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Sensitive Analysis of Microcystins in Aquatic Products by Nano-Flow Chip Liquid Chromatography-Mass Spectrometry
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摘要: 建立了一种芯片液相色谱-高分辨质谱测定3种微囊藻毒素（MC-RR、MC-YR和MC-LR）的分析方法.在最优条件下，3种藻毒素在16 min内实现基线分离，并选择电喷雾源正离子模式进行质谱测定.方法在2.5~2 000 ng/mL范围内线性良好，线性相关系数大于0.996 5，检测限介于0.5~2.0 ng/mL之间.鱼肉和虾肉样品经固相萃取法净化处理后未测得上述毒素残留，加标回收率为82.8%~120.3%.方法具有样品用量少、定性快速准确、灵敏度高等优点，适合用于水产品等生物样品中藻毒素的痕量监测.Abstract: A method for the simultaneous analysis of 3 microcystins (MC-RR, MC-YR and MC-LR) was set up by the nano-flow chip liquid chromatography-high resolution mass spectrometry. Under optimal conditions, the mentioned microcystins were baseline separated in 16 min with an ESI source in the positive ion mode. Good linear range in 2.5~2 000 ng/mL was obtained, with the correlation coefficient larger than 0.996 5 and the limit of detection between 0.5~2.0 ng/mL. Solid phase extraction method was used for the extraction and clean-up of fish and shrimp samples. No residue of the three microcystins was detected and the spiked recoveries were between 82.8%~120.3%. The developed method is sensitive, rapid and exact with low sample consumption, so it is reliable for the trace determination of microcystins in biological samples including aquatic products.
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Key words:
- microcystins /
- nano-flow chip liquid chromatography /
- mass spectrometry /
- aquatic product
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图 1 MCs的化学结构(a)和芯片示意图(b)
Figure 1. Chemical structure of MCs (a) and schematic diagram of chip (b)
图 2 不同流动相对分离的影响
(a)乙腈-H2O，(b)乙腈-0.05% FA，(c)乙腈-0.10% FA，(d)乙腈-0.15% FA
毛细管泵流速2 μL/min，10%乙腈等度洗脱；纳流泵流速600 nL/min，0~20 min，10-60% B相梯度洗脱进样体积l μL，进样冲洗体积为4 μLFigure 2. Effects of different mobile phase on separation
(1)MC-RR(0.5 μg/mL)，(2)MC-YR(2.0 μg/mL)，(3)MC-LR(1.0 μg/mL)
图 3 不同梯度洗脱条件对分离的影响
乙腈-0.15% FA；毛细管泵流速2 μL/min，10%乙腈等度洗脱；进样体积1 μL，进样冲洗体积为4 μL
Figure 3. Effects of different gradient elutions on separation
(1)MC-RR(0.5 μg/mL), (2)MC-YR(2.0 μg/mL), (3)MC-LR(1.0 μg/mL)
图 4 正离子检测模式下3种MCs的全扫描质谱图
Figure 4. Mass spectra of three MCs under full scan with positive mode
表 1 3种MCs的保留时间和特征离子
Table 1. Retention time and characteristic ions of three MCs
分析物 分子式 保留时间/min m/z[M+H]+ 偏差/δ m/z[M+2H]2+ 偏差/δ MC-RR C49H75N13O12 11.97 1 038.573 4 0.29 519.791 1 1.73 MC-YR C52H72N10O13 14.08 1 045.539 4 3.92 523.272 4 2.10 MC-LR C49H74N10O12 14.63 995.551 4 -4.62 498.283 3 3.21 
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表 2 3种MCs的分析参数
Table 2. Analytical parameters of three MCs
分析物 线性范围
/(ng/mL)相关系数 检测限
/(ng/mL)日内RSDs /% (n=7) 日间RSDs /% (n=3) 保留时间/min 峰面积 保留时间/min 峰面积 MC-RR 2.5~500.0 0.999 4 0.5 0.22 2.85 0.55 4.09 MC-YR 10.0~2 000.0 0.996 5 2.0 0.11 3.42 0.39 5.41 MC-LR 5.0~1 000.0 0.998 2 0.8 0.19 4.24 0.41 6.81
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表 3 水产品的加标回收率
Table 3. Spiked recoveries of aquatic products
分析物 加入质量浓度
/(ng/mL)虾 鱼 测得质量浓度/(ng/mL) 回收率/% 测得质量浓度/(ng/mL) 回收率/% MC-RR 75.0 75.1±1.8 100.1 78.1±1.4 104.1 MC-YR 300.0 324.0±4.3 108.0 248.4±5.2 82.8 MC-LR 150.0 149.3±4.7 99.5 180.5±5.4 120.3
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表 4 本法与HPLC相关方法比较
Table 4. Comparison of related HPLC methods
检测方法 前处理
方法进样体积
/μL线性范围
/(ng/mL)检测限
/(ng/mL)样品基质 参考文献 HPLC-UV 固相微萃取 10 1.0~200.0 0.15 (MC-RR), 0.45 (MC-LR) 水 [4] HPLC-UV 磁性固相萃取 20 0.25~146.5 0.011 (MC-LR) 水 [5] HPLC-MS/MS 基质固相分散 5 5.0~100.0 13.0 μg/kg (dw) 蔬菜 [6] HPLC-MS/MS 超声辅助萃取 5 2~500.0 0.78 (MC-RR), 0.12 (MC-YR), 0.20 (MC-LR) 螺类 [7] nano-flow chip HPLC-MS 固相萃取 1 2.5~2 000.0 0.5(MC-RR), 2.0 (MC-YR), 0.8 (MC-LR) 水产品 本文
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