1 引 言
羅丹明B常被違法用于調(diào)味品的染色,孔雀石綠和結(jié)晶紫因具有消毒和殺菌作用而常被違法應(yīng)用于水產(chǎn)品養(yǎng)殖中。三者結(jié)構(gòu)類似,都具有高毒、高殘留和致癌、致畸、致突變等特點(diǎn),色譜分離較難,且由于食品基質(zhì)復(fù)雜,對(duì)質(zhì)譜檢測(cè)影響較大。目前,單獨(dú)檢測(cè)3種物質(zhì)的方法分別有高效液相色譜法[1]、氣相色譜-質(zhì)譜法[2]、液相色譜-質(zhì)譜法[3]和液相色譜-串聯(lián)質(zhì)譜法[4],未見有應(yīng)用固相萃取-超高效液相色譜-串聯(lián)質(zhì)譜法對(duì)復(fù)雜食品基質(zhì)中上述3種物質(zhì)同時(shí)測(cè)定的報(bào)道。
2 實(shí)驗(yàn)部分
2.1 儀器、試劑與材料 超高效液相色譜-串聯(lián)質(zhì)譜儀(ACQUITY UPLC-Quattro Premier XE, Waters公司)。色譜柱: Acquity UPLCTM BEH C18柱(50 mm×2.1 mm, 1.7
SymbolmA@ m,Waters公司);固相萃取柱(OASIS MCX, 60 mg/3 mL, Waters公司)。羅丹明B、結(jié)晶紫、孔雀石綠(Sigma公司),用乙腈(色譜純,F(xiàn)isher公司)配制成標(biāo)準(zhǔn)溶液。
2.2 色譜-質(zhì)譜條件 流動(dòng)相A為乙腈,B為0.2%甲酸溶液,流速為0.2 mL/min;進(jìn)樣量:10
SymbolmA@ L;柱溫: 35 ℃,梯度洗脫: 0~1 min, 30%~40% A; 1~2 min, 40%~50% A; 2~3 min, 50%~70% A; 3~5 min, 70%~90% A; 5~6 min, 90%~70% A; 6~7 min, 70%~30% A。電噴霧離子源正離子多反應(yīng)監(jiān)測(cè),毛細(xì)管電壓:3.0 kV;離子源溫度:120 ℃;脫溶劑氣溫度:350 ℃;脫溶劑氣流量:800 L/h;錐孔反吹氣流量:50 L/h。其它質(zhì)譜參數(shù)見表1。
孔雀石綠Malachite329313.*208603834 * 定量離子對(duì)(Quantification ion pair)。
2.3 提取 稱取勻漿后的水產(chǎn)品配料調(diào)味醬2 g于具塞離心管中,依次加人1 mL 20%鹽酸羥胺,1 mL 0.1 mol/L對(duì)甲苯磺酸,2.5 mL 0.1 mol/L乙酸銨緩沖溶液和12 mL乙腈,漩渦振蕩提取5 min,低溫(5 ℃) 10000 r/min離心5 min。上清液移至另一離心管中,將殘?jiān)鼡v碎,用12 mL乙腈重復(fù)提取,合并上清液;加入5 g中性氧化鋁,漩渦振蕩提取5 min后,低溫(5 ℃) 1
SymboltB@ 104 r/min離心5 min,上清液轉(zhuǎn)移至分液漏斗中,加人15 mL二氯甲烷,10 mL水,振搖2 min,靜置分層,收集下層有機(jī)層,再用10 mL二氯甲烷萃取一次,合并有機(jī)相于40 ℃旋轉(zhuǎn)蒸發(fā)近干。
2.4 凈化 依次用甲醇、0.2%甲酸各3 mL活化MCX 固相萃取柱,提取物用6 mL乙腈溶解后過柱,柱流速低于1 mL/min。再用 3 mL 0.2%甲酸,3 mL 50%甲醇淋洗,最后用 3 mL 10%氨水-甲醇溶液洗脫,洗脫液于40 ℃旋轉(zhuǎn)蒸發(fā)近干,殘留物用1 mL流動(dòng)相定容,經(jīng)0.2
SymbolmA@ m濾膜過濾, 供超高效液相色譜-串聯(lián)質(zhì)譜測(cè)定。
3 結(jié)果與討論
3.1 超高效液相色譜條件的優(yōu)化 研究了水-甲醇和水-乙腈兩種體系中水相及有機(jī)相比、添加物的種類(甲酸和乙酸胺)及添加比例對(duì)目標(biāo)物分離的影響。結(jié)果表明,選用乙腈-0.2%甲酸做流動(dòng)相時(shí),3種染料有較好的分離度,且色譜峰峰型良好,混合標(biāo)準(zhǔn)溶液的MRM及總離子流色譜圖見圖1。
SymbolmA@ g/L)MRM、總離子流色譜圖
Fig.1 Multiple reaction monitoring,total ion curren chromatograms of Rhodamine B,crystal violet and malachite standard solution(10
SymbolmA@ g/L)
3.2 方法檢出限和定量限 對(duì)3種物質(zhì)混合標(biāo)準(zhǔn)溶液在5
SymboltB@ 10
Symbolm@@ 4 ~1.0 mg/L濃度范圍內(nèi)進(jìn)行分析, 線性相關(guān)系數(shù)r≥0.997。在空白樣品中添加目標(biāo)化合物,測(cè)得該方法的檢出限為0.18~0.48
SymbolmA@ g/kg。
3.3 回收率、精密度及實(shí)際樣品測(cè)定
在空白水產(chǎn)調(diào)味品中添加不同濃度(5.0, 20.0和100.0
SymbolmA@ g/kg)的標(biāo)準(zhǔn)溶液,測(cè)得平均回收率分別為羅丹明B 90.7%~96.8%、結(jié)晶紫78.4%~90.3%、孔雀石綠78.6%~88.9%;日內(nèi)相對(duì)標(biāo)準(zhǔn)偏差均<10%,日間相對(duì)標(biāo)準(zhǔn)偏差均<15%。用本方法對(duì)10份水產(chǎn)調(diào)味品樣品進(jìn)行檢測(cè), 其中一份樣品中羅丹明B呈陽性。
References
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4 Hurtaud-Pessel D, Couedor P, Verdon E. J. Chromatogr. A, 2011, 1218(12): 1632~1645
Simultaneous Determination of Rhodamine B,Crystal Violet
and Malachite in Aquatic Flavourings by Solid-phase
Extraction Coupled with Ultra Performance Liquid
Chromatography-Tandem Mass Spectrometry
ZHANG Zhen-Li1,2, ZHANG Pin.1, SHEN Da-Zhong*1
.1(College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China)
.2(Zibo Institute of Product Quality Supervision Inspection, Zibo 255033, China)
Abstract A method was developed for the simultaneous determination of rhodamine B, crystal violet and malachite in aquatic flavourings based on solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry. The samples were extracted with acetonitrile-buffer, partitioned against methylene dichloride and then cleaned up by a solid phase extraction cartridge. The target analytes were separated on a BEH C18 column with gradient elution using acetonitrile and water (containing 0.2% (V/V) formic acid) as mobile phases. The linear ranges were from 5×10
Symbolm@@ 4 to 1 mg/L with the limits of detection of 0.18-0.48
SymbolmA@ g/kg. The recoveries of the method were 90.7%-96.8%, 78.4%-90.3%, 78.6%-88.9%, respectively.
Keywords Ultra performance liquid chromatography-tandem mass spectrometry; Solid-phase extraction; Rhodamine B; Crystal violet; Malachite; Aquatic flavourings
(Received 15 September 2011; accepted 3 November 2011)