人尿中31种抗生素快速检测方法的建立及应用

Establishment and application of a rapid detection method for 31 antibiotics in human urine

目的基于自动化移液工作站和超高效液相色谱-三重四级杆串联质谱(ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry,UHPLC-QqQ-MS/MS),建立同时测定人体尿液中31种抗生素的方法,准确、快速地测定尿液中多种抗生素含量。方法根据样本基质特点和目标物质理化性质,对仪器分析参数和前处理条件进行了优化。取1.0 ml尿样于96孔样本收集盘中,加入乙酸铵缓冲液和β-葡萄糖醛苷酶于37℃水浴过夜。采用Oasis HLB 96孔固相萃取板对样本进行富集萃取,洗脱液氮吹浓缩后用30%甲醇溶液定容,ACQUITY UPLC HSS T3色谱柱分离,多反应监测模式下测定,采用内标法定量。结果31种抗生素在0.2~20.0μg/L的浓度范围内显示出良好的线性关系(r^(2)>0.990),方法检出限和定量限(limit of quantification,LOQ)范围分别为0.03~0.25μg/L和0.09~0.83μg/L。在3个不同浓度水平下,加标回收率范围为65.7%~115.4%,相对标准偏差(relative standard deviations,RSDs)为0.9%~17.4%。应用该方法对50份尿样进行检测,共检出13种抗生素,其中萘啶酸、磺胺二甲唑、强力霉素和红霉素检出率均>50%,浓度范围<LOQ~6.78μg/L。结论该方法实现了样本前处理过程的自动化,可同时检测多种抗生素指标,具有操作简便、回收率高和检出限低等优点,适合大批量人尿抗生素的检测。

Objective An ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry(UHPLC-QqQ-MS/MS)method for simultaneous determination of 31 antibiotics in human urine was established based on an automated pipetting workstation,to accurately and rapidly determine the content of multiple antibiotics in human urine.Methods According to the characteristics of the sample matrix and the physicochemical properties of target compounds,the instrumental analysis parameters and pretreatment conditions were optimized.A 1.0 ml urine sample was taken in a 96-well sample collection plate,added with ammonium acetate buffer andβ-glucuronidase at 37℃overnight.The sample was enriched and extracted using an Oasis HLB 96-well solidphase extraction plate,followed by nitrogen blow concentration of the eluent,and then reconstituted with a 30%methanol solution.The ACQUITY UPLC HSS T3 chromatographic column was used for separation,and the multiple reaction monitoring mode was used for detection.The quantification was performed using the internal standard.Results The results showed that the 31 antibiotics had good linear relationships within the concentration range of 0.2-20.0μg/L(r^(2)>0.990).The method detection and quantification limits ranged from 0.03 to 0.25μg/L and 0.09 to 0.83μg/L,respectively.The spiked recovery rates at the three different concentration levels ranged from 65.7%to 115.4%,with relative standard deviations(RSDs)ranging from 0.9%to 17.4%.When applied to the analysis of 50 urine samples,a total of 13 antibiotics were detected,among which the detection rates of nalidixic acid,sulfathiazole,doxycycline,and erythromycin were all greater than 50%,with concentration range<limits of quantification to 6.78μg/L.Conclusions This method automates the sample pretreatment process,enabling simultaneous detection of multiple antibiotic indicators,has the advantages of simple operation,high recovery rates,and low detection limits,and is suitable for detecting large quantities of human urine antibiotics.