阳离子交换在线固相萃取/液相色谱—串联质谱法检测牛奶中14种磺胺药物残留

顾欣等
摘 要 建立了阳离子交换模式在线固相萃取液相色谱串联质谱法检测牛奶中14种磺胺药物方法。取5 g样品用15 mL乙腈提取和除蛋白,提取液于50 ℃氮气吹干后,用1.00 mL 0.2%甲酸溶解,溶解液通过双三元液相色谱用阳离子在线固相萃取柱在线富集净化,2%氨水甲醇/0.2%甲酸(50∶50,V/V)洗脱。然后转移至C18色谱柱上进行分离,再用串联四级杆质谱检测。结果表明,14种磺胺类药物在0.1~10 μg/kg含量范围内线性良好(r≥0.999);方法的检出限为0.05 μg/kg,定量限为0.1 μg/kg;方法回收率在60%~90%范围内,批内和批间相对标准偏差都小于10%。本方法较传统固相萃取柱净化法更简捷、经济和稳定。
1 引 言
固相萃取(Solid phase extraction, SPE)利用固体吸附剂吸附液体样品中的目标化合物,与样品的基体和干扰化合物分离,然后再用洗脱液洗脱,达到分离和富集目标化合物的目的。样品中目标物质的分离和富集是色谱分析中必不可少的一部分,因此固相萃取技术作为一种传统的前处理手段已被广泛应用于色谱分析的各个领域。常用的固相萃取主要有分散相固相萃取和填料柱固相萃取这两种方式[1~4],通过这些手段可以达到去除试样中的干扰物质,使痕量组分得到富集便于检测和分离,保护色谱分析柱等目的。然而,传统离线固相萃取常需繁琐的手工操作才能完成,比色谱分离和数据处理等花费更多的时间。近年来,随着液相色谱仪技术的迅速发展,仪器的自动化程度越来越高,样品的前处理技术实现自动化成为了一种新趋势。在线固相萃取技术传统填料柱固相萃取和分散相固相萃取相比具有节省人力和耗材,提高前处理稳定性和缩短检测时间的优势。
磺胺类药物是具有对氨基苯磺酰胺结构的一类广谱抗菌药物的总称,兽医临床和畜牧养殖业中被广泛应用于预防和治疗细菌感染性疾病[11,12]。但是磺胺药会引起人过敏性反应,且可能有致癌性,其不合理使用使其在动物性食品中残留, 引起生态环境污染,危害人类健康。针对该情况开发了各类检测方法,其中较为常见的有酶联免疫法,HPLC法和UPLCMS/MS法等。据文献报道,采用酶联免疫法快速检测牛奶中的8种磺胺类药物[13],其检测限可达5 μg/L,灵敏度较高,检测通量大,可以用来做大规模筛查,但无法很好区分磺胺药物种类;使用高效液相色谱法结合基质固相分散前处理法检测牛奶中8种磺胺类药物,其方法灵敏度可达0.05 mg/L[14],但其分析时间较长且灵敏度偏低;有报道超高效液相色谱串联质谱法单针进样可在10 min内同时检测24种磺胺类药物[15],其检测灵敏度高,方法特异性好,但是前处理步骤过于简单,在检测大量样品时容易造成杂质对分析柱和质谱源区的污染。为保障目前市售牛奶的食品质量安全,防止磺胺类抗菌药在奶牛养殖环节的违法滥用,本实验采用在线固相萃取结合液质联用技术检测牛奶中14种常见磺胺类药物, 利用双三元液相的程序控制流路切换功能实现了在线固相萃取,解决了牛奶基质中乳脂和蛋白对仪器的干扰和污染;克服了使用传统固相萃取技术带来的耗材成本高,人力耗费大等缺点;提高了检测通量和稳定性,具有更简捷、经济和稳定的特点。
2 实验部分
2.1 仪器与试剂
实验表明,当源电压设为3500 kV时,大部分磺胺类化合物的[M+H\]+分子离子峰绝对响应达到最大值;之后,随着源电压上升,响应没有显著增加;到3800 kV之后甚至有下降趋势,且源区有微弱电弧光显现。推测可能是电压太高而造成雾化后的微液滴电荷过载,发生击穿放电现象,此现象导致进入离子传输通道的带电粒子数下降。因此,源电压选择3500 kV。另外,在被检测的化合物中,SPD与SDZ分子离子的质荷比仅差1,SMZ与ST之间,SBZ与SDMD与SMPD之间的质荷比仅差2,因此有必要提高Q1段的质量数分辨率,避免相互之间的干扰。将Q1段的质量数分辨率从原来的半峰宽0.7提高到0.4,这样可能会损失一定的响应值,但是可以很好地解决质量数接近的分子离子互相干扰问题,有效提高信噪比。
3.2 在线固相萃取条件的优化
在线固相萃取(Online SPE)是通过控制两台独立工作的液体传输泵和切换阀使用程序分别进行检品的富集净化和分析,需要有一台泵负责进样和富集净化功能,使目标化合物在在线固相萃取柱上实现保留富集和洗涤;通过阀切换,使用另一台泵进行样品的过柱分离和检测。选择合适的在线固相萃取柱和设计合理的富集净化和洗脱条件是在线固相萃取方法的关键,本方法选择Retain CX作为在线SPE柱,Retain CX的富集净化机理为阳离子交换结合反相机理,磺胺类药物大多为碱性化合物,使用阳离子交换结合反相模式的在线固相萃取柱具有更好的富集净化效果。在进样时进样泵的流路采用甲醇0.2%甲酸(10∶90, V/V)为流动相,可在富集磺胺类药物的同时清洗部分极性较大的杂质,通过在线分析柱后直接连接质谱检测器,发现采用此流动相并无目标化合物的流失(图2a)。选择5%氨水甲醇溶液作为洗脱液,通过阀切换,使用分析流路进行洗脱和分析,分析流路应在阀切换之前先用洗脱溶液充满流路,这将有助于更快地在阀切换之后集中将目标化合物洗脱,避免因为分析流动相造成在线净化柱上目标化合物的扩散而造成目标峰展宽。将流路中的分析柱拆去,用两通替代,可以获得目标化合物的洗脱时间约为4.2 min(图2b)。据此可以确定,阀切换的时间应该略晚于此时间,但不应过长而导致部分低极性杂质进入分析流路。由于5%氨水甲醇洗脱剂和0.2%甲酸乙睛(15∶85, V/V)分析流动相的差异较大,容易由于溶剂化效应而造成色谱峰展宽,因此洗脱时采用较低的流速0.4 mL/min, 减小进样溶剂的总量;此时分析流路使用较高流速3.3 检测限和定量限的测定
选择20个空白样品,按优化条件进行处理后上机测定,取与标准品图谱中相同保留时间的噪音信号平均值,以信噪比S/N≥3为检出限(LOD), S/N≥10为定量下限(LOQ),确定该方法对盐酸赛庚啶的检出限为0.05 μg/kg,定量下限为0.1 μg/kg。经过实际进样检测后,检测定量限附近峰型良好。
3.4 在线固相萃取与传统固相萃取方法检测14种磺胺类药物的回收率与精密度比较
为研究在线固相萃取和传统固相萃取法检测牛奶种14种磺胺类药物时方法回收率和精密度的差异,使用6批不同牛奶,每批称取等量6份,3份一组分两组,分别加入0.1,0.5 和5.0 μg/kg的14种磺胺类化合物,两组分别采用在线阳离子交换固相萃取法和传统阳离子交换固相萃取法,所得结果如表4所示。实验结果表明,在线阳离子交换固相萃取法检测牛奶中14种磺胺类化合物时回收率结果在60%~90%之间,在线阳离子交换固相萃取法与传统阳离子交换固相萃取法的批内和批间相对标准偏差也都在0~10%之间,所得结果基本一致,且具有简捷、经济和稳定的优点。
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Determination of 14 Sulfonamides Residue in Milk by Online
Solid Phase Extraction in Cation Exchange Mode/Liquid
ChromatographyTandem Mass Spectrometry
GU Xin, WU JianPing*, ZHANG Xing, LI DanNi, YAN Feng, ZHOU YueRong
(Shanghai Municipal Supervisory Institute of Veterinary Drugs and Feedstaff, Shanghai 201103, China)
Abstract To determine the residue of 14 sulfonamides in milk, a high performance liquid chromatography tandem mass spectrometry (HPLCMS/MS) method with online soild phase extraction (SPE) in cation exchange mode was established. 5 g of milk was extracted with 15 mL acetonitrile. Then the extraction was evaporated by 50 ℃ nitrogen and dissolved by 1.00 mL 0.2% formic acid. The dissolution was enriched and purified by MS/MS cation exchange online SPE column on a double ternary liquid chromatography, and eluted by the mixed solution of 2% ammonia methanol and 0.2% formic acid (50∶ 50, V/V). The compounds were separated by an octadecyl silica bonded column and determined by the tandem mass spectrometry. The results showed that the linearity of 14 sulfonamides was good in the range of 0.1–10 μg/kg (r≥0.9995). The LOD of the method was 0.05 μg/kg, while the LOQ was 0.1 μg/kg. The recoveries of the 14 sulfonamides were in the range of 60 %-90 %, while the interbatch and intrabatch RSDs were all lower than 10%. The method was proved to be more convenient, economical and stable than the traditional SPE column method.
Keywords Online solid phase extraction; Cation exchange; Double ternary liquid chromatographytandem mass spectrometry; Sulfonamides; Milk
(Received 25 August 2014; accepted 10 October 2014)
This work was supported by the Special Fund for Agroscientific Research in the Public Interest (No. 201203023)
GU Xin, WU JianPing*, ZHANG Xing, LI DanNi, YAN Feng, ZHOU YueRong
(Shanghai Municipal Supervisory Institute of Veterinary Drugs and Feedstaff, Shanghai 201103, China)
Abstract To determine the residue of 14 sulfonamides in milk, a high performance liquid chromatography tandem mass spectrometry (HPLCMS/MS) method with online soild phase extraction (SPE) in cation exchange mode was established. 5 g of milk was extracted with 15 mL acetonitrile. Then the extraction was evaporated by 50 ℃ nitrogen and dissolved by 1.00 mL 0.2% formic acid. The dissolution was enriched and purified by MS/MS cation exchange online SPE column on a double ternary liquid chromatography, and eluted by the mixed solution of 2% ammonia methanol and 0.2% formic acid (50∶ 50, V/V). The compounds were separated by an octadecyl silica bonded column and determined by the tandem mass spectrometry. The results showed that the linearity of 14 sulfonamides was good in the range of 0.1–10 μg/kg (r≥0.9995). The LOD of the method was 0.05 μg/kg, while the LOQ was 0.1 μg/kg. The recoveries of the 14 sulfonamides were in the range of 60 %-90 %, while the interbatch and intrabatch RSDs were all lower than 10%. The method was proved to be more convenient, economical and stable than the traditional SPE column method.
Keywords Online solid phase extraction; Cation exchange; Double ternary liquid chromatographytandem mass spectrometry; Sulfonamides; Milk
(Received 25 August 2014; accepted 10 October 2014)
This work was supported by the Special Fund for Agroscientific Research in the Public Interest (No. 201203023)
GU Xin, WU JianPing*, ZHANG Xing, LI DanNi, YAN Feng, ZHOU YueRong
(Shanghai Municipal Supervisory Institute of Veterinary Drugs and Feedstaff, Shanghai 201103, China)
Abstract To determine the residue of 14 sulfonamides in milk, a high performance liquid chromatography tandem mass spectrometry (HPLCMS/MS) method with online soild phase extraction (SPE) in cation exchange mode was established. 5 g of milk was extracted with 15 mL acetonitrile. Then the extraction was evaporated by 50 ℃ nitrogen and dissolved by 1.00 mL 0.2% formic acid. The dissolution was enriched and purified by MS/MS cation exchange online SPE column on a double ternary liquid chromatography, and eluted by the mixed solution of 2% ammonia methanol and 0.2% formic acid (50∶ 50, V/V). The compounds were separated by an octadecyl silica bonded column and determined by the tandem mass spectrometry. The results showed that the linearity of 14 sulfonamides was good in the range of 0.1–10 μg/kg (r≥0.9995). The LOD of the method was 0.05 μg/kg, while the LOQ was 0.1 μg/kg. The recoveries of the 14 sulfonamides were in the range of 60 %-90 %, while the interbatch and intrabatch RSDs were all lower than 10%. The method was proved to be more convenient, economical and stable than the traditional SPE column method.
Keywords Online solid phase extraction; Cation exchange; Double ternary liquid chromatographytandem mass spectrometry; Sulfonamides; Milk
(Received 25 August 2014; accepted 10 October 2014)
This work was supported by the Special Fund for Agroscientific Research in the Public Interest (No. 201203023)
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