AAPH,and inhibits or retards substrate oxidation (Walton,Lentle,Reynolds,Kruger,&Mcghie,2006).As mentioned in2.9,ﬁnal ORAC values were expressed as l mol of trolox equiva-lents(TE)/mg of ART,and a higher ORAC value indicated stronger antioxidant activity.As shown in Table1,The ORAC value of the tested ART extract was9.29±0.08l mol TE/mg,which was signiﬁ-cantly higher than that of ascorbic acid(1.79±0.03l mol TE/mg). The result indicated that ART exhibited very good antioxidant activity.3.3.HPLC analysisUnder the optimital HPLC separation condition,a satisfactory separation of the puriﬁed anthocyanin extract of R.tomentosa was obtained and the HPLC chromatogram is shown in Fig.3A.The Major anthocyanins represented about99%of the total peak area with regard to the UV–Vis spectrum taken on-line during HPLC and chromatographic features.However,other minor peaks were also detected which had percentage areas of less than1%. No UV absorbance maxima in the310–320nm range were de-tected,indicating no acylation of anthocyanins with aromatic acids (Giusti&Jing,2001).3.4.Identiﬁcation of compoundsThe detailed MS data,including retention times,molecular ion peaks,MS2fragments and percent area at520nm,of all anthocya-nins are summarized in Table2.Fig.4shows the electrospray mass spectrum and the structures of the isolated anthocyanins.Two major peaks,peak2and peak5(P2,P5)were obtained as amorphous red powder.As shown in Table2,P2and P5showed peaks at449m/z and463m/z from ESI–MS,which were in accor-dance with the mass calculated for C21H21O11(449.1)and C22H23O11(463),on the basis of the MS2detected mass fragments at m/z287and m/z301related to the loss of one hexose ([MÀ162]+)molecule,respectively,and the UV–Vis spectrum of the two compounds showed the visible k max to be516nm withTable1Antioxidant activities of anthocyanins(ART)extracted from Rhodomyrtus tomentosa and ascorbic acid using the DPPHÅassay,ABTSÅ+assay,reducing power assay and ORAC test.Samples IC50/DPPHÅ(l g/ml)A IC50/TEAC(l g/ml)B A700nm=0.5/reducing power(l g/ml)C ORAC value(l mol TE/mg)ART 6.27±0.25b90.3±1.52b51.7±0.74a9.29±0.08aAscorbic acid17.4±0.31a206±2.37a31.3±0.93b 1.79±0.03bAll the trials were performed in triplicate(n=3)and all the data represent the means±standard deviation(n>3).Data in the same column with different letters are signiﬁcantly different(P<0.05).A The antioxidant activity was calculated as the concentration of the test sample needed to decrease the absorbance at517nm by50%.B The antioxidant activity was evaluated as the concentration of the test sample required to decrease the absorbance at734nm by50%.C The antioxidant activity was evaluated as the concentration of the test sample needed to raise the absorbance at700nm to0.5.4 C.Cui et al./Food Chemistry139(2013)1–8the ratio of A 440nm /A k max exceeding 0.20,corresponding to cyani-din-3-O-glucoside and peonidin-3-O-glucoside (Cerezo et al.,2010;Zhang,Xue,Yang,Ji,&Jiang,2004).The NMR data of P2and P5were as follows:Peak 2(P2):1H NMR (CF 3COOD–CD 3OD):d 6.64(1H,d,J =1.2Hz,H-6),d 6.86(1H,d,J =1.2Hz,H-8),8.22(1H,dd,J =8.8,2.2Hz,H-60),8.02(1H,d,J =2.2Hz,H-20),7.04(1H,d,J =8.8Hz,H-50),8.98(1H,s,H-4);5.31(1H,d,J =7.8Hz,H-1glc),3.69(1H,m,H-2glc),3.56(2H,m,H-3,5glc),3.46(1H,m,H-4glc),3.73(1H,dd,J =12.2,5.6Hz,H-6b glc),3.96(1H,dd,J =12.2,2.2Hz,H-6a glc).13C NMR (CF 3COOD–CD 3OD):d 164.5(C-2),145.8(C-3),137.2(C-4),159.6(C-5),103.7(C-6),170.8(C-7),95.4(C-8),157.9(C-9),113.6(C-10),121.5(C-10),118.7(C-20),147.6(C-30),156.0(C-40),117.7(C-50),128.4(C-60),104.1(C-1glc),75.0(C-2glc),78.3(C-3glc),71.3(C-4glc),79.0(C-5glc),62.6(C-6glc).Peak 5(P5):1H NMR (CF 3COOD–CD 3OD):d 6.64(1H,d,J =1.2Hz,H-6),6.88(1H,d,J =1.2Hz,H-8),7.02(1H,d,J =8.4Hz,H-50),8.21(1H,dd,J =8.4,2.0Hz,H-60),8.16(1H,d,J =2.0Hz,H-20),8.99(1H,s,H-4),3.99(3H,s,OCH 3);5.31(1H,d,J =8.0Hz,H-1glc), 3.65(1H,m,H-2glc),3.56(2H,m,H-3,5glc),3.46(1H,m,H-4glc),3.73(1H,dd,J =12.0,4.0Hz,H-6b glc),3.94(1H,dd,J =12.0,2.0Hz,H-6a glc).13C NMR (CF 3COOD–CD 3OD):d 163.2(C-2),144.7(C-3),136.5(C-4),158.5(C-5),103.1(C-6),170.6(C-7),94.5(C-8),157.0(C-9),112.8(C-10),120.3(C-10),114.5(C-20),148.7(C-30),155.7(C-40),116.8(C-50),128.1(C-60),103.5(C-1glc),75.1(C-2glc),78.5(C-3glc),71.1(C-4glc),78.8(C-5glc),62.5(C-6glc),57.0(OCH 3).The 1H NMR spectra of P2showed the presence of two meta-coupled doublet (J =1.2Hz)protons on the A-ring at d 6.64and 6.86ppm,which were assigned to H-6and H-8,respectively.Two sets of doublet and one set of double doublet of an ABM sys-tem at d 7.04(1H,d,J =8.8Hz),8.22(1H,dd,J =8.8, 2.2Hz),8.02ppm (1H,d,J =2.2Hz)were observed which were characteris-tic of H-50,H-60and H-20,respectively.The ring C was a ﬂavanone moiety.The proton signal at d 3.46–5.31(H-1-glc–H-6-glc)showed a D -glucopyranose moiety which was assigned as -conﬁguration based on the large proton-coupling constants of its anomeric proton d 5.31ppm (1H,d,J =7.8Hz,H-10)(Byamukama,Kiremire,showing the six anthocyanin proﬁles of Rhodomyrtus tomentosa extract monitored at 520nm.(Table 2isolated from the fruits of Rhodomyrtus tomentosa .Table 2LC–MS characteristics of anthocyanins separated from Rhodomyrtus tomentosa :retention time,wavelengths of maximum absorption (k max ),molecular ion,fragmentation pattern and tentative identiﬁcation.Peak no.Elution time (min)Peak area (%)(520nm)k max (nm)Molecular ion [M +],m /z a Mass loss (M+H +)–MS 2m /z MS 2of [M +],m /z b Peak assignment Contents (mg/100g dry fruits)111.75 3.35523/277465À162302.9Delphinidin-3-O-glucoside2.07±0.03213.2047.27516/280449/286.9À162286.9Cyanidin-3-O-glucoside29.4±0.39313.73 5.42526/277479/316.9À162316.9Petunidin-3-O-glucoside3.51±0.14414.780.85516/281433/271.0À162271.0,415.1Pelargonidin-3-O-glucoside0.49±0.05515.4634.22506/279463/300.9À162300.9Peonidin-3-O-glucoside21.3±0.33615.908.86527/277493/331.0À162331.0Malvidin-3-O-glucoside5.69±0.16a,bThe fragment ions are shown in order of their relative abundance.Andersen,&Steigen,2005).The cross peak at d 5.31/145.8(H-1glc/C-3)in the HMBC spectrum of P2indicated that the sugar moiety was attached to C-3,which was conﬁrmed by Zarena and Sankar (2012).Based on the above results and literature (Lee &Choung,2011),P2was identiﬁed as cyanidin-3-O-glucopyranoside.Comparing the 1H and 13C NMR spectra of P5with those of P2,its spectral features were closely similar to those of P2,except for the excess of the three-proton signal at d 3.99(3H,s)in the 1H NMR spectra and one carbon signal at d 57.0in the 13C NMR spectra,which was due to a methyl group attached to oxygen.Further sup-port for this structure was obtained by the mass spectrum of P5displaying a [M]+ion peak at m /z 463,corresponding to the excess of an methylene moiety (14mass units)from P2([M]+).Based on the above evidence,the structure of P5,which differed from P2by a methyl group attached to the H-30position instead of a hydro-xyl group attached to the H-30position on the B ring entity,was de-duced to be peonidin-3-O-glucoside and the chemical structure of P2was further conﬁrmed by comparison of the NMR and MS data with the literature (Fossen,Slimestad,Øvstedal,&Andersen,2002).Peak 1(P1):amorphous red powder;ESI–MS m /z 465;1H NMR (CF 3COOD–CD 3OD):d 6.66(1H,d,J =1.2Hz,H-6), 6.87(1H,d,J =1.2Hz,H-8),7.74(2H,d,J =2.2Hz,H-20,60),8.93(1H,s,H-4);5.34(1H,d,J =7.8Hz,H-1glc),3.73(1H,m,H-2glc),3.60(2H,m,H-3,5glc),3.51(1H,m,H-4glc),3.79(1H,dd,J =12.2,5.4Hz,H-6b glc), 3.94(1H,dd,J =12.2, 2.0Hz,H-6a glc).13C NMR (CF 3COOD–CD 3OD):d 164.2(C-2),144.6(C-3),136.8(C-4),159.4(C-5),103.1(C-6),170.8(C-7),95.4(C-8),157.8(C-9),113.0(C-10),121.5(C-10),113.7(C-20),147.6(C-30),146.0(C-40),147.9Electrospray mass spectrum of identiﬁed anthocyanins.Peak 1:delphinidin-3-O-glucoside;peak 2:cyanidin-3-O-glucoside;peak 3:petunidin-3-O-glucoside;pelargonidin-3-O-glucoside;peak 5:peonidin-3-O-glucoside;peak 6:malvidin-3-O-glucoside.(C-50),113.4(C-60),103.5(C-1glc),74.8(C-2glc),78.0(C-3glc),71.2 (C-4glc),78.8(C-5glc),62.3(C-6glc).The MS analysis of P1(t R=11.75min)showed an[M]+ion at m/ z465and a major fragmentation in MS2at m/z303(À162amu) which would correspond to the monoglucoside of delphinidin(Cer-ezo et al.,2010),The UV–Vis spectrum of this compound showed the visible k max to be523nm;the ratio of absorbance at440nm to the absorbance at visible maximum wavelength(A440nm/A k max ratio)for peak1was found to be0.28,indicating that the com-pound is delphinidin-3-O-glucoside(Longo&Vasapollo,2006).The1H NMR spectra of P1showed the presence of two meta-coupled doublet(J=1.2Hz)protons on the A-ring at d6.66and 6.87ppm,which were assigned to H-6and H-8,respectively.One set of doublets of an AM system at d7.74(2H,d,J=2.2Hz)was ob-served which was characteristic of H-20and60.The analysis of the NMR spectral data also revealed only a single glucose moiety with a proton signal at d5.34(1H,d,J=7.8Hz)coupled with the C-3 aglycone at144.6ppm,indicating a D-glucopyranose moiety with a b-conﬁguration attached to the C-3position.Thus the structure of P1was elucidated to be delphinidin-3-O-glucoside and was in agreement with the report of Pazmiño-Durán,Giusti,Wrolstad, and Glória(2001).Peak3(P3):amorphous purple powder;ESI–MS m/z479;1H NMR(CF3COOD–CD3OD):d6.64(1H,d,J=1.2Hz,H-6),6.89(1H, d,J=1.2Hz,H-8),7.74(1H,d,J=2.2Hz,H-60),7.90(1H,d, J=2.2Hz,H-20),8.97(1H,s,H-4),3.99(3H,s,OCH3);5.33(1H,d, J=8.0Hz,H-1glc),3.69(1H,dd,m,H-2glc),3.57(2H,m,H-3,5 glc),3.42(1H,m,H-4glc),3.74(1H,dd,J=12.2,5.6Hz,H-6b glc),3.92(1H,dd,J=12.2,2.0Hz,H-6a glc).13C NMR(CF3COOD–CD3OD):d162.9(C-2),145.0(C-3),135.6(C-4),158.9(C-5),103.4 (C-6),170.6(C-7),95.5(C-8),157.1(C-9),113.3(C-10),119.7(C-10),109.6(C-20),149.5(C-30),146.0(C-40),147.7(C-50),113.0(C-60),103.5(C-1glc),74.5(C-2glc),78.0(C-3glc),71.0(C-4glc), 78.2(C-5glc),62.6(C-6glc),57.5(OCH3).The ESI–MS spectrum of peak3(t R=13.73min)was character-ized by an ion signal at m/z479with an MS2fragment at m/z 317([MÀ162]+)coinciding with the molecular formula C22H23O12 of petunidin aglycone linked with a glucose moiety(Lin,Harnly, Pastor-Corrales,&Luthria,2008).The UV–Vis spectrum of pigment 3,taken on-line during HPLC,showed a visible maximum at 526nm.Comparing the1H and13C NMR spectra of P3with those of P1, its spectral features were closely similar to those of P1,except for the excess of the three-proton signal at d3.99(3H,s)in1H NMR spectra and one carbon signal at d57.5in13C NMR spectra due to a methyl group attached to the H-30position instead of a hydro-xyl group attached to the H-30position on the B ring entity.By comparison with previous research data(Lee et al.,2009),P3was assigned as petunidin-3-O-glucoside.Peak4(P4):amorphous red powder;ESI–MS m/z433;1H NMR (CF3COOD–CD3OD):d6.67(1H,d,J=1.2Hz,H-6),6.94(1H,d, J=1.2Hz,H-8),7.05(2H,d,J=8.8Hz,H-30,50),8.60(2H,d, J=8.8Hz,H-20,60),9.08(1H,s,H-4), 5.34(1H,d,J=8.0Hz,H-1 glc),3.68(1H,m,H-2glc),3.54(2H,m,H-3,5glc),3.42(1H,m, H-4glc),3.80(1H,dd,J=12.2,6.0Hz,H-6b glc),4.04(1H,dd, J=12.2, 2.0Hz,H-6a glc).13C NMR(CF3COOD–CD3OD):d164.8 (C-2),145.8(C-3),137.4(C-4),159.9(C-5),103.7(C-6),170.9(C-7),95.5(C-8),158.1(C-9),114.5(C-10),121.5(C-10),136.8(C-20), 118.6(C-30),166.0(C-40),118.7(C-50),136.4(C-60),104.1(C-1 glc),75.3(C-2glc),78.5(C-3glc),71.3(C-4glc),79.0(C-5glc), 62.6(C-6glc).The molecular ion[M]+at m/z433received from the ESI–MS analysis of P4(t R=15.46min)conﬁrmed the molecular formula, C21H21O10,for glucoside derivatives of pelargonidin aglycone.The fragment ion[M+HÀ162]+at m/z271was consistent with the structure of pelargonidin with a loss of glucose moiety from pelargonidin-3-O-glucoside(Hong&Wrolstad,1990).The UV–Vis spectrum of this compound showed the visible k max at506nm.The1H NMR and13C NMR spectra suggested that P4contained a ﬂavanone moiety;the proton signals at d6.67(1H,d,J=1.2Hz)and 6.94(1H,d,J=1.2Hz)implied the presence of two meta-coupled doublet protons on the A-ring which were assigned to H-6and H-8,respectively.Two sets of doublets of an AB system at d7.05 (2H,d,J=8.8Hz)and8.60(2H,d,J=8.8Hz)were observed,which were characteristic of H-30,H-50and H-20,H-60,respectively.As P2, the large coupling constants(J=8Hz)for the anomeric protons (d5.34,1H,d,J=8.0Hz,H-1glc)conﬁrmed the presence of a b-D-glucosidic linkage in P4.By comparison with the literature,P4 was further conﬁrmed as pelargonidin-3-O-glucoside(Pedersen, Andersen,Aksnes,&Nerdal,1993).Peak6(P6):amorphous dark red powder;ESI–MS m/z493;1H NMR(CF3COOD–CD3OD):d6.74(1H,d,J=1.9Hz,H-6),7.06(1H, d,J=1.9Hz,H-8),7.96(2H,d,J=2.2Hz,H-20,60),8.96(1H,s,H-4), 3.90(6H,s,2ÂCH3);5.37(1H,d,J=7.8Hz,H-1glc),3.64(1H,m, H-2glc),3.74(1H,m,H-3glc),3.79(1H,m,H-5glc),3.43(1H, m,H-4glc),3.51(1H,dd,J=12.4,4.8Hz,H-6b glc),3.96(1H,dd, J=12.4, 2.2Hz,H-6a glc).13C NMR(CF3COOD–CD3OD):d163.0 (C-2),145.0(C-3),136.2(C-4),158.9(C-5),103.1(C-6),170.5(C-7),95.1(C-8),157.4(C-9),112.9(C-10),120.0(C-10),110.5(C-20), 149.1(C-30),146.7(C-40),149.2(C-50),111.0(C-60),103.8(C-1 glc),74.6(C-2glc),78.0(C-3glc),70.9(C-4glc),77.8(C-5glc), 67.3(C-6glc),57.3(2ÂOCH3).The last peak in the chromatogram was peak6,with the MS and MS2proﬁles from ESI–MS spectra showing strong ion peaks at m/z 493and331for peak6(t R=15.91min)coinciding with the molec-ular ion of malvidin-3-O-glucoside with a loss of a glucose moiety (Li et al.,2011).Based on the spectrum data and comparing with previously reported data(Alcalde-Eon,Escribano-Bailón,Santos-Buelga,&Rivas-Gonzalo,2006),P6was identiﬁed as malvidin-3-O-glucoside.As shown in Fig.3A,there are six principal anthocyanin peaks, and two major peaks(peak2and peak5)with concentrations of 47%(29.4±0.39mg/100g)and34%(21.3±0.33mg/100g)of the total peak area with retention times of13.20and15.46min,respec-tively,(Table2).The elution times of the other four peaks with the concentration of3.35%(peak1,2.07±0.03mg/100g),5.42%(peak 3, 3.51±0.14mg/100g),0.85%(peak4,0.49±0.05mg/100g), 8.86%(peak6,5.69±0.16mg/100g)were11.75(peak1),13.73 (peak3),14.78(peak4),15.90(peak6)min.Theﬁnal structures of the six compounds isolated and identiﬁed are shown in Fig.3B.Based on the available literature,there have been only a few re-ports in previous studies regarding the extraction and preliminary qualitative research of the anthocyanins in R.tomentosa fruits.This is theﬁrst time that all the major anthocyanins were systemati-cally isolated and identiﬁed from R.tomentosa fruits.4.ConclusionsThe total anthocyanin content of the puriﬁed R.tomentosa(Ait.) Hassk fruits extract was62.8±1.2mg/100g of freeze-dried weight of R.tomentosa fruits and it possessed excellent in vitro free radi-cal-scavenging activity.Pure anthocyanins were isolated and ana-lyzed by ESI–MS and NMR spectroscopy.The six anthocyanin structures characterized were delphinidin-3-O-glucoside,cyani-din-3-O-glucoside,petunidin-3-O-glucoside,pelargonidin-3-O-glucoside,peonidin-3-O-glucoside,and malvidin-3-O-glucoside. As a result,this study has systematically documented,for theﬁrst time,the presence of six anthocyanin derivatives from the tested extract.Fruits of R.tomentosa also contain important amounts of other phenolics,amino acids and ascorbic acid.These phytochemi-cals may partially explain the diverse bioactive properties of thisC.Cui et al./Food Chemistry139(2013)1–87。

Biomedical_Chromatography

Fingerprint analysis of Rhizoma chuanxiong867ORIGINAL RESEARCH ORIGINAL RESEARCHBIOMEDICAL CHROMATOGRAPHY Biomed. Chromatogr . 21: 867–875 (2007)Published online 12 April 2007 in Wiley InterScience () DOI: 10.1002/bmc.833Fingerprint analysis of Rhizoma chuanxiong by pressurizedcapillary electrochromatography and high-performance liquid chromatographyGuoxiang Xie,1 Aihua Zhao,1 Peng Li,2 Lu Li 2 and Wei Jia 1*1School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China 2Unimicro (Shanghai) Technologies Co. Ltd, Shanghai 201203, People’s Republic of ChinaReceived 25 December 2006; revised 8 February 2007; accepted 9 February 2007ABSTRACT: Pressurized capillary electrochromatography (pCEC) and high-performance liquid chromatography (HPLC) were used simultaneously to establish ﬁngerprints of Rhizoma chuanxiong . Ten batches of Rhizoma chuanxiong collected from different regions in China were used to obtain the characteristic pCEC and HPLC ﬁngerprints using a standardized procedure of sample preparation and analysis. A total of 22 common peaks were isolated within 60min by pCEC and 16 common peaks by HPLC within 65min. The ﬁngerprints of Rhizoma chuanxiong were then used to identify the raw herbs from different sources in China.The two proposed methods demonstrated good stability and reproducibility with RSD less than 5% for retention time in pCEC and in HPLC, respectively. Finally, the data from the analyses of 10 batches of Rhizoma chuanxiong by pCEC and HPLC were all processed with similarity analysis with two mathematical methods, correlation coefﬁcient and the included angle cosine. The ﬁngerprints of Rhizoma chuanxiong established with pCEC and HPLC are suitable to identify samples from different sources and can be used to control the quality of raw herbs. Copyright © 2007 John Wiley & Sons, Ltd.KEYWORDS: pressurized capillary electrochromatography; high-performance liquid chromatography; ﬁngerprint analysis;Rhizoma chuanxiongINTRODUCTIONTraditional Chinese medicines (TCMs) are gaining more and more attention in many ﬁelds because of their low toxicity and good therapeutic performance TCMs are composed of diverse of components and their contents vary with growing soil, climate and the growth stage when harvested (Zhou et al., 2002). This makes the quality control of crude drugs and their medical preparations extremely difﬁcult. Traditionally,the contents of active components in TCMs were used to evaluate the quality of raw herbal medicines. How-ever, according to this method, it is difﬁcult to insure the biologically active compounds, and to separate them from the large amounts of proteins, sugars and tannins that may contribute little to the pharmaceutical effects. Furthermore, all the components in TCMs are held to be responsible for the beneﬁcial effects and not just the few active compounds (Drug Administra-tion Bureau of China, 2000). Conventional research focuses mainly on the determination of the most activecomponents, while ﬁngerprinting can offer characteriza-tion of a complex system with a degree of quantitative reliability . In this respect, ﬁngerprinting has gained increasing attention for quality control systems (National Pharmacopoeia Committee, 2000; Gu et al.,2004). Fingerprint analysis has been introduced and accepted by the US Food and Drug Administration (FDA) as one of the requirements for botanical pre-parations (CDER/FDA, 2000) and by the European Agency for the Evaluation of Medicinal Products for herbal preparations (CPMP/CVMP, 2000). Further-more, ﬁngerprint analysis has been introduced and accepted by the World Health Organization (WHO) as a strategy for assessing herbal medicines (World Health Organization, 1991) and is also required by the State Food and Drug Administration Bureau of China for the standardization of injections made from TCMs or their raw materials (Drug Administration Bureau of China, 2000) Fingerprint analysis of medicinal herbs can be used for identifying and assessing the stability of the plants.Chromatography, including thin-layer chromato-graphy (TLC), high-performance liquid chromato-graphy (HPLC) and gas chromatography (GC), is recommended for ﬁngerprinting of TCMs in the Chinese Pharmacopoeia (National Pharmacopoeia *Correspondence to: Wei Jia, School of Pharmacy, Shanghai Jiao Tong University, No . 800 Dongchuan Road, Shanghai 200240,People’s Republic of China.E-mail: weijia@Abbreviations used: ACN, acetonitrile; EOF, electro-osmotic ﬂow;Copyright © 2007 John Wiley & Sons, Ltd.Biomed. Chromatogr . 21: 867–875 (2007)DOI: 10.1002/bmc868G. Xie et al.ORIGINALRESEARCH of years; it is fast and easy to operate, but of poor resolution . HPLC has high precision, sensitivity and reproducibility for ﬁngerprinting TCM, but HPLC is not suitable for analysis of some highly viscous samples. CE has high speed, efﬁciency, ultrasmall sample volume and minimal consumption of solvent, but the reproduc-ibility and the selectivity are not as good as in HPLC.CEC is a hybrid technique that combines the selectivity of HPLC and the separation efﬁciency of CE How-ever, in practice, when CEC is used without pressure,often on a commercial CE instrument, there were pro-blems associated with bubble formation in CEC, occur-ring initially in the unpacked section of the capillary,probably as a result of differences in velocity of the liquid eluent between the packed and unpacked sec-tions of the capillary (Poppe, 1997; Zhang et al., 2003)and column dry-out . The use of supplementary pre-ssure has proved effective to stabilize the ﬂow con-ditions. Compared with traditional HPLC and CE, the mobile phase in the pCEC system is driven by a pre-ssurized ﬂow and an electro-osmotic ﬂow (EOF) simul-taneously, reducing band broadening and improving separation efﬁciency. Now pCEC has become an attrac-tive technique for pharmaceutical analysis (Zhang et al.,2003; Lue et al., 2007) because of its combination of the inherent advantages of two major separation tech-niques. Therefore, we intend to develop a characteristic ﬁngerprint of Rhizoma chuanxiong using pCEC and HPLC simultaneously for identifying the raw herb .The ﬁngerprints can also help identify the geographical origins of the samples.Rhizoma chuanxiong , derived from the rhizome of Ligusticum chuanxiong Hort. (Umbelliferae ), is a well-known TCM herb with hemodynamic and analgesic effects (National Pharmacopoeia Committee, 2005). Its constituents include ferulic acid, chuanxiongzine (i .e .tetramethylpyrazine) and other compounds (Chen and Chen, 1992; Hu et al., 1990; Mouer et al., 1998; Ozaka and Ma, 1990; Sato et al., 1990; Ho et al., 1989). HPLC has been reported in analysis of components of Rhizoma Chuanxiong (Gong et al., 2003, 2004, 2005; Li et al., 2004; Wang et al., 2000), but these reports are mostly relevant to chemometric approaches for the con-struction of chromatographic ﬁngerprint analysis and do not offer sufﬁcient discrimination to reveal the dif-ferences between similar kinds of compounds. Finger-print analysis of TCMs such as Flos carthami by pCEC and HPLC has been carried out successfully in our studies (Xie et al., 2006). However, at present no report for ﬁngerprint chromatogram analysis of Rhizoma chuanxiong by pCEC was found and no batch compari-son data comparing the traditional technique (HPLC)with pCEC have been reported for evaluation of this TCM In this study, pCEC was applied to develop a ﬁngerprint of Rhizoma chuanxiong for the ﬁrst time,and the results were compared with those from HPLC.EXPERIMENTALApparatus.All pCEC separation was performed on a Trisep™-2100 capillary electrochromatography system (Unimicro Technologies Inc , Pleasanton, CA, USA) which comprised a Unimicro binary microsyringe pump, a high-voltage power supply (+30 and −30kV), a Valco six-port injection valve, a UV–vis variable wavelength detector equipped with a cell for on-column detection, and an Unimicro Trisep™ workstation 2003, as described in the literature (Jiang et al., 2001). A continuous mobile phase was generated by merging two solvent ﬂows in a mixer and entered the Valco six-port injection valve via a microsyringe pump. Injected samples were delivered to the injection valve and introduced in the internal 10nL sample loop, and then carried to the four-port split valve by the mobile phase ﬂow.After splitting in the four-port valve, the ﬂow entered a capillary column under constant pressure of 13,000kPa .Pressure was applied to the column inlet during the separa-tion . A positive voltage was applied to the outlet of the column, and the inlet of the column was connected to the split valve and grounded.The separation was performed using a reversed-phase column (EP-150-30/50-5-C18, Unimicro Technologies Inc .).The column was 50cm (of which 30cm was packed) ×150µm i d Detection windows (~2mm long) were burned into the column walls. A −10kV voltage was applied across the capillary to produce EOF. The ﬂow-rate was 0.08mL/min and the injection volume was 10nL. The data was collected directly from the UV detector employing a sample wave-length of 280nm and analyzed using the Unimicro Trisep™workstation 2003.The ﬁnal pCEC mobile phase employed water [0.02% (v/v)triﬂuoroacetic acid (TFA)] (A) and 95% aqueous methanol [0.02% (v/v) TFA] (B). This was ﬁltered using 0.22µm HPLC ﬁlters and separation was achieved using the following gradi-ent: 0–10min, 2–15% B; 10–40min, 15–40% B; 40–65min,40–95% B. Samples and pCEC mobile phases were sonicated prior to use using an ultrasonic bath for 10min at room temperature in order to remove any air bubbles.An Agilent 1100 liquid chromatography (Agilent, USA)equipped with quaternary gradient pump and a UV detection system was used. An HPLC method was developed using a reversed-phase column (Elite Symmetry C 18, 250 × 4.6mm i.d., 5µm). The binary gradient elution system consisted of A [water + 0.5% (v/v) H 3PO 4] and B (methanol) and separation was achieved using the following gradient: 0–3min, 15% B;3–55min, 15–95% B; 55–65min, 95% B . The column temperature was kept constant at 25°C The ﬂow-rate was 1mL/min and the injection volume was 10µL. The UV detec-tion wavelength was set at 280nm.Reagents and materials.Ferulic acid and chuanxiongzine (Fig . 1) were provided by the National Institute for the Control of Pharmaceutics and Biological Products, Beijing,China . Chromatographic-grade methanol (CH 3OH), aceto-nitrile (ACN) and other analytical-grade chemicals were used.Ultrapure water was prepared with the Millipore Milli-Q SP water puriﬁcation system (18.2M Ω, Milipore, Bedford, MA,USA) All aqueous solutions were prepared with ultrapure water Genuine Rhizoma chuanxiong were purchased fromCopyright © 2007 John Wiley & Sons, Ltd.Biomed. Chromatogr . 21: 867–875 (2007)DOI: 10.1002/bmcFingerprint analysis of Rhizoma chuanxiong 869ORIGINALRESEARCH Shanghai Huayu Chinese Herbs Co. Ltd, and collected from different producing areas of China (Table 1). They were veriﬁed by Professor Zhong Liu of Shanghai Jiao Tong University of China as the dried rhizome of Ligusticum chuanxiong Hort. (Umbelliferae ).Sample preparation.All samples of Rhizoma chuanxiong were kept in a desiccator. About 1.0g of dried samples were ground into powder accurately weighed and extracted with 10mL ethanol–water solution (75:25, v/v) in an ultrasonic water bath for 30min The extraction was repeated twice The extracted solution was combined and centrifuged for about 20min. The supernatant was concentrated by evapora-tion and then the residue was dissolved with methanol–water (75:25, v/v) by ultrasonication (250W) and transferred to a 25mL volumetric ﬂask The ﬂuid was ﬁltered through a syringe ﬁlter (0.22µm) and injected directly into the HPLC or pCEC system.Stand ard sample preparation.Standard samples of ferulic acid (0.19mg/mL) and chuanxiongzine (0.18mg/mL) were prepared with methanol.RESULTS AND DISCUSSIONEffect of the extraction methodSelection of an extraction method ﬁtted for the type of herb is always challenging and depends on the acces-sibility of plant material and the content of the analyte.A good extraction method for TCM ﬁngerprintingnot only requires more complete isolation of active components from herb, but also allows a comprehen-sive chemical proﬁle . In this work, the ferulic acid content, commonly used as quality index, and the number of peaks were studied by HPLC in order to evaluate the extraction efﬁciency.Water, 50% aqueous ethanol, 75% aqueous ethanol and 95% aqueous ethanol were chosen as extraction solvents and ultrasonication and reﬂuxing as extraction methods. It was found that the highest content of the ferulic acid in Rhizoma chuanxiong was obtained by ultrasonic extraction and reﬂuxing with 75% aqueous ethanol . The number of peaks in the ﬁngerprint chromatogram was also a key factor in choosing the extraction method and, in this respect, ultrasonic extraction was better than the reﬂuxing method The reproducibility of the full assay, including the whole extraction process, was evaluated and the RSD value was less than 2.5% (n = 3). Moreover, ultrasonication was simpler and faster than reﬂuxing and was therefore selected as the optimum extraction method.Identiﬁcation of ferulic acid and chuanxiongzine with pCECFerulic acid and chuanxiongzine were the active com-pounds isolated from Rhizoma chuanxiong . Therefore,the active constituents ferulic acid and chuanxiongzine were used as two marker compounds in the Rhizoma chuanxiong ﬁngerprint analysis Standard solutions of ferulic acid and chuanxiongzine were analyzed under the same pCEC conditions as the samples. The result-ing electrochromatogram is shown in Fig . 2(b). The peaks of the samples were identiﬁed based on their UV spectra and migration times of the peaks spiked with standard ferulic acid and chuanxiongzine. Peaks 4 and 6 were identiﬁed as ferulic acid and chuanxiongzine,respectively [Fig. 2(a)].Fingerprint development of Rhizoma chuanxiong by pCEC(a) The peak shape of ferulic acid and chuanxiongzine and the resolution between them and their neighboring peaks, as well as the resolutions between all the peaks,together with (b) the number of peaks in the whole electrochromatogram, and (c) the separation dura-tion for the process, were considered as the criteria for optimization of the separation conditions Several factors were studied to achieve good separation, includ-ing the mobile phase composition, buffer system and applied voltage.First, the composition of the mobile phase was tested during the separation. The mobile phases used were: (1) water:95% v/v CH 3OH; (2) water:0.02% v/v TFA:95% v/v CH 3OH; (3) water:0.02% v/v TFA:95%Figure 1. Structures of ferulic acid and chuanxiongzine.Table 1. The source of samples Sample no.Region1Mengyang, Pengzhou 2Longmu, Pengzhou 3Nanmu, Pengzhou 4Junle, Pengzhou5Guansheng, Chongzhou 6Zhongxing, Dujiangyan 7Liujie, Dujiangyan 8Shiyang, Dujiangyan 9a Sichuan 10aSichuanaSamples 9 and 10 were obtained from Shanghai Hua Yu Chinese Herbs Co. Ltd; they were from Sichuan but the detailed sources were not known.Copyright © 2007 John Wiley & Sons, Ltd.Biomed. Chromatogr . 21: 867–875 (2007)DOI: 10.1002/bmc870G. Xie et al.ORIGINALRESEARCH v/v CH 3OH:0.02% v/v TFA. The separation was per-formed with mobile phase 1 using the following gradi-ent: 0–3min, 15% B; 3–30min, 15–100% B. The peaks were eluted over 20–30min and a poor resolution of chuanxiongzine with its neighboring peaks was ob-served. However, the peak shapes and resolution were greatly improved with mobile phase 2. In addition,when using mobile phase 3 with the following gradient,the separation was completed within 60min with good peak shape and resolution: 0–10min, 2–15% B; 10–40min, 15–40% B; 40–65min, 40–95% B. The resolu-tion between ferulic acid, chuanxiongzine and their neighboring peaks was 2.74 and 3.24, respectively.The effect of using of different acids, such as acetic acid (HAC), formic acid (FA), phosphoric acid (PA)and triﬂuoroacetic acid (TFA), on the separation of samples was investigated. Addition of HAC resulted inFigure 2. Electrochromatograms of marker compound of ferulic acid and chuanxiongzine (b),and Rhizoma chuanxiong (sample 6) (a). Peak 4, ferulic acid; peak 6, chuanxiongzine.low peak sensitivity and resolution, and poor peak shape. In contrast, when the pH of mobile phase was adjusted with FA, PA and TFA, the sensitivity, resolu-tion and peak shapes became better and the best reso-lution was achieved with 0.02% TFA . The baseline noise was 40, 7, 5 and 4µV using HAC, FA, PA and TFA, respectively. To improve peak shape and resolu-tion, all other pCEC separation were performed using TFA as the acid modiﬁer Finally, water:002% TFA and 95% methanol:0.02% TFA were chosen as the mobile phase.The inﬂuence of an applied voltage (−15 to +15kV)was examined There were 55 peaks on the electro-chromatogram with an applied voltage of −10kV, while there were only 32 peaks at +10kV. As anticipated, a higher voltage reduced the retention time of peaks due to an increase in the net velocity.However, the pCECCopyright © 2007 John Wiley & Sons, Ltd.Biomed. Chromatogr . 21: 867–875 (2007)DOI: 10.1002/bmcFingerprint analysis of Rhizoma chuanxiong 871ORIGINALRESEARCH system became less stable due to resulting Joule heat of the high applied voltage. A decrease in resolution was observed when the applied voltage was at −15kV and at the same time, when the applied voltage was +15kV,small bubbles were observed due to the differences in velocity of the liquid eluent between the packed and unpacked sections of the capillary and the resulting Joule heat (Poppe, 1997; Zhang et al., 2003). The peak of the measured compound also varied when different voltages were applied; for example, ferulic acid exhi-bited a single peak when negative voltage was applied,and the single peak split into two peaks when positive voltage was applied . Therefore, a voltage of −10kV was used throughout the reminder of this study.To develop characteristic ﬁngerprints, all the experi-mental procedures, including the extracting and the analytical conditions and methods, must be standard-ized. The determinant of the analytical conditions and methods was described earlier. The method validation of ﬁngerprint analysis was performed based on the relative migration time (the ratio of peak migration time of sample constituents to the reference peak) and the relative peak area (the ratio of peak area of sample constituents to the reference peak).The sample solution was successively injected into the pCEC system and analyzed ﬁve times Precisions not exceeding 2.9 and 5.3% were obtained for relative migration times and relative areas of all peaks, respec-tively. The results of the precision tests indicated that the method was suitable for the analysis. The inter-day precisions of the proposed method, on the basis of analyzing ﬁve replicate samples on separate days,were below 3.1% for relative migration times and below 6.3% for relative peak areas. The stability test was performed with sample solutions extracted from Dujiangyan, Rhizoma chuanxiong , for 24h The rela-tive standard deviations (RSDs) of the relative migra-tion times and the relative peak areas were less than 5.8%. This indicated that the sample solution was stable for 24h and was ﬁt for the analysis. The repro-ducibility test—analysis of ﬁve samples that were pre-pared from the same batch of the dried ﬂower—was also satisﬁed by the result that the RSDs of the relative retention times and relative peak areas of all peaks were less than 5.0%.To standardize the characteristic ﬁngerprint of the raw herb, 10 batches of 1.0g Rhizoma chuanxiong samples each from Dujiangyan were mixed homo-geneously. A 1.0g mixture was taken out and analyzed with the developed procedure . The average electro-chromatogram from the 10 batches was regarded as the standardized characteristic ﬁngerprint of Rhizoma chuanxiong. Peaks that existed in all 10 electro-chromatograms were labeled as ‘common peaks’ for Rhizoma chuanxiong . There are 22 ‘common peaks’ in the ﬁngerprint [Fig . 2(a)]. The area of all 22 peaks accounted for more than 90% of total peak area. The relative migration time (the ratio of peak migration time of sample constituents to the reference peak)and the relative peak area (the ratio of peak area of sample constituents to the reference peak) are shown in Table 2. The whole electrochromatographic proﬁle,including the peaks, together with the marker com-pound ferulic acid and chuanxiongzine, could provide a useful means of identifying and assessing Rhizoma chuanxiong Using the relative peak areas of the 22common peaks in the electrochromatograms of the 10 samples, similarity analyses (National Institute for the Control of Pharmaceutics and Biological Products,2004; Wang et al., 2005) were conducted with two dif-ferent mathematical methods including the correlation coefﬁcient and the included angle cosine, and the results are shown in Table 4. As shown, the values of correlation coefﬁcient and the included angle cosine ofTable 2. The results of the ﬁngerprint analysis of Rhizoma chuanxiong by pressurized capillary electrochromatography Retention Peak Relative Relative Retention Peak Relative Relative Peak time area retention peak Peak time area retention peak no.(min)(uV s)time aarea bno.(min)(µV s)time aarea b1 3.98210,3080.180.301233.5233,016 1.510.09214.2824,0330.640.121340.747116,273 1.84 3.33315.08813,2300.680.381441.8877,191 1.890.21417.0953,0400.770.091543.52963,128 1.96 1.81518.46212,6530.830.361645.08916,468 2.030.47622.18334,894 1.00 1.001746.627264,906 2.107.59723.81913,276 1.070.381847.96414,328 2.160.41824.8375,193 1.120.151951.99611,946 2.340.34926.540118,156 1.20 3.392053.0195,436 2.390.161028.04821,046 1.260.602153.68218,434 2.420.531132.6834,2511.470.122257.6793,0042.600.09aRelative retention time: the ratio of peak migration time of sample constituents to the reference peak of chuanxiongzine (peak 6); b relative peak area: the ratio of peak area of sample constituents to the reference peak of chuanxiongzine (peak 6).Copyright © 2007 John Wiley & Sons, Ltd.Biomed. Chromatogr . 21: 867–875 (2007)DOI: 10.1002/bmc872G. Xie et al.ORIGINALRESEARCH Table 3. The results of the ﬁngerprint analysis of Rhizoma chuanxiong by HPLC Retention Peak Relative Relative Retention Peak Relative Relative Peak time area retention peak Peak time area retention peak no.(min)(mAU)time aarea bno.(min)(mAU)time aarea b113.737222.830.640.19935.7652880.85 1.67 2.44214.64914.450.680.781036.538415.73 1.700.35316.216189.210.760.161137.723132.62 1.760.11421.4361178.58 1.00 1.001239.7176972.92 1.85 5.92524.1414304.93 1.13 3.651340.616331.13 1.890.28624.758112.95 1.150.101445.365426.23 2.120.36725.684972.89 1.200.831546.1581093.92 2.150.93829.782203.011.390.171647.168187.82.200.16aRelative retention time: the ratio of peak migration time of sample constituents to the reference peak of ferulic acid (peak 4); b relative peak area: the ratio of peak area of sample constituents to the reference peak of ferulic acid (peak 4).the samples were more than 0.910 except for samples 2and 5. It is concluded that most of these samples are alike except for samples 2 and 5.Fingerprint d evelopment of Rhizoma chuanxiong by HPLC.In order to obtain good resolution and a large number of peaks, optimization of separation con-ditions in HPLC was done by investigating the inﬂu-ence of the mobile phase, the detection wavelength and the gradient mode Preliminary studies indicated that better separation and results were obtained using the mobile phase of A (water + 0.5% H 3PO 4) and B (methanol) than the same CEC eluent for HPLC for comparison between the techniques. Therefore, in this work, methanol and water were chosen as the mobile phase. A small amount of H 3PO 4 was added to the mo-bile phase in order to obtain better shape of peaks. The optimum mobile phase was A (water + 0.5% H 3PO 4)and B (methanol) in the gradient mode as follows: 0–3min, 15% B; 3–55min, 15–95% B; 55–65min, 95%B. The column temperature was kept constant at 25°C.The ﬂow-rate was 1mL/min and the injection volume was 10µL. In order to obtain a large number of peaks on the HPLC chromatogram, the solution of ferulic acid, chuanxiongzine and the sample of Rhizoma chuanxiong were scanned by UV from 190 to 600nm;280nm was selected as the best detection wavelength.Chromatograms of 10 samples were placed into one, shown in Fig . 3. There are about 50 peaks within 65min in the chromatogram . Among these,16 common peaks [shown in Fig . 3(c)] were found in the standard ﬁngerprint of Rhizoma chuanxiong .The total peak area of non-common peaks was less than 10%, which met the standards. The relative migra-tion time (the ratio of peak migration time of sample constituents to the reference peak) and the relative peak area (the ratio of peak area of sample con-stituents to the reference peak) are shown in Table 3.The method validation of ﬁngerprint analysis was performed based on the relative migration time and the relative peak area.The sample solution was successively injected into the HPLC system and analyzed ﬁve times. Precisions not exceeding 0.8 and 1.9% were obtained for relative migration times and relative peak areas of all peaks,respectively . The results of precision tests indicated that the method was good for the analysis. The inter-day precisions of the proposed method, on the basis of analyzing ﬁve replicate samples on separate days,were below 0.9% for relative migration times and within 2.3% for relative peak areas. The stability test was performed with sample solutions extracted from Dujiangyan Rhizoma chuanxiong for 24h. The relative standard deviations (RSDs) of the relative migration times and the relative peak areas were less than 2.8%.This indicated that the sample solution was stable for 24h and suitable for the analysis. The reproducibility test—the analysis of ﬁve samples that were prepared from the same batch of the dried ﬂower—was also satisﬁed by the results that the RSDs of the relative retention times and relative peak areas of all peaks were less than 2.5%.Sixteen common peaks, which appeared in the ﬁngerprint of the genuine herb, represented the char-acteristics of the herb’s constituents, and the result of the relative values of the 10 samples, the peaks,together with the index compound, ferulic acid, could provide a useful means of identifying and assessing Rhizoma chuanxiong . As shown in Table 4, the values of the correlation coefﬁcient and the included angle cosine with HPLC are slightly higher than that with pCEC . Sample 5, from Chongzhou Guansheng in China, is obviously different from the other samples,indicating that place of origin signiﬁcantly inﬂuences the kinds and content of components in crude TCMs,and hence affects their quality.Comparison of pCEC and HPLC ﬁngerprinting Both pCEC and HPLC could display the whole con-centration distribution of different kinds of compo-nents, which is the most important character of aCopyright © 2007 John Wiley & Sons, Ltd.Biomed. Chromatogr . 21: 867–875 (2007)DOI: 10.1002/bmcFingerprint analysis of Rhizoma chuanxiong 873ORIGINALRESEARCH Figure 3. HPLC chromatograms of marker compound of ferulic acid and chuanxiongzine (a), Rhizoma chuanxiong from different regions (b) and Rhizoma chuanxiong (sample 6) (c).Peak 4, ferulic acid. This ﬁgure is available in colour online at /journal/bmcﬁngerprint. Characteristics of pCEC and HPLC methods used to develop TCM ﬁngerprints are summarized in Table 5.The number of common peaks, the non-common peak area, the reproducibility, the resolution andsystem suitability were key factors. The sample loaded onto pCEC was very small (10nL), which led to rela-tively low stability . As shown in Table 5, HPLC is advantageous for precision and stability of the analysis,while pCEC is superior in resolution, sensitivity and。

高效液相色谱法测定生活饮用水中的苯并[a]芘

高效液相色谱法测定生活饮用水中的苯并[a]芘贺光秀;贾瑞宝;邓长江【摘要】建立高效液相色谱二极管阵列检测器检测生活饮用水苯并[a]芘的测定方法。

采用C18反相色谱柱(150 mm×4.6 mm，5μm)，在流动相为甲醇-水(体积比为90∶10)、流量1.0 mL/min、检测波长295 nm、柱温35℃、进样体积20μL的条件下测定生活饮用水中苯并[a]芘。

该方法检出限为6 ng/L，线性范围0～100 ng/mL，加标回收率为88.1%～93.4%，测定结果的相对标准偏差为1.06%(n=9)。

该法样品预处理简单，分离度高，分析时间短，适用于生活饮用水中苯并[a]芘的准确定性定量测定。

%A method was developed for determining benzo [a] pyrene in drinking water by HPLC-PDA detector. With column of C18(150 mm×4.6 mm,5μm),the mobile phase of methanol-water(volume ratio was 90∶10) and the flow rate of 1.0 mL/min,the determine wavelength of 295 nm,the column temperature of 35℃,and the injection volume of 20μL, the concentration of benzo [a] pyrene in drinking water was detected. The limit of detection was 6 ng/mL. The linear plots were obtained between 0-100 ng/mL. Overall recoveries were between 88.1%and 93.4%,and the relative standard deviation of determination results was 1.06%(n=9). This method has advantages of simple process of sample treatment, good isolation and short analysis time,and is suitable for the quantitative and qualitative determination of Benzo [a] pyrene in drinking water.【期刊名称】《化学分析计量》【年(卷),期】2014(000)003【总页数】3页(P50-52)【关键词】苯并a芘;高效液相色谱法;二极管阵列检测器;生活饮用水【作者】贺光秀;贾瑞宝;邓长江【作者单位】山东绿洁环境检测有限公司，济南 250101;山东省城市供排水水质监测中心，济南 250000;山东省国合循环经济研究中心，济南 250100【正文语种】中文【中图分类】O657.7苯并[a]芘(Bap)是一种公认的强致癌物质，主要由煤炭、石油、天然气、木材等不完全燃烧而产生，在土壤、空气、水等多种环境介质中广泛存在。

超高效液相色谱-串联质谱法同时测定水果中10种保鲜剂残留量

Determination of 10 Preservative Residues in Fruits by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry
CAO Xueqin1,2, YANG Fei1, CHEN Guotong1, MAO Qiongling1, LI Fang1, YANG Zhong1,*
(MRM) with an electrospray ionization source in the positive ion mode, and quantified by an external standard method
using a matrix-match standard solution. The calibration curves showed good linearity with correlation coefficients greater
Keywords: preservatives; QuEChERS; fruits; ultra-high performance liquid chromatography-tandem mass spectrometry
DOI:10.7506/spkx：建立超高效液相色谱-串联质谱法同时测定水果中咪鲜胺、多菌灵、甲基硫菌灵、噻菌灵、异菌脲、抑霉唑、苯菌灵、腐霉利、百菌清、嘧霉胺10 种保鲜剂残留方法。样品用QuEChERS方法处理，经 Acquity UPLC BEH C18色谱柱分离，乙腈-0.1%甲酸溶液为流动相梯度洗脱，经超高效液相色谱-串联质谱仪采用电喷雾电离源多反应监测模式检测，基质匹配标准溶液外标法定量。结果表明，10 种保鲜剂在各自质量浓度范围内线性良好（r＞0.992），在3 个加标水平下，回收率为75.0%～112%，相对标准偏差（n=6）不大于9.5%。10 种防腐保鲜剂的检出限（RSN≥3）为0.02～1.50 μg/kg，定量限（RSN≥10）为0.1～4.1 μg/kg。该方法操作简便，灵敏度高，准确可靠，适用于水果中10 种保鲜剂残留量的同时测定。关键词：保鲜剂；QuEChERS；水果；超高效液相色谱-串联质谱法

超高效液相色谱-串联质谱法测定化妆品中15种N-亚硝胺化合物

第42 卷第 11 期2023 年11 月Vol.42 No.111469~1478分析测试学报FENXI CESHI XUEBAO（Journal of Instrumental Analysis）超高效液相色谱-串联质谱法测定化妆品中15种N-亚硝胺化合物汪毅1，梁文耀1，何国山1，陈张好2，周智明2，吴谦1，席绍峰1，谭建华1*（1．广州质量监督检测研究院，国家化妆品质量检验检测中心（广州），广东广州511447；2．广东省药品检验所，广东广州510663）摘要：采用超高效液相色谱-串联质谱（UPLC-MS/MS）建立了化妆品中15种痕量N-亚硝胺化合物的分析方法。

水剂样品以水或乙腈分组超声提取，膏霜乳液样品采用亚铁氰化钾-乙酸锌溶液沉淀大分子或者饱和氯化钠-乙腈盐析分组处理后，以Agilent Poroshell 120 SB-Aq（100 mm×3.0 mm，2.7 μm）色谱柱分离，经大气压化学电离源（APCI）电离，多反应监测模式检测，以同位素内标法定量。

结果表明，15种N-亚硝胺化合物在相应质量浓度范围内线性关系良好（r2＞0.995），检出限和定量下限分别为5~15 ng/g和15~45 ng/g。

水、乳、膏霜3种化妆品基质在25、50、100 ng/g加标水平下的平均回收率为88.0%~111%，相对标准偏差（RSD，n=6）为1.4%~9.8%。

该方法用于市售化妆品检测，发现13批次样品检出N-亚硝基二乙醇胺（NDELA），其中1批次超限量值。

方法的专属性强，灵敏度高，精密度好，解决了N-亚硝胺化合物稳定性差、易被干扰等问题，适用于化妆品中15种N-亚硝胺化合物的痕量测定。

关键词：N-亚硝胺化合物；化妆品；超高效液相色谱-串联质谱法（UPLC-MS/MS）；大气压化学电离源中图分类号：O657.63；O623.732文献标识码：A 文章编号：1004-4957（2023）11-1469-10 Determination of Fifteen N-nitrosamine Compounds in Cosmetics by Ultra Performance Liquid Chromatography-TandemMass SpectrometryWANG Yi1，LIANG Wen-yao1，HE Guo-shan1，CHEN Zhang-hao2，ZHOU Zhi-ming2，WU Qian1，XI Shao-feng1，TAN Jian-hua1*（1．Guangzhou Quality Supervision and Testing Institute，National Quality Supervision and Testing Center for Cosmetics（Guangzhou），Guangzhou　511447，China；2．Guangdong Institute for Drug Control，Guangzhou　510663）Abstract：An ultra performance liquid chromatography-tandem mass spectrometric（UPLC-MS/MS）method was established for detecting 15 trace N-nitrosamine compounds in cosmetics. The final estab⁃lished method involved ultrasonic extraction of cosmetics using water or acetonitrile for different com⁃pounds. The samples were treated with potassium ferrocyanide-zinc acetate solution for precipitating macromolecules or saturated sodium chloride-acetonitrile for salting out.An Agilent Poroshell 120 SB-Aq（100 mm × 3.0 mm，2.7 μm） chromatography column was used for separation，followed by atmospheric pressure chemical ionization（APCI） source and multiple reaction monitoring mode detec⁃tion in the isotope internal standard method for quantification. The result showed good linearity（r2> 0.995） for the 15 N-nitrosamine compounds in their respective concentration ranges，with detection and quantitation limits of 5-15 ng/g and 15-45 ng/g，respectively.The average recoveries for the three cosmetic matrices（aqueous，emulsion，cream） at spiked levels of 25，50，100 ng/g were be⁃tween 88.0% and 111%，with relative standard deviations（RSD，n=6） of 1.4%-9.8%. The method was applied to the detection of commercial cosmetics and N-nitrosodiethanolamine（NDELA） was de⁃tected in 13 batches，with one batch exceeding the limit. The strong specificity，high sensitivity，and good precision made the method could solve the problems of poor stability and easy interference ofdoi：10.19969/j.fxcsxb.23051602收稿日期：2023－05－16；修回日期：2023－06－10基金项目：广东省药品监督管理局化妆品风险评估重点实验室专项（2021ZDZ03）；广东省市场监督管理局科技项目（2022CZ06）∗通讯作者：谭建华，博士，正高级工程师，研究方向：色谱－质谱检测技术研究，E-mail：tanjianhua0734@第 42 卷分析测试学报N-nitrosamine compounds，and was suitable for the trace determination of 15 N-nitrosamine com⁃pounds in cosmetics.Key words：N-nitrosamine compounds；cosmetics；ultra performance liquid chromatography-tan⁃dem mass spectrometry（UPLC-MS/MS）；atmospheric pressure chemical ionization（APCI） sourceN-亚硝胺化合物是一类具有N-亚硝基结构的化合物，因取代基的不同，形成了种类繁多的同系物，目前已发现超过300种［1］。

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