侯曉林，孫英健，吳國娟，苗 虹，吳永寧

(1.北京農(nóng)學(xué)院動(dòng)物科技系，北京 102206；2.中國疾病預(yù)防控制中心營養(yǎng)與食品安全所，北京 100050)

基質(zhì)固相分散術(shù)-UPLC法檢測(cè)禽蛋中對(duì)位紅和蘇丹紅

侯曉林1，孫英健1，吳國娟1，苗 虹2，吳永寧2

(1.北京農(nóng)學(xué)院動(dòng)物科技系，北京 102206；2.中國疾病預(yù)防控制中心營養(yǎng)與食品安全所，北京 100050)

建立禽蛋中對(duì)位紅和蘇丹紅Ⅰ～Ⅳ號(hào)5種色素的液相色譜檢測(cè)方法。采用基質(zhì)固相分散術(shù)，以氧化鋁作基質(zhì)分散基體，混合均勻的樣品用正己烷-丙酮(99.5:0.5，V/V)直接洗脫，吹干后定容測(cè)定，外標(biāo)法定量。5種色素的平均回收率為63.8%～96.2%，平均變異系數(shù)為0.52%～10.07%，檢測(cè)限為0.01～0.02mg/kg。在檢測(cè)的100個(gè)樣品中，1例檢出蘇丹紅Ⅳ號(hào)。

禽蛋；對(duì)位紅；蘇丹紅；高效液相色譜；基質(zhì)固相分散

蘇丹紅(sudan dyes)是一類人工合成的偶氮類、油溶性的化工染料，廣泛用于溶劑、油、蠟和汽油的增色以及鞋、地板的增光，蘇丹紅Ⅰ、Ⅱ、Ⅲ、Ⅳ已被國際癌癥研究機(jī)構(gòu)列為三類致癌物之一[1-2]，禁止作為色素添加劑在食品和飼料中使用，而其殘留在動(dòng)物體內(nèi)對(duì)食用者的健康和生命安全都存在潛在危害。對(duì)位紅化學(xué)結(jié)構(gòu)與蘇丹紅相似，具有相似毒性。對(duì)于該類化合物的檢測(cè)，主要檢測(cè)方法有液相色譜法和液相色譜-質(zhì)譜法[3-22]和氣相色譜法[5]。提取凈化方法集中于液相萃取和固相萃取。禽蛋及其制品黏性大，不易分散，利用國標(biāo)[3]或傳統(tǒng)有機(jī)溶劑萃取[4-22]，提取效率較低，往往需要耗費(fèi)大量溶劑來提高回收率。

基質(zhì)固相分散基本操作是將樣品(固態(tài)或液態(tài))直接與固相萃取材料一起混合研磨，使樣品均勻分散于固定相顆粒的表面，形成一個(gè)獨(dú)特的色譜固定相，將其裝柱后依靠所選定的溶劑洗脫樣品。它集合了傳統(tǒng)樣品的均質(zhì)、組織細(xì)胞裂解、提取、過濾、凈化等過程，使樣品的預(yù)處理變得簡便，同時(shí)也避免了目標(biāo)物的損失。由于該方法具有簡便、樣品和溶劑用量少等優(yōu)點(diǎn)而受到關(guān)注。本實(shí)驗(yàn)采用基質(zhì)固相分散術(shù)提取和凈化禽蛋中的蘇丹紅，UPLC對(duì)其進(jìn)行測(cè)定，旨在為其鑒定方法的建立提供一定的參考依據(jù)。

1 材料與方法

1.1 材料與試劑

對(duì)位紅及蘇丹紅Ⅰ、Ⅱ、Ⅲ、Ⅳ標(biāo)準(zhǔn)品 Sigma-Aldrich公司；乙腈、甲醇為色譜純；水為新制的雙蒸水；中性氧化鋁(100～200目) 上?；瘜W(xué)試劑公司，使用前120℃烘干3h，然后置于干燥器內(nèi)待用。

1.2 儀器與設(shè)備

超高效液相色譜UPLC-AQUITY系統(tǒng)(配備自動(dòng)進(jìn)樣器，二元泵和二極管陣列檢測(cè)器) Waters公司。

1.3 方法

1.3.1 標(biāo)準(zhǔn)貯備溶液配制

分別稱取對(duì)位紅、蘇丹紅Ⅰ、蘇丹紅Ⅱ、蘇丹紅Ⅲ及蘇丹紅Ⅳ各10.0mg(按實(shí)際含量折算)，用氯仿溶解并定容至10mL。

1.3.2 色譜條件

色譜儀：Waters ACQUITY UPLC配備Photodiode Array(PDA)檢測(cè)器。色譜柱：ACQUITY UPLC BEH C18(50mm×2.1mm，1.7μm)。流動(dòng)相：乙腈-水(80:20，V/V)。流速0.4mL/min、柱溫35℃、檢測(cè)波長500nm、進(jìn)樣體積5μL。

1.3.3 樣品處理

稱取2g氧化鋁置研缽中，稱取0.5g蛋黃置于氧化鋁上，將氧化鋁和蛋黃充分研磨混合成均勻的粉狀。后裝入5mL空注射器中，然后用35mL丙酮-正己烷(0.5:99.5，V/V)混合溶劑洗脫，收集洗脫液，并于45℃條件氮吹濃縮至近干。用乙酸-乙腈(1:1000，V/V)將殘?jiān)匦氯芙獠⒍ㄈ葜?mL，過0.25μm微孔濾膜，濾液供色譜分析。

2 結(jié)果與分析

2.1 樣品處理?xiàng)l件的選擇

2.1.1 萃取條件的選擇

由于禽蛋中蛋白質(zhì)的含量較多，用直接的液液萃取方法提取時(shí)不能有效地分散樣品，表面的一層蛋白質(zhì)先變性后會(huì)將樣品中心的色素包裹起來，即使通過振蕩等手段也很難將包被層破壞，因此不能充分地提取樣品。采用乙腈、正己烷和氯仿萃取的回收率均較低，有的甚至低于60%。因此，本實(shí)驗(yàn)針對(duì)蛋類食品的特點(diǎn)，選用了基質(zhì)固相分散技術(shù)，用氧化鋁與樣品一起研磨，達(dá)到均勻后再裝柱、洗脫。這樣能使樣品與氧化鋁，以及洗脫溶劑充分接觸，從而達(dá)到很好的提取效率。

2.1.2 洗脫劑種類的選擇

稱取10份空白樣品，添加100μg/kg的蘇丹紅混合標(biāo)準(zhǔn)工作液，分別用乙腈、三氯甲烷、丙酮正己烷作洗脫劑進(jìn)行洗脫。實(shí)驗(yàn)結(jié)果表明：氧化鋁對(duì)上述5種色素的結(jié)合不緊密，即使用正己烷淋洗，各色素均有洗脫。因此，本實(shí)驗(yàn)條件下，省略了淋洗凈化步驟，采用洗脫劑對(duì)各色素直接洗脫。

實(shí)驗(yàn)發(fā)現(xiàn)完全用正己烷洗脫，對(duì)位紅的回收率較低，即使將洗脫體積增加至40mL，回收率一般在40%～50%左右。因此，采用丙酮-正己烷(0.5:99.5，V/V)混合液進(jìn)行洗脫，5種色素的回收率均比較滿意。因此本研究初步確定用正己烷和丙酮的混合溶劑作為洗脫液。

2.1.3 洗脫劑體積的優(yōu)化

為了確保5種蘇丹紅類染料能全部洗脫下來，提高禽蛋中蘇丹紅染料的添加水平(200μg/kg)，采用丙酮-正己烷(0.5:99.5，V/V)洗脫，收集洗脫液，每5mL洗脫液收集，累計(jì)到35mL。結(jié)果表明，收集的洗脫液總量在35mL以后，洗脫液中不再檢出待測(cè)物。故洗脫劑用量確定為35mL。

2.2 校正曲線和線性范圍

取1.3.1節(jié)配制的標(biāo)準(zhǔn)貯備液分別用乙腈稀釋，配制成質(zhì)量濃度為0.025、0.05、0.10、0.20、0.40μg/mL的混合標(biāo)準(zhǔn)溶液，進(jìn)液相色譜儀，測(cè)定標(biāo)準(zhǔn)曲線及相關(guān)系數(shù)，如表2所示。所有相關(guān)系數(shù)均大于0.995，表明在0.025～0.40μg/mL質(zhì)量濃度范圍，5種色素的響應(yīng)具有很好的線性相關(guān)性(表1)。5種色素的濃度與色譜峰面積呈良好線性關(guān)系，以3倍信噪比為檢出限(LOD)，計(jì)算得出對(duì)位紅、蘇丹紅Ⅰ、Ⅱ、Ⅲ、Ⅳ的檢出限分別為0.01～0.02mg/kg。以10倍信噪比為定量下限(L O Q)，計(jì)算出對(duì)位紅、蘇丹紅Ⅰ、Ⅱ、Ⅲ、Ⅳ定量限分別為0.03～0.06mg/kg。

表1 5種色素的標(biāo)準(zhǔn)曲線、檢測(cè)限(LOD)和定量限(LOQ)Table 1 Standard curves, limit of detection, and limit of quantification for the five colorants

2.3 回收率與變異系數(shù)

圖1 UPLC-PDA的典型色譜圖Fig.1 UPLC chromatogram of blank sample, sudanⅠ－Ⅳ and para red standard, and blank sample spiked with the 5 compounds

表2 UPLC-PDA檢測(cè)方法的回收率、日內(nèi)變異系數(shù)(n=6)及日間變異系數(shù)Table 2 Recoveries, coefficients of variation for the UPLC-PDA method (n=6)

采用外標(biāo)法定量，以陰性樣品為測(cè)試樣品，添加不同質(zhì)量濃度水平的蘇丹紅，每個(gè)水平做6個(gè)平行樣，計(jì)算回收率和變異系數(shù)(CV)，結(jié)果見表2。5種色素的日內(nèi)回收率范圍為61.7%～98.7%，變異系數(shù)為1.53%～7.55%。日間平均回收率為63.8%～96.2%，變異系數(shù)為0.52%～10.07%。其中蘇丹紅Ⅳ的回收率略低。典型色譜圖見圖1。

2.4 實(shí)測(cè)樣本

應(yīng)用所建立的方法，在北京市超市隨機(jī)抽檢100個(gè)樣品，其中有一例樣品檢出蘇丹紅Ⅳ，其含量為0.3mg/kg。色譜圖見圖2。

圖2 UPLC-PDA方法測(cè)定的蘇丹紅Ⅳ陽性樣品Fig.2 UPLC chromatogram one sample containing sudan Ⅳ

3 結(jié) 論

采用氧化鋁為固相載體，利用固相基質(zhì)分散技術(shù)術(shù)，建立禽蛋中對(duì)位紅、蘇丹紅Ⅰ、Ⅱ、Ⅲ、Ⅳ號(hào)5種色素的液相色譜測(cè)定方法。該方法較為簡單、方便，節(jié)省時(shí)間和溶劑。回收率和穩(wěn)定性滿足該類色素測(cè)定的要求，建議推廣使用。

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Use of Matrix Solid-Phase Dispersion and Ultra Performance Liquid Chromatography for Determination of Para Red and SudanⅠ－Ⅳ in Egg Yolk

HOU Xiao-lin1，SUN Ying-jian1，WU Guo-juan1，MIAO Hong2，WU Yong-ning2
(1. Department of Animal Science, Beijing University of Agriculture, Beijing 102206, China；2. Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100050, China)

An ultra-performance liquid chromatography (UPLC) method combined with matrix solid-phase dispersion (MSPD)was established for determination of para red and sudanⅠ, Ⅱ, Ⅲ and Ⅳ in egg yolk. A total of 0.5 g egg yolk was mixed with 2 g of neutral alumina, then rinsed with 35 mL of hexane-acetone (0.5:99.5, V/V) through a 5 mL syringe. The eluent was heated to about 45℃ with nitrogen blowing to dryness and redissolved for UPLC analysis. Mean recovery for the five dyes ranged from 63.8% to 96.2%, with coefficient variation at 0.52%－10.07%. One sample was found containing sudan Ⅳ among the 100 samples.Key words：egg yolk；para red；sudanⅠ－Ⅳ；ltra-performance liquid chromatography；maxtrix solid-phase dispersion

O657.31

A

1002-6630(2010)24-0285-04

2010-01-07

北京市屬高等學(xué)校人才強(qiáng)教計(jì)劃項(xiàng)目(PXM2009_014207_077729)；國家“863”計(jì)劃項(xiàng)目(2007AA06Z404)

侯曉林(1973—)，男，副教授，博士，研究方向?yàn)楂F醫(yī)藥理與毒理學(xué)。E-mail：hxlsx@163.com