自动固相萃取-超高效液相色谱-串联质谱法同时测定暂养水中45种抗生素药物的含量

Simultaneous Determination of 45 Antibiotic Drugs in Temporary Aquaculture Water by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry with Automatic Solid Phase Extraction

提出了自动固相萃取-超高效液相色谱-串联质谱法(SPE-UPLC-MS/MS)同时测定水产品暂养水中45种抗生素药物含量的方法,包括4种四环素类、3种氯霉素类、15种喹诺酮类、17种磺胺类、4种呋喃类、孔雀石绿及隐色孔雀石绿。取40.0 mL暂养水,与10 mL乙二胺四乙酸二钠(Na_(2)EDTA)-Mcllvaine缓冲溶液混匀,加入0.05 mL混合内标溶液(其中氘代喹诺酮类、氘代磺胺类的质量浓度为1.00 mg·L^(-1),氘代孔雀石绿、氘代隐色孔雀石绿、氘代氯霉素类的质量浓度为100μg·L^(-1))。上述溶液用0.1 mol·L^(-1)盐酸溶液调节至pH 4,过HLB固相萃取柱(预先用3 mL甲醇、3 mL水活化),以2 mL水淋洗,用6 mL甲醇洗脱,收集洗脱液,于40℃氮吹至近干,用1.00 mL 20%(体积分数)甲醇溶液溶解残渣,经0.22μm滤膜过滤。滤液采用Agilent Eclipse Plus C_(18)RRHD色谱柱分离,以不同体积比的甲醇和0.1%(体积分数)甲酸溶液混合液为流动相进行梯度洗脱,以多反应监测(MRM)模式进行定性与定量分析。结果表明,氯霉素类和孔雀石绿工作曲线的线性范围为0.200~20.0μg·L^(-1),测定下限为5.0 ng·L^(-1);其他抗生素药物工作曲线的线性范围为1.00~100μg·L^(-1),测定下限为25 ng·L^(-1)。按照标准加入法进行回收试验,回收率为61.6%~115%,测定值的相对标准偏差(n=6)均小于9.0%。

A method for simultaneous determination of 45 antibiotic drugs in temporary aquaculture water,including 4 tetracyclines,3 chloramphenicols,15 quinolones,17 sulfonamides,4 furanes,malachite green and leucomalachite green,was proposed by ultra-high performance liquid chromatography tandem mass spectrometry with automatic solid phase extraction.40.O mL of temporary aquaculture water was taken and mixed well with 10 mL of Naz EDTA-Mellvaine buffer solution.The mixed internal standard(0.05 mL)was added,with 1.00 mg L^(-1)of deuterated quinolones and sulfonamides,and 100μg·L^(-1)of deuterated malachite green,leucomalachite green and chloramphenicols.The above solution was adjusted to pH 4 with o.1 mol·L^(-1)hydrochloric acid solution,and the solution was passed through HLB solid phase extraction column,which was pre-activated with 3 mL of methanol and 3 mL of water.Elution was carried out with 6 mL of methanol after rinsing with 2 mL of water,and the eluate was collected and blown to near dryness by nitrogen at 4o C.The residue was reconstituted in 1.00 mL of 20%(volume fraction)methanol solution,and the solution was passed through 0.2μm filter membrane:The filtrate was separated on Agilent Eclipse Plus Cis RRHD column,with the mixtures of methanol and 0.1%(volume fraction)formic acid solution at different volume ratios as the mobile phase for gradient elution,and multiple reaction monitoring(MRM)mode for the qualitative and quantitative analysis.As shown by the results,linear range of working curves for chloramphenicols and malachite green was 0.200-20.0μg·L^(-1),with lower limits of determination of 5.0 ng·L^(-1),while linear range of working curves for other antibiotic drugs was 1.00-100μg·L^(-1),with lower limits of determination of 25 ng·L^(-1).Test for recovery was made by standard addition method,giving results in the range of 61.6%-115%,with RSDs(n=6)of the determined values less than 9.0%.