基于高通量全自动固相萃取的超高效液相色谱-串联质谱法测定人尿中16种抗生素和4种β-受体激动剂

Determination of sixteen antibiotics and fourβ-agonists in human urine samples using ultra-performance liquid chromatography-tandem mass spectrometry based on high-throughput automatic solid-phase extraction

建立了基于高通量全自动固相萃取的人体尿液中大环内酯、四环素、喹诺酮、磺胺4类16种常见抗生素及特步他林、沙丁胺醇、莱克多巴胺、克伦特罗4种β-受体激动剂的超高效液相色谱-串联质谱(UPLC-MS/MS)分析方法。尿液样本于室温解冻后,取1 mL,然后加入内标、200μL乙酸铵缓冲液和20μL β-葡萄糖醛酸酶,于37℃条件下酶解过夜。采用全自动固相萃取设备对尿液中的目标物进行提取,考察了固相萃取板、淋洗液、洗脱液种类及体积,结果显示采用Oasis Prime HLB 96孔固相萃取板,以1.5 mL 10%(v/v)甲醇水溶液作为淋洗液、2 mL甲醇作为洗脱液时,20种目标物的回收率最为理想。于45℃氮吹浓缩,比较了不同氮吹条件(完全吹干、氮吹近干、氮吹至1 mL和洗脱液中加水作为保护剂)下目标物的回收率。结果表明,在洗脱液中加入水作为保护剂时,绝对回收率最为理想。研究优化了色谱分离条件,结果显示,以HSS T3(100 mm×3.0 mm, 1.8μm)作为分析柱,0.1%(v/v)甲酸水溶液-0.1%甲酸(v/v)乙腈溶液作为流动相,以0.3 mL/min流速梯度洗脱时,分离效果最好。比较了不同比例甲醇水溶液及初始流动相作为进样溶剂时的出峰情况,发现在30%(v/v)甲醇水溶液里目标物的峰形和信噪比最为理想。该方法标准曲线拟合度良好(相关系数>0.997),方法检出限为0.02~0.12 ng/mL,定量限为0.06~0.41 ng/mL。在0.25、2.5和12.5 ng/mL加标水平下,回收率为81.7%~120.0%(除四环素外),日内精密度和日间精密度(n=6)分别为1.1%~11.0%和1.2%~13.0%。基质效应考察结果表明,采取同位素内标校正后所有目标物均为弱基质效应。为考察该方法的正确度,采用BCR-503(含沙丁胺醇和克伦特罗)及内部质控样进行实验,结果显示沙丁胺醇和克伦特罗的测定值均在参考范围内,20种目标物在两种浓度内部质控样的7次测定浓度平均值范围分别为0.44~0.59 ng/mL(0.5 ng/mL)和1.72~2.16 ng/mL(2.0 ng/mL)。该方法采用自动化样品前处理设备结合96孔固相萃取板,有效提高了检测效率,具有操作简单、灵敏度高、回收率好、基质效应弱等优点,可满足人体尿液中16种抗生素和4种β-受体激动剂的同时测定需求。该研究为人体尿液中抗生素和β-受体激动剂的监测、暴露特征及健康风险评估提供了方法支撑。

An analytical method combining high-throughput automatic solid-phase extraction with ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)was developed to determine 16 antibiotics(macrolides,tetracyclines,quinolones,and sulfonamides)and 4β-agonists(terbutaline,salbutamol,ractopamine,and clenbuterol)in human urine samples.After thawing at room temperature,1 mL of urine was sampled and the internal standard was added,followed by the addition of 200μL ammonium acetate buffer and 20μLβ-glucuronidase,and the mixture was incubated at 37℃overnight.Automatic solid-phase extraction was used to extract the target compounds from the urine samples,and the recoveries were compared using different solid-phase extraction 96-well plates(PRiME MCX,Sep-Pak C 18,PRiME HLB),types and volumes of rinse solutions and eluents.Satisfactory recoveries of the 20 target compounds were obtained using the Oasis PRiME HLB 96-well plate,with 1.5 mL 10%(v/v)methanol aqueous solution and 2.0 mL methanol as the rinse solution and eluent,respectively.The eluent was concentrated under nitrogen gas at 45℃,and the recoveries of the target compounds were compared under different conditions(completely or almost dry,drying to 1 mL,and adding water as a protective agent),and the recovery rate was optimal when water was added as a protective agent.In this study,two types of analytical columns(ACQUITY BEH C 18 and ACQUITY HSS T 3)and different gradient elution procedures and mobile phases were compared.The optimal chromatographic effect was realized using an HSS T 3 column(100 mm×3.0 mm,1.8μm)and 0.1%(v/v)formic acid aqueous solution-0.1%(v/v)formic acid in acetonitrile as the mobile phase in gradient elution at a flow rate of 0.3 mL/min.Comparing the peaks observed using different proportions of methanol aqueous solution and the initial mobile phase as the injection solvent revealed that 30%(v/v)methanol aqueous solution was the optimal solution in terms of peak shape and signal-to-noise ratio.MS was conducted using positive electrospray ionization(ESI+)in multiple reaction monitoring(MRM)mode,and the MS parameters were optimized,including the curtain(CUR)and collision gases(CAD).The standard curve obtained using this method exhibited a good linearity(correlation coefficient>0.997),and the respective limits of detection and quantification were 0.02-0.12 ng/mL and 0.06-0.41 ng/mL.At spiked levels of 0.25,2.5,and 12.5 ng/mL,the recoveries were in the range of 81.7%-120.0%(except that of tetracycline),the intra-and inter-day RSDs(n=6)were 1.1%-11.0%and 1.2%-13.0%,respectively.Azithromycin,trimethoprim,terbutaline,salbutamol,ractopamine,and clenbuterol displayed moderate matrix effects,but all targets exhibited weak matrix effects after correction using the isotope internal standard.To evaluate the accuracy of this method,BCR-503(containing salbutamol and clenbuterol)and internal quality control samples were used and the concentrations of salbutamol and clenbuterol were within the reference ranges.Additionally,the mean concentrations of the 20 target compounds of two different internal quality control samples after 7 measurements were in the ranges of 0.44-0.59 ng/mL(0.5 ng/mL)and 1.72-2.16 ng/mL(2.0 ng/mL),respectively,which were satisfactory.In this study,the analytical method employed automatic sample pretreatment with a 96-well solid-phase extraction plate,and the detection efficiency was considerably improved.This method displays the advantages of simple operation,ideal recovery,a high sensitivity and weak matrix effect,which satisfies the requirements for the simultaneous determination of 16 antibiotics and 4β-agonists in human urine samples.This study provides a crucial method for use in monitoring antibiotics andβ-agonists in human urine and studying their exposure characteristics and health risks.