养殖水中孔雀石绿、隐色孔雀石绿、结晶紫、 隐色结晶紫、亚甲基蓝残留量的测定

目的建立一种高效液相色谱-串联质谱同时测定养殖水体中孔雀石绿、结晶紫及其代谢物和亚甲基蓝残留量的方法。方法将水样用盐酸羟胺-对甲苯磺酸溶液和乙腈提取,二氯甲烷萃取2次后浓缩,用1 mL乙腈-5 mmol/L乙酸铵(1:1,V:V)定容。以乙腈-5 mmol/L乙酸铵(85:15,V:V)混合溶液为流动相进行色谱分离,然后进入串联质谱仪,选用电喷雾离子源,在正离子、多反应监测扫描模式下进行定性,孔雀石绿和结晶紫通过内标法定量,亚甲基蓝通过外标法定量。结果在优化条件下,孔雀石绿、隐色孔雀石绿、结晶紫、隐色结晶紫在0.025~0.600 ng/mL浓度范围内,亚甲基蓝在0.05~1 ng/mL浓度范围内均满足线性关系,相关系数r0.99,方法检出限为0.2 ng/mL。加标浓度在0.2~2.0 ng/mL时,孔雀石绿、隐色孔雀石绿、结晶紫、隐色结晶紫和亚甲基蓝的平均回收率均在70%~110%之间,相对标准偏差均在10%以内。结论该方法简单快速、灵敏度高、前处理成本低、重现性好、回收率高,适用于同时检测养殖水体中孔雀石绿、隐色孔雀石绿、结晶紫、隐色结晶紫、亚甲基蓝残留量。

Determination of the malachite green, leucomalachite green, crystal violet, leucocrystal violet and methylene blue residues in aquaculture water

Objective To establish a method for determination and quantification of malachite green, leucomalachite green, crystal violet, leucocrystal violet and methylene blue residues in aquaculture water by high performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). Methods Water samples were extracted with hydroxylamine-p-toluenesulfonic acid solution and acetonitrile. After extracted with dichloromethane twice and dried by rotary evaporator, the extracts were dissolved with 1 mL acetonitrile and 5 mmol/L ammonium acetate (1:1, V:V). Chromatographic separation was achieved with acetonitrile and 5 mmol/L ammonium acetate (85:15, V:V) as mobile phase. Qualification was performed by electrospray ionization (ESI) in positive mode using MRM by tandem mass spectrometry. Malachite green and crystal violet were quantified by internal standard method and methylene blue was quantified by external standard method. Results Under optimized conditions, malachite green, leuco malachite green, crystal violet, leuco crystal violet in 0.025~0.600 ng/mL concentration range, methylene blue in 0.05~1 ng/mL concentration range, had good linear relationship, with correlation coefficient of more than 0.99 and the limit of detection was 0.2 ng/mL. When the standard concentration was 0.2~2.0 ng/mL, the average recoveries of malachite green, leucomalachite green, crystal violet, leucocrystal violet and methylene blue were 70%~110%, and the relative standard deviation was within 10%. Conclusion This method is simple and rapid, and has high sensitivity, low processing cost, good reproducibility and high recovery rate, which is suitable for the simultaneous detection of of malachite green, leuco malachite green, crystal violet, leuco crystal violet, methylene blue residue in aquaculture water.