超高效液相色谱-电喷雾串联质谱法测定水产品中亚甲基蓝及代谢物

建立了超高效液相色谱-电喷雾串联质谱法同时检测水产品中亚甲基蓝及其代谢物的分析方法。经过条件优化，试样用10 mL乙腈提取3次，并在体系中添加盐酸羟胺、对甲苯磺酸等提高提取效率，提取液经对丙磺酸固相萃取小柱净化，按V（乙腈）：V（乙酸铵，1 mol·L-1）=1：1洗脱，采用Thermo Hypersil C18色谱柱，以甲醇和0.2%甲酸为流动相进行梯度洗脱分离，电喷雾离子源（ESI），正离子模式，采用选择反应监测（SRM）扫描模式。结果表明，亚甲基蓝及代谢物（天青A、天青B、天青C）在0.50-100.00 μg·L-1范围内线性关系良好，相关系数R〉0.99，在10 min内实现分离，3个加标水平（2 μg·kg-1、4 μg·kg-1、10 μg·kg-1）的平均回收率为74.23%-94.40%（n=6），相对标准偏差（RSD）为1.13%-10.28%，检测限（LOD）为0.40~0.60 μg·kg-1，定量限（LOQ）为2.00 μg·kg-1。该方法简便快捷、准确度高，易于推广应用，可作为快速测定水产品中亚甲基蓝及其代谢物的有效方法。

Determination of methylene blue and its metabolites in aquatic products by UPLC/ESI-MS/MS

We established a method for simultaneous determination of methylene blue and its metabolites in aquatic products using ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry （UPLC/ESI-MS/MS）. We extracted the sample with acetonitrile for three times and added hydroxylamine hydrochloride and paratoluenesulfonic acid for efficiency in the meantime, purified it by a PRS SPE column, and eluted by V（Acetonitrile）：V（Ammonium acetate, 1mol·L-1）=1：1. Next, based on Thermo Hypersil Gold C18 column, we used a methanol and 2% formic acid as gradient elution separation to deal with the sample, with the electrospray of mass spectrometry in positive ionization mode and the identifying of methylene blue and its metabolites in selected reaction monitoring （SRM mode）. The results show that methylene blue and its metabolites have a good linear relationship within 0.50-100.00 μg·L-1（R〉0.99）. The separation is finished within 10 min. Average recovery （n=6） of the target compounds at 3 levels （2 μg·kg-1, 4 μg·kg-1, 10 μg·kg-1） is 78.11%~94.40%, while the relative standard deviation （RSD） is 2.12%~8.13%. Besides, the limit of detection （LOD） ranges from 0.40 to 0.60 μg·kg-1, and the limit of quantity （LOQ） is 2.00 μg·kg-1. The method is convenient, accurate, easily be applied and popularized, which is suitable for determination and confirmation of methylene blue and its metabolites in aquatic products.