时间:2022-10-11 10:58:38
1 引 言
罗丹明B常被违法用于调味品的染色,孔雀石绿和结晶紫因具有消毒和杀菌作用而常被违法应用于水产品养殖中。三者结构类似,都具有高毒、高残留和致癌、致畸、致突变等特点,色谱分离较难,且由于食品基质复杂,对质谱检测影响较大。目前,单独检测3种物质的方法分别有高效液相色谱法[1]、气相色谱-质谱法[2]、液相色谱-质谱法[3]和液相色谱-串联质谱法[4],未见有应用固相萃取-超高效液相色谱-串联质谱法对复杂食品基质中上述3种物质同时测定的报道。
2 实验部分
2.1 仪器、试剂与材料 超高效液相色谱-串联质谱仪(ACQUITY UPLC-Quattro Premier XE, Waters公司)。色谱柱: 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)。罗丹明B、结晶紫、孔雀石绿(Sigma公司),用乙腈(色谱纯,Fisher公司)配制成标准溶液。
2.2 色谱-质谱条件 流动相A为乙腈,B为0.2%甲酸溶液,流速为0.2 mL/min;进样量:10
SymbolmA@ L;柱温: 35 ℃,梯度洗脱: 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。电喷雾离子源正离子多反应监测,毛细管电压:3.0 kV;离子源温度:120 ℃;脱溶剂气温度:350 ℃;脱溶剂气流量:800 L/h;锥孔反吹气流量:50 L/h。其它质谱参数见表1。
孔雀石绿Malachite329313.*208603834 * 定量离子对(Quantification ion pair)。
2.3 提取 称取匀浆后的水产品配料调味酱2 g于具塞离心管中,依次加人1 mL 20%盐酸羟胺,1 mL 0.1 mol/L对甲苯磺酸,2.5 mL 0.1 mol/L乙酸铵缓冲溶液和12 mL乙腈,漩涡振荡提取5 min,低温(5 ℃) 10000 r/min离心5 min。上清液移至另一离心管中,将残渣捣碎,用12 mL乙腈重复提取,合并上清液;加入5 g中性氧化铝,漩涡振荡提取5 min后,低温(5 ℃) 1
SymboltB@ 104 r/min离心5 min,上清液转移至分液漏斗中,加人15 mL二氯甲烷,10 mL水,振摇2 min,静置分层,收集下层有机层,再用10 mL二氯甲烷萃取一次,合并有机相于40 ℃旋转蒸发近干。
2.4 净化 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乙腈溶解后过柱,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-甲醇溶液洗脱,洗脱液于40 ℃旋转蒸发近干,残留物用1 mL流动相定容,经0.2
SymbolmA@ m滤膜过滤, 供超高效液相色谱-串联质谱测定。
3 结果与讨论
3.1 超高效液相色谱条件的优化 研究了水-甲醇和水-乙腈两种体系中水相及有机相比、添加物的种类(甲酸和乙酸胺)及添加比例对目标物分离的影响。结果表明,选用乙腈-0.2%甲酸做流动相时,3种染料有较好的分离度,且色谱峰峰型良好,混合标准溶液的MRM及总离子流色谱图见图1。
SymbolmA@ g/L)MRM、总离子流色谱图
Fig.1 Multiple reaction monitoring,total ion curren chromatograms of Rhodamine B,crystal violet and malachite standard solution(10
SymbolmA@ g/L)
3.2 方法检出限和定量限 对3种物质混合标准溶液在5
SymboltB@ 10
Symbolm@@ 4 ~1.0 mg/L浓度范围内进行分析, 线性相关系数r≥0.997。在空白样品中添加目标化合物,测得该方法的检出限为0.18~0.48
SymbolmA@ g/kg。
3.3 回收率、精密度及实际样品测定
在空白水产调味品中添加不同浓度(5.0, 20.0和100.0
SymbolmA@ g/kg)的标准溶液,测得平均回收率分别为罗丹明B 90.7%~96.8%、结晶紫78.4%~90.3%、孔雀石绿78.6%~88.9%;日内相对标准偏差均<10%,日间相对标准偏差均<15%。用本方法对10份水产调味品样品进行检测, 其中一份样品中罗丹明B呈阳性。
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)